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Sample records for tenella derived tlr

  1. A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

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    Daniel M Appledorn

    2010-03-01

    Full Text Available Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+ and CD8(- T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

  2. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

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    Yin, Guangwen; Qin, Mei [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Liu, Xianyong [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Suo, Jingxia; Tang, Xinming; Tao, Geru [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Han, Qian [Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 (United States); Suo, Xun [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Wu, Wenxue, E-mail: labboard@126.com [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China)

    2013-10-25

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

  3. Tetraspanin-3 regulates protective immunity against Eimera tenella infection following immunization with dendritic cell-derived exosomes

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    The effects of immunization with dendritic cell (DC) exosomes, which had been incubated or non-incubated with an anti-tetraspanin-3 (Tspan-3) blocking antibody (Ab), were studied using an experimental model of Eimeria tenella avian coccidiosis. Purified exosomes from cecal tonsil and splenic DCs exp...

  4. Pam2 lipopeptides systemically increase myeloid-derived suppressor cells through TLR2 signaling

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    Maruyama, Akira; Shime, Hiroaki, E-mail: shime@med.hokudai.ac.jp; Takeda, Yohei; Azuma, Masahiro; Matsumoto, Misako; Seya, Tsukasa, E-mail: seya-tu@pop.med.hokudai.ac.jp

    2015-02-13

    Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that exhibit potent immunosuppressive activity. They are increased in tumor-bearing hosts and contribute to tumor development. Toll-like receptors (TLRs) on MDSCs may modulate the tumor-supporting properties of MDSCs through pattern-recognition. Pam2 lipopeptides represented by Pam2CSK4 serve as a TLR2 agonist to exert anti-tumor function by dendritic cell (DC)-priming that leads to NK cell activation and cytotoxic T cell proliferation. On the other hand, TLR2 enhances tumor cell progression/invasion by activating tumor-infiltrating macrophages. How MDSCs respond to TLR2 agonists has not yet been determined. In this study, we found intravenous administration of Pam2CSK4 systemically up-regulated the frequency of MDSCs in EG7 tumor-bearing mice. The frequency of tumor-infiltrating MDSCs was accordingly increased in response to Pam2CSK4. MDSCs were not increased by Pam2CSK4 stimuli in TLR2 knockout (KO) mice. Adoptive transfer experiments using CFSE-labeled MDSCs revealed that the TLR2-positive MDSCs survived long in tumor-bearing mice in response to Pam2CSK4 treatment. Since the increased MDSC population sustained immune-suppressive properties, our study suggests that Pam2CSK4-triggered TLR2 activation enhances the MDSC potential and suppress antitumor immune response in tumor microenvironment. - Highlights: • Pam2CSK4 administration induces systemic accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • TLR2 is essential for Pam2CSK4-induced accumulation of CD11b{sup +}Gr1{sup +} MDSCs. • Pam2CSK4 supports survival of CD11b{sup +}Gr1{sup +} MDSCs in vivo.

  5. Induction of protective immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina infections using DC-derived exosomes

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    This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs) in the absence of soluble Ag. Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from E...

  6. The rhoptry proteome of Eimeria tenella sporozoites

    KAUST Repository

    Oakes, Richard D.

    2013-02-01

    Proteins derived from the rhoptry secretory organelles are crucial for the invasion and survival of apicomplexan parasites within host cells. The rhoptries are club-shaped organelles that contain two distinct subpopulations of proteins that localise to separate compartments of the organelle. Proteins from the neck region (rhoptry neck proteins, RON) are secreted early in invasion and a subset of these is critical for the formation and function of the moving junction between parasite and host membranes. Proteins from the bulb compartment (rhoptry protein, ROP) are released later, into the nascent parasitophorous vacuole where they have a role in modifying the vacuolar environment, and into the host cell where they act as key determinants of virulence through their ability to interact with host cell signalling pathways, causing an array of downstream effects. In this paper we present the results of an extensive proteomics analysis of the rhoptry organelles from the coccidian parasite, Eimeria tenella, which is a highly pathogenic parasite of the domestic chicken causing severe caecal coccidiosis. Several different classes of rhoptry protein have been identified. First are the RON proteins that have varying degrees of similarity to proteins of Toxoplasma gondii and Neospora caninum. For some RON families, E. tenella expresses more than one gene product and many of the individual RON proteins are differentially expressed between the sporozoite and merozoite developmental stages. The E. tenella sporozoite rhoptry expresses only a limited repertoire of proteins with homology to known ROP proteins from other coccidia, including just two secreted ROP kinases, both of which appear to be equipped for catalytic activity. Finally, a large number of hitherto undescribed proteins that map to the sporozoite rhoptry are identified, many of which have orthologous proteins encoded within the genomes of T. gondii and N. caninum. © 2012 .

  7. Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7.

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    Coleman, Leon G; Zou, Jian; Crews, Fulton T

    2017-01-25

    Toll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration. TLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles. We report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs. Our results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.

  8. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

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    Herzmann, Nicole; Salamon, Achim [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany); Fiedler, Tomas [Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany)

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  9. Candida albicans induces selective development of macrophages and monocyte derived dendritic cells by a TLR2 dependent signalling.

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    Alberto Yáñez

    Full Text Available As TLRs are expressed by haematopoietic stem and progenitor cells (HSPCs, these receptors may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that in in vitro defined conditions inactivated yeasts and hyphae of Candida albicans induce HSPCs proliferation and differentiation towards the myeloid lineage by a TLR2/MyD88 dependent pathway. In this work, we showed that C. albicans invasive infection with a low virulence strain results in a rapid expansion of HSPCs (identified as LKS cells: Lin(- c-Kit(+ Sca-1(+ IL-7Rα(-, that reach the maximum at day 3 post-infection. This in vivo expansion of LKS cells in TLR2(-/- mice was delayed until day 7 post- infection. Candidiasis was, as expected, accompanied by an increase in granulopoiesis and decreased lymphopoiesis in the bone marrow. These changes were more pronounced in TLR2(-/- mice correlating with their higher fungal burden. Accordingly, emigration of Ly6C(high monocytes and neutrophils to spleen was increased in TLR2(-/- mice, although the increase in macrophages and inflammatory macrophages was completely dependent on TLR2. Similarly, we detected for the first time, in the spleen of C. albicans infected control mice, a newly generated population of dendritic cells that have the phenotype of monocyte derived dendritic cells (moDCs that were not generated in TLR2(-/- infected mice. In addition, C. albicans signalling through TLR2/MyD88 and Dectin-1 promotes in vitro the differentiation of Lin(- cells towards moDCs that secrete TNF-α and are able to kill the microorganism. Therefore, our results indicate that during infection C. albicans can directly stimulate progenitor cells through TLR2 and Dectin-1 to generate newly formed inflammatory macrophages and moDCs that may fulfill an essential role in defense mechanisms against the pathogen.

  10. Hybrid Cells Derived from Human Breast Cancer Cells and Human Breast Epithelial Cells Exhibit Differential TLR4 and TLR9 Signaling

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    Songül Tosun

    2016-05-01

    Full Text Available TLRs are important receptors of cells of the innate immune system since they recognize various structurally conserved molecular patterns of different pathogens as well as endogenous ligands. In cancer, the role of TLRs is still controversial due to findings that both regression and progression of tumors could depend on TLR signaling. In the present study, M13SV1-EGFP-Neo human breast epithelial cells, MDA-MB-435-Hyg human breast cancer cells and two hybrids M13MDA435-1 and -3 were investigated for TLR4 and TLR9 expression and signaling. RT-PCR data revealed that LPS and CpG-ODN induced the expression of pro-inflammatory cytokines, like IFN-β, TNF-α, IL-1β and IL-6 in hybrid cells, but not parental cells. Interestingly, validation of RT-PCR data by Western blot showed detectable protein levels solely after LPS stimulation, suggesting that regulatory mechanisms are also controlled by TLR signaling. Analysis of pAKT and pERK1/2 levels upon LPS and CpG-ODN stimulation revealed a differential phosphorylation pattern in all cells. Finally, the migratory behavior of the cells was investigated showing that both LPS and CpG-ODN potently blocked the locomotory activity of the hybrid cells in a dose-dependent manner. In summary, hybrid cells exhibit differential TLR4 and TLR9 signaling.

  11. Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs induce inflammatory responses in avian macrophages.

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    Yock-Ping Chow

    Full Text Available At least 19 glycosylphosphatidylinositol (GPI-anchored surface antigens (SAGs are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown.Ten SAGs, belonging to two previously defined multigene families (A and B, were expressed as soluble recombinant (r fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1β mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity.In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12 may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages.

  12. Immunomodulatory properties of Alternanthera tenella Colla aqueous extracts in mice

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    Guerra R.N.M.; Pereira H.-A.W.; Silveira L.M.S.; Olea R.S.G.

    2003-01-01

    Plants from the genus Alternanthera are thought to possess antimicrobial and antiviral properties. In Brazilian folk medicine, the aqueous extract of A. tenella Colla is used for its anti-inflammatory activity. The present study investigated the immunomodulatory property of A. tenella extract by evaluating the antibody production in male albino Swiss mice weighing 20-25 g (10 per group). The animals received standard laboratory diet and water ad libitum. The effect of A. tenella extract (5 an...

  13. Systemic inflammation and liver injury following hemorrhagic shock and peripheral tissue trauma involve functional TLR9 signaling on bone marrow-derived cells and parenchymal cells.

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    Gill, Roop; Ruan, Xiangcai; Menzel, Christoph L; Namkoong, Seung; Loughran, Patricia; Hackam, David J; Billiar, Timothy R

    2011-02-01

    Hemorrhagic shock due to trauma (HS/T) induces an inflammatory response that can contribute to end-organ injury. The pathways involved in the initiation and propagation of HS/T-induced inflammation are incompletely understood. Here, we hypothesized that the DNA sensor TLR9 would have a role in inflammatory signaling after HS/T. Using mice expressing a nonfunctional, mutant form of TLR9, we identified a role of TLR9 in driving the initial cytokine response and liver damage in a model of hemorrhagic shock and bilateral femur fracture. Circulating DNA levels were found to correlate with the degree of tissue damage. Experiments using chimeric mice show that TLR9 on both bone marrow-derived cells and parenchymal cells are important for the TLR9-mediated liver and tissue damage, as well as systemic inflammation after HS/T. These data suggest that release of DNA may be a driver of the inflammatory response to severe injury as well as a marker of the extent of tissue damage. One of the sensors of DNA in the setting of HS/T seems to be TLR9.

  14. Neuroprotective effect of a novel gastrodin derivative against ischemic brain injury: involvement of peroxiredoxin and TLR4 signaling inhibition

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    Mao, Xiao-Na; Zhou, Hong-Jing; Yang, Xiao-Jia; Zhao, Li-Xue; Kuang, Xi; Chen, Chu; Liu, Dong-Ling; Du, Jun-Rong

    2017-01-01

    The inhibition of extracellular inflammatory peroxiredoxin (Prx) signaling appears to be a potential therapeutic strategy for neuroinflammatory injury after acute ischemic stroke. Gastrodin (Gas) is a phenolic glycoside that is used for the treatment of cerebral ischemia, accompanied by regulation of the autoimmune inflammatory response. The present study investigated the neuroprotective effects of Gas and its derivative, Gas-D, with a focus on the potential mechanism associated with inflammatory Prx–Toll-like receptor 4 (TLR4) signaling. Gas-D significantly inhibited Prx1-, Prx2-, and Prx4-induced inflammatory responses in RAW264.7 macrophages and H2O2-mediated oxidative injury in SH-SY5Y nerve cells. In rats, intraperitoneal Gas-D administration 10 h after reperfusion following 2-h middle cerebral artery occlusion (MCAO) ameliorated neurological deficits, brain infarction, and neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, T-lymphocyte invasion, and lipid peroxidation. Delayed Gas-D treatment significantly inhibited postischemic Prx1/2/4 expression and spillage, TLR4 signaling activation, and inflammatory mediator production. In contrast, Gas had no significant effects in either cell model or in MCAO rats under the same conditions. These results indicate that Gas-D may be a drug candidate with an extended therapeutic time window that blocks inflammatory responses and attenuates the expression and secretome of inflammatory Prxs in acute ischemic stroke. PMID:29207618

  15. Lignin-rich Enzyme Lignin (LREL), a Cellulase-treated Lignin-Carbohydrate Derived from Plants, Activates Myeloid Dendritic Cells via Toll-like Receptor 4 (TLR4)

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    Tsuji, Ryohei; Koizumi, Hideki; Aoki, Dan; Watanabe, Yuta; Sugihara, Yoshihiko; Matsushita, Yasuyuki; Fukushima, Kazuhiko; Fujiwara, Daisuke

    2015-01-01

    Lignin-carbohydrates, one of the major cell wall components, are believed to be the structures that form chemical linkage between lignin and cell wall polysaccharides. Due to the molecular complexity of lignin-containing substances, their isolation and the assignment of their biological activities have so far remained a difficult task. Here, we extracted two lignin-containing carbohydrates, lignin-rich enzyme lignin (LREL) and pure enzyme lignin (PEL), from barley husk and demonstrated that they act as immune stimulators of dendritic cells (DCs), which are particularly important in linking innate and adaptive immunity. Thioacidolysis, acid hydrolysis, and mild alkali hydrolysis of both LREL and PEL revealed that their immunostimulatory activities depended on the lignin structure and/or content, neutral sugar content (especially the characteristic distribution of galactose and mannose), and presence of an ester bond. Furthermore, we showed that the immunostimulatory potency of the lignin-carbohydrate depended on its molecular weight and degree of polymerization. We also demonstrated that the LREL-induced activation of DCs was mediated via TLR4. Thus, LREL-induced increases in the expression levels of several cell surface marker proteins, production of inflammatory cytokines IL-12p40 and TNF-α, and activation and nuclear translocation of transcription factors, as was observed in the WT DCs, were completely abrogated in DCs derived from the TLR4−/− mice but not in DCs derived from the TLR2−/−, TLR7−/−, and TLR9−/− mice. We further demonstrated that LRELs isolated from other plant tissues also activated DCs. These immunostimulatory activities of lignin-carbohydrates, extracted from edible plant tissues, could have potential relevance in anti-infectious immunity and vaccine adjuvants. PMID:25548274

  16. Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngenic lymphocyte-derived DNA immunization.

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    Chen, Min; Zhang, Weijuan; Xu, Wei; Zhang, Feng; Xiong, Sidong

    2011-07-01

    We previously established a systemic lupus erythematosus (SLE) animal model in non-susceptible BALB/c mice by immunizing with activated syngeneic lymphocyte-derived DNA (ALD-DNA), manifested by high level of anti-double-stranded DNA (dsDNA) antibodies (Abs), proteinuria, glomerular deposition of immune complex and glomerulonephritis. The production of anti-dsDNA Abs is closely related with the renal inflammation and damage in this model. However, recognition of ALD-DNA and its signaling pathway within antigen-presenting cells (APC) remains not fully clarified. Herein, in this study, Toll-like receptor 9 (TLR9), a well-known pattern-recognition receptor for dsDNA with CpG motif, was found to be dynamically up-regulated in B cells during the process of the SLE disease. Knockdown of TLR9 by short interfering RNA (siRNA) in B cells in vitro and in vivo reduced the production of anti-dsDNA antibody and consequently ameliorated the SLE syndrome in mice while the affinity and isotype of the antibody remained the same. Our results implied that TLR9 signaling of B cells might play an important role in the production of anti-dsDNA Abs triggered by auto dsDNA, which would extend our understanding of TLR9 immune recognition in the pathogenesis of SLE disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Genetic variation in TLR10 is not associated with chronic Q fever, despite the inhibitory effect of TLR10 on Coxiella burnetii-induced cytokines in vitro

    NARCIS (Netherlands)

    Ammerdorffer, Anne; Stappers, Mark H.T.; Oosting, Marije; Roest, Hendrik Jan

    2016-01-01

    Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10

  18. Attenuation of LPS-induced inflammation by ICT, a derivate of icariin, via inhibition of the CD14/TLR4 signaling pathway in human monocytes.

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    Wu, Jinfeng; Zhou, Junmin; Chen, Xianghong; Fortenbery, Nicole; Eksioglu, Erika A; Wei, Sheng; Dong, Jingcheng

    2012-01-01

    To evaluate the anti-inflammatory potential of ICT in LPS stimulated human innate immune cells. 3, 5, 7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3- methylbutyl)-flavone (ICT) is a novel derivative of icariin, the major active ingredient of Herba Epimedii, an herb used in traditional Chinese medicine. We previously demonstrated its anti-inflammatory potential in a murine macrophage cell line as well as in mouse models. We measured TNF-α production by ELISA, TLR4/CD14 expression by flow cytometry, and NF-κB and MAPK activation by western blot all in LPS-stimulated PBMC, human monocytes, or THP-1 cells after treatment with ICT. ICT inhibited LPS-induced TNF-α production in THP-1 cells, PBMCs and human monocytes in a dose-dependent manner. ICT treatment resulted in down-regulation of the expression of CD14/TLR4 and attenuated NF-κB and MAPK activation induced by LPS. We illustrate the anti-inflammatory property of ICT in human immune cells, especially in monocytes. These effects were mediated, at least partially, via inhibition of the CD14/TLR4 signaling pathway. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. CXCL4 Exposure Potentiates TLR-Driven Polarization of Human Monocyte-Derived Dendritic Cells and Increases Stimulation of T Cells.

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    Silva-Cardoso, Sandra C; Affandi, Alsya J; Spel, Lotte; Cossu, Marta; van Roon, Joel A G; Boes, Marianne; Radstake, Timothy R D J

    2017-07-01

    Chemokines have been shown to play immune-modulatory functions unrelated to steering cell migration. CXCL4 is a chemokine abundantly produced by activated platelets and immune cells. Increased levels of circulating CXCL4 are associated with immune-mediated conditions, including systemic sclerosis. Considering the central role of dendritic cells (DCs) in immune activation, in this article we addressed the effect of CXCL4 on the phenotype and function of monocyte-derived DCs (moDCs). To this end, we compared innate and adaptive immune responses of moDCs with those that were differentiated in the presence of CXCL4. Already prior to TLR- or Ag-specific stimulation, CXCL4-moDCs displayed a more matured phenotype. We found that CXCL4 exposure can sensitize moDCs for TLR-ligand responsiveness, as illustrated by a dramatic upregulation of CD83, CD86, and MHC class I in response to TLR3 and TLR7/8-agonists. Also, we observed a markedly increased secretion of IL-12 and TNF-α by CXCL4-moDCs exclusively upon stimulation with polyinosinic-polycytidylic acid, R848, and CL075 ligands. Next, we analyzed the effect of CXCL4 in modulating DC-mediated T cell activation. CXCL4-moDCs strongly potentiated proliferation of autologous CD4(+) T cells and CD8(+) T cells and production of IFN-γ and IL-4, in an Ag-independent manner. Although the internalization of Ag was comparable to that of moDCs, Ag processing by CXCL4-moDCs was impaired. Yet, these cells were more potent at stimulating Ag-specific CD8(+) T cell responses. Together our data support that increased levels of circulating CXCL4 may contribute to immune dysregulation through the modulation of DC differentiation. Copyright © 2017 by The American Association of Immunologists, Inc.

  20. Immunomodulatory properties of Alternanthera tenella Colla aqueous extracts in mice

    Directory of Open Access Journals (Sweden)

    R.N.M. Guerra

    2003-09-01

    Full Text Available Plants from the genus Alternanthera are thought to possess antimicrobial and antiviral properties. In Brazilian folk medicine, the aqueous extract of A. tenella Colla is used for its anti-inflammatory activity. The present study investigated the immunomodulatory property of A. tenella extract by evaluating the antibody production in male albino Swiss mice weighing 20-25 g (10 per group. The animals received standard laboratory diet and water ad libitum. The effect of A. tenella extract (5 and 50 mg/kg, ip was evaluated in mice immunized with sheep red blood cells (SRBC 10%, ip as T-dependent antigen, or in mice stimulated with mitogens (10 µg, Escherichia coli lipopolysaccharide, LPS, ip. The same doses (5 and 50 mg/kg, ip of A. tenella extract were also tested for antitumor activity, using the Ehrlich ascites carcinoma as model. The results showed that 50 mg/kg A. tenella extract ip significantly enhanced IgM (64% and IgG2a (50% antibody production in mice treated with LPS mitogen. The same dose had no effect on IgM-specific response, whereas the 5 mg/kg treatment caused a statiscally significant reduction of anti-SRBC IgM-specific antibodies (82%. The aqueous extract of A. tenella (50 mg/kg increased the life span (from 16 ± 1 to 25 ± 1 days and decreased the number of viable tumor cells (59% in mice with Ehrlich ascites carcinoma. The present findings are significant for the development of alternative, inexpensive and perhaps even safer strategies for cancer treatment.

  1. Combined TLR2 and TLR4 ligation in the context of bacterial or helminth extracts in human monocyte derived dendritic cells: Molecular correlates for Th1/Th2 polarization

    NARCIS (Netherlands)

    Riet, E. van; Everts, B.; Retra, K.; Phylipsen, M.; Hellemond, J.J. van; Tielens, A.G.M.; Kleij, D. van der; Hartgers, F.C.; Yazdanbakhsh, M.

    2009-01-01

    Background: Recognition of pathogens by dendritic cells (DCs) through interaction with pattern recognition receptors, including Toll like receptors (TLR), is crucial for the initiation of appropriate polarized T helper (Th) cell responses. Yet, the characteristics and differences in molecular

  2. Combined TLR2 and TLR4 ligation in the context of bacterial or helminth extracts in human monocyte derived dendritic cells: Molecular correlates for Th1/Th2 polarization

    NARCIS (Netherlands)

    E. van Riet (Elly); B. Everts (Bart); K. Retra (Kim); M. Phylipsen (Marion); J.J. van Hellemond (Jaap); A.G.M. Tielens (Aloysius); D. van der Kleij (Desiree); F.C. Hartgers (Franca); M. Yazdanbakhsh (Maria)

    2009-01-01

    textabstractBackground: Recognition of pathogens by dendritic cells (DCs) through interaction with pattern recognition receptors, including Toll like receptors (TLR), is crucial for the initiation of appropriate polarized T helper (Th) cell responses. Yet, the characteristics and differences in

  3. Single-stranded DNA oligonucleotides inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques

    NARCIS (Netherlands)

    Sköld, A.; Hasan, M.; Vargas, L.; Saidi, H.; Bosquet, N.; Grand, R. le; Smith, C.I.; Spetz, A.L.

    2012-01-01

    TLR3 is a key receptor for recognition of double-stranded RNA and initiation of immune responses against viral infections. However, hyperactive responses can have adverse effects, such as virus-induced asthma. Strategies to prevent TLR3-mediated pathology are therefore desired. We investigated the

  4. Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing.

    Science.gov (United States)

    Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

    2016-01-01

    TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2-/- mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

  5. Genetic variation in TLR10 is not associated with chronic Q fever, despite the inhibitory effect of TLR10 on Coxiella burnetii-induced cytokines in vitro.

    Science.gov (United States)

    Ammerdorffer, Anne; Stappers, Mark H T; Oosting, Marije; Schoffelen, Teske; Hagenaars, Julia C J P; Bleeker-Rovers, Chantal P; Wegdam-Blans, Marjolijn C; Wever, Peter C; Roest, Hendrik-Jan; van de Vosse, Esther; Netea, Mihai G; Sprong, Tom; Joosten, Leo A B

    2016-01-01

    Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. CD4+CD25+ Treg induction by an HSP60-derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent.

    Science.gov (United States)

    Wang, Xuefeng; Zhou, Sha; Chi, Ying; Wen, Xiaoyun; Hoellwarth, Jason; He, Lei; Liu, Feng; Wu, Calvin; Dhesi, Shawn; Zhao, Jiaqing; Hu, Wei; Su, Chuan

    2009-11-01

    Chronic schistosome infection results in the suppression of host immune responses, allowing long-term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4(+)CD25(+) T cells by Schistosoma japonicum HSP60 (SjHSP60)-derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4(+)CD25(+) T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4(+)CD25(+) T-cell populations. Release of IL-10 and TGF-beta by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1-induced CD4(+)CD25(+) T cells inhibited delayed-type hypersensitivity in BALB/c mice. Additionally, SJMHE1-treated APC were tolerogenic and induced CD4(+) cells to differentiate into suppressive CD4(+)CD25(+) Treg. Furthermore, our data support a role for TLR2 in SJMHE1-mediated CD4(+)CD25(+) Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum-host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.

  7. Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

    Science.gov (United States)

    Han, Young Woo; Choi, Jin Young; Uyangaa, Erdenebelig; Kim, Seong Bum; Kim, Jin Hyoung; Kim, Bum Seok; Kim, Koanhoi; Eo, Seong Kug

    2014-09-01

    Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/-) and TLR4(-/-) mice, i.e. TLR3(-/-) mice were highly susceptible to JE, whereas TLR4(-/-) mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+)Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/-) mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/-) myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+) and CD8(+) T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+)Ly-6C(high) monocytes) and CD4(+)Foxp3(+) Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components

  8. Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

    Directory of Open Access Journals (Sweden)

    Young Woo Han

    2014-09-01

    Full Text Available Japanese encephalitis (JE is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9. Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/- and TLR4(-/- mice, i.e. TLR3(-/- mice were highly susceptible to JE, whereas TLR4(-/- mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/- mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5, transcription factors (IRF-3, IRF-7, and IFN-dependent (PKR, Oas1, Mx and independent ISGs (ISG49, ISG54, ISG56 by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/- myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+ and CD8(+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+Ly-6C(high monocytes and CD4(+Foxp3(+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components

  9. TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

    Science.gov (United States)

    Zambirinis, Constantinos P; Levie, Elliot; Nguy, Susanna; Avanzi, Antonina; Barilla, Rocky; Xu, Yijie; Seifert, Lena; Daley, Donnele; Greco, Stephanie H; Deutsch, Michael; Jonnadula, Saikiran; Torres-Hernandez, Alejandro; Tippens, Daniel; Pushalkar, Smruti; Eisenthal, Andrew; Saxena, Deepak; Ahn, Jiyoung; Hajdu, Cristina; Engle, Dannielle D; Tuveson, David; Miller, George

    2015-11-16

    Modulation of Toll-like receptor (TLR) signaling can have protective or protumorigenic effects on oncogenesis depending on the cancer subtype and on specific inflammatory elements within the tumor milieu. We found that TLR9 is widely expressed early during the course of pancreatic transformation and that TLR9 ligands are ubiquitous within the tumor microenvironment. TLR9 ligation markedly accelerates oncogenesis, whereas TLR9 deletion is protective. We show that TLR9 activation has distinct effects on the epithelial, inflammatory, and fibrogenic cellular subsets in pancreatic carcinoma and plays a central role in cross talk between these compartments. Specifically, TLR9 activation can induce proinflammatory signaling in transformed epithelial cells, but does not elicit oncogene expression or cancer cell proliferation. Conversely, TLR9 ligation induces pancreatic stellate cells (PSCs) to become fibrogenic and secrete chemokines that promote epithelial cell proliferation. TLR9-activated PSCs mediate their protumorigenic effects on the epithelial compartment via CCL11. Additionally, TLR9 has immune-suppressive effects in the tumor microenvironment (TME) via induction of regulatory T cell recruitment and myeloid-derived suppressor cell proliferation. Collectively, our work shows that TLR9 has protumorigenic effects in pancreatic carcinoma which are distinct from its influence in extrapancreatic malignancies and from the mechanistic effects of other TLRs on pancreatic oncogenesis. © 2015 Zambirinis et al.

  10. Eimeria tenella: in vitro development in irradiated bovine kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Crane, M.St.J.; Schmatz, D.M.; Stevens, S.; Habbersett, M.C.; Murray, P.K. (Merck Sharp and Dohme Research Labs., Rahway, NJ (USA))

    1984-06-01

    The initial infection and first-generation development of Eimeria tenella was quantified using a cloned MDBK (Madin-Darby Bovine Kidney) cell line, irradiated with gamma radiation prior to infection, as the host cell. Irradiated cell cultures were found to be more susceptible to infection and had a greater capacity to support parasite development than non-irradiated cultures. It was suggested that the larger proportion of cells in the G/sub 2/ phase of the cell cycle, the larger individual cell size and the inhibition of cell division in the irradiated cultures were all factors contributing to the increased susceptibility to infection and capacity to support parasite growth and development. The application of this technique (host cell irradiation) to the cultivation of other intracellular, protozoan parasites is discussed.

  11. Activated human neonatal CD8+ T cells are subject to immunomodulation by direct TLR2 or TLR5 stimulation.

    LENUS (Irish Health Repository)

    McCarron, Mark

    2012-02-01

    In conditions of optimal priming, the neonate possesses competency to mount quantitatively adult-like responses. Vaccine formulations containing sufficiently potent adjuvants may overcome the neonate\\'s natural tendency for immunosuppression and provoke a similarly robust immune response. TLR expression on T cells represents the possibility of directly enhancing T cell immunity. We examined the ex vivo responsiveness of highly purified human cord blood-derived CD8(+) T cells to direct TLR ligation by a repertoire of TLR agonists. In concert with TCR stimulation, only Pam(3)Cys (palmitoyl-3-Cys-Ser-(Lys)(4)) and flagellin monomers significantly enhanced proliferation, CD25(+) expression, IL-2, IFN-gamma, TNF-alpha, and intracellular granzyme B expression. TLR2 and TLR5 mRNA was detected in the CD8(+) T cells. Blocking studies confirmed that the increase in IFN-gamma production was by the direct triggering of surface TLR2 or TLR5. The simultaneous exposure of CD8(+) T cells to both TLR agonists had an additive effect on IFN-gamma production. These data suggest that a combination of the two TLR ligands would be a potent T cell adjuvant. This may represent a new approach to TLR agonist-based adjuvant design for future human neonatal vaccination strategies requiring a CD8(+) component.

  12. Salicylic acid on antioxidant activity and betacyan in production from leaves of Alternanthera tenella

    Directory of Open Access Journals (Sweden)

    Isabel Rodrigues-Brandão

    2014-10-01

    Full Text Available This research investigates effects of salicylic acid (an abiotic elicitor on the antioxidant activity and betacyan production from leaves of Alternanthera tenella cultured in vitro was evaluated. Plants were grown in a liquid MS medium and vermiculite substrate. After 35 days salicylic acid was added to the medium. Content of betacyanins, total phenols and flavonoids and non-enzymatic antioxidant capacity were determined in leaves of A. tenella after 0, 12, 36 and 48h of treatment. After 36h, concentration of betacyanins and total phenols increased. On the other hand, the increase of the treatment time caused a slight decrease in total flavonoids and reduced the DPPH free radical activity. As result the antioxidant activity of the leaves of A. tenella is promoted by salicylic acid and can be attributed to the increase in betacyanin content, which are compounds with recognized antioxidant action.

  13. Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

    Science.gov (United States)

    Zhai, Qi; Huang, Bing; Dong, Hui; Zhao, Qiping; Zhu, Shunhai; Liang, Siting; Li, Sha; Yang, Sihan; Han, Hongyu

    2016-01-01

    The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

  14. Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella.

    Directory of Open Access Journals (Sweden)

    Qi Zhai

    Full Text Available The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE based on the expressed sequence tag (EST. The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites. The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.

  15. Effects of Separate and Concomitant TLR-2 and TLR-4 Activation in Peripheral Blood Mononuclear Cells of Newborn and Adult Horses.

    Directory of Open Access Journals (Sweden)

    Johannes Cornelis Vendrig

    Full Text Available Deficient innate and adaptive immune responses cause newborn mammals to be more susceptible to bacterial infections than adult individuals. Toll-like receptors (TLRs are known to play a pivotal role in bacterial recognition and subsequent immune responses. Several studies have indicated that activation of certain TLRs, in particular TLR-2, can result in suppression of inflammatory pathology. In this study, we isolated peripheral blood mononuclear cells (PBMCs from adult and newborn horses to investigate the influence of TLR-2 activation on the inflammatory response mediated by TLR-4. Data were analysed in a Bayesian hierarchical linear regression model, accounting for variation between horses. In general, cytokine responses were lower in PBMCs derived from foals compared with PBMCs from adult horses. Whereas in foal PBMCs expression of TLR-2, TLR-4, and TLR-9 was not influenced by separate and concomitant TLR-2 and TLR-4 activation, in adult horse PBMCs, both TLR ligands caused significant up-regulation of TLR-2 and down-regulation of TLR-9. Moreover, in adult horse PBMCs, interleukin-10 protein production and mRNA expression increased significantly following concomitant TLR-2 and TLR-4 activation (compared with sole TLR-4 activation. In foal PBMCs, this effect was not observed. In both adult and foal PBMCs, the lipopolysaccharide-induced pro-inflammatory response was not influenced by pre-incubation and co-stimulation with the specific TLR-2 ligand Pam3-Cys-Ser-Lys4. This indicates that the published data on other species cannot be translated directly to the horse, and stresses the necessity to confirm results obtained in other species in target animals. Future research should aim to identify other methods or substances that enhance TLR functionality and bacterial defence in foals, thereby lowering susceptibility to life-threatening infections during the first period of life.

  16. Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

    Science.gov (United States)

    Krüger, Anne; Oldenburg, Marina; Chebrolu, Chiranjeevi; Beisser, Daniela; Kolter, Julia; Sigmund, Anna M; Steinmann, Jörg; Schäfer, Simon; Hochrein, Hubertus; Rahmann, Sven; Wagner, Hermann; Henneke, Philipp; Hornung, Veit; Buer, Jan; Kirschning, Carsten J

    2015-12-01

    Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  17. Dietary Curcuma longa enhances resistance against Eimeria maxima and Eimeria tenella infections in chickens

    Science.gov (United States)

    The effects of dietary supplementation with an organic extract of Curcuma longa on systemic and local immune responses to experimental Eimeria maxima and E. tenella infections were evaluated in commercial broiler chickens. Infected chickens given the C. longa-containing diet had increased body weig...

  18. In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals

    NARCIS (Netherlands)

    Burt, S.A.|info:eu-repo/dai/nl/140114432; Tersteeg-Zijderveld, M.H.G.; Jongerius-Gortemaker, B.G.M.; Vervelde, L.|info:eu-repo/dai/nl/134923391; Vernooij, J.C.M.|info:eu-repo/dai/nl/340304596

    2013-01-01

    Resistance to coccidiostats and possible future restrictions on their use raise the need for alternative methods of reducing coccidiosis in poultry. The aim of this study was to evaluate the effect of selected phytochemicals on Eimeria tenella sporozoite invasion in vitro. Four phytochemicals were

  19. Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

    Science.gov (United States)

    Guo, Yiqi; Audry, Magali; Ciancanelli, Michael; Alsina, Laia; Azevedo, Joana; Herman, Melina; Anguiano, Esperanza; Sancho-Shimizu, Vanessa; Lorenzo, Lazaro; Pauwels, Elodie; Philippe, Paul Bastard; Pérez de Diego, Rebeca; Cardon, Annabelle; Vogt, Guillaume; Picard, Capucine; Andrianirina, Zafitsara Zo; Rozenberg, Flore; Lebon, Pierre; Plancoulaine, Sabine; Tardieu, Marc; Valérie Doireau; Jouanguy, Emmanuelle; Chaussabel, Damien; Geissmann, Frederic; Abel, Laurent

    2011-01-01

    Autosomal dominant TLR3 deficiency has been identified as a genetic etiology of childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE). This defect is partial, as it results in impaired, but not abolished induction of IFN-β and -λ in fibroblasts in response to TLR3 stimulation. The apparently normal resistance of these patients to other infections, viral illnesses in particular, may thus result from residual TLR3 responses. We report here an autosomal recessive form of complete TLR3 deficiency in a young man who developed HSE in childhood but remained normally resistant to other infections. This patient is compound heterozygous for two loss-of-function TLR3 alleles, resulting in an absence of response to TLR3 activation by polyinosinic-polycytidylic acid (poly(I:C)) and related agonists in his fibroblasts. Moreover, upon infection of the patient’s fibroblasts with HSV-1, the impairment of IFN-β and -λ production resulted in high levels of viral replication and cell death. In contrast, the patient’s peripheral blood mononuclear cells responded normally to poly(I:C) and to all viruses tested, including HSV-1. Consistently, various TLR3-deficient leukocytes from the patient, including CD14+ and/or CD16+ monocytes, plasmacytoid dendritic cells, and in vitro derived monocyte-derived macrophages, responded normally to both poly(I:C) and HSV-1, with the induction of antiviral IFN production. These findings identify a new genetic etiology for childhood HSE, indicating that TLR3-mediated immunity is essential for protective immunity to HSV-1 in the central nervous system (CNS) during primary infection in childhood, in at least some patients. They also indicate that human TLR3 is largely redundant for responses to double-stranded RNA and HSV-1 in various leukocytes, probably accounting for the redundancy of TLR3 for host defense against viruses, including HSV-1, outside the CNS. PMID:21911422

  20. Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation

    Directory of Open Access Journals (Sweden)

    Lindenmaier Werner

    2010-04-01

    Full Text Available Abstract Background Toll-like receptor (TLR 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (αT2ib which was generated from an antagonistic monoclonal antibody (mAb towards human and murine TLR2 (T2.5 to inhibit the function of TLR2. αT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser3 amino acid sequence. Results Coexpression of αT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with αT2ib indicated interaction of αT2ib with its cognate antigen within cells. αT2ib inhibited NF-κB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding αT2ib into HEK293 cells demonstrated high efficiency of the TLR2-αT2ib interaction. The αT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV-αT2ib. Transduction with AdVαT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM. Furthermore, TLR2 activation dependent TNFα mRNA accumulation, as well

  1. The effects of Ostertagia occidentalis somatic antigens on ovine TLR2 and TLR4 expression

    Directory of Open Access Journals (Sweden)

    Hassan BORJI

    2015-10-01

    Full Text Available Background: Recognition of helminth-derived pathogen associated molecular patterns (PAMPs by pattern recognition receptors (PRRs, including toll like recep­tors (TLRs is the first step towards initiating anti–helminth immune re­sponses.Methods: Using somatic antigens of Ostertagia occidentalis, an important abomasal parasite of ruminants, the expression of ovine TLR2 and TLR4 in peripheral blood mononuclear cells (PBMCs was analyzed by real-time quatitative reverse-transcrip­tion polymerase chain reaction (qRT-PCR. Somatic antigens of O. occidentalis were prepared to stimulate ovine PBMCs in a time and dose dependent manner.Results: A high expression of TLR2 and TLR4 was observed in PBMCs cultured with somatic antigens of the parasites specially when PBMCs were cultured with 100 µg/ml of somatic antigens and incubated for 2h. Up-regulation of TLR2 expres­sion was more pronounced and evident in our study.Conclsusion: Somatic antigens of O. occidentalis have immunostimulatory and domi­nant role on peripheral immune cells. This study provide for the first time evidence of induction of TLRs in ovine PBMCs by somatic antigen of O. occidentalis

  2. Anticoccidial efficacy of toltrazuril and halofuginone against Eimeria tenella infection in broiler chickens in Egypt.

    Science.gov (United States)

    Ramadan, A; Abo el-Sooud, K; el-Bahy, M M

    1997-01-01

    The anticoccidial activities of toltrazuril and halofuginone against Eimeria tenella were tested in broiler chickens. Comparisons were made between ummedicated infected and uninfected control birds in addition to infected groups given either toltrazuril at 37.5, 75 and 150 ppm in the drinking water, or halofuginone at 1.5, 3 and 6 ppm in the feed. Both drugs were highly efficacious against E tenella. Treatment improved the bodyweight gain and survival percentage in comparison with the unmedicated, infected group. Intestinal lesions, faecal and oocyst scores and oocyst shedding in dropping were significantly reduced by both drugs. Toltrazuril gave better protection than halofuginone; 75 and 150 ppm toltrazuril in drinking water gave good protection when administered four and five days after inoculation.

  3. Quantitative proteomic studies in resistance mechanisms of Eimeria tenella against polyether ionophores.

    Science.gov (United States)

    Thabet, Ahmed; Honscha, Walther; Daugschies, Arwid; Bangoura, Berit

    2017-05-01

    Polyether ionophores are widely used to treat and control coccidiosis in chickens. Widespread use of anticoccidials resulted in worldwide resistance. Mechanisms of resistance development and expansion are complex and poorly understood. Relative proteomic quantification using LC-MS/MS was used to compare sensitive reference strains (Ref-1, Ref-2) with putatively resistant and moderately sensitive field strains (FS-R, FS-mS) of Eimeria tenella after isotopic labelling with tandem mass tags (TMT). Ninety-seven proteins were identified, and 25 of them were regulated. Actin was significantly upregulated in resistant strains in comparison with their sensitive counterparts. On the other hand, microneme protein (MIC4) was downregulated in resistant strains. Optimization of labelling E. tenella sporozoites by TMT might identify further proteins that play a role in the obvious complex mechanism leading to resistance against Monensin.

  4. Assessment of humoral immunity to Eimeria tenella sporozoites in chickens by ELISA

    Directory of Open Access Journals (Sweden)

    S. Saravanan

    2014-07-01

    Full Text Available Aim: To assess the humoral immune response of Eimeria tenella sporozoites in broiler chickens by a developed enzyme linked immunosorbent assay (ELISA and the efficacy in terms of bodyweight, lesion score and oocysts excretion in immunized broilers. Materials and Methods: Purified live E. tenella sporozoites were administered subcutaneously in neck region of broiler chickens in the early life (first week at different concentrations. The potency of the sporozoite vaccine as assessed by IgG levels and the performance in immunized broilers as assessed by body weight, lesion score and oocysts excretion in faeces after challenge with 10, 000 live E. tenella oocysts at 49 days of age were evaluated. Results: The chickens of group (T4 immunized with 20 µg of antigen on day 6 showed an increase in IgG levels (0.161±0.004 two weeks post immunization (PI peaking (0.399± 0.016 at 5 weeks PI. The mean weekly weight gain (g after challenge, at 56 days of age was high in T4 (148±4.751 g with a low mean lesion score (2.5±0.22 and mean oocyst output (x103 oocytes per gram (OPG in faeces (100.3± 45.72 when compared to unimmunised infected controls. Conclusion: An early but partial immune response against caecal coccidiosis could be achieved by immunization with E. tenella specific sporozoites in chickens of less than a week old. Moreover, the performance of immunized chickens as indicated by weight gain, lesion score and oocyst output was found to be superior to the unimmunized infected controls.

  5. The Role of TLR2, TLR4, and TLR9 in the Pathogenesis of Atherosclerosis

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    Mohsin H. K. Roshan

    2016-01-01

    Full Text Available Toll-like receptors (TLRs are key players in the pathogenesis of inflammatory conditions including coronary arterial disease (CAD. They are expressed by a variety of immune cells where they recognize pathogen-associated molecular patterns (PAMPs. TLRs recruit adaptor molecules, including myeloid differentiation primary response protein (MYD88 and TIRF-related adaptor protein (TRAM, to mediate activation of MAPKs and NF-kappa B pathways. They are associated with the development of CAD through various mechanisms. TLR4 is expressed in lipid-rich and atherosclerotic plaques. In TLR2−/− and TLR4−/− mice, atherosclerosis-associated inflammation was diminished. Moreover, TLR2 and TLR4 may induce expression of Wnt5a in advanced staged atheromatous plaque leading to activation of the inflammatory processes. TLR9 is activated by CpG motifs in nucleic acids and have been implicated in macrophage activation and the uptake of oxLDL from the circulation. Furthermore, TLR9 also stimulates interferon-α (INF-α secretion and increases cytotoxic activity of CD4+ T-cells towards coronary artery tunica media smooth muscle cells. This review outlines the pathophysiological role of TLR2, TLR4, and TLR9 in atherosclerosis, focusing on evidence from animal models of the disease.

  6. Anticoccidial activity of hydrochloric acid (HCl against Eimeria tenella in broiler chickens

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    Rao Z Abbas

    2011-05-01

    Full Text Available The present study was planned to evaluate the anticoccidial activity of the different concentrations of the HCl against Eimeria tenella infection in broiler chickens in comparison with the amprolium anticoccidial. For this purpose, a total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations of HCl (1000ppm, 2000ppm and 3000ppm and amproilum (at the dose rate of 125ppm were given to the experimental groups in drinking water from 10 to 19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. At the 12th day of age, all the groups were inoculated orally with 75,000 sporulated oocysts except non infected non medicated control. Anticoccidial activity was evaluated on the basis of performance (weight gain, feed conversion ratio and pathogenic (oocyst score, lesion score and mortality %age parameters. Among HCl medicated groups, the maximum anticoccidial effect was seen in the group medicated with 1000ppm HCl followed by 2000ppm and 3000ppm HCl medicated groups. Amprolium and 1000ppm HCl were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella challenge. In summary, the lower doses of HCl have the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. It is therefore suggested that further studies should be carried out to determine the possible minimum safe levels of HCl with least toxic effects to be used as anticoccidial.

  7. Anticoccidial effects of Aloe secundiflora leaf extract against Eimeria tenella in broiler chicken.

    Science.gov (United States)

    Kaingu, Fredrick; Liu, Dandan; Wang, Lele; Tao, Jianping; Waihenya, Rebecca; Kutima, Helen

    2017-04-01

    Anticoccidial effects of Aloe secundiflora crude leaf extract was tested in broiler chickens following oral infection with Eimeria tenella. Sixty 22-day-old birds were divided into six groups of ten birds each. Three treatment groups A, B, and C were fed with the extract (100, 250, and 500 mg/day, respectively) mixed in feed for 10 days, and three control groups: group D (drug control) administered 300 mg/l of sulfachloropyrazine sodium soluble powder in drinking water for 5 days, group E (infected/non-medicated positive control), and group F (uninfected/non-medicated negative control). Except for group F, all groups were orally inoculated with 75,000 sporulated oocysts of E. tenella. The effects of the extract on E. tenella infection were evaluated by severity of bloody diarrhea, body weight (BW) gain, oocyst output, and lesion score. No bird in the treated groups died of coccidiosis, and severity of bloody diarrhea was milder than in the positive control group. BW gains in the treated groups were significantly higher than in group E (p Aloe dosage. The findings of this study suggest that Aloe secundiflora extract presents an alternative anticoccidial agent for the control of avian coccidiosis.

  8. Gametogenesis and fecundity of Acropora tenella (Brook 1892) in a mesophotic coral ecosystem in Okinawa, Japan

    Science.gov (United States)

    Prasetia, Rian; Sinniger, Frederic; Harii, Saki

    2016-03-01

    Mesophotic coral ecosystems (below 30-40 m depth) host a large diversity of zooxanthellate coral communities and may play an important role in the ecology and conservation of coral reefs. Investigating the reproductive biology of mesophotic corals is important to understand their life history traits. Despite an increase in research on mesophotic corals in the last decade, their reproductive biology is still poorly understood. Here, gametogenesis and fecundity of the Indo-Pacific mesophotic coral , Acropora tenella, were examined in an upper mesophotic reef (40 m depth) in Okinawa, Japan for the first time. Acropora tenella is a hermaphrodite with a single annual gametogenic cycle, and both oogenesis and spermatogenesis occurring for 11-12 and 5-6 months, respectively. Timing of spawning of this species was similar to other shallow Acropora spp. in the region. However, colonies had longer gametogenic cycles and less synchronous gamete maturation compared to shallow acroporids with spawning extended over consecutive months. Both the polyp fecundity (number of eggs per polyp) and gonad index (defined as the number of eggs per square centimeter) of A. tenella were lower than most acroporids. Our findings contribute to understanding of the life history of corals on mesophotic reefs and suggest that the reproductive biology of upper mesophotic corals is similar to that of shallow-water corals.

  9. Monitoring of local CD8 β-expressing cell populations during Eimeria tenella infection of naïve and immune chickens

    DEFF Research Database (Denmark)

    Wattrang, Eva; Thebo, Per; Lunden, Anna

    2016-01-01

    The purpose of this study was to monitor abundance and activation of local CD8β-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor (TCR) γ...

  10. Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella.

    Directory of Open Access Journals (Sweden)

    Ziwen Wang

    Full Text Available Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4, a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4 inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The

  11. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  12. A novel recombinant BCG vaccine encoding eimeria tenella rhomboid and chicken IL-2 induces protective immunity against coccidiosis.

    Science.gov (United States)

    Wang, Qiuyue; Chen, Lifeng; Li, Jianhua; Zheng, Jun; Cai, Ning; Gong, Pengtao; Li, Shuhong; Li, He; Zhang, Xichen

    2014-06-01

    A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4(+) and CD8(+) cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

  13. Profil Saraf Nitrergik Sekum Ayam Pedaging yang Diinfeksi Eimeria tenella (THE PROFILE OF NEORAL NITRERGIC IN THE COECUM OF BROILER INFECTED WITH Emeria tenella

    Directory of Open Access Journals (Sweden)

    Amelia Hana

    2016-01-01

    Full Text Available Neurotransmitter found in the enteric nervous system that plays an important role in a variety ofenteropathies, including inflammatory bowel disease. Alteration of nitrergic neurons has been reported to bedependent on the manner by which inflammation is caused. This study was performed to determine theprofile of neural nitrergic with Nicotinamide Adenine Dinucleotide Phosphate-diaphorase (NADPH-dstaining in the cecum of broiler infected by Eimeria tenella (E. tenella. Thirty six health free of coccidiosisbroiler one-day old chickens (DOC used in this study. All of broiler adapted for 7 days with food and drinkgiven ad libitum then divided into three groups, each group consisted of 12 broilers. The 1st group wascontrol only given orally 1.0 ml of distilled water/ heads , while 2nd group was infected with a single dose of5x103 oocysts/ head orally, and 3rd group was infected with a single dose of 2x104 oocysts/ head orally. Onday 7 post infection, all of chickens were fasted for 12 hours then were euthanized and cecum was taken.Lesion score of cecum was assessed. Furthermore, tissue of the coecum was prepared for hisochemicalusing NADPH-d staining to determine morphology, and morhometric of nitrergic neurons. The resultshown that cecum lesion score of group I is 0, group II is +2, group III is +3. Neuron density in the cecum ofgroup I is 2794.96+4.65 neuron/cm2, group II is 4930.36+63.73 neuron/cm2 and group III is 7892.31+44.97 neuron/cm2. Neuron density of group II and III increased signifcantly (p<001 than group I. Nitrergic neurondesity of group III increased significantly (p<0.05 than that of group II. It was concluded that the infectionof E.tenella led to increase the number of neutrl mienteric nitrergic of the cecum. The higher lesion score ofcecum led to increase the number of mienteric nitrergic neuron.

  14. The Role of TLR4 in Chemotherapy-Driven Metastasis.

    Science.gov (United States)

    Ran, Sophia

    2015-06-15

    Tumor resistance to cytotoxic drugs is one of the main obstacles to successful cancer therapy. Emerging evidence suggests that chemoresistance is promoted by substances released from dead and damaged cells that activate the host repair program orchestrated by Toll-like receptor-4 (TLR4). TLR4 is often overexpressed in malignant and tumor-infiltrating immune cells. In addition to endogenous ligands released by therapy-induced tumor destruction, TLR4 is directly activated by paclitaxel, one of the most commonly used chemotherapeutic drugs against various human cancers. TLR4 activation promotes local and systemic inflammation, leading to induction of multiple circuits that create a regenerative environment favoring local recurrence and metastasis. Of particular importance is TLR4-mediated recruitment of endothelial progenitors derived from immature myeloid cells. These cells play a major role in rebuilding tumor-associated lymphatic and blood vessels, thereby promoting lymphatic and hematogenous metastasis. The latter is further enhanced by the premetastatic niche generated by mobilization of myeloid provascular cells to distant organs. This review summarizes the recent evidence demonstrating that paclitaxel and other clinically used anticancer drugs actively induce metastasis even while shrinking the primary tumor. Better understanding of the mechanisms underlying TLR4-dependent chemotherapy-driven metastasis might be the key to overcoming challenges of cancer eradication. ©2015 American Association for Cancer Research.

  15. Structural Basis of TLR5-Flagellin Recognition and Signaling

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Sung-il; Kurnasov, Oleg; Natarajan, Venkatesh; Hong, Minsun; Gudkov, Andrei V.; Osterman, Andrei L.; Wilson, Ian A. (Scripps); (Roswell); (Sanford-Burnham)

    2012-03-01

    Toll-like receptor 5 (TLR5) binding to bacterial flagellin activates signaling through the transcription factor NF-{kappa}B and triggers an innate immune response to the invading pathogen. To elucidate the structural basis and mechanistic implications of TLR5-flagellin recognition, we determined the crystal structure of zebrafish TLR5 (as a variable lymphocyte receptor hybrid protein) in complex with the D1/D2/D3 fragment of Salmonella flagellin, FliC, at 2.47 angstrom resolution. TLR5 interacts primarily with the three helices of the FliC D1 domain using its lateral side. Two TLR5-FliC 1:1 heterodimers assemble into a 2:2 tail-to-tail signaling complex that is stabilized by quaternary contacts of the FliC D1 domain with the convex surface of the opposing TLR5. The proposed signaling mechanism is supported by structure-guided mutagenesis and deletion analyses on CBLB502, a therapeutic protein derived from FliC.

  16. Eimeria tenella oocyst shedding and output in cecal or fecal contents following experimental challenge in broilers.

    Science.gov (United States)

    Jordan, A; Caldwell, D J; Klein, J; Coppedge, J; Pohl, S; Fitz-Coy, S; Lee, J T

    2011-05-01

    Two experiments were conducted to determine whether Eimeria tenella oocyst output in cecal and fecal contents, lesion development, and performance characteristics were affected by ad libitum versus restricted feeding and challenge level. In experiment 1, 144 Cobb 500 males were placed in battery cages with 6 chicks/pen. On d 20, half of the battery pens were placed on feed restriction and all broilers were orally challenged with Eimeria tenella oocysts at one of 3 challenge levels (0, 5,000, or 20,000 sporulated oocysts). Cecal and fecal material were collected separately from d 4 postchallenge through d 10 postchallenge for oocysts output (oocysts shed/g) determination. Six days postchallenge, 3 broilers from each pen were removed and subjected to necropsy for lesion assessment. In experiment 2, 96 Cobb 500 males were placed in identical battery pens with 8 chicks/pen. On d 14, restricted feeding was initiated and broilers were challenged with Eimeria tenella oocysts at one of 3 challenge levels (1,000, 5,000, or 20,000 oocysts). Twenty-four hour collections of cecal and fecal material were obtained separately from d 4 postchallenge through d 10 postchallenge for oocysts per gram and total output determination. Six days postchallenge, 4 broilers from each pen were removed and subjected to necropsy for lesion assessment. In both experiments, BW gain was not affected by challenge dose in either the ad libitum-fed or restrict-fed broilers. Increased lesion development was observed with increasing challenge levels, and oocyst shedding peaked between d 7 and 9 postchallenge in both experiments. Oocyst concentration was higher in cecal droppings compared with fecal material throughout peak shedding; however, total oocyst output for the challenge period was similar between fecal material and cecal droppings.

  17. Potassium 4-(hydroxymethyl)-benzenosulfonate: a novel metabolite isolated from the marine red alga Bostrychia tenella (Rhodomelaceae, ceramiales); 4-(Hidroximetil)-Benzenossulfonato de potassio: metabolito inedito isolado da alga marinha Bostrychia tenella (Rhodomelaceae, ceramiales)

    Energy Technology Data Exchange (ETDEWEB)

    Felicio, Rafael de; Debonsi, Hosana Maria [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Ciencias Farmaceuticas. Dept. de Fisica e Quimica]. E-mail: hosana@fcfrp.usp.br; Yokoya, Nair Sumie [Instituto de Botanica de Sao Paulo, SP (Brazil). Secao de Ficologia

    2008-07-01

    Chemical investigation of the dichloromethane/methanol extract of the marine alga Bostrychia tenella has led to the isolation of two aromatic compounds, the new sulfate metabolite potassium 4-(hydroxymethyl)-benzenosulfonate (1) and the compound 1-methoxyphenethyl alcohol (2), described previously as a synthetic product. Their structures were determined by spectroscopic methods including NMR, MS, IR and by comparison with literature data. (author)

  18. TLR4 plays a crucial role in MSC-induced inhibition of NK cell function

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Ying [No. 307 Hospital of the Chinese People' s Liberation Army, Beijing (China); Liu, Jin; Liu, Yang; Qin, Yaru [Beijing Institute of Radiation Medicine, Beijing (China); Luo, Qun [No. 307 Hospital of the Chinese People' s Liberation Army, Beijing (China); Wang, Quanli, E-mail: 13691110351@163.com [No. 307 Hospital of the Chinese People' s Liberation Army, Beijing (China); Duan, Haifeng, E-mail: duanhf0720@163.com [Beijing Institute of Radiation Medicine, Beijing (China)

    2015-08-21

    Mesenchymal stem cells (MSC) are a kind of stromal cell within the tumor microenvironment. In our research, MSC derived from acute myeloid leukemia patients' bone marrow (AML-MSC) and lung cancer tissues (LC-MSC) as well as normal bone marrow-derived MSC (BM-MSC) cultured in conditioned medium of HeLa cells were found to have higher expressions of Toll-like receptor (TLR4) mRNA compared with BM-MSC. The sorted TLR4-positive MSC (TLR4+ MSC) differed in cytokine (interleukin-6, interleukin-8, and monocyte chemoattractant protein-1) secretion from those of unsorted MSC. MSC was reported to inhibit natural killer (NK) cell proliferation and function. In this research, we confirmed that TLR4+ MSC aggravate this suppression. Furthermore, when TLR4 in the sorted cells were stimulated by LPS or following blocked by antibody, the suppression on NK cell proliferation and cytotoxicity were more intensive or recovered respectively. Compared to unsorted MSC, NKG2D receptor expression on NK cells were also inhibited by TLR4+ MSC. These findings suggest that activation of TLR4 pathway is important for TLR4+ MSC and MSC to obstruct anti-tumor immunity by inhibiting NK cell function, which may provide a potential stroma-targeted tumor therapy. - Highlights: • TLR4+ MSC inhibit NK cell proliferation in vivo and in vitro. • TLR4+ MSC inhibit NKG2D expression on NK cells and NK cell cytotoxicity. • The distinguished cytokine expression of TLR4+ MSC may contribute to the inhibition on NK cell function.

  19. Anticoccidial efficacy testing: In vitro Eimeria tenella assays as replacement for animal experiments.

    Science.gov (United States)

    Thabet, Ahmed; Zhang, Runhui; Alnassan, Alaa-Aldin; Daugschies, Arwid; Bangoura, Berit

    2017-01-15

    Availability of an accurate in vitro assay is a crucial demand to determine sensitivity of Eimeria spp. field strains toward anticoccidials routinely. In this study we tested in vitro models of Eimeria tenella using various polyether ionophores (monensin, salinomycin, maduramicin, and lasalocid) and toltrazuril. Minimum inhibitory concentrations (MIC 95 , MIC 50/95 ) for the tested anticoccidials were defined based on a susceptible reference (Houghton strain), Ref-1. In vitro sporozoite invasion inhibition assay (SIA) and reproduction inhibition assay (RIA) were applied on sensitive laboratory (Ref-1 and Ref-2) and field (FS-1, FS-2, and FS-3) strains to calculate percent of inhibition under exposure of these strains to the various anticoccidials (%I SIA and%I RIA, respectively). The in vitro data were related to oocyst excretion, lesion scores, performance, and global resistance indices (GI) assessed in experimentally infected chickens. Polyether ionophores applied in the RIA were highly effective at MIC 95 against Ref-1 and Ref-2 (%I RIA ≥95%). In contrast, all tested field strains displayed reduced to low efficacy (%I RIA animal model (p89%) against all strains used in this study. However, adjusted GI (GI adj ) for toltrazuril-treated groups exhibited differences between reference and field strains which might indicate varying sensitivity. RIA is a suitable in vitro tool to detect sensitivity of E. tenella towards polyether ionophores, and may thus help to reduce, replace, or refine use of animal experimentation for in vivo sensitivity assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene.

    Science.gov (United States)

    Bogado, A L G; Martins, G F; Sasse, J P; Guimarães, J da S; Garcia, J L

    2017-03-15

    Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.

  1. TLR4 plays a crucial role in MSC-induced inhibition of NK cell function.

    Science.gov (United States)

    Lu, Ying; Liu, Jin; Liu, Yang; Qin, Yaru; Luo, Qun; Wang, Quanli; Duan, Haifeng

    2015-08-21

    Mesenchymal stem cells (MSC) are a kind of stromal cell within the tumor microenvironment. In our research, MSC derived from acute myeloid leukemia patients' bone marrow (AML-MSC) and lung cancer tissues (LC-MSC) as well as normal bone marrow-derived MSC (BM-MSC) cultured in conditioned medium of HeLa cells were found to have higher expressions of Toll-like receptor (TLR4) mRNA compared with BM-MSC. The sorted TLR4-positive MSC (TLR4+ MSC) differed in cytokine (interleukin-6, interleukin-8, and monocyte chemoattractant protein-1) secretion from those of unsorted MSC. MSC was reported to inhibit natural killer (NK) cell proliferation and function. In this research, we confirmed that TLR4+ MSC aggravate this suppression. Furthermore, when TLR4 in the sorted cells were stimulated by LPS or following blocked by antibody, the suppression on NK cell proliferation and cytotoxicity were more intensive or recovered respectively. Compared to unsorted MSC, NKG2D receptor expression on NK cells were also inhibited by TLR4+ MSC. These findings suggest that activation of TLR4 pathway is important for TLR4+ MSC and MSC to obstruct anti-tumor immunity by inhibiting NK cell function, which may provide a potential stroma-targeted tumor therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Enhancement of the Immunostimulatory Activity of a TLR7 Ligand by Conjugation to Polysaccharides.

    Science.gov (United States)

    Shinchi, Hiroyuki; Crain, Brian; Yao, Shiyin; Chan, Michael; Zhang, Shannon S; Ahmadiiveli, Alast; Suda, Yasuo; Hayashi, Tomoko; Cottam, Howard B; Carson, Dennis A

    2015-08-19

    Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands

  3. Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection.

    Science.gov (United States)

    Song, Xiaokai; Xu, Lixin; Yan, Ruofeng; Huang, Xinmei; Li, Xiangrui

    2015-08-15

    The search for effective vaccines against chicken coccidiosis remains a challenge because of the complex organisms with multiple life cycle stages of Eimeria. Combination of T-cell epitopes from different stages of Eimeria life cycle could be an optimal strategy to overcome the antigen complexity of the parasite. In this study, 4 fragments with concentrated T-cell epitopes from the sporozoite antigen SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic expression vector pVAX1 in different forms, with or without chicken cytokines IL-2 or IFN-γ genes as genetic adjuvants, to construct multistage, multi-epitope DNA vaccines against Eimeria tenella. Transcription and expression of the multi-epitope DNA vaccines in vivo were detected by reverse transcription-PCR (RT-PCR) and Western blot. On the basis of survival rate, lesion score, body weight gain, oocyst decrease ratio and the anti-coccidial index (ACI), Animal experiments were carried out to evaluate the protective efficacy against Eimeria tenella. Results showed the constructed DNA vaccines were transcribed and translated successfully in vivo. Animal experiment showed that the multi-epitopes DNA vaccines were more effective to stimulate immune response than single fragment. Compared with the DNA vaccines composed with less T-cell epitopes, DNA vaccine pVAX1-m1-m2-s1-s2 containing 4 fragments with concentrated T-epitopes provided the highest ACI of 180.39. DNA vaccines composed of antigens from two developmental stages were more effective than the single-stage ones. Especially DNA vaccine pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39. Furthermore, cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope DNA vaccines significantly. Overall, pVAX1-m1-m2-s1-s2-IFN-γ provided the most effective protection with the ACI of 189.92. The multi-epitope DNA vaccines revealed in this study provide new candidates for Eimeria vaccine development

  4. Protection Efficacy of Multivalent Egg Yolk Immunoglobulin against Eimeria Tenella Infection in Chickens

    Directory of Open Access Journals (Sweden)

    JJ Xu

    2013-09-01

    Full Text Available Background: To control avian coccidiosis with drug-independent strategy effec­tively and safely, multivalent hyperimmune egg yolk immunoglobulin (IgY was prepared and its ability to protect against Eimeria tenella infection was evaluated.Methods: Hens were orally immunized with live oocysts of 5 species of Eimeria for six times, antibody titers in serum and yolk were monitored by indirect enzyme-linked immunosorbent assay. The specific IgY was isolated, purified and lyophi­lized. IgY powder was orally administrated as dietary supplement in newly hatched chicks at various dosages. Birds were orally challenged with 10000 sporulated oo­cysts of E. tenella at 10 days of age, weighed and killed at 8 days post challenge, and the protective effect was assessed.Results: The averge yeid of IgY was 9.2 mg/ml yolk, the antibody titer of IgY reached to 1:163840 per mg with the purity up to 98%. Chickens fed IgY resulted in reduced mortality, increased body weight gain (BWG, reduced oocyst shedding, reduced caecal lesion score and increased anti-coccidial index. In terms of BWG and caecal lesion, IgY significantly enhanced the resistance of bird at ≥ 0.05% of IgY in the diet when compared with the challenged control group (P0.05.Conclusion: Supplementing newly hatched chicks with Eimeria-specific IgY represents a promising strategy to prevent avian coccidiosis.

  5. IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis.

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    Del Cacho, Emilio; Gallego, Margarita; Lillehoj, Hyun Soon; Quílez, Joaquín; Lillehoj, Erik P; Ramo, Ana; Sánchez-Acedo, Caridad

    2014-02-26

    Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

  6. In vitro inhibition of Eimeria tenella sporozoite invasion into host cells by probiotics.

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    Hessenberger, S; Schatzmayr, G; Teichmann, K

    2016-10-15

    The aim was to study the effects of probiotics isolated from the intestinal tract of livestock animals on Eimeria tenella invasion into Madin-Darby bovine kidney (MDBK) cells in vitro. E. tenella sporozoites were purified and labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester before seeding on cell cultures, and invasion was evaluated by fluorescence microscopy. Two protocols (A and B) were used. In protocol A, Enterococcus faecium # 589 or Lactobacillus salivarius subsp. salivarius # 505 were added together with sporozoites to MDBK cell cultures and invasion was evaluated after incubation for approximately 20h. Viable, dead, or spent culture supernatants of probiotics were tested. In protocol B, viable probiotics were incubated with MDBK cells for one hour before sporozoites were added and invasion was evaluated after two more hours of incubation. Parasite invasion of viable, dead, or spent culture supernatant of E. faecium # 589 was assessed. Using protocol A, it was shown that parasite invasion was inhibited by viable (80%) or dead (75%) E. faecium # 589. While inhibition by viable L. salivarius subsp. salivarius # 505 was not valid at the highest concentration and not significant at the other test concentrations, dead cells inhibited parasite invasion up to 45%. Spent culture supernatants of both probiotics had no influence on parasite invasion. Using protocol B, it was shown that viable Bifidobacterium animalis subsp. animalis # 503, E. faecium # 497, E. faecium # 589, L. reuteri # 514, L. salivarius subsp. salivarius # 505, and Bacillus subtilis # 588 inhibited parasite invasion into MDBK cells up to 80%. Anticoccidial activity was strain-specific for E. faecium strains, and the strongest effect was shown by E. faecium # 589. Anticoccidial effects of some of the tested probiotics have already been shown in vivo, which makes them candidates to prevent coccidiosis. These findings have now been confirmed in vitro. The used parasite invasion

  7. TLR3 and TLR4 expression in healthy and diseased human endometrium

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    Kimmig Rainer

    2008-09-01

    Full Text Available Abstract Background Toll-like receptors (TLRs play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases. Methods TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3. The expression studies applied quantitative RT-PCR and immunolabelling of both proteins. Results TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3. Conclusion Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.

  8. TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes.

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    Marije Oosting

    Full Text Available After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs, such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.

  9. TLR1/TLR2 heterodimers play an important role in the recognition of Borrelia spirochetes.

    Science.gov (United States)

    Oosting, Marije; Ter Hofstede, Hadewych; Sturm, Patrick; Adema, Gosse J; Kullberg, Bart-Jan; van der Meer, Jos W M; Netea, Mihai G; Joosten, Leo A B

    2011-01-01

    After infection with Borrelia species, the risk for developing Lyme disease varies significantly between individuals. Recognition of Borrelia by the immune system is mediated by pattern recognition receptors (PRRs), such as TLRs. While TLR2 is the main recognition receptor for Borrelia spp., little is known about the role of TLR1 and TLR6, which both can form functionally active heterodimers with TLR2. Here we investigated the recognition of Borrelia by both murine and human TLR1 and TLR6. Peritoneal macrophages from TLR1- and TLR6- gene deficient mice were isolated and exposed to Borrelia. Human PBMCs were stimulated with Borrelia with or without specific TLR1 and TLR6 blocking using specific antibodies. Finally, the functional consequences of TLR polymorphisms on Borrelia-induced cytokine production were assessed. Splenocytes isolated from both TLR1-/- and TLR6-/- mice displayed a distorted Th1/Th2 cytokine balance after stimulation with B.burgdorferi, while no differences in pro-inflammatory cytokine production were observed. In contrast, blockade of TLR1 with specific neutralizing antibodies led to decreased cytokine production by human PBMCs after exposure to B.burgdorferi. Blockade of human TLR6 did not lead to suppression of cytokine production. When PBMCs from healthy individuals bearing polymorphisms in TLR1 were exposed to B.burgdorferi, a remarkably decreased in vitro cytokine production was observed in comparison to wild-type controls. TLR6 polymorphisms lead to a minor modified cytokine production. This study indicates a dominant role for TLR1/TLR2 heterodimers in the induction of the early inflammatory response by Borrelia spirochetes in humans.

  10. TLR2- and 4-independent immunomodulatory effect of high molecular weight components from Ascaris suum.

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    Favoretto, Bruna C; Silva, Sandriana R; Jacysyn, Jacqueline F; Câmara, Niels O S; Faquim-Mauro, Eliana L

    2014-03-01

    Components of high molecular-weight (PI) obtained from Ascaris suum extract down-regulate the Th1/Th2-related immune responses induced by ovalbumin (OVA)-immunization in mice. Furthermore, the PI down-modulates the ability of dendritic cells (DCs) to activate T lymphocytes by an IL-10-mediated mechanism. Here, we evaluated the role of toll like receptors 2 and 4 (TLR2 and 4) in the modulatory effect of PI on OVA-specific immune response and the PI interference on DC full activation. An inhibition of OVA-specific cellular and humoral responses were observed in wild type (WT) or in deficient in TLR2 (TLR2(-/-)) or 4 (TLR4(-/-)) mice immunized with OVA plus PI when compared with OVA-immunized mice. Low expression of class II MHC, CD40, CD80 and CD86 molecules was observed in lymph node (LN) cells from WT, TLR2(-/-) or TLR4(-/-) mice immunized with OVA plus PI compared with OVA-primed cells. We also verified that PI was able to modulate the activation of DCs derived from bone marrow of WT, TLR2(-/-) or TLR4(-/-) mice induced in vitro by agonists of TLRs, as observed by a decreased expression of class II MHC and costimulatory molecules and by low secretion of pro-inflammatory cytokines. Its effect was accompanied by IL-10 synthesis. In this sense, the modulatory effect of PI on specific-immune response and DC activation is independent of TLR2 or TLR4. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. IFNβ produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response.

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    Núñez, Nicolás Gonzalo; Andreani, Virginia; Crespo, María Inés; Nocera, David Andrés; Breser, María Laura; Morón, Gabriel; Dejager, Lien; Libert, Claude; Rivero, Virginia; Maccioni, Mariana

    2012-02-01

    Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNβ was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNβ-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response. ©2012 AACR.

  12. Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae aqueous extracts

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    Carla de Agostino Biella

    2008-09-01

    Full Text Available Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE and hot (HAE aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p. with each extract (50, 100 or 200 mg/kg. Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg. Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

  13. Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts.

    Science.gov (United States)

    Biella, Carla de Agostino; Salvador, Marcos José; Dias, Diones Aparecida; Dias-Baruffi, Marcelo; Pereira-Crott, Luciana Simon

    2008-09-01

    Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

  14. Fruit and seed biometry of cambuí (Myrciaria tenella O. Berg = Biometria de fruto e semente de cambuí (Myrciaria tenella O. Berg

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    Ana Veruska Cruz da Silva

    2012-12-01

    Full Text Available The cambuí tree (Myrciaria tenella O. Berg is native to Brazil and its fruits are harvested by extraction for fresh consumption in the state of Sergipe (Brazil. Because of the regional importance of the species, this work aims to characterize the cambuí fruits and seeds. The fruits were harvested mature from native trees in the Reserva do Caju Experimental Field, on Itaporanga d’Ajuda (Sergipe, Brazil, belonging to Embrapa Tabuleiros Costeiros. Two hundred fruits and seeds were biometrically analyzed. In addition, we evaluated the fruit, pulp, and seed color based on RHS Color Chart. Two different skin colors were found: 1. yellow [orange - red (group 32A] with yellow pulp [yellow - orange (group 17A]; and 2. purple [violet -blue (group 93A] with red pulp [red - purple (group 60A]. The seeds have only one color, green [yellow-green (group 152A] with dark stripes [Brown (group 200C]. The fruits mean diameter was 9.23 mm.fruit-1, and average width 8.50 mm.fruit-1. The seeds mean diameter was 5.34 mm.seed-1, mean width 6.52 mm.seed-1 and thickness 5.08 mm.seed-1. The cambuí is a little red fruit. The biometry had a high variability as expected, because it is a native species without any type of management.=Resumo - O cambuí (Myrciaria tenella O. Berg é nativo do Brasil e os frutos são colhidos por extrativismo para consumo in natura no estado de Sergipe (Brasil. Por causa da importância regional da espécie, objetivou-se com este trabalho caracterizar os frutos e sementes de cambuí. Os frutos foram colhidos maduros provenientes de plantas nativas do Campo Experimental Reserva do Caju, no município de Itaporanga d’Ajuda (Sergipe, Brasil pertencente a Embrapa Tabuleiros Costeiros. Foram analisados 200 frutos e sementes biometricamente. Além disso, caracterizou-se a cor da fruta, da polpa, e da semente com base na cartela de cores RHS. Foram identificados duas cores diferentes para a fruta: 1. de cor amarela [laranja - vermelho (grupo32A

  15. Temperatura e luz na germinação das sementes de apaga-fogo (Alternanthera tenella Effect of temperature and light on joyweed (Alternanthera tenella seed germination

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    R.S. Canossa

    2008-01-01

    Full Text Available A biologia de plantas daninhas tropicais ainda é, em grande parte, desconhecida, especialmente em relação aos fatos que podem ser aproveitados do ponto de vista do controle cultural. Objetivou-se com este trabalho avaliar a influência da temperatura e da luz na germinação das sementes de Alternanthera tenella. Os tratamentos consistiram em quatro temperaturas (20, 25, 30 e 35 ºC na ausência de luz e na manutenção da temperatura constante de 25ºC na presença e ausência de luz. O teste de germinação foi constituído por quatro repetições de 100 sementes para cada tratamento, colocadas para germinar em caixas plásticas do tipo gerbox e mantidas em câmaras de germinação do tipo BOD. As avaliações de germinação foram diárias e no mesmo horário, computando-se as plântulas normais, quando apresentavam radícula e folhas cotiledonares visíveis. Calcularam-se a germinação total e o índice de velocidade de germinação (IVG após o período de 34 dias, e os resultados correspondem à média de dois experimentos. Foi observado que o aumento da temperatura proporcionou aumento na porcentagem e na velocidade de germinação das sementes até 28,2 ºC, decrescendo a partir dessa temperatura. Na temperatura de 25 ºC, verificou-se que a luz causou incremento na porcentagem e na velocidade de germinação das sementes de apaga-fogo.Biology of tropical weeds is still mostly unknown, especially in relation to facts that could be used as cultural tools for weed management. The present work was aimed at evaluating the infuence of temperature and light on germination of Alternanthera tenella seeds. Treatments consisted of four constant temperature regimes (20, 25, 30 and 35 ºC in absence of light, and constant temperature of 25 ºC in the presence and absence of light. The germination test was conducted with four replicates of 100 seeds per treatment, placed in plastic boxes (gerbox and kept in BOD Germinator. Germination evaluations

  16. Synthetic Toll-Like Receptor 4 (TLR4) and TLR7 Ligands Work Additively via MyD88 To Induce Protective Antiviral Immunity in Mice.

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    Goff, Peter H; Hayashi, Tomoko; He, Wenqian; Yao, Shiyin; Cottam, Howard B; Tan, Gene S; Crain, Brian; Krammer, Florian; Messer, Karen; Pu, Minya; Carson, Dennis A; Palese, Peter; Corr, Maripat

    2017-10-01

    We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic

  17. Innate immune receptors in human airway smooth muscle cells: activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 agonists.

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    Anne Månsson Kvarnhammar

    Full Text Available BACKGROUND: Pattern-recognition receptors (PRRs, including Toll-like receptors (TLRs, NOD-like receptors (NLRs and RIG-I-like receptors (RLRs, recognize microbial components and trigger a host defense response. Respiratory tract infections are common causes of asthma exacerbations, suggesting a role for PRRs in this process. The present study aimed to examine the expression and function of PRRs on human airway smooth muscle cells (HASMCs. METHODS: Expression of TLR, NLR and RLR mRNA and proteins was determined using real-time RT-PCR, flow cytometry and immunocytochemistry. The functional responses to ligand stimulation were investigated in terms of cytokine and chemokine release, cell surface marker expression, proliferation and proteins regulating the contractile state. RESULTS: HASMCs expressed functional TLR2, TLR3, TLR4, TLR7 and NOD1. Stimulation with the corresponding agonists Pam3CSK4, poly(I:C, LPS, R-837 and iE-DAP, respectively, induced IL-6, IL-8 and GM-CSF release and up-regulation of ICAM-1 and HLA-DR, while poly(I:C also affected the release of eotaxin and RANTES. The proliferative response was slightly increased by LPS. Stimulation, most prominently with poly(I:C, down-regulated myosin light chain kinase and cysteinyl leukotriene 1 receptor expression and up-regulated β2-adrenoceptor expression. No effects were seen for agonist to TLR2/6, TLR5, TLR8, TLR9, NOD2 or RIG-I/MDA-5. CONCLUSION: Activation of TLR2, TLR3, TLR4, TLR7 and NOD1 favors a synthetic phenotype, characterized by an increased ability to release inflammatory mediators, acquire immunomodulatory properties by recruiting and interacting with other cells, and reduce the contractile state. The PRRs might therefore be of therapeutic use in the management of asthma and infection-induced disease exacerbations.

  18. Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation.

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    Sophia Maschalidi

    Full Text Available Intracellular Toll-like receptors (TLRs expressed by dendritic cells recognize nucleic acids derived from pathogens and play an important role in the immune responses against the influenza virus (IAV, a single-stranded RNA sensed by different receptors including TLR7. However, the importance of TLR7 processing in the development of anti-viral immune responses is not known. Here we report that asparagine endopeptidase (AEP deficient mice are unable to generate a strong anti-IAV response, as demonstrated by reduced inflammation, cross presentation of cell-associated antigens and priming of CD8(+ T cells following TLR7-dependent pulmonary infection induced by IAV. Moreover, AEP deficient lung epithelial- or myeloid-cells exhibit impaired TLR7 signaling due to defective processing of this receptor. Indeed, TLR7 requires a proteolytic cleavage by AEP to generate a C-terminal fragment competent for signaling. Thus, AEP activity is critical for TLR7 processing, opening new possibilities for the treatment of influenza and TLR7-dependent inflammatory diseases.

  19. TLR3 deficiency increases voluntary alcohol consumption.

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    Jang, Yujin; Lee, Min Hee; Park, Jong-Hwan; Han, Seung-Yun; Kim, Dong Kwan

    2016-03-23

    Toll-like receptors (TLRs) are innate immunity-related receptors. Many studies have indicated the involvement of TLRs in neurophysiology and neuropathology. One study showed that TLR3 regulates hippocampal memory and is highly expressed in the mesolimbic dopamine system, suggesting that TLR3 signaling may regulate alcohol consumption. The present study assessed the potential role of TLR3 in alcohol intake pattern. We used adult BalbC wild-type mice and TLR3 knockout mice and tested two-bottle alcohol preference over 15 days and one-bottle 2 or 4 h drinking in the dark over 4 consecutive days. The 10% alcohol consumption rate of TLR3 knockout mice increased on the 24 h free-choice test. Our findings support a potential regulatory role of TLR3 in alcohol consumption.

  20. Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

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    Lopetuso, Loris R; Jia, Ruo; Wang, Xiao-Ming; Jia, Li-Guo; Petito, Valentina; Goodman, Wendy A; Meddings, Jon B; Cominelli, Fabio; Reuter, Brian K; Pizarro, Theresa T

    2017-03-01

    A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.

  1. Structure Based Modeling of Small Molecules Binding to the TLR7 by Atomistic Level Simulations

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    Francesco Gentile

    2015-05-01

    Full Text Available Toll-Like Receptors (TLR are a large family of proteins involved in the immune system response. Both the activation and the inhibition of these receptors can have positive effects on several diseases, including viral pathologies and cancer, therefore prompting the development of new compounds. In order to provide new indications for the design of Toll-Like Receptor 7 (TLR7-targeting drugs, the mechanism of interaction between the TLR7 and two important classes of agonists (imidazoquinoline and adenine derivatives was investigated through docking and Molecular Dynamics simulations. To perform the computational analysis, a new model for the dimeric form of the receptors was necessary and therefore created. Qualitative and quantitative differences between agonists and inactive compounds were determined. The in silico results were compared with previous experimental observations and employed to define the ligand binding mechanism of TLR7.

  2. Immunological Evaluation of Two Local Isolates of Eimeria tenella Gametocytes against Coccidiosis in Poultry

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    Masood Akhtar*, M. Irfan Anwar, Zafar Iqbal, Faqir Muhammad1, Mian Muhammad Awais, Ahsan ul Haq2 and Elzbieta Hiszczynska-Sawicka3

    2012-01-01

    Full Text Available Two local isolates of Eimeria tenella gametocytes against coccidiosis were immunologically evaluated in chickens. Cell mediated immune response was detected by modified splenic cell migration inhibition assay (MSCMIA and data were expressed in terms of per cent migration index. No significant difference in per cent migration index was detected for the chickens immunized either with Vaccine-I (local isolate-I or with Vaccine-II (local isolate-II; however per cent migration index was comparatively lower in chickens immunized with Vaccine-II as compared to Vaccine-I; indicating a somewhat higher cell mediated immune (CMI response. Humoral immune response was monitored by ELISA in vaccinated and control chickens. Significantly elevated (P<0.05 antibody titer (IgG in chickens immunized with Vaccine-II as compared to Vaccine-I was detected. Significantly higher protection (67% in chickens immunized with Vaccine-II followed by vaccine-I (49% was recorded. Further, oocyst count was significantly lower (P<0.05 in chickens immunized with Vaccine-II as compared to those immunized with Vaccine-I. It was concluded that vaccinal strain (Vaccine-II contained additional protein of high molecular weight (49.23 kDa in its gametocytes provided cross protection and can be used to prepare commercial vaccine against coccidiosis in poultry.

  3. Immunization of young chicks using graded dose of wild strain of Eimeria tenella

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    E.N. Kimbita

    2005-09-01

    Full Text Available A wild strain of Eimeria tenella was isolated and utilized for immunization studies. Its optimal sporulation was attained at room temperature 24-25 °C after 24-48 h. Two groups of chicks were immunized by dosing a graded dose of five oocysts/chick/day for 6 days followed by 50 oocysts/chick/day for 7 days. A third group was not immunized and served as a negative control. Immunized chicks gained mass at the same rate as unimmunized ones, but when challenged with 200 000 oocysts/chick, mass gains declined in the unimmunized group. The growth rate of immunized chicks was not affected by challenge (P > 0.05. Upon challenge, unimmunized chicks produced significantly more oocysts than immunized chicks (P < 0.005. Immunized chicks withstood a challenged with 200 000 oocysts/chick without developing any clinical signs whereas the unimmunized chicks developed typical clinical signs of coccidiosis. Unimmunized chicks had significantly more severe lesions in the caecum than any other group (P > 0.005 and also produced significantly more oocysts than any other group (P > 0.005.

  4. Dietary Curcuma longa enhances resistance against Eimeria maxima and Eimeria tenella infections in chickens.

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    Kim, Duk Kyung; Lillehoj, Hyun S; Lee, Sung Hyen; Jang, Seung I; Lillehoj, Erik P; Bravo, David

    2013-10-01

    The effects of dietary supplementation with an organic extract of Curcuma longa on systemic and local immune responses to experimental Eimeria maxima and Eimeria tenella infections were evaluated in commercial broiler chickens. Dietary supplementation with C. longa enhanced coccidiosis resistance as demonstrated by increased BW gains, reduced fecal oocyst shedding, and decreased gut lesions compared with infected birds fed a nonsupplemented control diet. The chickens fed C. longa-supplemented diet showed enhanced systemic humoral immunity, as assessed by greater levels of serum antibodies to an Eimeria microneme protein, MIC2, and enhanced cellular immunity, as measured by concanavalin A-induced spleen cell proliferation, compared with controls. At the intestinal level, genome-wide gene expression profiling by microarray hybridization identified 601 differentially expressed transcripts (287 upregulated, 314 downregulated) in gut lymphocytes of C. longa-fed chickens compared with nonsupplemented controls. Based on the known functions of the corresponding mammalian genes, the C. longa-induced intestinal transcriptome was mostly associated with genes mediating anti-inflammatory effects. Taken together, these results suggest that dietary C. longa could be used to attenuate Eimeria-induced, inflammation-mediated gut damage in commercial poultry production.

  5. Effect of anaerobic digestion on oocysts of the protozoan Eimeria tenella.

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    Lee, M R; Shih, J C

    1988-10-01

    The effect of anaerobic digestion of poultry waste on oocysts of the protozoan Eimeria tenella, a common enteric pathogen that causes coccidiosis in poultry, was investigated in this study. Thermophilic (50 degrees C) and mesophilic (35 degrees C) anaerobic digestors, with poultry manure as the substrate, were inoculated with the oocysts. The oocysts were damaged during anaerobic digestion, as determined by morphological change and loss of their ability to sporulate. The recovered oocysts were tested for their infectivity in young chicks, as measured by body weight gain, mortality, and cecal lesions. Oocysts lost all their infectivity during thermophilic digestion, while oocysts subjected to mesophilic digestion remained moderately infective in comparison with untreated oocysts, which produced severe coccidiosis, high mortality, and low body weight gain in chicks. Oocysts were inactivated at 50 degrees C when they were suspended in digestor fluid or saline. Inactivation at 35 degrees C was significantly stronger in the digestor fluid than in the saline, which implied that factors other than temperature were involved in the lethal effect of anaerobic digestion on protozoan oocysts. In this study we demonstrated that the treatment of animal waste by anaerobic digestion, especially at a thermophilic temperature, has the benefits of pathogen control and protection of human and animal health in a farm environment.

  6. Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1.

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    Lianlian Jiang

    Full Text Available Apical membrane antigen-1 (AMA1 is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites. The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1 and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development.

  7. ABEJAS VISITANTES DE Aspilia tenella (KUNTH S. F. BLAKE (ASTERACEAE: COMPORTAMIENTO DE FORRAJEO Y CARGAS POLÍNICAS BEES VISITING Aspilia tenella (KUNTH S .F. BLAKE (ASTERACEAE: FORAGING BEHAVIOR AND POLLEN LOADS

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    Clara Isabel Aguilar Sierra

    2008-12-01

    Full Text Available En cuatro localidades de la zona de influencia del embalse Porce II (Antioquia, Colombia, se observó el comportamiento de 23 taxones de abejas durante sus visitas a Aspilia tenella (Kunth S. F. Blake; se registró el número de individuos y el tiempo total de visita y se midieron la temperatura y la humedad relativa. Los análisis de varianza indican que hubo una relación estadísticamente significativa en cuanto a los atributos considerados para las especies de abejas y una covariación significativa con las variables ambientales. Las pruebas de rangos múltiples muestran que Trigona nigerrima es la especie con los mayores valores promedio para el número de individuos y el tiempo de visita. En el estudio de las cargas polínicas se encontraron 30 tipos polínicos, dentro de los cuales A. tenella presentó el mayor porcentaje; Piper aduncum, Miconia minuti flora, Psidium guajava, Rapanea sp., Mimosa pudica y Psychotria sp., en su orden, son otras fuentes de polen importantes para varias de las especies de abejas. El análisis de agrupamiento para las abejas capturadas permitió diferenciar dos grupos: el primero incluye a 18 especies cuyas cargas polínicas que poseían una abundancia relativa de granos de polen de A. tenella por encima de 77%; el segundo grupo estaba conformado por cinco especies de abejas que recolectaron porcentajes mayores de otros tipos polínicos: Exomalopsis sp. 1 (90,4% de M. minuti flora; Lasioglossum sp. 1 y Coelioxys aff. mexicana (77,4% y 64,1% de P. aduncum, respectivamente; Exomalopsis sp. 2 (67,1% de P. guajava y Auglochloropsis vesta (55,5% de Rapanea sp.. Trigona fulviventris y Apis mellifera presentaron los valores más altos de riqueza de tipos polínicos en las cargas de polen; ello evidencia sus nichos tróficos amplios y su comportamiento generalista de visita.We observed the behavior of 23 species of wild bees visiting Aspilia tenella (Kunth S .F. Blake in four different localities of the Porce II dam

  8. TLR5 risk-associated haplotype for canine inflammatory bowel disease confers hyper-responsiveness to flagellin.

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    Aarti Kathrani

    Full Text Available Single nucleotide polymorphisms (SNP in the TLR5 gene have been associated with human inflammatory bowel disease (IBD and animal models of this disease. We recently demonstrated a significant association between three non-synonymous SNPs in the canine TLR5 gene and IBD in German shepherd dogs (GSDs. However, so far, no direct link between these SNPs and a disturbance in TLR5 function was shown. In the present study, we determined the functional significance of the canine TLR5 SNPs by transfecting the identified risk-protective and risk-associated haplotype into human embryonic kidney cells (HEK and assessed nuclear factor-kappa B (NF-κB activation and CXCL8 production after stimulation. In addition, a whole blood assay for TLR5 activation was developed using blood derived from carrier dogs of either haplotype. There was a significant increase in NF-kB activity when cells transfected with the risk-associated TLR5 haplotype were stimulated with flagellin compared to the cells expressing the risk-protective TLR5 haplotype. This difference in NFkB activation correlated with CXCL8 expression in the supernatant measured by ELISA. Furthermore, whole blood taken from carrier dogs of the risk-associated TLR5 haplotype produced significantly more TNF after stimulation with flagellin compared to that taken from carriers of the risk-protective haplotype. Thus, we show for the first time a direct functional impact of the canine IBD risk-associated TLR5 haplotype, which results in hyper-responsiveness to flagellin compared to the IBD risk-protective TLR5 haplotype. Our data potentially suggest that similarly to human IBD and experimental models, TLR5 may also play a role in canine IBD. Blocking the hyper-responsive receptor found in susceptible dogs with IBD may alleviate the inappropriate inflammation seen in this disease.

  9. Consumption of Bifidobacterium animalis subsp. lactis BB-12 in yogurt reduced expression of TLR-2 on peripheral blood-derived monocytes and pro-inflammatory cytokine secretion in young adults.

    Science.gov (United States)

    Meng, Huicui; Ba, Zhaoyong; Lee, Yujin; Peng, Jiayu; Lin, Junli; Fleming, Jennifer A; Furumoto, Emily J; Roberts, Robert F; Kris-Etherton, Penny M; Rogers, Connie J

    2017-03-01

    Probiotic bacteria modulate immune parameters and inflammatory outcomes. Emerging evidence demonstrates that the matrix used to deliver probiotics may influence the efficacy of probiotic interventions in vivo. The aims of the current study were to evaluate (1) the effect of one species, Bifidobacterium animalis subsp. lactis BB-12 at a dose of log10 ± 0.5 CFUs/day on immune responses in a randomized, partially blinded, 4-period crossover, free-living study, and (2) whether the immune response to BB-12 differed depending on the delivery matrix. Healthy adults (n = 30) aged 18-40 years were recruited and received four treatments in a random order: (A) yogurt smoothie alone; smoothie with BB-12 added (B) before or (C) after yogurt fermentation, or (D) BB-12 given in capsule form. At baseline and after each 4-week treatment, peripheral blood mononuclear cells (PBMCs) were isolated, and functional and phenotypic marker expression was assessed. BB-12 interacted with peripheral myeloid cells via Toll-like receptor 2 (TLR-2). The percentage of CD14 + HLA-DR + cells in peripheral blood was increased in male participants by all yogurt-containing treatments compared to baseline (p = 0.0356). Participants who consumed yogurt smoothie with BB-12 added post-fermentation had significantly lower expression of TLR-2 on CD14 + HLA-DR + cells (p = 0.0186) and reduction in TNF-α secretion from BB-12- (p = 0.0490) or LPS-stimulated (p = 0.0387) PBMCs compared to baseline. These findings not only demonstrate a potential anti-inflammatory effect of BB-12 in healthy adults, but also indicate that the delivery matrix influences the immunomodulatory properties of BB-12.

  10. Modulation of regulatory T cell function by monocyte-derived dendritic cells matured through electroporation with mRNA encoding CD40 ligand, constitutively active TLR4, and CD70.

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    Pen, Joeri J; De Keersmaecker, Brenda; Maenhout, Sarah K; Van Nuffel, An M T; Heirman, Carlo; Corthals, Jurgen; Escors, David; Bonehill, Aude; Thielemans, Kris; Breckpot, Karine; Aerts, Joeri L

    2013-08-15

    Regulatory T cells (Tregs) counteract anticancer immune responses through a number of mechanisms, limiting dendritic cell (DC)-based anticancer immunotherapy. In this study, we investigated the influence of various DC activation stimuli on the Treg functionality. We compared DCs activated by electroporation with mRNA encoding constitutively active TLR4 (caTLR4) and CD40 ligand (DiMix-DCs), or these factors together with mRNA encoding the costimulatory molecule CD70 (TriMix-DCs) with DCs maturated in the presence of a mixture of inflammatory cytokines (DCs maturated with a combination of the cytokines IL-1β, IL-6, TNF-α, and PGE2) for their ability to counteract Tregs on different levels. We first demonstrated that there was no difference in the extent of Treg induction starting from CD4(+)CD25(-) T cells under the influence of the different DC maturation stimuli. Second, we showed that both DiMix- and TriMix-DCs could partly alleviate Treg inhibition of CD8(+) T cells. Third, we observed that CD8(+) T cells that had been precultured with DiMix-DCs or TriMix-DCs were partially protected against subsequent Treg suppression. Finally, we showed that Tregs cocultured in the presence of TriMix-DCs, but not DiMix-DCs, partially lost their suppressive capacity. This was accompanied by a decrease in CD27 and CD25 expression on Tregs, as well as an increase in the expression of T-bet and secretion of IFN-γ, TNF-α, and IL-10, suggesting a shift of the Treg phenotype toward a Th1 phenotype. In conclusion, these data suggest that TriMix-DCs are not only able to suppress Treg functions, but moreover could be able to reprogram Tregs to Th1 cells under certain circumstances.

  11. Immune protection duration and efficacy stability of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 against coccidiosis.

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    Song, Xiaokai; Zhao, Xiaofang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2017-04-01

    In our previous study, an effective DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 was constructed. In the present study, the immunization dose of the DNA vaccine pVAX1.0-TA4-IL-2 was further optimized. With the optimized dose, the dynamics of antibodies induced by the DNA vaccine was determined using indirect ELISA. To evaluate the immune protection duration of the DNA vaccine, two-week-old chickens were intramuscularly immunized twice and the induced efficacy was evaluated by challenging with E. tenella at 5, 9, 13, 17 and 21weeks post the last immunization (PLI) separately. To evaluate the efficacy stability of the DNA vaccine, two-week-old chickens were immunized with 3 batches of the DNA vaccine, and the induced efficacy was evaluated by challenging with E. tenella. The results showed that the optimal dose was 25μg. The induced antibody level persisted until 10weeks PPI. For the challenge time of 5 and 9weeks PLI, the immunization resulted in ACIs of 182.28 and 162.23 beyond 160, showing effective protection. However, for the challenge time of 13, 17 and 21weeks PLI, the immunization resulted in ACIs below 160 which means poor protection. Therefore, the immune protection duration of the DNA vaccination was at least 9weeks PLI. DNA immunization with three batches DNA vaccine resulted in ACIs of 187.52, 191.57 and 185.22, which demonstrated that efficacies of the three batches DNA vaccine were effective and stable. Overall, our results indicate that DNA vaccine pVAX1.0-TA4-IL-2 has the potential to be developed as effective vaccine against coccidiosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Effect of Sapindus rarak powder as feed additive on performance and lipid profile of broiler chicken infected by Eimeria tenella

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    Tiurma Pasaribu

    2014-12-01

    Full Text Available Sapindus rarak fruits have bioactive compound named saponin which are potential to inhibit growth of protozoa and reduce cholesterol. This study was conducted to evaluate effect of lerak (Sapindus rarak powder as microparticle on performance and lipid profile of chicken broiler that was infected by Eimeria tenella. A total of150headsof DOCwere assign to recive on of5 treatments: T1 (K+, TAk, TSRa, TIE, T2 (K-, TAk, TSRa, IE, T3 (Sal, IE, T4 (Sra 2,5, IE, and T5 (Sra 1,25, IE; each treatment consisted of 6replications with 5birds per replications in battery cage for 34 days. All chickens except those in control positive treatment (non-infected, non-medicated were inoculated orally with 6000 oocysts E. tenella on the 14th day of age. Variables measured were body weight gain, carcass, OPG (oocysts pergram of faeces, lipid profile, and mortality. Treatment with S. rarak, dose of 1.25g/kg was not significantly different from the salinomycin treatment on BWG and feed conversion. Percentage of carcassand organs and blood cholesterol concentration were not significantly differentin all treatments, but blood triglyceride at S. rarak addition with doseof 2.5, 1.25g/kg and salinomycin addition with dose of 0.5g/kg, and negative control were lower than positive control. While number of OPG in faeces (days 14-34 showed that S. rarak powder with dose of 2.5 and 1.25g/kg suppressed the development of oocystsof E. tenella. In conclusion, S. rarak microparticle (75µm can be used as feed additive to replace salinomycinas coccidiostat.

  13. Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

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    Kuo-Ching Sheng

    2013-01-01

    Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

  14. The production of reactive oxygen species in TLR-stimulated granulocytes is not enhanced by hyperglycemia in diabetes.

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    Fagundes-Netto, F S; Anjos, P M F; Volpe, C M O; Nogueira-Machado, J A

    2013-11-01

    Toll-like receptors (TLRs) play an important role in several inflammatory diseases such as diabetes. The present study was to determine whether hyperglycemia in diabetes interferes in reactive oxygen species (ROS) production in granulocytes stimulated with either TLR2/zymosan, TLR4/lipopolysaccharide (LPS) or TLR2,4,9/concanavalin A (ConA). NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and mitogen-activated protein kinase (MAPK) signaling pathways associated with ROS generation in TLR-stimulated granulocytes were evaluated. Our results demonstrate that ROS generation in resting granulocytes derived from patients suffering from Type 2 diabetes mellitus (T2DM) is significantly higher than that observed in equivalent cells from healthy controls. However, ROS formed by TLR-stimulated granulocytes from T2DM patients and healthy subjects were comparable. ROS production by TLR4,9 depends on NADPH-oxidase and MAPK signaling pathways. In contrast, the activation of TLR2 leads to ROS production by a mechanism that is dependent on NADPH oxidase but independent of the MAPK. In conclusion our results suggest that hyperglycemia in diabetes may prime cells metabolically for ROS generation but does not exert any significant effect on TLR-stimulated ROS production and possibly does not aggravate the development of ROS-dependent diabetic complications. © 2013 Elsevier B.V. All rights reserved.

  15. Rationally Designed TLR4 Ligands for Vaccine Adjuvant Discovery

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    Kelsey A. Gregg

    2017-05-01

    Full Text Available Adjuvant properties of bacterial cell wall components like MPLA (monophosphoryl lipid A are well described and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS, altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential sources among bacterial strains, the number of useable compounds within this promising class of adjuvants are few. We have developed bacterial enzymatic combinatorial chemistry (BECC as a method to generate rationally designed, functionally diverse lipid A. BECC removes endogenous or introduces exogenous lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis to develop structurally distinct LOS molecules that elicit differential Toll-like receptor 4 (TLR4 activation. Using reporter cell lines that measure NF-κB activation, BECC-derived molecules were screened for the ability to induce a lower proinflammatory response than Escherichia coli LOS. Their structures exhibit varied, dose-dependent, TLR4-driven NF-κB activation with both human and mouse TLR4 complexes. Additional cytokine secretion screening identified molecules that induce levels of tumor necrosis factor alpha (TNF-α and interleukin-8 (IL-8 comparable to the levels induced by phosphorylated hexa-acyl disaccharide (PHAD. The lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary blood mononuclear cells (PBMCs, and human monocyte-derived dendritic cells (DCs. This newly described system allows directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates.

  16. Ameliorative effect of mycofix plus 3.0 in reducing intensity of Eimeria tenella infection during aflatoxicosis in broiler chicks

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    A. M. Shareef

    2008-01-01

    Full Text Available One hundred and sixty male broiler chicks were fed at one day of age aflatoxin (AF at a rate of 3.5 mg/kg alone, or with groups injected with Eimeria tenella sporulated oocysts (40000 at 14 days of age. Adsorbent (Mycofix® plus 3.0 was incorporated at a rate of 0.25% in the above mentioned groups from one day of age till the end of the experiment. The study was conducted to reveal the effect of a aforementioned different diets and treatments on live body weight, feed consumption, feed conversion ratio, blood parameters (total red blood cells, hemoglobin, packed cell volume, biochemical profile of serum (alkaline phosphatase and β-carotin, liver weights, bursal and thymus indexes, caecal lesion scores and mortalities. The results indicated that AF was responsible for a significant (P<0.05 reduction. in body weigh gain (BWG, feed consumption, and an increase in feed conversion ratio. Afllatoxin was also responsible for reduction in blood parameters, β-Carotin, bursal and thymus indexes. While relative liver weight and alkaline phosphatase level were significantly (P<0.05 increased. Groups that fed AF at a rate of 3.5 mg/kg feed and exposed to sporulated oocysts of Eimeria tenella show a high significant (P<0.05 reduction in BWG, feed consumption and an increase in feed conversion ratio. Aflatoxin was also responsible for significant blood parameter, β-carotin, and also a significant (P<0.05 increase in caecal lesion scores, mortality, alkaline phosphatase level and relative liver weight, while they showed significant (P<0.05 decrease in bursal and thymus indexes in comparison with injected groups with E-tenella sporulated oocysts alone. The study approved that the groups maintained on mycofix plus 3.0 (0.25% and contaminated with aflatoxin 3.5 mg/kg, revealed a positive noticeable effects in amelioration on BWG, feed consumption and feed conversion, blood parameter, β-carotin, alkaline phosphatase level, relative liver weight, bursal and

  17. Telomere-mediated chromosomal instability triggers TLR4 induced inflammation and death in mice.

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    Rabindra N Bhattacharjee

    Full Text Available BACKGROUND: Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC-/- mice display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: We examined the function of TLR4 in telomerase deficient mTERC-/- mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC+/+, mTERC+/- and mTERC-/- mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFalpha and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC-/- mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-kappaB binding to its promoter by down-regulating ATF-3 in mTERC-/- macrophages. CONCLUSIONS/SIGNIFICANCE: Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-kappaB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.

  18. Regulation of intestinal immune responses through TLR activation: implications for pro- and prebiotics

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    Sander eDe Kivit

    2014-02-01

    Full Text Available The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC and resident immune cells in the lamina propria. Many diseases, including chronic intestinal inflammation (e.g. inflammatory bowel disease, irritable bowel syndrome (IBS, allergic gastroenteritis (e.g. eosinophilic gastroenteritis and allergic IBS and infections are nowadays associated with a deregulated microbiota. The microbiota may directly interact with TLR. In addition, differences in intestinal TLR expression in health and disease may suggest that TLR play an essential role in disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is involved in either maintaining intestinal homeostasis or the induction of an inflammatory response. This mini review provides an overview of the current knowledge regarding the contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting intestinal inflammation, with a focus on food allergy. We will also highlight a potential role of the microbiota in regulating gut immune responses, especially through TLR activation.

  19. Natural selection in the TLR-related genes in the course of primate evolution.

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    Nakajima, Toshiaki; Ohtani, Hitoshi; Satta, Yoko; Uno, Yasuhiro; Akari, Hirofumi; Ishida, Takafumi; Kimura, Akinori

    2008-12-01

    The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pressure in the course of primate evolution. We compared the nucleotide sequences of 16 TLR-related genes, including TLRs (TLR1-10), MYD88, TILAP, TICAM1, TICAM2, MD2, and CD14, among seven primate species. Analysis of the non-synonymous/synonymous substitution ratio revealed the presence of both strictly conserved and rapidly evolving regions in the TLR-related genes. The genomic segments encoding the intracellular Toll/interleukin 1 receptor domains, which exhibited lower rates of non-synonymous substitution, have undergone purifying selection. In contrast, TLR4, which carried a high proportion of non-synonymous substitutions in the part of extracellular domain spanning 200 amino acids, was found to have been the suggestive target of positive Darwinian selection in primate evolution. However, sequence analyses from 25 primate species, including eight hominoids, six Old World monkeys, eight New World monkeys, and three prosimians, showed no evidence that the pressure of positive Darwinian selection has shaped the pattern of sequence variations in TLR4 among New World monkeys and prosimians.

  20. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

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    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-07-25

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16(INK4a) was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16(INK4a) post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis.

  1. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16INK4a protein expression

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    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16INK4a was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16INK4a post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis. PMID:27454079

  2. A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

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    Anthony Ryan

    2011-06-01

    Full Text Available Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4⁻/⁻ and Myd88⁻/⁻, but not TRIF⁻/⁻ mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

  3. A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins.

    LENUS (Irish Health Repository)

    Ryan, Anthony

    2011-06-01

    Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H\\/HeN and C3H\\/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H\\/HeJ mice and failed to induce a subsequent Th cell response. TLR4(-\\/-) and Myd88(-\\/-), but not TRIF(-\\/-) mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

  4. TLR2 and TLR4 triggering exerts contrasting effects with regard to HIV-1 infection of human dendritic cells and subsequent virus transfer to CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Fromentin Rémi

    2009-05-01

    Full Text Available Abstract Background Recognition of microbial products through Toll-like receptors (TLRs initiates inflammatory responses orchestrated by innate immune cells such as dendritic cells (DCs. As these cells are patrolling mucosal surfaces, a portal of entry for various pathogens including human immunodeficiency virus type-1 (HIV-1, we investigated the impact of TLR stimulation on productive HIV-1 infection of DCs and viral spreading to CD4+ T cells. Results We report here that engagement of TLR2 on DCs increases HIV-1 transmission toward CD4+ T cells by primarily affecting de novo virus production by DCs. No noticeable and consistent effect was observed following engagement of TLR5, 7 and 9. Additional studies indicated that both HIV-1 infection of DCs and DC-mediated virus transmission to CD4+ T cells were reduced upon TLR4 triggering due to secretion of type-I interferons. Conclusion It can thus be proposed that exposure of DCs to TLR2-binding bacterial constituents derived, for example, from pathogens causing sexually transmissible infections, might influence the process of DC-mediated viral dissemination, a phenomenon that might contribute to a more rapid disease progression.

  5. Anticoccidial effects of acetic acid on performance and pathogenic parameters in broiler chickens challenged with Eimeria tenella

    Directory of Open Access Journals (Sweden)

    Rao Z. Abbas

    2011-02-01

    Full Text Available The objective of the present study was to evaluate the anticoccidial effect of the different concentrations of the acetic acid in the broiler chickens in comparison with the amprolium anticoccidial. A total of 198 chicks were placed 11 per pen with three pens per treatment. The different concentrations (1%, 2% and 3% of acetic acid and amproilum (at the dose rate of 125ppm were given to the experimental groups in drinking water from 10-19th days of age. One group was kept as infected non medicated control and one as non infected non medicated control. All the groups were inoculated orally with 75,000 sporulated oocysts at the 12th day of age except non infected non medicated control. Anticoccidial effect was evaluated on the basis of performance (weight gain, feed conversion ratio and pathogenic (oocyst score, lesion score and mortality %age parameters. Among acetic acid medicated groups, the maximum anticoccidial effect was seen in the group medicated with 3% acetic acid followed by 2% and 1% acetic acid medicated groups. Amprolium and 3% acetic acid were almost equivalent in suppressing the negative performance and pathogenic effects associated with coccidiosis (Eimeria tenella challenge. In summary, acetic acid has the potential to be used as alternative to chemotherapeutic drugs for Eimeria tenella control. Concentration-dependent anticoccidial effect of acetic acid suggests that further studies should be carried out to determine the possible maximum safe levels of acetic acid with least toxic effects to be used as anticoccidial.

  6. Anti-idiotypic antibody with potential use as an Eimeria tenella sporozoite antigen surrogate for vaccination of chickens against coccidiosis.

    Science.gov (United States)

    Bhogal, B S; Nollstadt, K H; Karkhanis, Y D; Schmatz, D M; Jacobson, E B

    1988-01-01

    Anti-idiotypic antibodies were raised in rabbits against four monoclonal antibodies with specificity for the surface antigenic determinants of Eimeria tenella sporozoites, the infective stage of the coccidial parasite. Two of the monoclonal antibodies (1073 and 15-1) transferred passive protection in chickens against E. tenella infection. The polyclonal anti-idiotype antibody preparations against protective monoclonal antibodies contained specificities for the paratope-associated idiotypes of these monoclonal antibodies, as assessed by the competitive inhibition of binding of the homologous idiotype-anti-idiotype by the sporozoite antigen. Competitive inhibition of binding of homologous idiotype-anti-idiotype by the parasite antigen was not observed when the anti-idiotype antibody preparations against monoclonal antibodies 1546 and 1096 were tested. The anti-idiotype 1073 and 15-1 antibodies functioned as surrogate antigens in vivo when used for vaccination of young chickens, as evidenced by the induction of partial protective immunity against subsequent challenge infection with virulent parasites and induction of antisporozoite antibodies. These data clearly support the view that anti-idiotypic antibodies raised against the paratope-associated idiotypes can mimic pathogen antigens and therefore can provide a possible alternative approach for the vaccination of chickens against coccidiosis. PMID:3258583

  7. Plasma biochemical indices at various stages of infection with a field isolate of Eimeria tenella in broiler chicken

    Directory of Open Access Journals (Sweden)

    Dipak Kumar Mondal

    Full Text Available Coccidiosis has a major impact on poultry industry as it affects broiler and layer birds of all age groups. Caecal coccidiosis, caused by Eimeria tenella is a very devastating enteric disease in broiler,which involves huge economic loss In present study, experimental infective dose of Eimeria tenella isolated from field was determined in broiler chicken and subsequent alterations in different plasma biochemical constituents were evaluated at interval of 5th, 7th and 9th day of post inoculation (PI with the selected dose of 20000-25000 sporulated oocyst per bird. The dose was selected based on titration. A significant increase in plasma glucose, total cholesterol level and aspartate aminotransferase (AST activity were observed where as a significant decrease in the level of total plasma protein, albumin, globulin, triglyceride and alanine aminotransferase (ALT activity were evident during infection. Highest degree of infection was found on 7th day PI. Onward 9th day of PI onward clinical recovery was confirmed on the basis of pathognomonic caecal lesion score, clinical signs and symptoms. [Vet. World 2011; 4(9.000: 404-409

  8. Pharmacodynamic evaluation of a reference and a generic toltrazuril preparation in broilers experimentally infected with Eimeria tenella or E. acervulina.

    Science.gov (United States)

    Alcala-Canto, Y; Ramos-Martinez, E; Tapia-Perez, G; Gutierrez, L; Sumano, H

    2014-02-01

    Abstract 1. The aim of this study was to investigate the effects on pigmentation, faecal oocyst output, immune responsiveness and reactive oxygen species (ROS) generation following treatment with either the reference toltrazuril (Baycox) or a generic preparation (gen-TTZ), during an experimental Eimeria tenella (Et) or E. acervulina (Ea) infection of 210 Ross broiler chickens. 2. Results showed a significant difference on the anticoccidial efficacy 6 d after treating infected animals with Baycox (Et: 99.69% and Ea: 99.52%) or gen-TTZ (Et:85.71% and Ea 81.81%). 3. Gen-TTZ-treated animals were less strongly carotenoid-pigmented than Baycox-treated broilers. Mean plasma carotenoid concentrations were significantly higher in groups treated with Baycox than in broilers given gen-TTZ. 4. Treatment of animals with Baycox led to a significant decrease in ability of the peripheral blood mononuclear cells to produce ROS in contrast to gen-TTZ-treated groups. Baycox, but not generic toltrazuril, increased IL-10 and decreased tumour necrosis factor alpha (TNF-α) concentrations in chickens infected with E. tenella and E. acervulina. 5. It is suggested that differences in anticoccidial efficacy may be observed when using a generic toltrazuril product. Hence, in addition to plasma profiles of drugs, standardised clinical control tests may be necessary for generic formulations, particularly if other parameters are important to achieve a better control of coccidiosis.

  9. Association of TLR polymorphisms with bronchopulmonary dysplasia.

    Science.gov (United States)

    Malash, Amr Hosny; Ali, Aliaa Adel; Samy, Rania Mohamed; Shamma, Radwa Ahmed

    2016-10-30

    Bronchopulmonary dysplasia (BPD) remains a leading cause of morbidity and mortality during infancy. Evidence suggests that the Toll-like receptor (TLR) signaling pathway plays an integral role in lung inflammation and injury. This study aimed to detect single nucleotide polymorphisms (SNPs) in TLR pathway genes [TLR5 and Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP)] among preterm neonates and to determine their association with the development and severity of bronchopulmonary dysplasia. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion.

    Science.gov (United States)

    Kolter, Julia; Feuerstein, Reinhild; Spoeri, Evelyne; Gharun, Kourosh; Elling, Roland; Trieu-Cuot, Patrick; Goldmann, Tobias; Waskow, Claudia; Chen, Zhijian J; Kirschning, Carsten J; Deshmukh, Sachin D; Henneke, Philipp

    2016-03-15

    Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to β-hemolytic streptococci. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Cholesterol Oxidase Binds TLR2 and Modulates Functional Responses of Human Macrophages

    Directory of Open Access Journals (Sweden)

    Katarzyna Bednarska

    2014-01-01

    Full Text Available Cholesterol oxidase (ChoD is considered to be an important virulence factor for Mycobacterium tuberculosis (Mtb, but its influence on macrophage activity is unknown. Here we used Nocardia erythropolis ChoD, which is very similar to the Mtb enzyme (70% identity at the amino-acid level, to evaluate the impact of bacterial ChoD on the activity of THP-1-derived macrophages in vitro. We found that ChoD decreased the surface expression of Toll-like receptor type 2 (TLR2 and complement receptor 3 (CR3 on these macrophages. Flow cytometry and confocal microscopy showed that ChoD competed with lipoteichoic acid for ligand binding sites on TLR2 but not on CR3, suggesting that ChoD signaling is mediated via TLR2. Binding of ChoD to the membrane of macrophages had diverse effects on the activity of macrophages, activating p38 mitogen activated kinase and stimulating production of a large amount of interleukin-10. Moreover, ChoD primed macrophages to enhance the production of reactive oxygen species in response to the phorbol myristate acetate, which was reduced by “switching off” TLR-derived signaling through interleukin-1 receptor-associated kinases 1 and 4 inhibition. Our study revealed that ChoD interacts directly with macrophages via TLR2 and influences the biological activity of macrophages during the development of the initial response to infection.

  12. SNP marker discovery in koala TLR genes.

    Directory of Open Access Journals (Sweden)

    Jian Cui

    Full Text Available Toll-like receptors (TLRs play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

  13. SNP marker discovery in koala TLR genes.

    Science.gov (United States)

    Cui, Jian; Frankham, Greta J; Johnson, Rebecca N; Polkinghorne, Adam; Timms, Peter; O'Meally, Denis; Cheng, Yuanyuan; Belov, Katherine

    2015-01-01

    Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

  14. The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

    Science.gov (United States)

    Gupta, N; Sinha, R; Krasnodembskaya, A; Xu, X; Nizet, V; Matthay, M A; Griffin, J H

    2018-02-02

    Bone marrow derived mesenchymal stromal cells (BM-MSCs) have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on MSC survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 -/- and R41Q-PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC survival when exposed to cytotoxic stimuli in vitro. An E. coli pneumonia model was utilized to assess the effect of these specific pathways on MSC therapeutic activity in vivo. Our results showed that mutation of either the TLR4 or PAR1 pathways in MSCs impaired cell survival under conditions of inflammatory stress in vitro, and eliminated their therapeutic efficacy in vivo. Also, stimulation of the TLR4 pathway on MSCs led to secretion of low levels of prothrombin by MSCs, while disrupting the TLR4 pathway impaired canonical signaling through PAR1 in response to thrombin. Therefore, this study demonstrates that both TLR4 and PAR1 are required for MSC survival under inflammatory conditions in vitro and therapeutic capacity in vivo, and that the TLR4 pathway regulates signaling through PAR1 on MSCs. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

  15. TLR2, TLR4 and CD86 gene polymorphisms in recurrent aphthous stomatitis.

    Science.gov (United States)

    Karasneh, Jumana; Bani-Hani, Maisoun; Alkhateeb, Asem; Hassan, Ahmad; Alzoubi, Firas; Thornhill, Martin

    2015-11-01

    Recurrent aphthous stomatitis (RAS) is an inflammatory disease induced by genetic and environmental factors. Toll-like receptor (TLR) and CD86 are essential components for innate immunity and cellular immune response. We aimed to determine whether inheritance of specific TLR2, TLR4and CD86 gene polymorphisms are associated with RAS. Ninety-six patients with RAS and 153 controls were studied. Eight SNPs were genotyped using PCR-RFLP technique; four in TLR2 gene: rs4696480, rs3804100, rs121917864, rs5743708; three in TLR4 gene: rs10759931, rs4986790 rs1927911; and one in CD86 gene rs17281995. Association was assessed by logistic regression analysis. Linkage disequilibrium (LD) was assessed using the Haploview program. Significant increase in inheritance of A allele (OR = 1.6, P = 0.01) and AA genotype (OR = 3.89, P = 0.01) of TLR4 rs10759931 was observed in cases. TLR4rs1927911 C allele and CC genotype were also increased (OR = 1.60 and 2.78 respectively); however, this was not statistically significant (P = 0.02 and 0.03 respectively). TLR2 and CD86 did not show association with RAS. This is the first study to investigate the association of TLR and CD86 with RAS. We found a significant association between TLR4 rs10759931 polymorphism and RAS. Confirmatory studies in other populations and functional investigations are needed to determine the role of TLR4 in RAS. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Molecular and functional characterization of Toll-like receptor (Tlr)1 and Tlr2 in common carp (Cyprinus carpio).

    Science.gov (United States)

    Fink, Inge R; Pietretti, Danilo; Voogdt, Carlos G P; Westphal, Adrie H; Savelkoul, Huub F J; Forlenza, Maria; Wiegertjes, Geert F

    2016-09-01

    Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Palti, Yniv; Gahr, Scott A.; Purcell, Maureen K.; Hadidi, Sima; Rexroad, Caird E.; Wiens, Gregory A.

    2010-01-01

    Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

  18. TLR4 activates NF-{kappa}B in human ovarian granulosa tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Woods, Dori C., E-mail: dwoods2@partners.org [Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114 (United States); White, Yvonne A.R. [Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Service, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02114 (United States); Dau, Caroline [University of California, San Francisco, School of Dentistry, San Francisco, CA 94143 (United States); Johnson, A.L. [Center for Reproductive Biology and Health, The Pennsylvania State University, University Park, PA 16802 (United States)

    2011-06-17

    Highlights: {yields} TLR4 is expressed in human ovarian granulosa tumor cells. {yields} Acting through TLR4, LPS and HSP60 induce a NF{kappa}B signaling cascade in human ovarian granulosa tumor cells. {yields} NF{kappa}B activation or inhibition did not alter chemosensitivity to TRAIL or cisplatin. -- Abstract: Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-{kappa}B) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to I{kappa}B degradation and activation of NF-{kappa}B. NF-{kappa}B activation was confirmed by nuclear localization of NF-{kappa}B p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-{kappa}B signaling attenuated LPS-induced TNF{alpha} plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-{kappa}B signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-{kappa}B does not sensitize GCTs to TRAIL or cisplatin.

  19. Comparison of TLR-2, TLR-4, and antimicrobial peptide levels in different lesions of acne vulgaris.

    Science.gov (United States)

    Ozlu, Emin; Karadag, Ayse Serap; Ozkanli, Seyma; Oguztuzun, Serpil; Kilic, Murat; Zemheri, Ebru; Akbulak, Ozge; Akdeniz, Necmettin

    2016-12-01

    Recent studies have shown that tolls like receptors (TLRs) and antimicrobial peptides (hBD-1, cathelicidin) play an important role in the pathogenesis of acne vulgaris (AV). To evaluate and report the expression of TLR-2, TLR-4, hBD-1 and cathelicidin in different regions of skin in AV. This study was performed in 80 patients with AV and a control group of 20 healthy individuals. Skin biopsies were performed from 20 papular, 20 pustular, 20 comedonal and 20 nodular lesions of patients and 20 healthy volunteers. Expression levels of TLR-2, TLR-4, hBD-1 and cathelicidin in four separate areas (epidermis, dermis, inflammation region and skin appendages) were evaluated by immunohistochemical method. Further, these parameters were compared between different skin lesions. A significant difference was found between the levels of staining of TLR-2, TLR-4 and hBD-1 from the epidermis, inflammation region, dermis and skin appendages (p pathogenesis of AV and in the development of different acne types. We think that, better results could be obtained in treatment of AV with different treatment options targeted in regulation of TLR-2, TLR-4, hBD-1 and cathelicidin release.

  20. TLR2 and TLR9 Synergistically Control Herpes Simplex Virus Infection in the Brain

    DEFF Research Database (Denmark)

    Sørensen, Louise Nørgaard; Reinert, Line; Malmgaard, Lene

    2008-01-01

    Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few st...

  1. TLR2 and TLR9 synergistically control herpes simplex virus infection in the brain

    DEFF Research Database (Denmark)

    Sørensen, Louise N; Reinert, Line S; Malmgaard, Lene

    2008-01-01

    Viruses are recognized by the innate immune system through pattern recognition receptors (PRRs). For instance, HSV virions and genomic DNA are recognized by TLR2 and TLR9, respectively. Although several viruses and viral components have been shown to stimulate cells through TLRs, only very few st...

  2. The function of TLR4 in interferon gamma or interleukin-13 exposed and lipopolysaccharide stimulated gingival epithelial cell cultures.

    Science.gov (United States)

    Beklen, A; Sarp, A S; Uckan, D; Tsaous Memet, G

    2014-10-01

    Gingival epithelial cells are part of the first line of host defense against infection. Toll-like receptors (TLRs) serve important immune and nonimmune functions. We investigated how interferon gamma (INF-γ) and interleukin 13 (IL-13) are involved in the TLR4 ligand-induced regulation of interleukin-8 (IL-8) effects on gingival epithelial cells. We used immunohistochemistry to localize TLR4 in ten healthy and ten periodontitis tissue specimens. Gingival epithelial cells then were primed with Th1 cytokine (INF-γ) or Th2 cytokine (IL-13) before stimulation with Escherichia coli-derived lipopolysaccharide (LPS) and enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of IL-8 secretion in cell culture supernatants. Although both healthy and periodontitis gingival tissue samples expressed TLR4, the periodontitis samples showed more intense expression on gingival epithelial cells. Gingival epithelial cell cultures were primed with either INF-γ or IL-13 before stimulation with TLR4 ligand. Supernatants from co-stimulated epithelial cells exhibited IL-8 production in opposite directions, i.e., as one stimulates the release, the other reduces the release. INF-γ significantly increased TLR4 function, whereas IL-13 significantly decreased TLR4 function, i.e., production of IL-8. Pathogen associated molecular pattern-LPS, shared by many different periodonto-pathogenic bacteria, activates the gingival epithelial cells in a TLR-dependent manner. Diminished or increased TLR function in gingival epithelial cells under the influence of different Th cell types may protect or be harmful due to the altered TLR signaling.

  3. Association of TLR2 and TLR4 polymorphisms with risk of cancer: a meta-analysis.

    Science.gov (United States)

    Zhu, Longbiao; Yuan, Hua; Jiang, Tao; Wang, Ruixia; Ma, Hongxia; Zhang, Shuangyue

    2013-01-01

    The activation of Toll-like receptors (TLRs) may be an important event in the immune evasion of tumor cell. Recently, numerous studies have investigated the associations between TLR2 -196 to -174 del and two SNPs of TLR4 (rs4986790 and rs4986791) and the susceptibility to different types of cancer; however, the results remain conflicting. The aim of this study was to assess the association between TLR2 and TLR4 polymorphisms and cancer risk in a meta-analysis with eligible published studies. A dataset composed of 14627 cases and 17438 controls from 34 publications were included in a meta-analysis to evaluate the association between overall cancer risk or cancer-specific risk and three SNPs of TLRs (TLR2 -196 to -174 del, TLR4 rs4986790 and rs4986791). The results showed that all of these three polymorphisms were significantly associated with the increased cancer risk (dominant model: OR = 1.64, 95% CI: 1.04-2.60 for TLR2 -196 to -174 del; OR = 1.19, 95% CI: 1.01-1.41 for TLR4 rs4986790; and OR = 1.47, 95% CI: 1.120-1.80 for TLR4 rs4986791; respectively). In stratified analysis, we found the effect of TLR2 -196 to -174 del on cancer risk remained significant in the subgroup of Caucasians and South Asians, but not in East Asians. However, the association between rs4986791 and cancer risk was significant in both South Asians and East Asians, but not in Caucasians. Furthermore, the association between rs4986790 and cancer risk was statistically significant in digestive cancers (dominant model: OR = 1.76, 95% CI: 1.13-2.73) and female-specific cancers (dominant model: OR = 1.50, 95% CI: 1.16-1.94). However, no significant association with risk of digestive system cancers was observed for TLR2 -196 to -174 del and TLR4 rs4986791. This meta-analysis presented additional evidence for the association between TLR2 and TLR4 polymorphisms and cancer risk. Further well-designed investigations with large sample sizes are required to confirm this conclusion.

  4. Gene conversion limits divergence of mammalian TLR1 and TLR6

    Science.gov (United States)

    Kruithof, Egbert KO; Satta, Nathalie; Liu, Jia Wei; Dunoyer-Geindre, Sylvie; Fish, Richard J

    2007-01-01

    Background Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris). Results The N-terminal sequences of the orthologous proteins showed greater similarity than corresponding paralog sequences. However, we identified a region of 300 amino acids towards the C-terminus of TLR1 and TLR6, where paralogs had a greater degree of sequence identity than orthologs. Preservation of DNA sequence identity of paralogs in this region was observed in all nine mammalian species investigated, and is due to independent gene conversion events. The regions having undergone gene conversion in each species are almost identical and encode the leucine-rich repeat motifs 16 to 19, the C-terminal cap motif, the transmembrane domain and most of the intracellular Toll/interleukin-1 receptor (TIR) domain. Conclusion Our results show that, for a specific conserved region, divergence of TLR1 and TLR6 is limited by gene conversion, most likely because of the need for co-evolution with multiple intracellular and extracellular binding partners. Thus, gene conversion provides a mechanism for limiting the divergence of functional regions of protein paralogs, while allowing other domains to evolve diversified functions. PMID:17727694

  5. Gene conversion limits divergence of mammalian TLR1 and TLR6

    Directory of Open Access Journals (Sweden)

    Dunoyer-Geindre Sylvie

    2007-08-01

    Full Text Available Abstract Background Toll-like receptors (TLR recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris. Results The N-terminal sequences of the orthologous proteins showed greater similarity than corresponding paralog sequences. However, we identified a region of 300 amino acids towards the C-terminus of TLR1 and TLR6, where paralogs had a greater degree of sequence identity than orthologs. Preservation of DNA sequence identity of paralogs in this region was observed in all nine mammalian species investigated, and is due to independent gene conversion events. The regions having undergone gene conversion in each species are almost identical and encode the leucine-rich repeat motifs 16 to 19, the C-terminal cap motif, the transmembrane domain and most of the intracellular Toll/interleukin-1 receptor (TIR domain. Conclusion Our results show that, for a specific conserved region, divergence of TLR1 and TLR6 is limited by gene conversion, most likely because of the need for co-evolution with multiple intracellular and extracellular binding partners. Thus, gene conversion provides a mechanism for limiting the divergence of functional regions of protein paralogs, while allowing other domains to evolve diversified functions.

  6. Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to Toxoplasma gondii profilin

    OpenAIRE

    Raetz, Megan; Kibardin, Alexey; Sturge, Carolyn R.; Pifer, Reed; Li, Haiying; Burstein, Ezra; Ozato, Keiko; Larin, Sergei; Yarovinsky, Felix

    2013-01-01

    Toll-like receptors play a central role in the innate recognition of pathogens and the activation of dendritic cells (DCs). Here, we established that, in addition to TLR11, TLR12 recognizes the profilin protein of the protozoan parasite Toxoplasma gondii and regulates IL-12 production by DCs in response to the parasite. Similar to TLR11, TLR12 is an endolysosomal innate immune receptor that colocalizes and interacts with UNC93B1. Biochemical experiments revealed that TLR11 and TLR12 directly ...

  7. TLR7 expression is decreased during tumour progression in transgenic adenocarcinoma of mouse prostate mice and its activation inhibits growth of prostate cancer cells.

    Science.gov (United States)

    Han, Ju-Hee; Park, Shin-Young; Kim, Jin-Bum; Cho, Sung-Dae; Kim, Bumseok; Kim, Bo-Yeon; Kang, Min-Jung; Kim, Dong-Jae; Park, Jae-Hak; Park, Jong-Hwan

    2013-10-01

    Although various Toll-like receptors (TLRs) have been associated with immune response and tumorigenesis in the prostate cells, little is known about the role of TLR7. Accordingly, we examined the expression of TLR7 during tumour progression of TRMAP (transgenic mouse model for prostate cancer) mice and its role on cell growth. Toll-like receptor7 expression was examined by RT-polymerase chain reaction (PCR), Western blot, and immunohistochemistry. Cell growth was examined by MTT assay. Colony formation was investigated by crystal violet staining. Strong expression of TLR7 was detected in the normal prostate epithelia of Wild-type (WT) mice, but not in TLR7-deficient mice. In contrast, TLR7 expression was weak in transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 cells, as compared with murine bone marrow-derived macrophages (BMDMs). Moreover, TLR7 mRNA was markedly expressed in RWPE-1 cells (non-cancerous prostate epithelial cells), but not in PC3 and DU145 (prostate cancer cells). Immunohistochemically, TLR7 expression gradually decreased in TRAMP mice depending on the pathologic grade of the prostate cells. TLR7 agonists increased both the gene and protein expression of TLR7 and promoted production of proinflammatory cytokines/chemokines and IFN-β gene expression in prostate cancer cell lines. Moreover, loxoribine inhibited the growth and colony formation of TRAMP-C2 cells dependent of TLR7. These findings suggest that TLR7 may participate in tumour suppression in the prostate cells. © 2013 John Wiley & Sons Ltd.

  8. Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

    Science.gov (United States)

    Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

    2014-09-05

    Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Molecular identification and expression analysis of TLR5M and TLR5S from orange-spotted grouper (Epinepheluscoioides).

    Science.gov (United States)

    Bai, Jian-Shan; Li, Yan-Wei; Deng, Yan; Huang, Yan-Qiong; He, Shu-Hua; Dai, Jin; Zhao, Shang-Zhi; Dan, Xue-Ming; Luo, Xiao-Chun

    2017-04-01

    Toll-like receptor 5 (TLR5) is an important receptor that interacts with bacterial flagellin and regulates host immune response in mammal. Recent studies demonstrate that piscine contains two types of TLR5, namely membrane form of TLR5 (TLR5M) and soluble form of TLR5 (TLR5S), and both of which perform crucial role in flagellin response. In the present study, a TLR5M and a TLR5S sequence was cloned from orange-spotted grouper (Epinepheluscoioides), and their ORFs are respectively 2466 bp (821 aas) and 1935 bp (644 aas). EcTLR5M has the typical TLR structure of a LRR domain, a transmembrane region and a TIR domain, while EcTLR5S only contains a LRR domain like other species' TLR5S. Both molecules have 23 LRR motifs, a LRR-NT and a LRR-CT in the LRR domain, similar to those of other species. Phylogenetic and sequence alignment indicated that both EcTLR5s respectively displayed closer relationship and higher sequence identity with those in other fish species. In healthy grouper, EcTLR5M was highly expressed in the skin, head kidney and spleen, while EcTLR5S was mainly detected in the liver. Ciliate Cryptocaryon irritans infection could significantly up-regulate the expression level of EcTLR5s in the gill and spleen from day 1 to day 3, and higher expression fold change was observed in the spleen. Taken together, the present studies contributed to understanding the function of piscine TLR5M/S and clarify their possible role in fish immune response against ciliate infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. TLR2/TLR4 activation induces Tregs and suppresses intestinal inflammation caused by Fusobacterium nucleatum in vivo.

    Directory of Open Access Journals (Sweden)

    Yin-Ping Jia

    Full Text Available Toll-like receptors (TLRs 2 and 4 play critical roles in intestinal inflammation caused by Fusobacterium nucleatum (F. nucleatum infection, but the role of TLR2/TLR4 in regulation of proinflammatory cytokines remains unknown. In this study, through microarray analysis and qRT-PCR, we showed that TLR2/TLR4 are involved in the F. nucleatum-induced inflammatory signaling pathway in Caco-2 cells, C57BL/6 mice and human clinical specimens. In TLR2-/- and TLR4-/- mice, F. nucleatum infection resulted in increased colonization of the bacteria and production of the proinflammatory cytokines IL-8, IL-1β and TNF-α. In addition, the ratio of Foxp3+ CD4+ T cells in the total CD4+ T cells in TLR2-/- and TLR4-/- mice was less than that in wild-type mice, and the ratio in hybrid mice was more than that in knockout mice, which suggested that TLR2/TLR4 mediated the number of Tregs. Furthermore, it was observed that inflammatory cytokine levels were reduced in TLR2-/- mice after Treg transfer. Thus, these data indicate that TLR2/TLR4 regulate F. nucleatum-induced inflammatory cytokines through Tregs in vivo.

  11. DMPD: TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 2007 Feb 1. (.png) (.svg) (.html) (.csml) Show TLR signaling. PubmedID 17275323 Title TLR signaling. Author...ng) SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csml file with CIOP

  12. Heterogeneity of host TLR2 stimulation by Staphylocoocus aureus isolates.

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    Dina Hilmi

    Full Text Available High lipoprotein expression and potent activation of host Toll-like receptor-2 (TLR2 are characteristic features of the staphylococcal species. Expression of TLR2 in the host is important for clearance of Staphylococcus aureus infection and host survival. Thus, we hypothesized that bacterial regulation of its intrinsic TLR2-stimulatory capacity could represent a means for immune evasion or host adaptation. We, therefore, compared clinical S. aureus isolates in regards to their TLR2 activation potential and assessed the bacterial factors that modulate TLR2-mediated recognition. S. aureus isolates displayed considerable variability in TLR2-activity with low to absent TLR2-activity in 64% of the isolates tested (68/106. Notably, strain-specific TLR2-activity was independent of the strain origin, e.g. no differences were found between strains isolated from respiratory specimen from cystic fibrosis patients or those isolated from invasive disease specimen. TLR2-activity correlated with protein A expression but not with the agr status. Capsule expression and small colony variant formation had a negative impact on TLR2-activity but any disruption of cell wall integrity enhanced TLR2 activation. Altogether, heterogeneity in host TLR2-activity reflects differences in metabolic activity and cell wall synthesis and/or remodeling.

  13. Postnatal TLR2 activation impairs learning and memory in adulthood.

    Science.gov (United States)

    Madar, Ravit; Rotter, Aviva; Waldman Ben-Asher, Hiba; Mughal, Mohamed R; Arumugam, Thiruma V; Wood, W H; Becker, K G; Mattson, Mark P; Okun, Eitan

    2015-08-01

    Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. TLR4 as a negative regulator of keratinocyte proliferation.

    Directory of Open Access Journals (Sweden)

    Guergana Iotzova-Weiss

    Full Text Available TLR4 is an innate immune receptor with expression in human skin, keratinocytes as well as squamous cell carcinoma (SCC of the skin. In the present study we investigate the role of TLR4 as a negative regulator of keratinocyte proliferation. We present here that the expression of TLR4 increased with the differentiation of cultured keratinocytes in a passage-dependent manner or under calcium-rich conditions. Moreover, the down-regulation of TLR4 by specific knockdown increased the proliferation of HaCaT keratinocytes in vitro. In addition, subcutaneously injected HaCaT keratinocytes with shTLR4 formed growing tumors in nude mice. In contrast, we observed lower proliferation and increased migration in vitro of the SCC13 cell line stably overexpressing TLR4 in comparison to SCC13 TLR4 negative cells. In vivo, SCC13 TLR4-overexpressing tumors showed delayed growth in comparison to TLR4 negative tumors. The overexpression of TLR4 in SCC13 tumor cells was followed by phosphorylation of ERK1/2 and JNK and increased expression of ATF3. In gene expression arrays, the overexpression of TLR4 in tumor cells correlated with gene expression of ATF-3, IL-6, CDH13, CXCL-1 and TFPI. In summary, TLR4 negatively regulates the proliferation of keratinocytes and its overexpression reduces tumor growth of SCC cells.

  15. TLR4 as a negative regulator of keratinocyte proliferation.

    Science.gov (United States)

    Iotzova-Weiss, Guergana; Freiberger, Sandra N; Johansen, Pål; Kamarachev, Jivko; Guenova, Emmanuella; Dziunycz, Piotr J; Roux, Guillaume A; Neu, Johannes; Hofbauer, Günther F L

    2017-01-01

    TLR4 is an innate immune receptor with expression in human skin, keratinocytes as well as squamous cell carcinoma (SCC) of the skin. In the present study we investigate the role of TLR4 as a negative regulator of keratinocyte proliferation. We present here that the expression of TLR4 increased with the differentiation of cultured keratinocytes in a passage-dependent manner or under calcium-rich conditions. Moreover, the down-regulation of TLR4 by specific knockdown increased the proliferation of HaCaT keratinocytes in vitro. In addition, subcutaneously injected HaCaT keratinocytes with shTLR4 formed growing tumors in nude mice. In contrast, we observed lower proliferation and increased migration in vitro of the SCC13 cell line stably overexpressing TLR4 in comparison to SCC13 TLR4 negative cells. In vivo, SCC13 TLR4-overexpressing tumors showed delayed growth in comparison to TLR4 negative tumors. The overexpression of TLR4 in SCC13 tumor cells was followed by phosphorylation of ERK1/2 and JNK and increased expression of ATF3. In gene expression arrays, the overexpression of TLR4 in tumor cells correlated with gene expression of ATF-3, IL-6, CDH13, CXCL-1 and TFPI. In summary, TLR4 negatively regulates the proliferation of keratinocytes and its overexpression reduces tumor growth of SCC cells.

  16. TLR4- and TLR9-dependent effects on cytokines, cell viability, and invasion in human bladder cancer cells.

    Science.gov (United States)

    Olbert, Peter J; Kesch, Claudia; Henrici, Marcus; Subtil, Florentine S; Honacker, Astrid; Hegele, Axel; Hofmann, Rainer; Hänze, Jörg

    2015-03-01

    Adjuvant immunotherapy of bladder cancer by instillation of bacillus Calmette-Guérin (BCG) is highly recommended within certain groups of non-muscle-invasive stages but only partially effective. Toll-like receptors (TLRs) TLR4 and TLR9 likely mediate BCG effects by triggering innate systemic immune cell responses. In addition, TLR4 and TLR9 expressed in bladder cancer cells may contribute to the outcome of BCG treatment. Here, we studied the expression and function of TLR4 and TLR9 in human bladder cancer cell lines. TLR4 and TLR9 messenger RNA and protein levels were determined by real-time reverse transcription polymerase chain reaction and Western blot. Selected cell lines were analyzed with respect to cytokine induction, proliferation, and cell invasion after addition of BCG, TLR4-specific agonist lipopolysaccharide (LPS), or TLR9 agonist (CpG-oligodeoxynucleotide [ODN]). TLR4 and TLR9 were expressed quite heterogeneously in human bladder cancer cells. BCG caused induction of interleukin (IL)-6 or IL-8 in BFTC905 and T24 cells as representatives for TLR4-/TLR9-expressing cells. The study aimed to dissect TLR4- and TLR9-mediated effects. For functional analysis of TLR4 with LPS, we selected T24 and BFTC905 cells with high and undetectable TLR4 levels, respectively. For TLR9 analysis with CpG-ODN, we selected UMUC3 and RT112 cells with high and low TLR9 levels, respectively. Addition of LPS caused significant induction of TNFα and IL-6 messenger RNA in T24 cells but not in BFTC905 cells. Addition of CpG-ODN induced interferon ß (INFß), IL-8, tumor necrosis factor α (TNFα) and the angiogenic factors vascular endothelial growth factor-A and placental growth factor in UMUC3 cells; whereas in RT112 cells, induction of IL-8 and TNFα was noticed. Interestingly, addition of CpG-ODN significantly reduced cell viability and increased cell invasion in UMUC3 and RT112 cells. Our findings demonstrate that bladder cancer cell lines express functional TLR4 and TLR9 with

  17. TLR7 agonist induced repression of hepatocellular carcinoma via the TLR7-IKK-NF-?B-IL6 signaling pathway

    OpenAIRE

    REN, XINGBIN; WANG, FEI; JI, BAOJU; GAO, CHUNHAI

    2016-01-01

    Toll-like receptors (TLRs) are key members of innate immunity, involved in the defense against diseases, and evidence has revealed that TLR4/5 is involved in the carcinogenesis of hepatic cancer. TLR7 belongs to the TLR family, and its roles in immune-associated hepatic diseases have been well characterized; however, the consequences of agonist targeting of TLR7 in hepatic cancer have not previously been reported. The present study aimed to investigate the effects and underlying mechanisms of...

  18. QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi × Leghorn F2 cross, using a medium-density SNP panel

    Science.gov (United States)

    2014-01-01

    Background Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F2 cross. The 860 F2 animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied. Results Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL. Conclusions The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between

  19. QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi × Leghorn F₂ cross, using a medium-density SNP panel.

    Science.gov (United States)

    Bacciu, Nicola; Bed'Hom, Bertrand; Filangi, Olivier; Romé, Hélène; Gourichon, David; Répérant, Jean-Michel; Le Roy, Pascale; Pinard-van der Laan, Marie-Hélène; Demeure, Olivier

    2014-02-19

    Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F2 cross. The 860 F2 animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied. Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL. The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between different models and marker densities.

  20. Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.

    Directory of Open Access Journals (Sweden)

    Soo-Ho Choi

    Full Text Available Oxidation of low-density lipoprotein (LDL is one of the major causative mechanisms in the development of atherosclerosis. In previous studies, we showed that minimally oxidized LDL (mmLDL induced inflammatory responses in macrophages, macropinocytosis and intracellular lipid accumulation and that oxidized cholesterol esters (OxCEs were biologically active components of mmLDL. Here we identified a specific OxCE molecule responsible for the biological activity of mmLDL and characterized signaling pathways in macrophages in response to this OxCE. Using liquid chromatography - tandem mass spectrometry and biological assays, we identified an oxidized cholesteryl arachidonate with bicyclic endoperoxide and hydroperoxide groups (BEP-CE as a specific OxCE that activates macrophages in a TLR4/MD-2-dependent manner. BEP-CE induced TLR4/MD-2 binding and TLR4 dimerization, phosphorylation of SYK, ERK1/2, JNK and c-Jun, cell spreading and uptake of dextran and native LDL by macrophages. The enhanced macropinocytosis resulted in intracellular lipid accumulation and macrophage foam cell formation. Bone marrow-derived macrophages isolated from TLR4 and SYK knockout mice did not respond to BEP-CE. The presence of BEP-CE was demonstrated in human plasma and in the human plaque material captured in distal protection devices during percutaneous intervention. Our results suggest that BEP-CE is an endogenous ligand that activates the TLR4/SYK signaling pathway. Because BEP-CE is present in human plasma and human atherosclerotic lesions, BEP-CE-induced and TLR4/SYK-mediated macrophage responses may contribute to chronic inflammation in human atherosclerosis.

  1. Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages.

    NARCIS (Netherlands)

    Ferwerda, G.; Meyer-Wentrup, F.; Kullberg, B.J.; Netea, M.G.; Adema, G.J.

    2008-01-01

    The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized

  2. (TLR-4) expression on polymorphonuclear neutrophil leukocytes ...

    African Journals Online (AJOL)

    To establish a foundation for further researches on the improvement of polymorphonuclear neutrophil leukocytes (PMN) functions in dairy cow during perinatal period, the counting of PMN, as well as the mRNA and protein expression of toll-like receptor-4 (TLR-4) on PMN was studied during this critical period.

  3. Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia

    Directory of Open Access Journals (Sweden)

    Yao Linli

    2013-02-01

    Full Text Available Abstract Background Hypoxia induces microglial activation which causes damage to the developing brain. Microglia derived inflammatory mediators may contribute to this process. Toll-like receptor 4 (TLR4 has been reported to induce microglial activation and cytokines production in brain injuries; however, its role in hypoxic injury remains uncertain. We investigate here TLR4 expression and its roles in neuroinflammation in neonatal rats following hypoxic injury. Methods One day old Wistar rats were subjected to hypoxia for 2 h. Primary cultured microglia and BV-2 cells were subjected to hypoxia for different durations. TLR4 expression in microglia was determined by RT-PCR, western blot and immunofluorescence staining. Small interfering RNA (siRNA transfection and antibody neutralization were employed to downregulate TLR4 in BV-2 and primary culture. mRNA and protein expression of tumor necrosis factor-alpha (TNF-α, interleukin-1 beta (IL-1β and inducible nitric oxide synthase (iNOS was assessed. Reactive oxygen species (ROS, nitric oxide (NO and NF-κB levels were determined by flow cytometry, colorimetric and ELISA assays respectively. Hypoxia-inducible factor-1 alpha (HIF-1α mRNA and protein expression was quantified and where necessary, the protein expression was depleted by antibody neutralization. In vivo inhibition of TLR4 with CLI-095 injection was carried out followed by investigation of inflammatory mediators expression via double immunofluorescence staining. Results TLR4 immunofluorescence and protein expression in the corpus callosum and cerebellum in neonatal microglia were markedly enhanced post-hypoxia. In vitro, TLR4 protein expression was significantly increased in both primary microglia and BV-2 cells post-hypoxia. TLR4 neutralization in primary cultured microglia attenuated the hypoxia-induced expression of TNF-α, IL-1β and iNOS. siRNA knockdown of TLR4 reduced hypoxia-induced upregulation of TNF-α, IL-1β, iNOS, ROS and

  4. Induction of Protective Immunity against Eimeria tenella Infection using Antigen-loaded Dendritic Cells (DC) and DC-derived Exosomes

    Science.gov (United States)

    Eimeria is a protozoan parasite which causes coccidiosis, a major parasitic disease of poultry that is indigenous to most regions of the world and causes intestinal damage with reduced body weight gain. Conventional disease control methods have relied on prophylactic chemotherapy, but alternative m...

  5. Microsatellite mapping of QTLs affecting resistance to coccidiosis (Eimeria tenella) in a Fayoumi × White Leghorn cross

    Science.gov (United States)

    Pinard-van der Laan, Marie-Hélène; Bed'hom, Bertrand; Coville, Jean-Luc; Pitel, Frédérique; Feve, Katia; Leroux, Sophie; Legros, Hélène; Thomas, Aurélie; Gourichon, David; Repérant, Jean-Michel; Rault, Paul

    2009-01-01

    Background Avian coccidiosis is a major parasitic disease of poultry, causing severe economical loss to poultry production by affecting growth and feed efficiency of infected birds. Current control strategies using mainly drugs and more recently vaccination are showing drawbacks and alternative strategies are needed. Using genetic resistance that would limit the negative and very costly effects of the disease would be highly relevant. The purpose of this work was to detect for the first time QTL for disease resistance traits to Eimeria tenella in chicken by performing a genome scan in an F2 cross issued from a resistant Fayoumi line and a susceptible Leghorn line. Results The QTL analysis detected 21 chromosome-wide significant QTL for the different traits related to disease resistance (body weight growth, plasma coloration, hematocrit, rectal temperature and lesion) on 6 chromosomes. Out of these, a genome-wide very significant QTL for body weight growth was found on GGA1, five genome-wide significant QTL for body weight growth, plasma coloration and hematocrit and one for plasma coloration were found on GGA1 and GGA6, respectively. Two genome-wide suggestive QTL for plasma coloration and rectal temperature were found on GGA1 and GGA2, respectively. Other chromosme-wide significant QTL were identified on GGA2, GGA3, GGA6, GGA15 and GGA23. Parent-of-origin effects were found for QTL for body weight growth and plasma coloration on GGA1 and GGA3. Several QTL for different resistance phenotypes were identified as co-localized on the same location. Conclusion Using an F2 cross from resistant and susceptible chicken lines proved to be a successful strategy to identify QTL for different resistance traits to Eimeria tenella, opening the way for further gene identification and underlying mechanisms and hopefully possibilities for new breeding strategies for resistance to coccidiosis in the chicken. From the QTL regions identified, several candidate genes and relevant

  6. Microsatellite mapping of QTLs affecting resistance to coccidiosis (Eimeria tenella in a Fayoumi × White Leghorn cross

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    Gourichon David

    2009-01-01

    Full Text Available Abstract Background Avian coccidiosis is a major parasitic disease of poultry, causing severe economical loss to poultry production by affecting growth and feed efficiency of infected birds. Current control strategies using mainly drugs and more recently vaccination are showing drawbacks and alternative strategies are needed. Using genetic resistance that would limit the negative and very costly effects of the disease would be highly relevant. The purpose of this work was to detect for the first time QTL for disease resistance traits to Eimeria tenella in chicken by performing a genome scan in an F2 cross issued from a resistant Fayoumi line and a susceptible Leghorn line. Results The QTL analysis detected 21 chromosome-wide significant QTL for the different traits related to disease resistance (body weight growth, plasma coloration, hematocrit, rectal temperature and lesion on 6 chromosomes. Out of these, a genome-wide very significant QTL for body weight growth was found on GGA1, five genome-wide significant QTL for body weight growth, plasma coloration and hematocrit and one for plasma coloration were found on GGA1 and GGA6, respectively. Two genome-wide suggestive QTL for plasma coloration and rectal temperature were found on GGA1 and GGA2, respectively. Other chromosme-wide significant QTL were identified on GGA2, GGA3, GGA6, GGA15 and GGA23. Parent-of-origin effects were found for QTL for body weight growth and plasma coloration on GGA1 and GGA3. Several QTL for different resistance phenotypes were identified as co-localized on the same location. Conclusion Using an F2 cross from resistant and susceptible chicken lines proved to be a successful strategy to identify QTL for different resistance traits to Eimeria tenella, opening the way for further gene identification and underlying mechanisms and hopefully possibilities for new breeding strategies for resistance to coccidiosis in the chicken. From the QTL regions identified, several

  7. DMPD: The Troll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17449723 The Troll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling. Sh...Show The Troll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling. PubmedID 17449723 Title The Tro...ll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling. Authors Sheedy F

  8. Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.

    Science.gov (United States)

    Fieber, Christina; Janos, Marton; Koestler, Tina; Gratz, Nina; Li, Xiao-Dong; Castiglia, Virginia; Aberle, Marion; Sauert, Martina; Wegner, Mareike; Alexopoulou, Lena; Kirschning, Carsten J; Chen, Zhijian J; von Haeseler, Arndt; Kovarik, Pavel

    2015-01-01

    Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13-/- cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.

  9. Innate Immune Response to Streptococcus pyogenes Depends on the Combined Activation of TLR13 and TLR2

    Science.gov (United States)

    Fieber, Christina; Janos, Marton; Koestler, Tina; Gratz, Nina; Li, Xiao-Dong; Castiglia, Virginia; Aberle, Marion; Sauert, Martina; Wegner, Mareike; Alexopoulou, Lena; Kirschning, Carsten J.; Chen, Zhijian J.; von Haeseler, Arndt; Kovarik, Pavel

    2015-01-01

    Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13−/− cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms. PMID:25756897

  10. TLR4 and NKT cell synergy in immunotherapy against visceral leishmaniasis.

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    Subir Karmakar

    Full Text Available NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR can be modulated by activated invariant Natural Killer T (iNKT cells. The terminal β-(1-4-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1-4-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic.

  11. The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease

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    Rolf Spirig

    2012-01-01

    Full Text Available Cardiovascular disease is a complex disorder involving multiple pathophysiological processes, several of which involve activation of toll-like receptors (TLRs of the innate immune system. As sentinels of innate immunity TLRs are nonclonally germline-encoded molecular pattern recognition receptors that recognize exogenous as well as tissue-derived molecular dangers signals promoting inflammation. In addition to their expression in immune cells, TLRs are found in other tissues and cell types including cardiomyocytes, endothelial and vascular smooth muscle cells. TLRs are differentially regulated in various cell types by several cardiovascular risk factors such as hypercholesterolemia, hyperlipidemia, and hyperglycemia and may represent a key mechanism linking chronic inflammation, cardiovascular disease progression, and activation of the immune system. Modulation of TLR signaling by specific TLR agonists or antagonists, alone or in combination, may be a useful therapeutic approach to treat various cardiovascular inflammatory conditions such as atherosclerosis, peripheral arterial disease, secondary microvascular complications of diabetes, autoimmune disease, and ischemia reperfusion injury. In this paper we discuss recent developments and current evidence for the role of TLR in cardiovascular disease as well as the therapeutic potential of various compounds on inhibition of TLR-mediated inflammatory responses.

  12. Estimation of genetic diversity in a natural population of cambui tree (Myrciaria tenella O. Berg) using ISSR markers.

    Science.gov (United States)

    Santana, J G S; Nascimento, A L S; Costa, T S; de Almeida, T M B; Rabbani, A R C; Silva, A V C

    2016-10-05

    Cambui (Myrciaria tenella O. Berg) is a native species from Brazil, which belongs to the family Myrtaceae. Molecular characterization is one of the most used tools for the study of the biotechnological potential of species because the diversity level between individuals can be inferred. Analysis of genetic diversity is fundamental to the direction of the strategies necessary to form and maintain a germplasm. This study aimed to evaluate the genetic diversity in a natural population of cambui using inter-simple sequence repeat (ISSR) molecular markers. The natural population, which provided the plant material, is found at the Private Reserve of Natural Heritage of Caju, which belongs to the experimental field of Embrapa Tabuleiros Costeiros, in the municipality of Itaporanga d'Ajuda, SE, Brazil. Young leaves of each individual were collected for DNA extraction and analysis of PCR-ISSR. Thirty primers were tested and the top 10 were selected. The use of these primers resulted in 71 fragments with 98.3% polymorphism. Similarity of individuals ranged between 0.30 and 0.92. The most similar individuals were C13 and C17 and the most distant were C1 and C41. Through UPGMA, six distinct groups were identified. This information may be used for conservation of these genetic resources, germplasm exchange, creation of germplasm bank and in future studies with this species.

  13. Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association.

    Science.gov (United States)

    Conti, Filippo; Boucherit, Nicolas; Baldassarre, Veronica; Trouplin, Virginie; Toman, Rudolf; Mottola, Giovanna; Mege, Jean-Louis; Ghigo, Eric

    2014-01-01

    To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

  14. Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association

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    Filippo eConti

    2015-01-01

    Full Text Available To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS, avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with Escherichia coli LPS or with the LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid activating macrophages by the disruption of the TLR-2 and TLR-4 association.

  15. In situ TLR2 and TLR4 expression in a murine model of mycetoma caused by Nocardia brasiliensis.

    Science.gov (United States)

    Millán-Chiu, Blanca Edith; Hernández-Hernández, Francisca; Pérez-Torres, Armando; Méndez-Tovar, Luis Javier; López-Martínez, Rubén

    2011-04-01

    Actinomycetoma caused by Nocardia brasiliensis is a common disease in tropical regions. This ailment is characterized by a localized chronic inflammation that mainly affects the lower limbs. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, inducing the production of proinflammatory mediators. The role of TLRs in the immune response against N. brasiliensis is unknown. The aim of this work was to locate and quantify in a murine model the expression of TLR2 and TLR4 in the infection site using reverse transcription-PCR and immunohistochemistry. The results showed that TLR2 expression increased in the infected tissue, whereas TLR4 expression decreased. The presence of TLR2 and TLR4 was demonstrated in different cell populations throughout the chronic infectious process. In the early stages of this process, TLR2 was expressed in neutrophils and macrophages in direct contact with the inoculum, whereas TLR4 was observed in mast cells. In the advanced stages of the infection, TLR2 was expressed in foam cells and fibroblasts and was likely associated with bacterial containment, while TLR4 was downregulated, probably resulting in an imbalance between the host immune response and the bacterial load that favoured chronic disease. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  16. Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation

    Science.gov (United States)

    Song, Jianwen; Guan, Ming; Zhao, Zhenwen; Zhang, Junjie

    2015-01-01

    Lysophosphatidic acid (LPA) is an important phospholipid mediator in inflammation and immunity. However, the mechanism of LPA regulation during inflammatory response is largely unknown. Autotaxin (ATX) is the key enzyme to produce extracellular LPA from lysophosphatidylcholine (LPC). In this study, we found that ATX was induced in monocytic THP-1 cells by TLR4 ligand lipopolysaccharide (LPS), TLR9 ligand CpG oligonucleotide, and TLR3 ligand poly(I:C), respectively. The ATX induction by TLR ligand was abolished by the neutralizing antibody against IFN-β or the knockdown of IFNAR1, indicating that type I IFN autocrine loop is responsible for the ATX induction upon TLR activation. Both IFN-β and IFN-α were able to induce ATX expression via the JAK-STAT and PI3K-AKT pathways but with different time-dependent manners. The ATX induction by IFN-β was dramatically enhanced by IFN-γ, which had no significant effect on ATX expression alone, suggesting a synergy effect between type I and type II IFNs in ATX induction. Extracellular LPA levels were significantly increased when THP-1 cells were treated with IFN-α/β or TLR ligands. In addition, the type I IFN-mediated ATX induction was identified in human monocyte-derived dendritic cells (moDCs) stimulated with LPS or poly(I:C), and IFN-α/β could induce ATX expression in human peripheral blood mononuclear cells (PBMCs) and monocytes isolated form blood samples. These results suggest that, in response to TLR activation, ATX is induced through a type I INF autocrine-paracrine loop to enhance LPA generation. PMID:26313906

  17. Type I Interferons Function as Autocrine and Paracrine Factors to Induce Autotaxin in Response to TLR Activation.

    Directory of Open Access Journals (Sweden)

    Jianwen Song

    Full Text Available Lysophosphatidic acid (LPA is an important phospholipid mediator in inflammation and immunity. However, the mechanism of LPA regulation during inflammatory response is largely unknown. Autotaxin (ATX is the key enzyme to produce extracellular LPA from lysophosphatidylcholine (LPC. In this study, we found that ATX was induced in monocytic THP-1 cells by TLR4 ligand lipopolysaccharide (LPS, TLR9 ligand CpG oligonucleotide, and TLR3 ligand poly(I:C, respectively. The ATX induction by TLR ligand was abolished by the neutralizing antibody against IFN-β or the knockdown of IFNAR1, indicating that type I IFN autocrine loop is responsible for the ATX induction upon TLR activation. Both IFN-β and IFN-α were able to induce ATX expression via the JAK-STAT and PI3K-AKT pathways but with different time-dependent manners. The ATX induction by IFN-β was dramatically enhanced by IFN-γ, which had no significant effect on ATX expression alone, suggesting a synergy effect between type I and type II IFNs in ATX induction. Extracellular LPA levels were significantly increased when THP-1 cells were treated with IFN-α/β or TLR ligands. In addition, the type I IFN-mediated ATX induction was identified in human monocyte-derived dendritic cells (moDCs stimulated with LPS or poly(I:C, and IFN-α/β could induce ATX expression in human peripheral blood mononuclear cells (PBMCs and monocytes isolated form blood samples. These results suggest that, in response to TLR activation, ATX is induced through a type I INF autocrine-paracrine loop to enhance LPA generation.

  18. Role of TLR9 in Oncogenic Virus-Produced Cancer.

    Science.gov (United States)

    Martínez-Campos, Cecilia; Burguete-García, Ana I; Madrid-Marina, Vicente

    2017-03-01

    Nucleic acid recognition by toll-like receptor 9 (TLR9) initiates signaling pathways that regulate the production of proinflammatory cytokines or type I interferons, as well as many other molecules required to initialize the immune response. The use of synthetic oligodeoxynucleotides (ODNs) has been crucial to emulate the recognition of DNA sequences by TLR9. Furthermore, ODN administration to mice has shown to confer protection against a wide range of viral, bacterial, and parasitic pathogens. In contrast, oncogenic DNA viruses like hepatitis B virus, Epstein-Barr virus, and human papilloma virus inhibit TLR9 expression, thus contributing to the establishment of chronic viral infections. In this review, we will focus on TLR9 signals initiated by ODN recognition, on the inhibition of TLR9 expression mediated by DNA oncogenic viruses, and on TLR9 expression as a relevant event in the progression to cancer, considering other functions of this receptor, aside from viral recognition.

  19. Innate immune sensing of modified vaccinia virus Ankara (MVA is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.

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    Julie Delaloye

    2009-06-01

    Full Text Available Modified vaccinia virus Ankara (MVA is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs. The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR and the IPS-1 adapter (also known as Cardif, MAVS or VISA. Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/- and MyD88(-/- BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/- BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus

  20. The preparation and physico-chemical characterization of aluminum hydroxide/TLR7a, a novel vaccine adjuvant comprising a small molecule adsorbed to aluminum hydroxide.

    Science.gov (United States)

    Malyala, Padma; Laera, Donatello; Cianetti, Simona; Bufali, Simone; Aggravi, Marianna; Ianni, Elvira; Judge, Casey; Otten, Gillis; Singh, Manmohan; O' Hagan, Derek T

    2018-02-05

    Adjuvants are necessary to enable vaccine development against a significant number of challenging pathogens for which effective vaccines are not available [1]. We engineered a novel small-molecule immune potentiator (SMIP), a benzonaphthyridine agonist targeting toll-like receptor 7 (TLR7), as a vaccine adjuvant. TLR7 agonist (TLR7a) was engineered to be adsorbed onto aluminum hydroxide (AlOH), and the resulting AlOH/TLR7a was evaluated as a vaccine adjuvant [2-6]. AlOH/TLR7a exploits the flexibility of AlOH formulations, has an application in many vaccine candidates, and induced good efficacy and safety profiles against all tested antigens (bacterial- and viral-derived protein antigens, toxoids, glycoconjugates, etc.) in many animal models, including non-human primates [7]. In this paper, we describe the outcome of the physico-chemical characterization of AlOH/TLR7a. Reverse phase ultra-performance liquid chromatography (RP-UPLC), confocal microscopy, flow cytometry (FC), zeta potential (ZP) and phosphophilicity assays were used as tools to demonstrate the association of TLR7a to AlOH and to characterize this novel formulation. Raman spectroscopy, nuclear magnetic resonance and mass spectroscopy were also used to investigate the interaction between TLR7a and AlOH (data not shown). This pivotal work paved the way for AlOH/TLR7a to progress into the clinic for evaluation as an adjuvant platform for vaccines against challenging preventable diseases. Copyright © 2018. Published by Elsevier Inc.

  1. Staphylococcus aureus recognition by hematopoietic stem and progenitor cells via TLR2/MyD88/PGE2 stimulates granulopoiesis in wounds

    Science.gov (United States)

    Granick, Jennifer L.; Falahee, Patrick C.; Dahmubed, Delsheen; Borjesson, Dori L.; Miller, Lloyd S.

    2013-01-01

    During bacterial infection, hematopoietic stem and progenitor cells (HSPCs) differentiate into polymorphonuclear leukocytes (PMNs) in the bone marrow. We reported that HSPCs recruited to Staphylococcus aureus–infected skin wounds in mice undergo granulopoiesis, whereas other authors have demonstrated their differentiation in vitro after Toll-like receptor 2 (TLR2)/MyD88 stimulation. Here, we examined this pathway in HSPC trafficking and granulopoiesis within S aureus–infected wounds. Lineage− HSPCs from TLR2- or MyD88-deficient mice injected into infected wounds of wild-type (WT) mice exhibited impaired granulopoiesis. However, HSPCs from WT mice produced similar numbers of PMNs whether transferred into wounds of TLR2-, MyD88-deficient, or WT mice. Prostaglandin E2 (PGE2), which stimulates HSPC survival and proliferation, was produced by HSPCs after TLR2 stimulation, suggesting that TLR2/MyD88 activation promotes granulopoiesis in part by production and autocrine activity of PGE2. Pretreatment of TLR2- or MyD88-deficient HSPCs with PGE2 rescued granulocytic differentiation in vivo. Finally, we demonstrate that bone marrow–derived lin−/Sca-1+/c-kit+ cells produced PGE2 and underwent granulopoiesis after TLR2 stimulation. lin−/Sca-1+/c-kit+ cells deficient in TLR2 or MyD88 produced PMNs after PGE2 treatment when transferred into uninfected wounds. We conclude that granulopoiesis in S aureus–infected wounds is induced by TLR2/MyD88 activation of HSPCs through a mechanism that involves autocrine production and activity of PGE2. PMID:23869087

  2. TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

    Science.gov (United States)

    Israely, Tomer; Melamed, Sharon; Achdout, Hagit; Erez, Noam; Politi, Boaz; Waner, Trevor; Lustig, Shlomo; Paran, Nir

    2014-01-01

    Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

  3. TLR3 and TLR9 agonists improve postexposure vaccination efficacy of live smallpox vaccines.

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    Tomer Israely

    Full Text Available Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV, a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs were less protective than the TLR3 agonist poly(I:C. We show that activation of type 1 IFN by poly(I:C and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

  4. Efficacy of chimeric DNA vaccines encoding Eimeria tenella 5401 and chicken IFN-γ or IL-2 against coccidiosis in chickens.

    Science.gov (United States)

    Song, Xiaokai; Huang, Xinmei; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

    2015-09-01

    Chimeric DNA vaccines encoding Eimeria tenella (E. tenella) surface antigen 5401 were constructed and their efficacies against E. tenella challenge were studied. The open reading frame (ORF) of 5401 was cloned into the prokaryotic expression vector pGEX-4T2 to express the recombinant protein and the expressed recombinant protein was identified by Western blot. The ORF of 5401 and chicken cytokine gene IFN-γ or IL-2 were cloned into the eukaryotic expression vector pVAX1 consecutively to construct DNA vaccines pVAX-5401-IFN-γ, pVAX-5401-IL-2 and pVAX-5401. The expression of aim genes in vivo was detected by reverse transcription-polymerase chain reaction and Western blot. Fourteen-day-old chickens were inoculated twice at an interval of 7 days with 100 µg of plasmids pVAX-5401, pVAX-5401-IFN-γ and pVAX-5401-IL-2 or 200 µg of recombinant 5401 protein by leg intramuscular injection, respectively. Seven days after the second inoculation, all chickens except the unchallenged control group were challenged orally with 5 × 10(4) sporulated oocysts of E. tenella. Seven days after challenge, all chickens were weighted and slaughtered to determine the effects of immunization. The results showed the recombinant protein was about 90 kDa and reacted with antiserum against soluble sporozoites. The animal experiment showed that all the DNA vaccines pVAX-5401, pVAX-5401-IFN-γ or pVAX-5401-IL-2 and the recombinant 5401 protein could obviously alleviate body weight loss and cecal lesions as compared with non-vaccinated challenged control and empty vector pVAX1control. Furthermore, pVAX-5401-IFN-γ or pVAX-5401-IL-2 induced anti-coccidial index (ACI) of 180.01 or 177.24 which were significantly higher than that of pVAX-5401. The results suggested that 5401 was an effective candidate antigen for vaccine. This finding also suggested that chicken IFN-γ or IL-2 could effectively improve the efficacies of DNA vaccines against avian coccidiosis. Copyright © 2015 Elsevier

  5. STUDIES OF INNATE IMMUNITY PARAMETERS (TLR2, TLR4 AND HBD1 OF URETHERAL MUCOUS EPITHELIUM IN CHRONIC BACTERIAL CYSTITIS

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    O. A. Svitich

    2016-01-01

    Full Text Available The study for quantitation of TLR2, TLR4 and HBD-1 gene expression in urethral mucosal epithelium from women with chronic bacterial cystitis and healthy females. Materials and methods: RNA was extracted from the samples of urethral mucosa followed by RT-PCR. Results: increased expression of TLR2, TLR4 (respectively, 6.9-, 2.6-fold, along with 13.6-fold decrease in HBD-1 was revealed in women with chronic bacterial cystitis, as compared with appropriate parameters in normal women. Six months later, after combined therapy with autologous immunopeptide complex, TLR2, TLR4 и HBD1 expression levels proved to be near-normal in the patients, being similar to those in healthy females. Conclusion: Imbalanced expression of genes controlling innate immunity was revealed in urethral mucosa of the patients with chronic cystitis, thus increasing risk for urinary tract infections.

  6. Conventional Dendritic Cells Confer Protection against Mouse Cytomegalovirus Infection via TLR9 and MyD88 Signaling

    Directory of Open Access Journals (Sweden)

    Franz Puttur

    2016-10-01

    Full Text Available Cytomegalovirus (CMV is an opportunistic virus severely infecting immunocompromised individuals. In mice, endosomal Toll-like receptor 9 (TLR9 and downstream myeloid differentiation factor 88 (MyD88 are central to activating innate immune responses against mouse CMV (MCMV. In this respect, the cell-specific contribution of these pathways in initiating anti-MCMV immunity remains unclear. Using transgenic mice, we demonstrate that TLR9/MyD88 signaling selectively in CD11c+ dendritic cells (DCs strongly enhances MCMV clearance by boosting natural killer (NK cell CD69 expression and IFN-γ production. In addition, we show that in the absence of plasmacytoid DCs (pDCs, conventional DCs (cDCs promote robust NK cell effector function and MCMV clearance in a TLR9/MyD88-dependent manner. Simultaneously, cDC-derived IL-15 regulates NK cell degranulation by TLR9/MyD88-independent mechanisms. Overall, we compartmentalize the cellular contribution of TLR9 and MyD88 signaling in individual DC subsets and evaluate the mechanism by which cDCs control MCMV immunity.

  7. Lipoproteins attenuate TLR2 and TLR4 activation by bacteria and bacterial ligands with differences in affinity and kinetics.

    Science.gov (United States)

    van Bergenhenegouwen, Jeroen; Kraneveld, Aletta D; Rutten, Lieke; Garssen, Johan; Vos, Arjan P; Hartog, Anita

    2016-10-28

    The small intestine is a specialized compartment were close interactions take place between host, microbes, food antigens and dietary fatty acids. Dietary fats get absorbed by epithelial cells and processed into a range of lipoprotein particles after which they are basolaterally secreted and collected in the lymphatics. In contrast to the colon, the small intestine is covered only by a thin mucus coat that allows for intimate interactions between host-cells and microbes. Lipoproteins have long been recognized as protective factors in infectious diseases via the neutralization of bacterial toxins like lipopolysaccharides. Much less attention has been given to the potential role of lipoproteins as factors contributing to the maintenance of small intestinal immune homeostasis via modulating bacteria-induced immune responses. Lipoproteins VLDL, LDL and HDL were found to neutralize TLR responses towards specific TLR-ligands or a selection of gram-negative and gram-positive bacteria. Attenuation of TLR2 activity was acute and only slightly improved by longer pre-incubation times of ligands and lipoproteins with no differences between bacterial-lipopeptides or bacteria. In contrast, attenuation of TLR4 responses was only observed after extensive preincubation of lipoproteins and LPS. Preincubation of bacteria and lipoproteins led only to a modest attenuation of TLR4 activity. Moreover, compared to TLR2, TLR4 activity could only be attenuated by lipoproteins over a small ligand dose range. These results demonstrate the ability of lipoproteins VLDL, LDL and HDL to inhibit TLR responses towards bacterial-ligands and bacteria. Presence of lipoproteins was found to modulate the MAMP-induced cytokine release by primary human monocytes measured as changes in the release of IL-6, TNFα, GM-CSF and IFNγ. Using TLR2 and TLR4-reporter cells, lipoproteins were found to inhibit TLR responses with differences in affinity and kinetics. These data establish a role for lipoproteins as

  8. 2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells.

    Science.gov (United States)

    Rimbach, Katharina; Kaiser, Steffen; Helm, Mark; Dalpke, Alexander H; Eigenbrod, Tatjana

    2015-01-01

    Microbial RNA is an important stimulator of innate immune responses. Differences in posttranscriptional RNA modification profiles enable the immune system to discriminate between self and non-self nucleic acids. This principle may be exploited by certain bacteria to circumvent immune cell activation. In this regard, 2'-O-methylation of Escherichia coli tRNATyr at position 18 (Gm18) has recently been described to inhibit TLR7-mediated IFN-α production in human plasmacytoid dendritic cells (pDCs). Extending these findings, we now demonstrate that Gm18 also potently inhibits TLR7-independent human monocyte activation by RNA derived from a variety of bacterial strains. The half minimal inhibitory concentration values were similar to those found for IFN-α inhibition in pDCs. Mechanistically, 2'-O-methylated RNA impaired upstream signalling events, including MAP kinase and NFx03BA;B activation. Our results suggest that antagonizing effects of Gm18-modified RNA are due to competition with stimulatory RNA for receptor binding. The antagonistic effect was specific for RNA because the small molecule TLR7/8 agonist R848 was not inhibited. Despite the striking phenotype in human cells, 2'-O-methylated RNA did not interfere with TLR13 activation by bacterial 23S rRNA in murine DC and BMDM. Thus, we identify here Gm18 in E. coli tRNA(Tyr) as a universal suppressor of innate immune activation in the human but not the murine system. © 2015 S. Karger AG, Basel.

  9. Autonomous cure of damaged human intestinal epithelial cells by TLR2 and TLR4-dependent production of IL-22 in response to Spirulina polysaccharides.

    Science.gov (United States)

    Tominaga, Akira; Konishi, Yuko; Taguchi, Takahiro; Fukuoka, Satoshi; Kawaguchi, Tokuichi; Noda, Tetsuo; Shimizu, Keiji

    2013-12-01

    In order to analyze the damage of human epithelial cells, we used human quasi-normal FPCK-1-1 cells derived from a colonic polyp in a patient with familial adenomatous polyposis as a monolayer, which is co-cultured with peptidoglycan (PGN)-stimulated THP-1 cells. Co-cultured FPCK-1-1 cells showed a decreased transepithelial electrical resistance (TER) and the lower level of claudin-2. When Spirulina complex polysaccharides were added one day before the start of the co-culture, there was no decrease of TER and claudin-2 (early phase damage). In contrast, when Spirulina complex polysaccharides were added to FPCK-1-1 cells after the level of TER had decreased, there was no recovery at the level of claudin-2, though the TER level recovered (late phase damage). The mucosa reconstitution is suggested to be involved in the recovery from the damaged status. Interestingly, autonomous recovery of FPCK-1-1 cells from both the early and late phase damage requires the production of IL-22, because anti-IL-22 antibodies inhibited recovery in these cases. Antibodies against either TLR2 or TLR4 inhibited the production of IL-22 from FPCK-1-1 colon epithelial cells, suggesting that signals through TLR2 and TLR4 are necessary for autonomous recovery of FPCK-1-1 colon epithelial cells by producing IL-22. In conclusion, we have established a useful model for the study of intestinal damage and recovery using human colon epithelial cells and our data suggest that damage to human colon epithelial cells can, at least in part, be recovered by the autonomous production of IL-22 in response to Spirulina complex polysaccharides. © 2013.

  10. mRNA TLR2 AND TLR4 EXPRESSION IN THE ENDOMETRIUM TISSUE IN WOMEN WITH ENDOMETRIOSIS ASSOSIATED WITH INFERTILITY.

    Science.gov (United States)

    Koval, H; Chopiak, V; Kamyshnyi, А

    2015-01-01

    Endometriosis is an important medical and social problem as it causes stable pelvic pains, afflicts women of the reproductive age, provokes infertility characterized by poor outcome of treatment. In recent times much attention is paid to the mechanisms of congenital immunity as possible mediators of the development of endometriosis and targets of therapy. The work deals with the investigation of the levels of mRNA TLR2 and TLR4 expression in the tissue of eutopic endometrium in women with endometriosis and infertility in comparison with women afflicted with infertility of a tubular character with the aim to define the role of TLR2 and TLR4 in the development of infertility in case of endometriosis. The study was conducted by means of polymerase chain reaction (PCR) real-time method. The results of the study are indicative of an increased TLR2 and TLR4 expression (especially TLR2) in the endometrium in women with endometriosis. The results obtained may be indicative of an important role of TLR2 and TLR4 in the development of endometrioid ectopia and should be considered while treating infertility in women with endometriosis.

  11. Distinctive responses of brain tumor cells to TLR2 ligands.

    Science.gov (United States)

    Yoon, Hee Jung; Jeon, Sae-Bom; Koh, Han Seok; Song, Jae-Young; Kim, Sang Soo; Kim, In-Hoo; Park, Eun Jung

    2015-05-01

    Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll-like receptors (TLR)-2-mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2-deficient, interferon (IFN)-γ-deficient, and IFNγ-receptor-1-deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2-dependent pathway, not IFNγ-receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2-dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2-associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors. © 2015 Wiley Periodicals, Inc.

  12. Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis.

    Science.gov (United States)

    Sun, Hui; Wang, Longjiang; Wang, Tiantian; Zhang, Jie; Liu, Qing; Chen, Peipei; Chen, Zhengtao; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

    2014-04-01

    S. cerevisiae is generally regarded as safe and benign organism and its surface display system may be used as a unique eukaryotic expression system that is suitable for expressing eukaryotic antigen. In addition to the convenience of vaccine delivery, the yeast cell wall has been shown to enhance the innate immunity when immunized with the yeast live oral vaccine. In the present study, we expressed the chicken coccidian E. tenella EtMic2, a microneme protein, on the surface of the S. cerevisiae and evaluated it as a potential oral vaccine for chicken against E. tenella challenge. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the live oral vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. In addition, the yeast oral vaccine could stimulate humoral as well as cell mediate immune responses. These results suggested that EtMic2 displayed on the cell surface of S. cerevisiae could be used as potential live vaccine against chicken coccidiosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. TLR-2 and TLR-4 expression in monocytes of newborns with late-onset sepsis,

    Directory of Open Access Journals (Sweden)

    Ana C.C. Redondo

    2014-09-01

    Full Text Available Objetivos: Analisar a expressão dos TLR-2 e TLR-4 em monócitos de recém-nascidos com sepse tardia. Métodos: Trata-se de um estudo prospectivo com 27 recém-nascidos a termo entre 8 e 29 dias de vida com diagnóstico clínico e laboratorial de sepse tardia dos quais dez (37% apresentaram cultura positiva. As citocinas foram determinadas por teste de CBA em sangue periférico enquanto que a expressão e MFI (mediana de intensidade de fluorescência dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em monócitos de sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. O grupo usado para comparação foi de adultos saudáveis. Resultados: Microrganismos foram identificados em 37% dos pacientes e estes juntamente com os pacientes com sepse clínica tiveram níveis elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1β e de citocina anti-inflamatória (IL-10 corroborando o processo inflamatório/infeccioso. No monócito, a frequência de expressão do TLR-4 foi mais elevada (p = 0,01. Conclusões: Este estudo analisou a resposta imune inata no recém-nascido com sepse. Recémnascidos sépticos que dependem quase exclusivamente do sistema imune inato apresentaram pouca resposta in vivo na ativação de monócitos o que sugere uma resposta imune deficiente e maior susceptibilidade à infecção.

  14. TLR4 and TLR2 Expression in Biopsy Specimens from Antral and Corporal Stomach Zones in Helicobacter pylori Infections

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Khakzad

    2014-10-01

    Full Text Available Background: It is not yet known which types of Toll-like receptors (TLRs are most effective in Helicobacter pylori (H. pylori recognition. It is also not known which gastric zones have the most prominent roles in TLR-mediated bacterial recognition. The aim of this work was to analyze the expression of TLR2 and TLR4 in biopsy specimens from H. pylori-infected patients. Methods: Thirty-eight patients with gastrointestinal disorders were divided into four groups in this study. The groups were: (A H. pylori infection and peptic ulcer (n=15, (B peptic ulcer only (n=5, (C H. pylori infection only (n=10 and (D control, with neither H. pylori infection nor peptic ulcer (n=8. Biopsy specimens from sites of redness or atrophic mucosa from gastric antrum and body in patients with gastritis were collected. RNAs from the antrum and body specimens were isolated. TLR2 and TLR4 mRNA expression was assessed by RT-PCR and quantified as densitometric ratios of TLR2 and TLR4/β-actin mRNA. Results: In the antral zones of H. pylori-infected patients (Groups A and C TLR2 and TLR4 expression was significantly greater than in uninfected patients (Groups B and D regardless of peptic ulcers (p < 0.05. In the gastric body samples TLR2 expression was significantly greater in Group C (H. pylori infection only than in Group B (peptic ulcer only and TLR4 expression was significantly greater in group A (H. pylori infection and peptic ulcer than in Group B (peptic ulcer only (p < 0.05. No significant differences in expression of TLR4 and TLR2 were observed between samples from the antrum and body in same groups. Conclusions: We conclude that H. pylori infection leads to significant increase in TLR2 and TLR4 molecules expression in antral region related to the control group. Considering the stimulatory effect of H. pylori on TLRs expression in the gastric tissue, we assume that colonization of H. pylori infection might occurs more in the gastric antral region than in the gastric

  15. Analysis of TLR polymorphisms in typhoid patients and ...

    African Journals Online (AJOL)

    Ilakkia Sivaji

    2016-01-20

    Jan 20, 2016 ... Subjects and methods: TLR genes were amplified by PCR from peripheral blood leukocytes of schoolchildren with typhoid (n = 20) or asymptomatic typhoid carrier (n = 30) state, and normal healthy individuals (n = 50). The RFLP analyses for TLR1, 2, 4 and 5 genes using restriction enzymes such as AluI, ...

  16. TLR1 polymorphisms in Europeans and spontaneous pregnancy loss.

    NARCIS (Netherlands)

    Crisan, T.O.; Farcas, M.F.; Trifa, A.P.; Plantinga, T.S.; Militaru, M.S.; Pop, I.V.; Netea, M.G.; Popp, R.A.

    2012-01-01

    Toll-like receptors (TLRs) are critical components of the pathogen recognition by the host innate immune system. Recently it has been shown that TLR1 is under evolutionary pressure in Europeans. This involves the positive selection of the nonsynonymous TLR1 1805G variant in Europeans, although this

  17. Hantaan virus triggers TLR4-dependent innate immune responses.

    Science.gov (United States)

    Yu, Hai-Tao; Jiang, Hong; Zhang, Ye; Nan, Xue-Ping; Li, Yu; Wang, Wei; Jiang, Wei; Yang, Dong-Qiang; Su, Wen-Jing; Wang, Jiu-Ping; Wang, Ping-Zhong; Bai, Xue-Fan

    2012-10-01

    The innate immune response induced by Hantavirus is responsible for endothelial cell dysfunction and viral pathogenicity. Recent studies demonstrate that TLR4 expression is upregulated and mediates the secretion of several cytokines in Hantaan virus (HTNV)-infected endothelial cells. To examine viral interactions with host endothelial cells and characterize the innate antiviral responses associated with Toll-like receptors, we selected TLR4 as the target molecule to investigate anti-hantavirus immunity. TLR4 mRNA-silenced EVC-304 (EVC-304 TLR4-) cells and EVC-304 cells were used to investigate signaling molecules downstream of TLR4. The expression of the adaptor protein TRIF was higher in HTNV-infected EVC-304 cells than in EVC-304 TLR4- cells. However, there was no apparent difference in the expression of MyD88 in either cell line. The transcription factors for NF-κB and IRF-3 were translocated from the cytoplasm into the nucleus in HTNV-infected EVC-304 cells, but not in HTNV-infected EVC-304 TLR4- cells. Our results demonstrate that TLR4 may play an important role in the antiviral immunity of the host against HTNV infection through an MyD88-independent signaling pathway.

  18. Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease

    Directory of Open Access Journals (Sweden)

    Nadigel Jessica

    2011-11-01

    Full Text Available Abstract Background Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD, an inflammatory lung disorder. COPD is characterized by an increase in CD8+ T cells within the central and peripheral airways. We hypothesized that the CD8+ T cells in COPD patients have increased Toll-like receptor (TLR expression compared to control subjects due to the exposure of cigarette smoke in the airways. Methods Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8+ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology. Results No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8+ T cells. Exposure of CD8+ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8+ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10. Conclusions Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8+ T cells in COPD. CD8+ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8+ T cells in COPD.

  19. Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages.

    Science.gov (United States)

    Chakraborty, Deep Chandan; Mukherjee, Gayatri; Banerjee, Pallavi; Banerjee, Kalyan K; Biswas, Tapas

    2011-10-07

    Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the β-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

  20. Up-regulation of TLR2 and TLR4 in high mobility group Box1-stimulated macrophages in pulpitis patients

    Science.gov (United States)

    Mahmoudi, Javad; Sabermarouf, Babak; Baradaran, Behzad; Sadat-Hatamnezhad, Leila; Shotorbani, Siamak Sandoghchian

    2017-01-01

    Objective(s): High Mobility Group Box1 (HMGB1) is a nonhistone, DNA-binding protein that serves a crucial role in regulating gene transcription and is involved in a variety of proinflammatory, extracellular activities. The aim of this study was to explore whether HMGB1 stimulation can up-regulate the expression of Toll-like Receptor 2 (TLR2) and Toll-like Receptor 4 (TLR4) on macrophages from pulpitis and to clarify the subsequent events involving Th17 cells and Th17 cell-associated cytokine changes. Materials and Methods: Having prepared dental pulp tissues of pulpitis and healthy controls, macrophage were isolated and cultured. Macrophages were thereafter stimulated by HMGB1 time course. RT-QPCR, flowcytometer, immunofluorescence, Western blotting, and ELISA techniques were used in the present research. Results: Our results showed that the expression of TLR2 and TLR4 on macrophages stimulated with HMGB1 increased in pulpitis compared with controls (macrophages without HMGB1 stimulation) with a statistical significance (Ppulpitis increased, and NF-kB, the downstream target of TLR2 and TLR4, also showed a marked elevation after macrophages’ stimulation by HMGB1. Conclusion: The evidence from the present study suggests that the enhanced TLR2 and TLR4 pathways and Th17 cell polarization may be due to HMGB1 stimulation in pulpitis. PMID:28293399

  1. Acanthamoeba infection in lungs of mice expressed by toll-like receptors (TLR2 and TLR4).

    Science.gov (United States)

    Derda, Monika; Wojtkowiak-Giera, Agnieszka; Kolasa-Wołosiuk, Agnieszka; Kosik-Bogacka, Danuta; Hadaś, Edward; Jagodziński, Paweł P; Wandurska-Nowak, Elżbieta

    2016-06-01

    Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznań, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP). Copyright © 2016 Elsevier Inc. All rights reserved.

  2. TLR 2 and 4 responsiveness from isolated peripheral blood mononuclear cells from rats and humans as potential chronic pain biomarkers.

    Directory of Open Access Journals (Sweden)

    Yuen H Kwok

    Full Text Available BACKGROUND: Chronic pain patients have increased peripheral blood mononuclear cell Interkeukin-1β production following TLR2 and TLR4 simulation. Here we have used a human-to-rat and rat-to-human approach to further investigate whether peripheral blood immune responses to TLR agonists might be suitable for development as possible systems biomarkers of chronic pain in humans. METHODS AND RESULTS: Study 1: using a graded model of chronic constriction injury in rats, behavioral allodynia was assessed followed by in vitro quantification of TLR2 and TLR4 agonist-induced stimulation of IL-1β release by PBMCs and spinal cord tissues (n = 42; 6 rats per group. Statistical models were subsequently developed using the IL-1β responses, which distinguished the pain/no pain states and predicted the degree of allodynia. Study 2: the rat-derived statistical models were tested to assess their predictive utility in determining the pain status of a published human cohort that consists of a heterogeneous clinical pain population (n = 19 and a pain-free population (n = 11. The predictive ability of one of the rat models was able to distinguish pain patients from controls with a ROC AUC of 0.94. The rat model was used to predict the presence of pain in a new chronic pain cohort and was able to accurately predict the presence of pain in 28 out of the 34 chronic pain participants. CONCLUSIONS: These clinical findings confirm our previous discoveries of the involvement of the peripheral immune system in chronic pain. Given that these findings are reflected in the prospective graded rat data, it suggests that the TLR response from peripheral blood and spinal cord were related to pain and these clinical findings do indeed act as system biomarkers for the chronic pain state. Hence, they provide additional impetus to the neuroimmune interaction to be a drug target for chronic pain.

  3. Association of human TLR1 and TLR6 deficiency with altered immune responses to BCG vaccination in South African infants.

    Directory of Open Access Journals (Sweden)

    April Kaur Randhawa

    2011-08-01

    Full Text Available The development of effective immunoprophylaxis against tuberculosis (TB remains a global priority, but is hampered by a partially protective Bacillus Calmette-Guérin (BCG vaccine and an incomplete understanding of the mechanisms of immunity to Mycobacterium tuberculosis. Although host genetic factors may be a primary reason for BCG's variable and inadequate efficacy, this possibility has not been intensively examined. We hypothesized that Toll-like receptor (TLR variation is associated with altered in vivo immune responses to BCG. We examined whether functionally defined TLR pathway polymorphisms were associated with T cell cytokine responses in whole blood stimulated ex vivo with BCG 10 weeks after newborn BCG vaccination of South African infants. In the primary analysis, polymorphism TLR6_C745T (P249S was associated with increased BCG-induced IFN-γ in both discovery (n = 240 and validation (n = 240 cohorts. In secondary analyses of the combined cohort, TLR1_T1805G (I602S and TLR6_G1083C (synonymous were associated with increased IFN-γ, TLR6_G1083C and TLR6_C745T were associated with increased IL-2, and TLR1_A1188T was associated with increased IFN-γ and IL-2. For each of these polymorphisms, the hypo-responsive allele, as defined by innate immunity signaling assays, was associated with increased production of TH1-type T cell cytokines (IFN-γ or IL-2. After stimulation with TLR1/6 lipopeptide ligands, PBMCs from TLR1/6-deficient individuals (stratified by TLR1_T1805G and TLR6_C745T hyporesponsive genotypes secreted lower amounts of IL-6 and IL-10 compared to those with responsive TLR1/6 genotypes. In contrast, no IL-12p70 was secreted by PBMCs or monocytes. These data support a mechanism where TLR1/6 polymorphisms modulate TH1 T-cell polarization through genetic regulation of monocyte IL-10 secretion in the absence of IL-12. These studies provide evidence that functionally defined innate immune gene variants are associated with the

  4. Microglia are required for astroglial toll-like receptor 4 response and for optimal TLR2 and TLR3 response

    DEFF Research Database (Denmark)

    Holm, Thomas Hellesøe; Draeby, Dina; Owens, Trevor

    2012-01-01

    Within the central nervous system, astrocytes and microglia are the primary responders to endogenous ligands released upon injury and stress, as well as to infectious pathogens. Toll-like receptors (TLRs) are implicated in recognition of both types of stimulus. Whether astrocytes respond as stron......Within the central nervous system, astrocytes and microglia are the primary responders to endogenous ligands released upon injury and stress, as well as to infectious pathogens. Toll-like receptors (TLRs) are implicated in recognition of both types of stimulus. Whether astrocytes respond...... astrocytes from mixed glial cultures and measured their response to TLR agonists. Our results show that the response of astrocytes to TLR2 and TLR3 agonists is greatly enhanced by, and response to TLR4 agonists is completely dependent on, the presence of functional microglia. In the case of the TLR4 response...

  5. Association analysis identifies TLR7 and TLR8 as novel risk genes in asthma and related disorders

    DEFF Research Database (Denmark)

    Møller-Larsen, Steffen; Nyegaard, Mette; Haagerup, Annette

    2008-01-01

    BACKGROUND: Toll-like receptors (TLRs) are structurally and functionally related and play important roles in the innate and adaptive immune system. By genome scanning, evidence of linkage between chromosome Xp22 and asthma and related atopic disorders has previously been obtained. Xp22 harbours...... the TLR7 and TLR8 genes. METHODS: The involvement of TLR7 and TLR8 in the aetiology of asthma and related disorders was investigated by a family based association analysis of two independently ascertained family samples comprising 540 and 424 individuals from 135 and 100 families, respectively. Ten......). CONCLUSION: The results provide strong evidence that TLR7 and 8 may confer susceptibility to asthma and related atopic disorders and highlight these receptors as interesting targets for individualised, causally directed treatment....

  6. Association analysis identifies TLR7 and TLR8 as novel risk genes in asthma and related disorders

    DEFF Research Database (Denmark)

    Møller-Larsen, Steffen; Nyegaard, Mette; Haagerup, Annette

    2008-01-01

    BACKGROUND: Toll-like receptors (TLRs) are structurally and functionally related and play important roles in the innate and adaptive immune system. By genome scanning, we have previously obtained evidence of linkage between chromosome Xp22 and asthma and related atopic disorders. Xp22 harbours...... the TLR7 and TLR8 genes. METHODS: We investigated the involvement of TLR7 and TLR8 in the aetiology of asthma and related disorders by a family based association analysis of two independently ascertained family samples comprising 540 and 424 individuals from 135 and 100 families, respectively. Ten......). CONCLUSION: The results presented, provide strong evidence that TLR7 and 8 may confer susceptibility to asthma and related atopic disorders and highlight these receptors as interesting targets for individualized, causally directed treatment....

  7. Optimal oligonucleotide sequences for TLR9 inhibitory activity in human cells: lack of correlation with TLR9 binding.

    Science.gov (United States)

    Ashman, Robert F; Goeken, J Adam; Latz, Eicke; Lenert, Petar

    2011-03-01

    Toll-like receptor (TLR)9 performs our innate response to bacterial DNA, warning us of the presence of infection. Inhibitory oligodeoxyribonucleotides (INH-ODN) have been developed that selectively block activation of mouse TLR9. Their inhibitory motif consisting of CCx(not-C)(not-C)xxGGG (x = any base) also reduces anti-DNA antibodies in lupus mice. The current study demonstrates that this motif also provides the sequences required to block TLR9 in human B cells and human embryonic kidney (HEK) cells transfected with human TLR9. However, extending the sequence by four to five bases at the 5' end enhanced activity and this enhancement was greater when a phosphorothioate (pS) backbone replaced the native phosphodiester (pO) backbone. A series of pO-backbone INH-ODN representing a 500-fold range of activity in biologic assays was shown to cover less than a 2.5-fold range of avidity for binding human TLR9-Ig fusion protein, eliminating TLR9 ectodomain binding as the explanation for sequence-specific differences in biologic activity. With few exceptions, the relative activity of INH-ODN in Namalwa cells and HEK/human TLR9 cells was similar to that seen in mouse B cells. INH-ODN activity in human peripheral blood B cells correlated significantly with the cell line data. These results favor the conclusion that although the backbone determines strength of TLR9 binding, critical recognition of the INH-ODN sequence necessary for biologic activity is performed by a molecule that is not TLR9. These studies also identify the strongest INH-ODN for human B cells, helping to guide the selection of INH-ODN sequences for therapeutics in any situation where inflammation is enhanced by TLR9.

  8. Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection.

    Directory of Open Access Journals (Sweden)

    Jincheng Chen

    2015-07-01

    Full Text Available Dengue virus (DV infection is the most prevalent mosquito-borne viral disease and its manifestation has been shown to be contributed in part by the host immune responses. In this study, pathogen recognition receptors, Toll-like receptor (TLR 2 and TLR6 were found to be up-regulated in DV-infected human PBMC using immunofluorescence staining, flow cytometry and Western blot analyses. Using ELISA, IL-6 and TNF-α, cytokines downstream of TLR2 and TLR6 signaling pathways were also found to be up-regulated in DV-infected PBMC. IL-6 and TNF-α production by PBMC were reduced when TLR2 and TLR6 were blocked using TLR2 and TLR6 neutralizing antibodies during DV infection. These results suggested that signaling pathways of TLR2 and TLR6 were activated during DV infection and its activation contributed to IL-6 and TNF-α production. DV NS1 protein was found to significantly increase the production of IL-6 and TNF-α when added to PBMC. The amount of IL-6 and TNF-α stimulated by DV NS1 protein was reduced when TLR2 and TLR6 were blocked, suggesting that DV NS1 protein is the viral protein responsible for the activation of TLR2 and TLR6 during DV infection. Secreted alkaline phosphatase (SEAP reporter assay was used to further confirm activation of TLR2 and TLR6 by DV NS1 protein. In addition, DV-infected and DV NS1 protein-treated TLR6-/- mice have higher survivability compared to DV-infected and DV NS1 protein-treated wild-type mice. Hence, activation of TLR6 via DV NS1 protein could potentially play an important role in the immunopathogenesis of DV infection.

  9. Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

    Directory of Open Access Journals (Sweden)

    Radhashree Maitra

    2008-06-01

    Full Text Available With the advancement of biomedical technology, artificial materials have been developed to replace diseased, damaged or nonfunctional body parts. Among such materials, ultra high molecular weight alkane or modified alkyl polymers have been extensively used in heart valves, stents, pacemakers, ear implants, as well as total joint replacement devices. Although much research has been undertaken to design the most non-reactive biologically inert polyethylene derivatives, strong inflammatory responses followed by rejection and failure of the implant have been noted.Purification of the alkane polymers from the site of inflammation revealed extensive "in vivo" oxidation as detected by fourier transformed infra-red spectroscopy. Herein, we report the novel observation that oxidized alkane polymers induced activation of TLR1/2 pathway as determined by ligand dependent changes in intrinsic tyrosine fluorescence intensity and NF-kappaB luciferase gene assays. Oxidized polymers were very effective in activating dendritic cells and inducing secretion of pro-inflammatory cytokines. Molecular docking of the oxidized alkanes designated ligand specificity and polymeric conformations fitting into the TLR1/2 binding grooves.This is the first report of a synthetic polymer activating immune responses through TLR binding.

  10. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-α induction

    OpenAIRE

    Uematsu, Satoshi; Sato, Shintaro; Yamamoto, Masahiro; Hirotani, Tomonori; Kato, Hiroki; Takeshita, Fumihiko; Matsuda, Michiyuki; Coban, Cevayir; Ishii, Ken J.; Kawai, Taro; Takeuchi, Osamu; Akira, Shizuo

    2005-01-01

    Toll-like receptors (TLRs) recognize microbial pathogens and trigger innate immune responses. Among TLR family members, TLR7, TLR8, and TLR9 induce interferon (IFN)-α in plasmacytoid dendritic cells (pDCs). This induction requires the formation of a complex consisting of the adaptor MyD88, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and IFN regulatory factor (IRF) 7. Here we show an essential role of IL-1 receptor-associated kinase (IRAK)-1 in TLR7- and TLR9-mediated IRF7...

  11. Innate Immune Responses to TLR2 and TLR4 Agonists Differ between Baboons, Chimpanzees and Humans

    Science.gov (United States)

    Brinkworth, Jessica F.; Pechenkina, Ekaterina A.; Silver, Jack; Goyert, Sanna M.

    2012-01-01

    Background African catarrhine primates differ in bacterial disease susceptibility. Methods Human, chimpanzee, and baboon blood was stimulated with TLR-detected bacterial agonists and cytokine/chemokine induction assessed by real-time pcr. Results Humans and chimpanzees shared similar cytokine/chemokine responses, while baboon cytokine/chemokine induction differed. Generally, responses were agonist-independent. Conclusions These primates tend to generate species rather than agonist–specific responses to bacterial agonists. PMID:22978822

  12. MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis.

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    Dawn M E Bowdish

    2009-06-01

    Full Text Available Virtually all of the elements of Mycobacterium tuberculosis (Mtb pathogenesis, including pro-inflammatory cytokine production, granuloma formation, cachexia, and mortality, can be induced by its predominant cell wall glycolipid, trehalose 6,6'-dimycolate (TDM/cord factor. TDM mediates these potent inflammatory responses via interactions with macrophages both in vitro and in vivo in a myeloid differentiation factor 88 (MyD88-dependent manner via phosphorylation of the mitogen activated protein kinases (MAPKs, implying involvement of toll-like receptors (TLRs. However, specific TLRs or binding receptors for TDM have yet to be identified. Herein, we demonstrate that the macrophage receptor with collagenous structure (MARCO, a class A scavenger receptor, is utilized preferentially to "tether" TDM to the macrophage and to activate the TLR2 signaling pathway. TDM-induced signaling, as measured by a nuclear factor-kappa B (NF-kappaB-luciferase reporter assay, required MARCO in addition to TLR2 and CD14. MARCO was used preferentially over the highly homologous scavenger receptor class A (SRA, which required TLR2 and TLR4, as well as their respective accessory molecules, in order for a slight increase in NF-kappaB signaling to occur. Consistent with these observations, macrophages from MARCO(-/- or MARCO(-/-SRA(-/- mice are defective in activation of extracellular signal-related kinase 1/2 (ERK1/2 and subsequent pro-inflammatory cytokine production in response to TDM. These results show that MARCO-expressing macrophages secrete pro-inflammatory cytokines in response to TDM by cooperation between MARCO and TLR2/CD14, whereas other macrophage subtypes (e.g. bone marrow-derived may rely somewhat less effectively on SRA, TLR2/CD14, and TLR4/MD2. Macrophages from MARCO(-/- mice also produce markedly lower levels of pro-inflammatory cytokines in response to infection with virulent Mtb. These observations identify the scavenger receptors as essential binding

  13. Control of a line of E. tenella, partly resistant to monensin, by including toltrazuril discontinuously in the drinking water of chickens.

    Science.gov (United States)

    Chapman, H D

    1987-01-01

    Discontinuous medication with toltrazuril, given in the drinking water at a concentration of 50 ppm, was effective in controlling infection resulting from repeated inoculation of oocysts of a line of E. tenella partly resistant to monensin. Treatment was very effective when the drug was given for periods of 3 days, on two or three occasions at weekly intervals. Monensin, at a concentration of 120 ppm, given continuously in the feed was only partially effective. Following treatment with toltrazuril, birds inoculated with high doses of oocysts were immune to subsequent challenge. It is suggested that discontinuous medication with toltrazuril in the drinking water might provide an alternative to the current practice of incorporating drugs continuously in the feed.

  14. Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

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    Merk Martina

    2011-09-01

    Full Text Available Abstract Background Active dendritic cell (DC immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML. Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR agonists in intensively pretreated patients with AML. Methods Four different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls. Results Maturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C, induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70, showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells. Conclusions Our results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

  15. DMPD: TLR ignores methylated RNA? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16111629 TLR ignores methylated RNA? Ishii KJ, Akira S. Immunity. 2005 Aug;23(2):111-3. (.png) (.svg) (.html... SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csml file with CIOPlay

  16. SNP Marker Discovery in Koala TLR Genes: e0121068

    National Research Council Canada - National Science Library

    Jian Cui; Greta J Frankham; Rebecca N Johnson; Adam Polkinghorne; Peter Timms; Denis O'Meally; Yuanyuan Cheng; Katherine Belov

    2015-01-01

    .... Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study...

  17. Immune effects of beta-glucan are determined by combined effects on Dectin-1, TLR2, 4 and 5

    NARCIS (Netherlands)

    Kanjan, Pochanart; Sahasrabudhe, Neha M.; de Haan, Bart J.; de Vos, Paul

    2017-01-01

    Particulate beta-glucans enhanced NF-kappa B expression in cell-lines co-expressing Dectin-1A-TLR4 and Dectin1B-TLR4, while soluble beta-glucans only synergistically acted on Dectin-IA-TLR4. This was different with Dectin-1 co-expressing TLR2 and TLR5, which inhibited activation after particulate

  18. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

    Directory of Open Access Journals (Sweden)

    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  19. Alcohol, TLR4-TGF-β Antagonism, and Liver Cancer

    Science.gov (United States)

    Tsukamoto, Hidekazu; Mishra, Lopa; Machida, Keigo

    2016-01-01

    Alcohol abuse and obesity are two known risk factors for hepatocellular carcinoma (HCC) that also synergistically promote HBV/HCV-related carcinogenesis. TLR4, the PAMP for endotoxin participates in inflammatory processes such as M1 activation of hepatic macrophages in alcoholic liver disease. However its role in liver carcinogenesis via ectopic expression and activation, has only recently been revealed in alcohol/HCV-associated HCC models. Alcohol feeding to mice expressing the HCV Ns5a in a hepatocyte specific manner, aggravates liver inflammation via activation of overexpressed TLR4 in the parenchymal cells. Long-term alcohol feeding produces liver tumors in these transgenic mice in a manner dependent on TLR4. From these mice, CD133+/CD49f+ tumor initiating stem cell-like cells (TICs) have been isolated. These TICs exhibit self-renewal and tumorigenic activities driven by TLR4-dependent upregulation of the stem cell factor NANOG. Defective TGF-β tumor suppressor pathway is identified in the TICs and mediated by NANOG target genes Igf2bp3 and Yap1. This TGF-β pathway antagonism is responsible in part for TIC’s tumorigenic activity and chemoresistance. Conversely, mice with attenuated TGF-β pathway due to haploinsufficiency of β2-Spectrin, spontaneously develop liver tumors and alcohol-feeding increases tumor incidence in a TLR4 dependent manner. This reciprocal antagonism between TLR4 and TGF-β pathways may serve as a novel therapeutic target for HCC. PMID:26201318

  20. TLR ligands stimulation protects MSC from NK killing.

    Science.gov (United States)

    Giuliani, Massimo; Bennaceur-Griscelli, Annelise; Nanbakhsh, Arash; Oudrhiri, Noufissa; Chouaib, Salem; Azzarone, Bruno; Durrbach, Antoine; Lataillade, Jean-Jacques

    2014-01-01

    Mesenchymal stem cells (MSCs) play a fundamental role in allograft rejection and graft-versus-host disease through their immunosuppressive abilities. Recently, Toll-like receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and natural killer (NK) cells. Our results show that TLR-primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL-2-activated NK-induced killing. Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility complex class I chain A and ULBP3 and DNAM-1 ligands by TLR-primed MSC. These results indicate that MSCs are able to adapt their immuno-behavior in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4-primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC-mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders. © 2013 AlphaMed Press.

  1. The discriminative capacity of soluble Toll-like receptor (sTLR)2 and sTLR4 in inflammatory diseases

    NARCIS (Netherlands)

    Oever, J. ten; Kox, M.; Veerdonk, F.L. van de; Mothapo, K.M.; Slavcovici, A.; Jansen, T.L.Th.A.; Tweehuysen, L.; Giamarellos-Bourboulis, E.J.; Schneeberger, P.M.; Wever, P.C.; Stoffels, M.; Simon, A.; Meer, J. van der; Johnson, M.D.; Kullberg, B.J.; Pickkers, P.; Pachot, A.; Joosten, L.; Netea, M.G.

    2014-01-01

    BackgroundThe extracellular domains of cytokine receptors are released during inflammation, but little is known about the shedding of Toll-like receptors (TLR) and whether they can be used as diagnostic biomarkers.MethodsThe release of sTLR2 and sTLR4 was studied in in-vitro stimulations, as well as

  2. Characterization of TLR4-mediated auto-antibody production in a mouse model of histidyl-tRNA synthetase-induced myositis.

    Science.gov (United States)

    Harlow, Lisa; Fernandez, Irina; Soejima, Makoto; Ridgway, William M; Ascherman, Dana P

    2012-12-01

    We have previously shown that intramuscular immunization with a recombinant fragment of murine histidyl-tRNA synthetase (HRS) in the absence of exogenous adjuvant generates Ag-specific, IgG class switched Abs a murine model of myositis. Markedly diminished IgG anti-HRS auto-Ab responses in TLR4 signaling-deficient C3H/HeJ mice indicate that TLR4 is required for auto-Ab formation and/or class switching in this system. Comparative time course assessment of HRS-immunized C3H/HeOuJ (wild type) and C3H/HeJ (TLR4 mutant) mice shows here that despite significant impairment of class switched IgG anti-HRS responses in TLR4-deficient C3H/HeJ mice, production of IgM anti-HRS auto-Abs is relatively preserved-suggesting that TLR4-mediated signals modulate IgG class switching rather than auto-Ab formation in this genetic background. In C57BL/6-derived knockout mice lacking either MyD88 (B6.MyD88(-/-)) or TRIF (B6.TRIF(-/-)) adaptor molecules, immunization studies indicate that TRIF exerts a dominant role in the generation of HRS-specific IgG auto-Abs. Complementing these analyses, in vitro stimulation of unfractionated, as well as T cell-depleted, C3H/HeOuJ splenocytes with recombinant murine HRS reveals that TLR4-mediated generation of class switched auto-Abs can occur independently of T cell help. Overall, these findings support a broader role for TLR4 in the breakdown of immune tolerance and development of autoimmunity.

  3. Regional mucosa-associated microbiota determine physiological expression of TLR2 and TLR4 in murine colon.

    Directory of Open Access Journals (Sweden)

    Yunwei Wang

    2010-10-01

    Full Text Available Many colonic mucosal genes that are highly regulated by microbial signals are differentially expressed along the rostral-caudal axis. This would suggest that differences in regional microbiota exist, particularly mucosa-associated microbes that are less likely to be transient. We therefore explored this possibility by examining the bacterial populations associated with the normal proximal and distal colonic mucosa in context of host Toll-like receptors (TLR expression in C57BL/6J mice housed in specific pathogen-free (SPF and germ-free (GF environments. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP and clone library analysis revealed significant differences in the community structure and diversity of the mucosa-associated microbiota located in the distal colon compared to proximal colon and stool, the latter two clustering closely. Differential expression of colonic TLR2 and TLR4 along the proximal-distal axis was also found in SPF mice, but not in GF mice, suggesting that enteric microbes are essential in maintaining the regional expression of these TLRs. TLR2 is more highly expressed in proximal colon and decreases in a gradient to distal while TLR4 expression is highest in distal colon and a gradient of decreased expression to proximal colon is observed. After transfaunation in GF mice, both regional colonization of mucosa-associated microbes and expression of TLRs in the mouse colon were reestablished. In addition, exposure of the distal colon to cecal (proximal microbiota induced TLR2 expression. These results demonstrate that regional colonic mucosa-associated microbiota determine the region-specific expression of TLR2 and TLR4. Conversely, region-specific host assembly rules are essential in determining the structure and function of mucosa-associated microbial populations. We believe this type of host-microbial mutualism is pivotal to the maintenance of intestinal and immune homeostasis.

  4. Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis.

    Science.gov (United States)

    Cambier, Ludivine C; Heinen, Marie-Pierre A-L; Bagut, Elena T; Antoine, Nadine A; Mignon, Bernard R

    2016-04-01

    Polymorphonuclear neutrophils (PMNs), along with macrophages, are the first leukocytes recruited to the site of infection in dermatophytoses and are responsible for the in fine elimination of the fungus. It has been demonstrated that feline PMNs produce pro-inflammatory cytokines after stimulation with Microsporum canis. The activation of these cells results from the recognition of specific PAMPs (pathogen associated molecular patterns) from M. canis by PRRs (pattern recognition receptors) of PMNs. The C-type lectin receptors (CLRs) and toll-like receptors (TLRs) are the two main PRRs in phagocytic cells that recognize fungal components. The aim of this study was to evaluate the expression of TLR-2, TLR-4 and dectin-1 mRNA in feline PMNs exposed to different components from M. canis. Feline PMNs were stimulated for 2 h or 4 h with either live arthroconidia, heat-killed arthroconidia or secreted components from M. canis. The levels of TLR-2, TLR-4 and dectin-1 mRNA were assessed by RT-qPCR. Results showed an increase of TLR-2 and TLR-4 mRNA levels in feline PMNs stimulated with live and heat-killed arthroconidia, but not in those stimulated with the secreted components from M. canis. No significant variation in dectin-1 mRNA expression was observed in PMNs stimulated with the different fungal components. The overexpression of TLR-2 and TLR-4 mRNAs in stimulated feline PMNs suggests that these receptors are involved in the host immune response through the recognition of M. canis PAMPs. © 2016 ESVD and ACVD.

  5. Immunomodulation of TLR2 and TLR4 by G2013 (alfa-L-Guluronic acid in CVID Patients

    Directory of Open Access Journals (Sweden)

    Laleh Sharifi

    2017-07-01

    Full Text Available Background: Common variable immunodeficiency (CVID is a primary immune disorder associated with hypogammaglobulinemia, recurrent infections and autoimmune diseases. CVID patients are frequently in contact with infectious pathogens leading to the activation of innate immunity through Toll-like receptors (TLR affecting adaptive immunity. The aim of the present study was to test the immunomedulatory effect of small molecule G2013, a novel designed non-steroidal anti-inflammatory agent in CVID. Materials and Methods: After blood sampling from 16 CVID patients and 16 age- and sex-matched healthy controls, peripheral blood mononuclear cells (PBMCs were isolated and treated with/without lipopolysaccharide (LPS, lipopolyteichoic acid (LTA, and G2013. Assessing the immunomodulatory effect of G2013, flowcytometry was done for quantify the protein expression of TLR2 and TLR4. Gene expressions of signaling molecules involved in the TLR2 and TLR4 pathways were assessed by real-time PCR. ELISA performed assessing the production of IL-1b and IL-6. Results: G2013 significantly decreased the intensity of TLR2 expression in CVID PBMCs (p=0.001 also G2013 decreased significantly the NF-kB gene expression in PBMCs of CVID patients (p=0.006. Conclusion: These results indicated that G2013 had immunomodulatory effect at least in part via TLR2 and NF-kB expression. G2013 by decreasing MFI of TLR2 expression and NFkB gene expression provide the possibility of designing new drugs for preventing or controlling autoimmunity in CVID patients.

  6. TLR2-TLR4/CD14 polymorphisms and predisposition to severe invasive infections by Neisseria meningitidis and Streptococcus pneumoniae.

    Science.gov (United States)

    Tellería-Orriols, J J; García-Salido, A; Varillas, D; Serrano-González, A; Casado-Flores, J

    2014-01-01

    Streptococcus pneumoniae and Neisseria meningitidis are major causes of severe invasive bacterial infections in some individuals. Apparently the genetic is a major susceptibility determinant to these infectious diseases. We study if the functional polymorphisms within genes of the innate immune system (TLR2-TLR4 and CD14) are related to the predisposition to severe invasive infections caused by S. pneumoniae and N. meningitidis. Prospective descriptive study. Sixty-six Caucasian healthy children and 173 consecutive Caucasian children with invasive bacterial infections by N. meningitidis (n=59) and S. pneumoniae (n=114) were enrolled between January 1, 2008 and December 31, 2010. All blood samples were genotyped with description of the coding polymorphisms in p.R753Q of TLR2 gene and p.D299G of TLR4 gene as well as the promotor polymorphism c.-159C>T of the CD14 gene. Compared to the controls the p.753Q allele of TLR2 and the allele c.-159T of CD14 were more frequent in patients with S. pneumoniae (pGenetical variations in the innate immune system by polymorphisms in the TLR2 and CD14, could be related with an increases susceptibility to severe invasive infections by S. pneumoniae and N. meningitidis. Copyright © 2013 Elsevier España, S.L. and SEMICYUC. All rights reserved.

  7. Evaluation de l'efficacité des extraits aqueux de graines de papaye (Carica papaya L. dans le traitement de la coccidiose caecale à Eimeria tenella chez le poulet de chair

    Directory of Open Access Journals (Sweden)

    Akoa Etoa, JM.

    2003-01-01

    Full Text Available Evaluation of the Efficiency of Aqueous Extracts of Papaw Seeds (Carica papaya L. for the Treatment of Caecum Eimeria tenella Coccidiosis in Broiler Chicken. A study was carried out to study the efficiency of aqueous extracts of papaw seeds for the treatment of caecum Eimeria tenella coccidiosis in broiler chicken. Eighty eight 37-day old caged ISA15 VEDETTE broiler chickens were inoculated with a suspension of 3500 ± 1050 E. tenella oocysts/ml. The birds then divided into 4 groups of 22 birds received 10 days later either 0 (D0 dose, 10 (D10 dose, 20 (D20 dose or 40 g/l (D40 dose of aqueous extracts of papaw seeds. Mortality rates of 45.5%, 34.8%, 18.2% and 9.1% were recorded for D0, D10, D20 and D40 groups respectively. As compared to infestation rate before treatment, the reduction rate of the number of oocysts/g of faeces was respectively 6.6%, 42.8%, 73.6% and 91.8% for D0, D10, D20 and D40. Average daily weight gain was 42.7 g, 47.8 g, 69.8 g and 86 g for treatments D0, D10, D20 and D40, respectively while average weekly feed efficiency ratio values of 3.9, 4.1, 2.4 and 1.9 were respectively recorded for D0, D10, D20 and D40 treatments. Aqueous extracts of papaw seeds seemed to be efficient in the treatment of caecum E. tenella coccidiosis in broiler chickens.

  8. Pathogenic strains of Acanthamoeba are recognized by TLR4 and initiated inflammatory responses in the cornea.

    Directory of Open Access Journals (Sweden)

    Hassan Alizadeh

    Full Text Available Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK, a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05 CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

  9. TLR7 influences germinal center selection in murine SLE.

    Science.gov (United States)

    Boneparth, Alexis; Huang, Weiqing; Bethunaickan, Ramalingam; Woods, Megan; Sahu, Ranjit; Arora, Shitij; Akerman, Meredith; Lesser, Martin; Davidson, Anne

    2015-01-01

    TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa) cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa) or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

  10. TLR7 influences germinal center selection in murine SLE.

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    Alexis Boneparth

    Full Text Available TLR7 enhances germinal center maturation and migration of B cells to the dark zone where proliferation and somatic hypermutation occur. Our goal was to determine how Tlr7 dose influences selection of the autoreactive B cell repertoire in NZW/BXSB. Yaa mice bearing the site-directed heavy chain transgene 3H9 that encodes for the TLR7 regulated anti-CL response. To create a physiologic setting in which autoreactive B cells compete for survival with non-autoreactive B cells, we generated bone marrow chimeras in which disease onset occurred with similar kinetics and the transferred 3H9+ female non-Yaa, male Yaa or male TLR7(-/Yaa cells could be easily identified by positivity for GFP. Deletion of 3H9 B cells occurred in the bone marrow and the remaining 3H9 follicular B cells manifested a decrease in surface IgM. Although there were differences in the naïve repertoire between the chimeras it was not possible to distinguish a clear pattern of selection against lupus related autoreactivity in TLR7(-/Yaa or female chimeras. By contrast, preferential expansion of 3H9+ B cells occurred in the germinal centers of male Yaa chimeras. In addition, although all chimeras preferentially selected 3H9/Vκ5 encoded B cells into the germinal center and plasma cell compartments, 3H9 male Yaa chimeras had a more diverse repertoire and positively selected the 3H9/Vκ5-48/Jκ4 pair that confers high affinity anti-cardiolipin activity. We were unable to demonstrate a consistent effect of Tlr7 dose or Yaa on somatic mutations. Our data show that TLR7 excess influences the selection, expansion and diversification of B cells in the germinal center, independent of other genes in the Yaa locus.

  11. Critical role of TLR7 signaling in the priming of cross-protective cytotoxic T lymphocyte responses by a whole inactivated influenza virus vaccine.

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    Natalija Budimir

    Full Text Available Current influenza vaccines fail to induce protection against antigenically distinct virus strains. Accordingly, there is a need for the development of cross-protective vaccines. Previously, we and others have shown that vaccination with whole inactivated virus (WIV induces cross-protective cellular immunity in mice. To probe the mechanistic basis for this finding, we investigated the role of TLR7, a receptor for single-stranded RNA, in induction of cross-protection. Vaccination of TLR7-/- mice with influenza WIV failed to protect against a lethal heterosubtypic challenge; in contrast, wild-type mice were fully protected. The lack of protection in TLR7-/- mice was associated with high viral load and a relative paucity of influenza-specific CD8+ cytotoxic T lymphocyte (CTL responses. Dendritic cells (DCs from TLR7-/- mice were unable to cross-present WIV-derived antigen to influenza-specific CTLs in vitro. Similarly, TLR7-/- DCs failed to mature and become activated in response to WIV, as determined by the assessment of surface marker expression and cytokine production. Plasmacytoid DCs (pDCs derived from wild-type mice responded directly to WIV while purified conventional DCs (cDCs did not respond to WIV in isolation, but were responsive in mixed pDC/cDC cultures. Depletion of pDCs prior to and during WIV immunization resulted in reduced numbers of influenza-specific CTLs and impaired protection from heterosubtypic challenge. Thus, TLR7 plays a critical role in the induction of cross-protective immunity upon vaccination with WIV. The initial target cells for WIV appear to be pDCs which by direct or indirect mechanisms promote activation of robust CTL responses against conserved influenza epitopes.

  12. Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

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    Vania Sammartino Mariano

    Full Text Available TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

  13. Recognition of TLR2 N-Glycans: Critical Role in ArtinM Immunomodulatory Activity

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    da Silva, Thiago Aparecido; Ruas, Luciana Pereira; Nohara, Lilian L.; de Almeida, Igor Correia; Roque-Barreira, Maria Cristina

    2014-01-01

    TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties. PMID:24892697

  14. Fungal chitin dampens inflammation through IL-10 induction mediated by NOD2 and TLR9 activation.

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    Jeanette Wagener

    2014-04-01

    Full Text Available Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.

  15. Both TLR2 and TRIF contribute to interferon-β production during Listeria infection.

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    Camille Aubry

    Full Text Available Synthesis of interferon-β (IFN-β is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression.

  16. DAMP-TLR-cytokine axis dictates the fate of tumor.

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    Patidar, Ashok; Selvaraj, Sathishkumar; Sarode, Aditya; Chauhan, Prashant; Chattopadhyay, Debprasad; Saha, Bhaskar

    2017-10-09

    Random mutations leading to loss of cell cycle control is not a rare occurrence in an organism but the mutated cells are recognized and eliminated preventing the development of a tumor. These potentially tumorigenic cells release damage-associated molecular patterns (DAMPs), which are recognized by toll-like receptors (TLRs) on macrophages and dendritic cells. The initial TLR-DAMP interactions lead to different responses such as altered antigen presentation and cytokine release that directly affect T cell activation and removal of the tumorigenic cells. The indirect effects of TLR-DAMP interaction include chemokine-directed altered T cell trafficking, angiogenesis for both T cell infiltration and tumor cell metastasis, and alteration of intra-tumoral milieu contributing to the development of tumor cells heterogeneity. Thus, the initial TLR-DAMP interaction has a set of local effects that modulate tumor cell growth and heterogeneity and a disseminating set of central effects that dynamically affect T cell trafficking and functions. Herein, we argue that the DAMP-TLR-cytokine axis in the tumor microenvironment serves as the mainstay that orchestrates and regulates the pro- and anti-tumor elements which dynamically interact between themselves eventuating in tumor regression or growth. The knowledge of this TLR-based immuno-surveillance framework is a key to developing a novel immunotherapy against cancer. Copyright © 2017. Published by Elsevier Ltd.

  17. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS.

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    West, A Phillip; Brodsky, Igor E; Rahner, Christoph; Woo, Dong Kyun; Erdjument-Bromage, Hediye; Tempst, Paul; Walsh, Matthew C; Choi, Yongwon; Shadel, Gerald S; Ghosh, Sankar

    2011-04-28

    Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.

  18. TLR signaling augments macrophage bactericidal activity through mitochondrial ROS

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    West, A. Phillip; Brodsky, Igor E.; Rahner, Christoph; Woo, Dong Kyun; Erdjument-Bromage, Hediye; Tempst, Paul; Walsh, Matthew C.; Choi, Yongwon; Shadel, Gerald S.; Ghosh, Sankar

    2012-01-01

    Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria, and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase (Phox) machinery1. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to macrophage bactericidal activity, although the mechanisms linking innate immune signaling to mitochondria for mROS generation remain unclear2-4. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of the TLR signaling adapter tumor necrosis factor receptor-associated factor 6 (TRAF6) to mitochondria where it engages evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a protein implicated in mitochondrial respiratory chain assembly5. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT and TRAF6 depleted macrophages exhibit decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results therefore reveal a novel pathway linking innate immune signaling to mitochondria, implicate mROS as important components of antibacterial responses, and further establish mitochondria as hubs for innate immune signaling. PMID:21525932

  19. Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase.

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    Wegiel, Barbara; Gallo, David; Csizmadia, Eva; Roger, Thierry; Kaczmarek, Elzbieta; Harris, Clair; Zuckerbraun, Brian S; Otterbein, Leo E

    2011-11-15

    The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.

  20. Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes.

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    Kim, Min; Osborne, Naomi R; Zeng, Weiguang; Donaghy, Heather; McKinnon, Kay; Jackson, David C; Cunningham, Anthony L

    2012-05-01

    NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.

  1. Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss)

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    Palti, Yniv; Rodriguez, M. Fernanda; Gahr, Scott A.; Purcell, Maureen K.; Rexroad, Caird E.; Wiens, Gregory D.

    2010-01-01

    Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam2CSK4) and triacylated lipoprotein (Pam3CSK4). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are

  2. Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway.

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    Igor Landais

    2015-05-01

    Full Text Available Human Cytomegalovirus (HCMV encodes multiple microRNAs (miRNAs whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2. We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8 upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These

  3. Antibodies against Marinobacter algicola and Salmonella typhimurium flagellins do not cross-neutralize TLR5 activation.

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    Raul Terron-Exposito

    Full Text Available Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA-derived flagellins (F and FR as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF flagellins (FljB and FliC. We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2 and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure

  4. The MSHA strain of Pseudomonas aeruginosa activated TLR pathway and enhanced HIV-1 DNA vaccine immunoreactivity.

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    Jue Hou

    Full Text Available The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells and antigen presenting cells (APCs. Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells.

  5. TLR4 deficiency promotes autophagy during cigarette smoke-induced pulmonary emphysema

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    An, Chang Hyeok; Wang, Xiao Mei; Lam, Hilaire C.; Ifedigbo, Emeka; Washko, George R.; Ryter, Stefan W.

    2012-01-01

    Toll-like receptors (TLRs) exert important nonimmune functions in lung homeostasis. TLR4 deficiency promotes pulmonary emphysema. We examined the role of TLR4 in regulating cigarette smoke (CS)-induced autophagy, apoptosis, and emphysema. Lung tissue was obtained from chronic obstructive lung disease (COPD) patients. C3H/HeJ (Tlr4-mutated) mice and C57BL/10ScNJ (Tlr4-deficient) mice and their respective control strains were exposed to chronic CS or air. Human or mouse epithelial cells (wild-type, Tlr4-knockdown, and Tlr4-deficient) were exposed to CS-extract (CSE). Samples were analyzed for TLR4 expression, and for autophagic or apoptotic proteins by Western blot analysis or confocal imaging. Chronic obstructive lung disease lung tissues and human pulmonary epithelial cells exposed to CSE displayed increased TLR4 expression, and increased autophagic [microtubule-associated protein-1 light-chain-3B (LC3B)] and apoptotic (cleaved caspase-3) markers. Beas-2B cells transfected with TLR4 siRNA displayed increased expression of LC3B relative to control cells, basally and after exposure to CSE. The basal and CSE-inducible expression of LC3B and cleaved caspase-3 were elevated in pulmonary alveolar type II cells from Tlr4-deficient mice. Wild-type mice subjected to chronic CS-exposure displayed airspace enlargement;, however, the Tlr4-mutated or Tlr4-deficient mice exhibited a marked increase in airspace relative to wild-type mice after CS-exposure. The Tlr4-mutated or Tlr4-deficient mice showed higher levels of LC3B under basal conditions and after CS exposure. The expression of cleaved caspase-3 was markedly increased in Tlr4-deficient mice exposed to CS. We describe a protective regulatory function of TLR4 against emphysematous changes of the lung in response to CS. PMID:22983353

  6. Development of pristane induced mice model for lupus with atherosclerosis and analysis of TLR expression.

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    Chen, Xiaoqing; Cui, RanRan; Li, Rongda; Lin, Huili; Huang, Ziyang; Lin, Ling

    2016-01-01

    This study was designed to establish a murine model of lupus with atherosclerosis, and to investigate the expression of Toll-like receptors (TLRs) in the aorta and kidney. The 9-week-old female ApoE-/- and C57BL/6 mice were randomly divided into a ApoE-/- pristane treated group (group A), ApoE-/- control group (group B), C57BL/6 pristane treated group (group C) and C57BL/6 control group (group D). Each mouse was given either a single intraperitoneal injection of 0.5 ml pristane or saline. We observed that group A mice specifically had poor spirit, less activity, obvious hair loss, splenomegalia and renomegaly. Levels of ANA, anti-ds-DNA and anti-Sm antibodies were significantly higher than those in other groups. The group A and B mice generally displayed intimal hyperplasia and atherosclerosis mottling in the lumen of the aorta. The kidney tissues from group A, B and C mice showed increased expression levels of TLR2, TLR4, TLR7 and TLR9 proteins in comparison to group D. However, Group A mice did not show any significant difference in TLR2 and TLR4 protein expression levels when compared to group B and C, but displayed higher TLR7 expression than group B and higher TLR9 expression than group B and C mice. In contrast, the group A and B mice apparently expressed TLR2 and TLR4. We concluded that pristane treated apoE-/- mice exhibited lupus-like phenotype and developed atherosclerosis. The pristane treatment also induced abnormally high expression of TLR2 and TLR4 in the aorta and TLR2, TLR4, TLR7 and TLR9 in the kidney of apoE-/- mice.

  7. Comparison of relative TLR-2 and TLR-4 expression level of disease and healthy gingival tissue of smoking and non-smoking patients and periodontally healthy control patients.

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    Fatemi, K; Radvar, M; Rezaee, A; Rafatpanah, H; Azangoo khiavi, H; Dadpour, Y; Radvar, N

    2013-09-01

    Toll-like receptors are a family of sensor proteins that induce the inflammatory diseases in context of microbial infection. The role of these proteins in periodontal disease and their interaction with smoking status are yet to be investigated. The aim of the present study was to evaluate the effect of smoking on gene expression of toll like receptor 2 (TLR-2) and toll-like receptor 4 (TLR-4) in patients with periodontitis. RNAs were extracted from gingival biopsies of healthy sites (no bleeding on probing and pocket depth ≤3 mm) as well as diseased sites (with bleeding on probing and pocket depth ≥5 mm) of 20 smoker and 17 non-smoker subjects with chronic periodontitis. Gingival biopsies from eight periodontally healthy, never-smoker subjects served as control. Real-time PCR was carried out to evaluate the relative quantities of TLR-2 and TLR-4 mRNA concentrations. Regardless of smoking status, the relative expression levels of TLR-2 and TLR-4 were significantly greater (about 3 fold) at diseased sites compared to healthy sites of patients with periodontitis and healthy controls (p periodontitis, smoking caused an increase of about 6.5 fold in mRNA levels of TLR-4 in gingival tissue (p periodontitis might significantly increase TLR-2 and TLR-4 gene expression in gingival tissues, smoking habit in periodontitis subjects could selectively potentiate TLR-4 gene expression. © 2013 Australian Dental Association.

  8. Estudos anatômicos de folhas de espécies de plantas daninhas de grande ocorrência no Brasil: IV - Amaranthus deflexus, Amaranthus spinosus, Alternanthera tenella e Euphorbia heterophylla Leaf anatomical studies in weed species widely common in Brazil: IV - Amaranthus deflexus, Amaranthus spinosus, Alternanthera tenella and Euphorbia heterophylla

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    E.A. Ferreira

    2003-08-01

    Full Text Available Neste trabalho é apresentado um estudo da anatomia das folhas de espécies de plantas daninhas de grande ocorrência no Brasil: Amaranthus deflexus, Amaranthus spinosus, Alternanthera tenella e Euphorbia heterophylla, visando melhor compreensão das barreiras que cada espécie impõe à penetração dos herbicidas e outros compostos utilizados em aplicações foliares. As folhas completamente expandidas do terceiro ao quinto nó foram coletadas de plantas de ocorrência espontânea no campo. Das folhas de cada espécie foram obtidas três amostras da região mediana, com aproximadamente 1 cm², as quais foram utilizadas em estudos da estrutura, clarificação e em observações em microscópio eletrônico de varredura. Todas as espécies avaliadas são anfiestomáticas. As principais barreiras foliares potenciais à penetração de herbicidas observadas nas plantas daninhas A. deflexus e A. spinosus foram, respectivamente, grande espessura da cutícula da face adaxial e da cutícula das duas faces. Já em relação a A. tenella, grande espessura da cutícula das duas faces, elevado teor de cera epicuticular e alta densidade tricomática foram os principais obstáculos potenciais detectados. E. heterophylla apresentou como possíveis principais barreiras foliares à penetração de agroquímicos o alto teor de cera epicuticular, a elevada densidade de laticíferos e a grande espessura da cutícula da face adaxial.This research aimed to study the leaf anatomy of the weed species Amaranthus deflexus, Amaranthus spinosus, Alternanthera tenella and Euphorbia heterophylla, widely known in Brazil, to acquire a better understanding of the barriers each species imposes to herbicide penetration, and to other substances used for leaf spraying. Completely expanded leaves from the third to the fifth nodes were collected from spontaneous plants in the field. Three samples approximately 1cm², were removed from the medium portion of the leaves, from each

  9. Toll-Like Receptor 4 in Bone Marrow-Derived Cells Contributes to the Progression of Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Hui Wang

    2014-01-01

    Full Text Available Diabetic retinopathy (DR is a major microvascular complication in diabetics, and its mechanism is not fully understood. Toll-like receptor 4 (TLR4 plays a pivotal role in the maintenance of the inflammatory state during DR, and the deletion of TLR4 eventually alleviates the diabetic inflammatory state. To further elucidate the mechanism of DR, we used bone marrow transplantation to establish reciprocal chimeric animals of TLR4 mutant mice and TLR4 WT mice combined with diabetes mellitus (DM induction by streptozotocin (STZ treatment to identify the role of TLR4 in different cell types in the development of the proinflammatory state during DR. TLR4 mutation did not block the occurrence of high blood glucose after STZ injection compared with WT mice but did alleviate the progression of DR and alter the expression of the small vessel proliferation-related genes, vascular endothelial growth factor (VEGF, and hypoxia inducible factor-1α (HIF-1α. Grafting bone marrow-derived cells from TLR4 WT mice into TLR4 mutant mice increased the levels of TNF-α, IL-1β, and MIP-2 and increased the damage to the retina. Similarly, VEGF and HIF-1α expression were restored by the bone marrow transplantation. These findings identify an essential role for TLR4 in bone marrow-derived cells contributing to the progression of DR.

  10. Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

    Directory of Open Access Journals (Sweden)

    Randi M Sommerfelt

    Full Text Available Rheumatoid arthritis (RA is an autoimmune disease characterized by chronic synovitis leading to destruction of cartilage and bone. PLA2 enzymes are key players in inflammation regulating the release of unsaturated fatty acids such as arachidonic acid (AA, a precursor of pro-inflammatory eicosanoids. Several lines of evidence point to toll-like receptors (TLRs as drivers of synovitis and joint destruction in RA. However, few studies have addressed the implication of PLA2 activity downstream TLR activation in the synovium. Here, we aimed to characterize PLA2 enzyme involvement in TLR2-induced signaling in synovial fibroblast-like cells. TLRs1-7 and a range of sPLA2, iPLA2 and cPLA2 enzymes were found to be transcriptionally expressed in cultured synoviocytes. Activation of TLR2/1 and TLR2/6 led to phosphorylation of cPLA2α at Ser505, and induced AA release and PGE2 production; effects that were attenuated by cPLA2α inhibitors. In contrast, sPLA2 inhibitors did not affect AA or PGE2 release. cPLA2α inhibitors furthermore attenuated TLR-induced expression of IL-6, IL-8 and COX2. COX1/2 inhibitors attenuated TLR2/6-induced IL-6 transcription and protein production comparable to cPLA2α inhibition. Moreover, exogenously PGE2 added alone induced IL-6 production and completely rescued IL-6 transcription when added simultaneously with FSL-1 in the presence of a cPLA2α inhibitor. Our results demonstrate for the first time that cPLA2α is involved in TLR2/1- and TLR2/6-induced AA release, PGE2 production and pro-inflammatory cytokine expression in synoviocytes, possibly through COX/PGE2-dependent pathways. These findings expand our understanding of cPLA2α as a modulator of inflammatory molecular mechanisms in chronic diseases such as RA.

  11. Cloning and SNP screening of the TLR4 gene and the association ...

    African Journals Online (AJOL)

    Cloning and SNP screening of the TLR4 gene and the association between its polymorphism and somatic cell score in dairy cattle. ... Human models, in particular, suggest TLR4 to be a candidate gene associated with resistance to sepsis, immunodeficiencies, atherosclerosis and asthma. In this study the bovine TLR4 ...

  12. DMPD: LPS, TLR4 and infectious disease diversity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15608698 LPS, TLR4 and infectious disease diversity. Miller SI, Ernst RK, Bader MW.... Nat Rev Microbiol. 2005 Jan;3(1):36-46. (.png) (.svg) (.html) (.csml) Show LPS, TLR4 and infectious disease... diversity. PubmedID 15608698 Title LPS, TLR4 and infectious disease diversity. Authors Miller SI, Ernst RK,

  13. Genetic variation at Exon2 of TLR4 gene and its association with ...

    African Journals Online (AJOL)

    This study was conducted to analyze the polymorphisms of chicken Toll-like receptors 4(TLR4) gene and aimed to provide a theoretical foundation for a further research on correlation between chicken TLR4 gene and disease resistance. Genetic variations at exon 2 of TLR4 gene in 14 chicken breeds and the red jungle ...

  14. LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs.

    Directory of Open Access Journals (Sweden)

    Yvonne Lai

    Full Text Available Toll-like Receptor 3 (TLR3 detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.Using a human bronchial epithelial cell line (BEAS2B and human embryonic kidney cells (HEK 293T transiently transfected with TLR3, we found that LL37 enhanced poly(I:C-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

  15. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1.

    Science.gov (United States)

    Lin, Kuo-I; Kao, Yeong-Yi; Kuo, Hui-Kai; Yang, Wen-Bin; Chou, Alice; Lin, Hsin-Hung; Yu, Alice L; Wong, Chi-Huey

    2006-08-25

    The polysaccharides of Ganoderma lucidum (Reishi) possess immunomodulation activities; however, their mode of molecular action in regulating each cellular subset in the immune system is still not clear. Here, we investigate the function of the main polysaccharide fraction of Reishi (Reishi-F3) in B lymphocyte activation/differentiation. We find that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering the changes of a cascade of gene expression during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on the Toll-like receptors TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 block Reishi-F3-mediated induction of Blimp-1 mRNA and Ig secretion. We have shown that interaction of Reishi-F3 with TLR4/TLR2 followed by signaling through p38 MAPK is involved in the induction of Blimp-1 mRNA, whereas signaling through ERK, p38 MAPK, JNK, and IKK complex is involved in Reishi-F3-mediated Ig secretion. Furthermore, the differential mechanism of Reishi-F3 in mouse and human B cell activation is probably due to the presence of Blimp-1 regulatory site in human CD86 promoter. These results establish the signaling and molecular mechanisms of Reishi-F3 on promoting antibody secretion.

  16. TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

    Science.gov (United States)

    He, Aiqin; Ji, Rui; Shao, Jia; He, Chenyun; Jin, Ming; Xu, Yunzhao

    2016-02-01

    The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

  17. Morphology of the minute larva of Ptinella tenella, with special reference to effects of miniaturisation and the systematic position of Ptiliidae (Coleoptera: Staphylinoidea).

    Science.gov (United States)

    Grebennikov, Vasily V; Beutel, Rolf G

    2002-11-01

    External and internal features of minute reared ex ovo larvae of Ptinella tenella Erichson with the head widths ranging from 108 to 138mum are described. In total 139 setae (trichoid sensilla), 18 pores (non-trichoid sensilla), and 60 muscles were identified. The complexity of the chaetotaxy, the musculature of the head, the tentorium, and the digestive tract are not affected by the extremely small size of the larvae. Specific features, which may have resulted from miniaturisation are the elongate shape of the brain reaching the mesothorax posteriorly, the reduced condition of the thoracic sclerites and endosternites, the simplified musculature of the thorax and abdomen, and the loss of the abdominal spiracles. The monophyly of Ptiliidae is supported by the absence of head sutures, the presence of a setiferous protuberance close to the mandibular base, and the absence of a lacinia. Absence of abdominal spiracles and complete absence of eyes suggest the monophyly of a ptiliid subgroup which does not comprise Nossidium. Presence of a fimbriate galea is shared by larvae of Agyrtidae, Leiodidae, Ptiliidae, and Hydraenidae. The presence of a complex apical appendage on the distal maxillary palpomere and of hooks on abdominal segment X are possible synapomorphies of Ptiliidae and Hydraenidae. A description of the rearing procedure is provided and comments on feeding habits are made.

  18. TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity.

    Science.gov (United States)

    Johnson, Teresa R; Rao, Srinivas; Seder, Robert A; Chen, Man; Graham, Barney S

    2009-05-18

    Agonists for TLR7, TLR8, and TLR9 have been shown to enhance vaccine immunogenicity. We evaluated the impact of TLR activation on RSV disease in a murine model by administering TLR7/8 and TLR9 agonists during FI-RSV immunization or RSV infection. CpG administered during immunization reduced disease following challenge as evidenced by decreased lung pathology, illness, and cytokines. In marked contrast, TLR7/8 agonist had little impact. To evaluate potential therapeutic use, TLR agonists were administered during primary infection. Although type 2 cytokine responses decreased and type 1 cytokines and MIP-1-alpha/beta increased, both TLR7/8 and TLR9 agonists increased clinical symptoms and pulmonary inflammation when administered during primary infection. Thus, TLR9-induced signaling during FI-RSV immunization reduced vaccine-enhanced disease whereas immunostimulatory properties of TLR agonists enhanced disease severity when used during RSV infection. Immunomodulation elicited by TLR9 agonist confirms the adjuvant potential of TLR agonists during RSV immunization. However, in contrast to work done with HIV-1 vaccines, the inability of TLR7/8 agonist to boost type 1 vaccine-induced RSV immunity demonstrates pathogen-TLR specificity. These data reveal that the timing of administration of immunomodulatory agents is critical. Furthermore, these data underscore that amplification of anti-viral immune responses may result in immunopathology rather than immune-mediated protection.

  19. Identification and characterization of three TLR1 subfamily members from the orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Li, Yan-Wei; Xu, Dong-Dong; Li, Xia; Mo, Ze-Quan; Luo, Xiao-Chun; Li, An-Xing; Dan, Xue-Ming

    2016-08-01

    Toll-like receptors (TLRs), which play important roles in host defense against pathogen infection, are the most intensively studied pattern recognition receptors (PRRs). In this study, we identified three novel TLR1 subfamily members, including TLR1 (EcTLR1b), TLR2 (EcTLR2b) and TLR14 (EcTLR14), from the orange-spotted grouper (Epinephelus coioides). EcTLR1b and EcTLR2b displayed low sequence identity with the previously reported grouper TLR1 (EcTLR1a) and TLR2 (EcTLR2a), respectively. The open reading frames (ORFs) of EcTLR1b, EcTLR2b and EcTLR14 contain 2484 bp, 2394 bp and 2640 bp, which encode the corresponding 827 amino acids (aa), 797 aa and 879 aa, respectively. All three TLRs have leucine-rich repeat (LRR) domains (including an LRR-NT (except for EcTLR1b), several LRR motifs and an LRR-CT), a trans-membrane region and a Toll/interleukin-1 receptor (TIR) domain. The TIR domains of the three TLRs exhibited conserved boxes, namely box1, box2 and box3, and their 3D models were similar to those of human TLR1 or TLR2. Sequence alignment demonstrated that the TIR domains of the three TLRs shared higher sequence identity with those of other species than the full-length receptors. Phylogenetic analysis indicated that EcTLR1s and EcTLR2s are characterized by their differing evolutionary status, whereas EcTLR14 was found to be in the same group as other piscine TLR14/18s. The three TLRs were ubiquitously expressed in seven tested tissues of healthy groupers, although their expression profiles were different. Post Cryptocaryon irritans infection, TLR1s expression was up-regulated in the gills. The expression of TLR2b was mainly increased in the spleen, but decreased in the gills, which was similar to the expression pattern of TLR2a post C. irritans infection. Unlike EcTLR1b and EcTLR2b, however, the grouper TLR14 transcript was substantially induced in both tissues post challenge. These findings may be helpful in understanding the innate immune mechanism of host

  20. [Changes of FoxP3, CD4(+)CD25(+) regulatory T cells, TLR2 and TLR9 in children with infectious mononucleosis].

    Science.gov (United States)

    Wang, Qiang; Wang, Zuo-Feng; Cao, Mei; Wang, Zhi-Ying

    2013-04-01

    The aim of this study was to investigate the effects of TLR2, TLR9, CD4(+)CD25(+) regulatory T cells (Treg) and transcription factor FoxP3 in the pathogenesis of children with infectious mononucleosis (IM). Thirty-five acute IM patients admitted in our hospital from April 2010 to January 2011 were enrolled in this study. Thirty-five healthy subjects were taken as control. The thirty-five patients before treatment were considered as patients in acute stage, after treatment and without clinical symptom they were thought as patients in recovery stage. The expression levels of TLR2, TLR9 and FoxP3 mRNA were detected by real time PCR using SYBR Green I. The expression of T lymphocyte subset CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative levels of TLR2 mRNA (4.03 ± 0.56), TLR9 mRNA (8.88 ± 1.56) in peripheral blood mononuclear cells of IM patients in acute stage were significantly higher than those of the controls [TLR2 mRNA (2.22 ± 0.57), TLR9 mRNA (3.63 ± 1.30)] and IM patients in recovery stage [TLR2 mRNA (2.76 ± 0.83), TLR9 mRNA (5.34 ± 1.60)] (P 0.05). It is concluded that the expression of CD4(+)CD25(+)regulatory T cells is reduced, and its special transcription factor FoxP3 mRNA is down-regulated, but expression levels of TLR2 mRNA, TLR9 mRNA are up-regulated in IM patients of acute stage.

  1. Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells.

    Science.gov (United States)

    Eriksson, Mia; Peña-Martínez, Pablo; Ramakrishnan, Ramprasad; Chapellier, Marion; Högberg, Carl; Glowacki, Gabriella; Orsmark-Pietras, Christina; Velasco-Hernández, Talía; Lazarević, Vladimir Lj; Juliusson, Gunnar; Cammenga, Jörg; Mulloy, James C; Richter, Johan; Fioretos, Thoas; Ebert, Benjamin L; Järås, Marcus

    2017-10-24

    Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34+CD38- cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in MLL-AF9-driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine Trp53-/-MLL-AF9 AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine AML1-ETO9a-driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to MLL-rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the MLL-AF9 AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.

  2. DMPD: TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16410796 TLR signaling. Kawai T, Akira S. Cell Death Differ. 2006 May;13(5):816-25. (.png) (.svg) (.html...kira S. Publication Cell Death Differ. 2006 May;13(5):816-25. Pathway - PNG File (.png) SVG File (.svg) HTML File (.html

  3. Flavonoids Affect Host-Microbiota Crosstalk through TLR Modulation

    Directory of Open Access Journals (Sweden)

    Francisco J. Pérez-Cano

    2014-10-01

    Full Text Available Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

  4. Endothelial TLR4 and the microbiome drive cerebral cavernous malformations.

    Science.gov (United States)

    Tang, Alan T; Choi, Jaesung P; Kotzin, Jonathan J; Yang, Yiqing; Hong, Courtney C; Hobson, Nicholas; Girard, Romuald; Zeineddine, Hussein A; Lightle, Rhonda; Moore, Thomas; Cao, Ying; Shenkar, Robert; Chen, Mei; Mericko, Patricia; Yang, Jisheng; Li, Li; Tanes, Ceylan; Kobuley, Dmytro; Võsa, Urmo; Whitehead, Kevin J; Li, Dean Y; Franke, Lude; Hart, Blaine; Schwaninger, Markus; Henao-Mejia, Jorge; Morrison, Leslie; Kim, Helen; Awad, Issam A; Zheng, Xiangjian; Kahn, Mark L

    2017-05-18

    Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3-KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

  5. Domain combination of the vertebrate-like TLR gene family ...

    Indian Academy of Sciences (India)

    ... Refresher Courses · Symposia · Live Streaming. Home; Journals; Journal of Genetics; Volume 90; Issue 3. Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution. Baojun Wu Tianxiao Huan Jing Gong Pin Zhou Zengliang Bai. Research Article Volume 90 Issue 3 December ...

  6. Domain combination of the vertebrate-like TLR gene family ...

    Indian Academy of Sciences (India)

    (a) Unrooted NJ phylogenetic trees revealing the evolutionary relationship between V-TLR of Strongylocentrotus purpuratus and vertebrate TLRs. (b) Unrooted NJ phylogenetic ... The database accession IDs of these sequences are listed in table 2 of electronic supplementary material. Figure 4. Unrooted ML phylogenetic ...

  7. The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

    Science.gov (United States)

    Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

    2015-01-01

    TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

  8. Common Variants of TLR1 Associate with Organ Dysfunction and Sustained Pro-Inflammatory Responses during Sepsis

    Science.gov (United States)

    Pino-Yanes, Maria; Corrales, Almudena; Casula, Milena; Blanco, Jesús; Muriel, Arturo; Espinosa, Elena; García-Bello, Miguel; Torres, Antoni; Ferrer, Miguel; Zavala, Elizabeth

    2010-01-01

    Background Toll-like receptors (TLRs) are critical components for host pathogen recognition and variants in genes participating in this response influence susceptibility to infections. Recently, TLR1 gene polymorphisms have been found correlated with whole blood hyper-inflammatory responses to pathogen-associated molecules and associated with sepsis-associated multiorgan dysfunction and acute lung injury (ALI). We examined the association of common variants of TLR1 gene with sepsis-derived complications in an independent study and with serum levels for four inflammatory biomarkers among septic patients. Methodology/Principal Findings Seven tagging single nucleotide polymorphisms of the TLR1 gene were genotyped in samples from a prospective multicenter case-only study of patients with severe sepsis admitted into a network of intensive care units followed for disease severity. Interleukin (IL)-1β, IL-6, IL-10, and C-reactive protein (CRP) serum levels were measured at study entry, at 48 h and at 7th day. Alleles -7202G and 248Ser, and the 248Ser-602Ile haplotype were associated with circulatory dysfunction among severe septic patients (0.001≤p≤0.022), and with reduced IL-10 (0.012≤p≤0.047) and elevated CRP (0.011≤p≤0.036) serum levels during the first week of sepsis development. Additionally, the -7202GG genotype was found to be associated with hospital mortality (p = 0.017) and ALI (p = 0.050) in a combined analysis with European Americans, suggesting common risk effects among studies. Conclusions/Significance These results partially replicate and extend previous findings, supporting that variants of TLR1 gene are determinants of severe complications during sepsis. PMID:21048935

  9. L-selectin Promotes the Maturation of Dendritic Cells via Up-regulation the Expression of TLR4 in vitro.

    Science.gov (United States)

    Ye, Zhishuai; Liu, Jia; Zheng, Jie; Zhang, Jianing; Huang, Rongchong

    2017-08-01

    The relationship between dendritic cells (DCs) and L-selectin in the progress of atherosclerosis is unclear. Here, we used L-selectin co-cultured with DCs to investigate the effect of L-selectin on the maturation of DCs in vitro Monocytes derived DCs were isolated and cultured from human peripheral blood. After being stimulated with L-selectin and/or its antagonist for 24-48 hours, the feather of cells was observed by the electron microscope. The expression of mature antigens CD1a, CD80, CD83, and CD86 were investigated by flow cytometric analysis (FACS). RT-PCR and FACS were used to detect the mRNA and protein expression of Toll-like receptor 4(TLR-4). We found that only the cells of giving L-selectin have the mature special feature for irregular shapes. DCs which were stimulated by L-selectin have a larger number of expressing CD1a, CD80, CD83, and CD86 compared with non-stimulated and cultured with L-selectin antagonist. The transcript levels of TLR4 were significantly higher after L-selectin and lipopolysaccharide (LPS) stimulated. And the antagonist of L-selectin can deeply decrease the expression of CD1a, CD80, CD83, and CD86 on DCs appeared to coincide with the level of TLR4 transcription. The results demonstrate L-selectin can promote the maturation of DCs via up-regulation the expression of TLR4. © 2017 by the Association of Clinical Scientists, Inc.

  10. HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

    Directory of Open Access Journals (Sweden)

    James F Curtin

    2009-01-01

    Full Text Available Glioblastoma multiforme (GBM is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1, an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2 signaling on bone marrow-derived GBM-infiltrating DCs.Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad expressing Fms-like tyrosine kinase 3 ligand (Flt3L and thymidine kinase (TK delivered into the tumor mass, we demonstrated that CD4(+ and CD8(+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV] treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti

  11. Small Interference RNA Targeting TLR4 Gene Effectively Attenuates Pulmonary Inflammation in a Rat Model

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    Feixiang Wu

    2012-01-01

    Full Text Available Objective. The present study was to investigate the feasibility of adenovirus-mediated small interference RNA (siRNA targeting Toll-like receptor 4 (TLR4 gene in ameliorating lipopolysaccharide- (LPS- induced acute lung injury (ALI. Methods. In vitro, alveolar macrophages (AMs were treated with Ad-siTLR4 and Ad-EFGP, respectively, for 12 h, 24 h, and 48 h, and then with LPS (100 ng/mL for 2 h, and the function and expression of TLR4 were evaluated. In vivo, rats received intratracheal injection of 300 μL of normal saline (control group, 300 μL of Ad-EGFP (Ad-EGFP group, or 300 μL of Ad-siTLR4 (Ad-siTLR4 group and then were intravenously treated with LPS (50 mg/kg to induce ALI. Results. Ad-siTLR4 treatment significantly reduced TLR4 expression and production of proinflammatory cytokines following LPS treatment both in vitro and in vivo. Significant alleviation of tissue edema, microvascular protein leakage, and neutrophil infiltration was observed in the AdsiTLR4-treated animals. Conclusion. TLR4 plays a critical role in LPS-induced ALI, and transfection of Ad-siTLR4 can effectively downregulate TLR4 expression in vitro and in vivo, accompanied by alleviation of LPS-induced lung injury. These findings suggest that TLR4 may serve as a potential target in the treatment of ALI and RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  12. Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation.

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    Into, Takeshi; Kiura, Kazuto; Yasuda, Motoaki; Kataoka, Hideo; Inoue, Nobuo; Hasebe, Akira; Takeda, Kiyoshi; Akira, Shizuo; Shibata, Ken-ichiro

    2004-02-01

    Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll-like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-kappaB, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid. The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs. Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-kappa B activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant-negative forms also downregulated the activation of both NF-kappa B and caspase-8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2- and TLR6-mediated sequential bifurcate responses: NF-kappa B activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

  13. Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor.

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    Se Hwan Hwang

    Full Text Available The characteristics of mesenchymal stem cells (MSCs derived from human turbinates (hTMSCs have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.

  14. Evaluation of the persistence, integration, histopathology and environmental release of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2.

    Science.gov (United States)

    Song, Xiaokai; Zhang, Zeyang; Liu, Chang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2016-10-15

    In a previous study, the construction of the Eimeria tenella DNA vaccine pVAX1.0-TA4-IL-2 which provides effective protection against coccidiosis was described and the immunization procedure was optimized. However, the persistence, integration, histopathology and environmental release of the DNA vaccine remain unknown. In this study, the persistence, integration and histopathology of the DNA vaccine pVAX1.0-TA4-IL-2 was evaluated in chickens in the following immunization studies: (1) single-dose immunization in one-day-old chickens; (2) repeat-dose immunization in chickens; and (3) single-high-dose immunization of three batches of plasmid in chickens. The persistence, integration, histopathology of the DNA vaccine was also evaluated in mice. At 1, 1.5, 2-4 months post immunization, blood, duodenum, heart, liver, spleen, kidneys and the immunized muscle tissue were collected from ten animals of each group. Persistence and integration were evaluated using PCR with a confirmed sensitivity of 30 plasmid copies. Hematoxylin and eosin stained sections were examined for the presence of inflammation or abnormalities that may result from vaccination. Water and fecal samples were also collected from the chicken enclosures to evaluate the potential for environmental release of the DNA vaccine. Testing various tissues by PCR confirmed that plasmid DNA persisted 1.5 months in blood, heart, liver and spleen, 2 months in kidneys and muscle of injected site. Furthermore, the vaccine did not integrate with the host genome. The histopathological examinations did not show obvious inflammation or pathological damage in any tissue of the immunized chickens. Similar results were observed in mice. Moreover, the DNA vaccine was not released into the surrounding environment. These results indicate that the DNA vaccine pVAX1.0-TA4-IL-2 has potential as safe vaccine against coccidiosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. TLR-2 and TLR-4 transcriptions in unstimulated blood from dogs with leishmaniosis due to Leishmania infantum at the time of diagnosis and during follow-up treatment.

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    Montserrat-Sangrà, Sara; Alborch, Lorena; Ordeix, Laura; Solano-Gallego, Laia

    2016-09-15

    Innate immunity, in particular, the role of toll-like receptors (TLRs), has not been extensively studied in canine L. infantum infection. The main aim of this study was to determine the transcription of TLR2 and TLR4 in the blood of dogs with natural clinical leishmaniosis at the time of diagnosis and during treatment follow-up and subsequently correlate these findings with clinical, serological and parasitological data. Forty-six Leishmania-seropositive sick dogs with a high antibody level at the time of diagnosis were studied and compared with 34 healthy seronegative dogs. Twenty-two of these sick dogs were treated with meglumine antimoniate and allopurinol and followed-up at 30, 180 and 365days following the start of treatment. Clinical status was defined by a thorough physical examination, complete blood count, biochemistry profile, electrophoresis of serum proteins, and urinary protein/creatinine ratio (UPC). EDTA blood was stored in RNAlater(®) solution before RNA extraction and cDNA production were performed. TLR2, TLR4 and three reference genes (HPRT-1, CG14980 and SDHA) were studied in each blood sample by real time PCR. The relative quantification of TLR2 was higher (mean 3.5) in sick dogs when compared with seronegative healthy dogs (mean 1.3; P=0.0001) while the relative quantification of TLR4 was similar in both groups. In addition, the relative quantification of TLR2 significantly decreased during follow-up at all time points compared with day 0 whereas no changes were observed with TLR4 transcription. A significant positive correlation was noted between TLR2 and UPC, total protein, beta and gamma globulins, specific L. infantum antibodies and blood parasite load while a negative correlation was observed with albumin, albumin/globulin ratio, hematocrit and hemoglobin. TLR4 transcript did not correlate with any parameter. These findings indicate an up-regulation of TLR2 transcription in unstimulated blood in naturally infected sick dogs as compared to

  16. 1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo.

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    Bo Li

    Full Text Available 1,25-Dihydroxyvitamin D3 (1,25(OH2D3 suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH2D3 is in part mediated through an interplay between 1,25(OH2D3 and toll-like receptor (TLR7/8 signaling. 1,25(OH2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH2D3. To determine the molecular mechanism by which 1,25(OH2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β. In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH2D3 and may mediate the anti-inflammatory action of 1,25(OH2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.

  17. TLR7/TLR8 Activation Restores Defective Cytokine Secretion by Myeloid Dendritic Cells but Not by Plasmacytoid Dendritic Cells in HIV-Infected Pregnant Women and Newborns.

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    Cardoso, Elaine Cristina; Pereira, Nátalli Zanete; Mitsunari, Gabrielle Eimi; Oliveira, Luanda Mara da Silva; Ruocco, Rosa Maria S A; Francisco, Rossana Pulcineli Vieira; Zugaib, Marcelo; da Silva Duarte, Alberto José; Sato, Maria Notomi

    2013-01-01

    Mother-to-child transmission (MTCT) of HIV-1 has been significantly reduced with the use of antiretroviral therapies, resulting in an increased number of HIV-exposed uninfected infants. The consequences of HIV infection on the innate immune system of both mother-newborn are not well understood. In this study, we analyzed peripheral blood and umbilical cord blood (CB) collected from HIV-1-infected and uninfected pregnant women. We measured TNF-α, IL-10 and IFN-α secretion after the stimulation of the cells with agonists of both extracellular Toll-like receptors (TLRs) (TLR2, TLR4 and TLR5) and intracellular TLRs (TLR7, TLR7/8 and TLR9). Moreover, as an indicator of the innate immune response, we evaluated the responsiveness of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) to TLRs that are associated with the antiviral response. Our results showed that peripheral blood mononuclear cells (PBMCs) from HIV-1-infected mothers and CB were defective in TNF-α production after activation by TLR2, TLR5, TLR3 and TLR7. However, the TNF-α response was preserved after TLR7/8 (CL097) stimulation, mainly in the neonatal cells. Furthermore, only CL097 activation was able to induce IL-10 and IFN-α secretion in both maternal and CB cells in the infected group. An increase in IFN-α secretion was observed in CL097-treated CB from HIV-infected mothers compared with control mothers. The effectiveness of CL097 stimulation was confirmed by observation of similar mRNA levels of interferon regulatory factor-7 (IRF-7), IFN-α and TNF-α in PBMCs of both groups. The function of both mDCs and pDCs was markedly compromised in the HIV-infected group, and although TLR7/TLR8 activation overcame the impairment in TNF-α secretion by mDCs, such stimulation was unable to reverse the dysfunctional type I IFN response by pDCs in the HIV-infected samples. Our findings highlight the dysfunction of innate immunity in HIV-infected mother-newborn pairs. The activation of the TLR7/8 pathway

  18. Customized laboratory TLR4 and TLR2 detection method from peripheral human blood for early detection of doxorubicin-induced cardiotoxicity.

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    Pop-Moldovan, A L; Trofenciuc, N-M; Dărăbanţiu, D A; Precup, C; Branea, H; Christodorescu, R; Puşchiţă, M

    2017-05-01

    Cancer treatments can have significant cardiovascular adverse effects that can cause cardiomyopathy and heart failure with reduced survival benefit and considerable decrease in the use of antineoplastic therapy. The purpose of this study is to assess the role of TLR2 and TLR4 gene expression as an early marker for the risk of doxorubicin-induced cardiomyopathy in correlation with early diastolic dysfunction in patients treated with doxorubicin. Our study included 25 consecutive patients who received treatment with doxorubicin for hematological malignancies (leukemia, lymphomas or multiple myeloma), aged 18-65 years, with a survival probability>6 months and with left ventricular ejection fraction>50%. Exclusion criteria consisted of the following: previous anthracycline therapy, previous radiotherapy, history of heart failure or chronic renal failure, atrial fibrillation, and pregnancy. In all patients, in fasting state, a blood sample was drawn for the assessment of TLR2 and TLR4 gene expression. Gene expression was assessed by quantitative reverse transcription PCR (qRT-PCR) using blood collection, RNA isolation, cDNA reverse transcription, qRT-PCR and quantification of the relative expression. At enrollment, all patients were evaluated clinically; an ECG and an echocardiography were performed. The average amount of gene expression units was 0.113 for TLR4 (range 0.059-0.753) and 0.218 for TLR2 (range 0.046-0.269). The mean mRNA extracted quantity was 113 571 ng/μl. As for the diastolic function parameters, criteria for diastolic dysfunction were present after 6 months in 16 patients (64%). In these patients, the mean values for TLR4 were 0.1198625 and for TLR2 were 0.16454 gene expression units. As for the diastolic function parameters, criteria for diastolic dysfunction were present after 6 months in 16 patients (64%). In these patients, the mean value for TLR2 was 0.30±0.19 and for TLR4 was 0.15±0.04. The corresponding values for the patients who did not

  19. TLR9-based immunotherapy for the treatment of allergic diseases.

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    Farrokhi, Shokrollah; Abbasirad, Narjes; Movahed, Ali; Khazaei, Hossein Ali; Pishjoo, Masoud; Rezaei, Nima

    2017-03-01

    Toll-like receptors (TLRs), a family of pattern recognition receptors expressed on many cell types of innate immunity, recognize the pathogen-associated molecular patterns of microbes. The hygiene hypothesis suggests that a reduced microbial exposure in early childhood increases the susceptibility to allergic diseases due to deviation in development of the immune system. TLRs are key roles in the right and healthy direction of adaptive immunity with the induction of T-helper 2 toward Th1 immune responses and regulatory T cells. TLR ligand CpG-ODN-based immunomodulation is independent of allergen and it mainly affects innate immune system. While, CpG-oligodeoxynucleotide-based vaccination is allergen specific and induces adaptive immune system. The use of agonists of TLR9 in two distinct strategies of immunotherapy, immunomodulation and vaccination, could be presented as the curative method for the treatment of allergic diseases.

  20. Protection of ischemic post conditioning against transient focal ischemia-induced brain damage is associated with inhibition of neuroinflammation via modulation of TLR2 and TLR4 pathways.

    Science.gov (United States)

    Wang, Ying; Ge, Pengfei; Yang, Li; Wu, Chunyun; Zha, Hao; Luo, Tianfei; Zhu, Yuhong

    2014-01-24

    Ischemic postconditioning has been demonstrated to be a protective procedure to brain damage caused by transient focal ischemia/reperfusion. However, it is elusive whether the protection of postconditioning against brain damage and neuroinflammation is via regulating TLR2 and TLR4 pathways. In the present study, we examined the protection of ischemic postconditioning performed immediately prior to the recovery of cerebral blood supply on brain damage caused by various duration of ischemia and tested the hypothesis that its protection is via inhibition of neuroinflammation by modulating TLR2/TLR4 pathways. Brain damage in rats was induced by using the middle cerebral artery occlusion (MCAO) model. Ischemic postconditioning consisting of fivecycles of ten seconds of ischemia and reperfusion was performed immediately following theischemic episode Theduration of administration of ischemic postconditioning was examined by comparing its effects on infarction volume, cerebral edema and neurological function in 2, 3, 4, 4.5and 6 hour ischemia groups. The protective mechanism of ischemic postconditioning was investigated by comparing its effects on apoptosis, production of the neurotoxic cytokine IL-1β and the transcription and expression of TLR2, TLR4 and IRAK4 in the 2 and 4.5 hour ischemia groups. Ischemic postconditioning significantly attenuated cerebral infarction, cerebral edema and neurological dysfunction in ischemia groups of up to 4 hours duration, but not in 4.5and 6 hour ischemia groups. It also inhibited apoptosis, production of IL-1β, abnormal transcription and expression of TLR2, TLR4 and IRAK4 in the 2 hour ischemia group, but not in the 4.5 hour ischemia group. Ischemic postconditioning protected brain damage caused by 2, 3 and 4 hours of ischemia, but not by 4.5 and 6 hours of ischemia. The protection of ischemic postconditioning is associated with its inhibition of neuroinflammation via inhibition of TLR2 and TLR4 pathways.

  1. T cells exacerbate Lyme borreliosis in TLR2-deficient mice

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    Carrie E. Lasky

    2016-11-01

    Full Text Available Infection of humans with the spirochete, Borrelia burgdorferi, causes Lyme borreliosis and can lead to clinical manifestations such as, arthritis, carditis and neurological conditions. Experimental infection of mice recapitulates many of these symptoms and serves as a model system for the investigation of disease pathogenesis and immunity. Innate immunity is known to drive the development of Lyme arthritis and carditis, but the mechanisms driving this response remain unclear. Innate immune cells recognize B. burgdorferi surface lipoproteins primarily via Toll-like receptor (TLR2; however, previous work has demonstrated TLR2-/- mice had exacerbated disease and increased bacterial burden. We demonstrate increased CD4 and CD8 T cell infiltrates in B. burgdorferi-infected joints and hearts of C3H TLR2-/- mice. In vivo depletion of either CD4 or CD8 T cells reduced Borrelia-induced joint swelling and lowered tissue spirochete burden, while depletion of CD8 T cells alone reduced disease severity scores. Exacerbation of Lyme arthritis correlated with increased production of CXCL9 by synoviocytes and this was reduced with CD8 T cell depletion. These results demonstrate T cells can exacerbate Lyme disease pathogenesis and prolong disease resolution possibly through dysregulation of inflammatory responses and inhibition of bacterial clearance.

  2. Debugging of Web Applications with Web-TLR

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    María Alpuente

    2011-08-01

    Full Text Available Web-TLR is a Web verification engine that is based on the well-established Rewriting Logic--Maude/LTLR tandem for Web system specification and model-checking. In Web-TLR, Web applications are expressed as rewrite theories that can be formally verified by using the Maude built-in LTLR model-checker. Whenever a property is refuted, a counterexample trace is delivered that reveals an undesired, erroneous navigation sequence. Unfortunately, the analysis (or even the simple inspection of such counterexamples may be unfeasible because of the size and complexity of the traces under examination. In this paper, we endow Web-TLR with a new Web debugging facility that supports the efficient manipulation of counterexample traces. This facility is based on a backward trace-slicing technique for rewriting logic theories that allows the pieces of information that we are interested to be traced back through inverse rewrite sequences. The slicing process drastically simplifies the computation trace by dropping useless data that do not influence the final result. By using this facility, the Web engineer can focus on the relevant fragments of the failing application, which greatly reduces the manual debugging effort and also decreases the number of iterative verifications.

  3. TLR1-10, NF-κB and p53 expression is increased in oral lichenoid disease.

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    Peter Rusanen

    Full Text Available Toll-like receptors (TLRs and nuclear factor-κB (NF-κB in keratinocytes play an important role in dermatological autoimmune diseases. Tumour suppressor protein p53 regulates TLR expression. The aim of this study was to compare the expression of TLR1-TLR10, p53 and NF-κB in patients with oral lichenoid disease (OLD with healthy mucosa. Oral mucosal biopsies from 24 patients with OLD and 26 healthy controls (HC were analysed for the expression of TLR1-TLR10, NF-κB and p53 by immunohistochemistry. The expression of all TLRs was increased in OLD epithelia compared to HC samples and the difference was significant in TLR1, TLR3, TLR4, TLR5, TLR6 and TLR7. In the basement membrane zone, the immunoreactivity of TLR5 was significantly more intense in OLD compared to HC. In the intermediate layer, the immunoreactivity of NF-κB was significantly stronger in OLD, whereas the staining for p53 was more intense in all layers of OLD compared to HC samples. In OLD, a positive correlation between TLR2 and NF-κB in the basal layer and between TLR5, p53 and NF-κB in the intermediate layers was discovered. The expression of TLRs, p53 and NF-κB is increased in OLD, which may play a role in the pathogenesis of this chronic immune-mediated mucosal disease.

  4. TLR2 Expression Is Increased in Rosacea and Stimulates Enhanced Serine Protease Production by Keratinocytes

    Science.gov (United States)

    Yamasaki, Kenshi; Kanada, Kimberly; Macleod, Daniel T.; Borkowski, Andrew W.; Morizane, Shin; Nakatsuji, Teruaki; Cogen, Anna L.; Gallo, Richard L.

    2011-01-01

    A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea. PMID:21107351

  5. [Progress in studies on TLR4 signaling pathway and major depressive disorder].

    Science.gov (United States)

    Wang, Lu; Chen, Jindong

    2017-06-28

    TLR4 signaling pathway plays an important role in regulation of the innate immune response and the adaptive immune response. Studies have shown that TLR4 signaling pathway is closely associated with the immune inflammatory process in major depressive disorder, but the underlying mechanisms are not clear. The pathophysiological process of depression involves multiple molecule mechanisms and signal pathways. The change of TLR4 signaling pathways exists in the peripheral circulation system or the central nervous system of depressive patients and depression animal models. The activation of TLR4 signaling pathways is associated with "stress-inflammation-depression" approach and "leaky gut" hypothesis, however, the relationship of peripheral and central TLR4 signaling pathways is not clear yet. TLR4 signaling pathway may be potential targets for the anti-inflammatory treatment of depression.

  6. Effects of TLR agonists and viral infection on cytokine and TLR expression in Atlantic salmon (Salmo salar).

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    Arnemo, Marianne; Kavaliauskis, Arturas; Gjøen, Tor

    2014-10-01

    The development of efficient and cheap vaccines against several aquatic viruses is necessary for a sustainable fish farming industry. Toll-like receptor (TLR) ligands have already been used as good adjuvants in human vaccines. With more understanding of TLR expression, function, and ligand specificity in fish, more efficient adjuvants for fish viral vaccines can be developed. In this paper, we examine all known TLRs in Atlantic salmon (Salmo salar) and demonstrate that head kidney and spleen are the main organs expressing TLRs in salmon. We also show that adherent head kidney leucocytes from salmon are able to respond to many of the known agonists for human TLRs, and that viral infection can induce up-regulation of several TLRs. These findings substantiate these receptors' role in immune responses to pathogens in salmonids making their ligands attractive as vaccine adjuvant candidates. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

    Science.gov (United States)

    Paul, Joydeep; Naskar, Kshudiram; Chowdhury, Sayan; Alam, Md Nur; Chakraborti, Tapati; De, Tripti

    2014-12-01

    In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa β 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-κB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-κB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88-IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication.

  8. Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2.

    Science.gov (United States)

    Holden, James A; O'Brien-Simpson, Neil M; Lenzo, Jason C; Orth, Rebecca K H; Mansell, Ashley; Reynolds, Eric C

    2017-09-01

    Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2(-/-), and TLR4(-/-) macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals. Copyright © 2017 American Society for Microbiology.

  9. Helicobacter urease-induced activation of the TLR2/NLRP3/IL-18 axis protects against asthma.

    Science.gov (United States)

    Koch, Katrin N; Hartung, Mara L; Urban, Sabine; Kyburz, Andreas; Bahlmann, Anna S; Lind, Judith; Backert, Steffen; Taube, Christian; Müller, Anne

    2015-08-03

    Inflammasome activation and caspase-1-dependent (CASP1-dependent) processing and secretion of IL-1β and IL-18 are critical events at the interface of the bacterial pathogen Helicobacter pylori with its host. Whereas IL-1β promotes Th1 and Th17 responses and gastric immunopathology, IL-18 is required for Treg differentiation, H. pylori persistence, and protection against allergic asthma, which is a hallmark of H. pylori-infected mice and humans. Here, we show that inflammasome activation in DCs requires the cytoplasmic sensor NLRP3 as well as induction of TLR2 signaling by H. pylori. Screening of an H. pylori transposon mutant library revealed that pro-IL-1β expression is induced by LPS from H. pylori, while the urease B subunit (UreB) is required for NLRP3 inflammasome licensing. UreB activates the TLR2-dependent expression of NLRP3, which represents a rate-limiting step in NLRP3 inflammasome assembly. ureB-deficient H. pylori mutants were defective for CASP1 activation in murine bone marrow-derived DCs, splenic DCs, and human blood-derived DCs. Despite colonizing the murine stomach, ureB mutants failed to induce IL-1β and IL-18 secretion and to promote Treg responses. Unlike WT H. pylori, ureB mutants were incapable of conferring protection against allergen-induced asthma in murine models. Together, these results indicate that the TLR2/NLRP3/CASP1/IL-18 axis is critical to H. pylori-specific immune regulation.

  10. Endogenous IgG affects the cell biology of RPE cells and involves the TLR4 pathway.

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    Niu, Na; Zhang, Jie; McNutt, Michael A

    2013-10-25

    RPE is a key component of the blood-ocular barrier (BOB) and is equipped with immunological molecules such as toll-like receptors (TLRs) and complement receptors, which together orchestrate the innate and adaptive immunity of the eye. Immunoglobulin G (IgG) in the aqueous humor and vitreous body has traditionally been thought to be derived from serum via transcytosis across the BOB. Our previous work validated production of endogenous IgG by RPE cells locally. However, the function and role of this IgG in the intraocular immunity is poorly understood. After confirming IgG production in a human RPE cell line (ARPE-19) with immunofluorescence, in situ hybridization, and RT-PCR, we further investigated the function of endogenous IgG in RPE biology with MTS, flow cytometry, and cell invasion analysis after downregulation of IgG by siRNA. Involvement of the TLR4 pathway was also studied using Western blot, ELISA and confocal microscopy. Endogenous IgG is crucial for support of proliferation, mitosis, migration, and inhibition of apoptosis of RPE. Moreover, production of endogenous IgG by RPE is regulated by the TLR4 pathway in a concentration- and duration-dependent manner, and IgG affects the activation of the TLR4 pathway in a synergistic manner. Activation of the FcγR I pathway and production of IL-10 could be induced by IgG derived from RPE. These data suggest that endogenous IgG may be a molecule that is essential for the physiological function of RPE, and suggest IgG is important for regulating intraocular immune responses under physiologic and pathologic conditions.

  11. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

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    Eliran Moshe Reuven

    2014-08-01

    Full Text Available HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD of the HIV-1 envelope (ENV directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  12. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

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    Reuven, Eliran Moshe; Ali, Mohammad; Rotem, Etai; Schwarzer, Roland; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H; Shai, Yechiel

    2014-08-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  13. Myocyte TLR4 enhances enteric and systemic inflammation driving late murine endotoxic ileus

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    Buchholz, Bettina M.; Shapiro, Richard A.; Vodovotz, Yoram; Billiar, Timothy R.; Sodhi, Chhinder P.; Hackam, David J.

    2015-01-01

    Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4+/+, TLR4−/−, bmTLR4+/+/TLR4−/− chimera, SM22-Cre−/−TLR4flox/flox, and selective myocyte TLR4-deficient (SM22-Cre+/−TLR4flox/flox) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated

  14. TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis.

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    Yuan, Ming-Ming; Xu, Yu-Yin; Chen, Li; Li, Xing-Yu; Qin, Jing; Shen, Ying

    2015-04-09

    Toll-like receptor 3 (TLR3) plays a key role in innate immunity. In the present study, we analyzed tissues of patients with human hepatocellular carcinoma (HCC) to determine the significance of the relationship between TLR3 expression and cell proliferation, apoptosis, hepatitis B virus infections, angiogenesis and prognosis. We collected paraffin-embedded tissues from 85 patients with HCC who had complete histories and were followed for >5 years. The expression and intracellular localization of TLR3 and downstream proteins (TRIF, NF-κB, and IRF3) were detected using immunohistochemistry. Further, we determined the expression of proteins that mediate cell proliferation (Ki67, cyclin D1), apoptosis (survivin, bcl-2, caspases 3, 8, and 9), and angiogenesis (CD34, MMP-2) as well as the HBV proteins HBsAg and HBcAg. Apoptosis in HCC tissues was detected using TUNEL. We conducted dual-labeling immunohistochemical analyses of TLR3 expression and TUNEL activity. TLR3 expression was significantly lower in HCC tissues compared with adjacent tissues. TRIF, NF-κB, and IRF3 correlated positively with TLR3 expression. Survivin and Bcl-2 expression correlated negatively with TLR3. The frequencies of caspases 3, 8, and 9 expression correlated positively with TLR3 signaling proteins. Cytoplasmic TLR3 and serum levels of HBsAg correlated positively. The apoptotic index determined using the TUNEL method and correlated positively with TLR3 expression. TLR3 expression in the cytoplasm correlated positively with TUNEL-positive cells and HBsAg. Ki67 and cyclin D1 correlated negatively with TLR3 expression. MMP-2 expression, microvessel density (CD34(+)) and endothelial progenitor cells (EPCs) correlated negatively with TLR3 expression. Kaplan-Meier survival analysis shows that TLR3 expression correlated with longer survival. The expression of TLR3 in HCC tissues may exert a synergistic effect on apoptosis and inhibit the proliferation of HCC cells, MMP-2 expression, generation of EPCs

  15. Epithelial expression of TLR4 is modulated in COPD and by steroids, salmeterol and cigarette smoke

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    Dorscheid Delbert R

    2007-11-01

    Full Text Available Abstract The toll-like receptors (TLRs are a key component of host defense in the respiratory epithelium. Cigarette smoking is associated with increased susceptibility to infection, while COPD is characterised by bacterial colonisation and infective exacerbations. We found reduced TLR4 gene expression in the nasal epithelium of smokers compared with non-smoking controls, while TLR2 expression was unchanged. Severe COPD was associated with reduced TLR4 expression compared to less severe disease, with good correlation between nasal and tracheal expression. We went on to examine the effect of potential modulators of TLR4 expression in respiratory epithelium pertinent to airways disease. Using an airway epithelial cell line, we found a dose-dependent downregulation in TLR4 mRNA and protein expression by stimulation with cigarette smoke extracts. Treatment with the corticosteroids fluticasone and dexamethasone resulted in a dose-dependent reduction in TLR4 mRNA and protein. The functional significance of this effect was demonstrated by impaired IL-8 and HBD2 induction in response to LPS. Stimulation with salmeterol (10-6 M caused upregulation of TLR4 membrane protein presentation with no upregulation of mRNA, suggesting a post-translational effect. The effect of dexamethasone and salmeterol in combination was additive, with downregulation of TLR4 gene expression, and no change in membrane receptor expression. Modulation of TLR4 in respiratory epithelium may have important implications for airway inflammation and infection in response to inhaled pathogens.

  16. TLR3 and Rig-Like Receptor on Myeloid Dendritic Cells and Rig-Like Receptor on Human NK Cells Are Both Mandatory for Production of IFN-γ in Response to Double-Stranded RNA

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    Perrot, Ivan; Deauvieau, Florence; Massacrier, Catherine; Hughes, Nicola; Garrone, Pierre; Durand, Isabelle; Demaria, Olivier; Viaud, Nicolas; Gauthier, Laurent; Blery, Mathieu; Bonnefoy-Berard, Nathalie; Morel, Yannis; Tschopp, Jurg; Alexopoulou, Lena; Trinchieri, Giorgio; Paturel, Carine; Caux, Christophe

    2012-01-01

    Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-γ. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-γ production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-γ production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-γ production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I–specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation. PMID:20639488

  17. Release of IL-12 by dendritic cells activated by TLR ligation is dependent on MyD88 signaling, whereas TRIF signaling is indispensable for TLR synergy.

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    Krummen, Mathias; Balkow, Sandra; Shen, Limei; Heinz, Stefanie; Loquai, Carmen; Probst, Hans-Christian; Grabbe, Stephan

    2010-07-01

    Recently, it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study, we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules, thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines, particularly IL-12, whereas the expression of costimulatory molecules remained unchanged. Consequently, synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore, we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However, noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally, we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment, suggesting an autocrine action of type I IFNs.

  18. Susceptibility to SLE in South Indian Tamils may be influenced by genetic selection pressure on TLR2 and TLR9 genes.

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    Devaraju, Panneer; Gulati, Reena; Antony, Paul T; Mithun, C B; Negi, Vir S

    2015-03-01

    Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder with complex etiology. Genetics plays an important role in lupus pathogenesis through its influence on clinical and autoantibody phenotype of the disease. Toll like receptors (TLR) recognize molecular patterns of pathogens and activate the innate immune system. Their ability to identify nucleic acids makes them suitable candidates for investigation of their role in lupus pathogenesis. Hence, this study was carried out to analyze the G to A and C to T transitions in TLR2 and TLR9 genes respectively and to test their association with lupus susceptibility, clinical and autoantibody phenotypes in South Indian Tamils. Three hundred SLE patients fulfilling ACR 2012 criteria for SLE and 460 age, sex similar, ethnicity matched controls were recruited as cases and controls. TLR2 (R753Q) and TLR9 (-1237C/T) polymorphisms were analyzed by real time PCR. The TLR2 gene remained monomorphic in patients and controls, the frequency of the homozygous wild type allele being 100% and 99.6% respectively. Hence, it did not confer susceptibility to SLE. The more frequent T allele of TLR9 gene conferred a significant risk to develop SLE (p=0.011, OR 1.69, 95% CI 1.1-2.6). Both the polymorphisms did not influence clinical or autoantibody phenotype of the disease. Prevailing endemic infections in the Indian subcontinent may have exerted a selection pressure resulting in TLR2 gene remaining monomorphic and the TLR9 adapting to a mutation for its increased expression. These may have an additive effect in the presence of other genetic and environmental risk factors to confer susceptibility to SLE in South Indian Tamils. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Exercise Therapy Downregulates the Overexpression of TLR4, TLR2, MyD88 and NF-κB after Cerebral Ischemia in Rats

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    Lin Qiang

    2013-02-01

    Full Text Available Toll-like receptor 2 (TLR2 and Toll-like receptor 4 (TLR4 are considered to mediate the inflammatory reaction of cerebral ischemia injury, and exercise can inhibit the activity of the Toll-like receptor signaling pathway in the peripheral blood of humans. Although physical exercise has been demonstrated to be neuroprotective in both clinical and laboratory settings, the underlying mechanism remains unclear. To clarify this critical issue, this study investigated the effects of treadmill training on the recovery of neurological function and the expression of TLR2 and TLR4 and their main downstream targets, nuclear factor-kappaB (NF-κB and myeloid differentiation factor 88 (MyD88, in the ischemic rat brain after middle cerebral artery occlusion-reperfusion (MCAo/R. Rats were divided into seven groups: sham control without MCAo/R and five, nine and 16 days post-ischemic exercise or non-exercise. The neurological function and infarct volume were measured, and reverse transcription polymerase chain reaction (RT-PCR and Western blotting were used to detect the expression of TLR2, TLR4, NF-κB and MyD88 in ischemic brain tissue. The results indicated that treadmill training promoted functional recovery and reduced the overexpression of TLR2, TLR4, NF-κB and MyD88 in rat brain tissue after ischemia, a finding that may have implications for understanding the mechanism of exercise therapy after brain ischemia and indicating new therapeutic strategies for the pharmacological modulation of TLR signaling.

  20. Extract of Reishi polysaccharides induces cytokine expression via TLR4-modulated protein kinase signaling pathways.

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    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Lin, Chun-Cheng; Lin, Chun-Hung; Hsu, Jason; Wong, Chi-Huey

    2004-11-15

    We have demonstrated that an extract of Ganoderma lucidum (Reishi or Ling-Zhi) polysaccharides (EORP) exerts immunomodulating activities by stimulating the expression of inflammatory cytokines from mouse spleen cells. Interestingly, via responding to LPS in genetic variation of murine macrophage HeNC2 and GG2EE cell lines, and using TLR4 Ab blockage in human blood-derived monocytic macrophages, we have found that the TLR4, but not complement receptor type 3, is a putative receptor of EORP, mediating the consequent immunomodulating events associated with IL-1 gene expression. Based on our studies of reactive oxygen species production, polymyxin B inhibition, and protein tyrosine kinase (PTK) activity, we ruled out the possibility of LPS contamination in EORP. We have found that EORP differentially modulates the protein kinase (PK)-mediated signal transduction pathways associated with inflammatory cytokine IL-1. In human macrophages and murine macrophage J774A.1 cells, EORP was found to up-regulate IL-1 secretion and pro-IL-1 (precursor of IL-1) as well as IL-1-converting enzyme expression. Specifically, EORP rapidly stimulates PTK-mediated phosphorylation, followed by induction of PKs and activation of MAPKs: ERK, JNK, and p38. Using PK inhibitors in the kinase activity assays, Western blot analyses and IL-1 ELISA, we have extensively examined and dissected the role of individual PK in the regulation of pro-IL-1/IL-1. Our findings establish that EORP-mediated signaling pathways are involved in the pro-IL-1/IL-1 regulation: PTK/protein kinase C/MEK1/ERK and PTK/Rac1/p21-activated kinase/p38.

  1. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

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    Giselle A Funchal

    Full Text Available Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

  2. Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent.

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    Singleton, Theresa E; Massari, Paola; Wetzler, Lee M

    2005-03-15

    Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.

  3. TLR4 activates the β-catenin pathway to cause intestinal neoplasia.

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    Rebeca Santaolalla

    Full Text Available Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4, the receptor for bacterial lipopolysaccharide (LPS, is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice. We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased β-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner, increasing phosphorylation of β-catenin(Ser552, a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/β-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC.

  4. Hyperinsulinaemia down-regulates TLR4 expression in the mammalian heart

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    Melody ede Laat

    2014-07-01

    Full Text Available Toll-like receptors (TLR are key regulators of innate immune and inflammatory responses and their activation is linked to impaired glucose metabolism during metabolic disease. Determination of whether TLR4 signalling can be activated in the heart by insulin may shed light on the pathogenesis of diabetic cardiomyopathy, a process that is often complicated by obesity and insulin resistance. The aim of the current study was to determine if supraphysiological insulin concentrations alter the expression of TLR4, markers of TLR4 signalling and glucose transporters (GLUTs in the heart. First, the effect of insulin on TLR4 protein expression was investigated in vitro in isolated rat cardiac myocytes. Secondly, protein expression of TLR4, the pro-inflammatory cytokines IL-6 and TNF-α, suppressor of cytokine signalling 3 and GLUTs (1, 4, 8, 12 were examined in the equine ventricular myocardium following a prolonged, euglycaemic hyperinsulinaemic clamp. Down-regulation of TLR4 in plasma membrane-rich fractions of rat cardiac myocytes was observed after incubation with a supraphysiologic concentration of insulin as well as in the equine myocardium after prolonged insulin infusion. Further, cardiac TLR4 expression was negatively correlated with serum insulin concentration. Markers of cardiac TLR4 signalling and GLUT expression were not affected by hyperinsulinaemia and concomitant TLR4 down-regulation. Since TLRs are major determinants of the inflammatory response, our findings suggest that insulin infusion exerts an anti-inflammatory effect in the hearts of non-obese individuals. Understanding the regulation of cardiac TLR4 signalling during metabolic dysfunction will facilitate improved management of cardiac sequelae to metabolic syndrome and diabetes.

  5. The receptor for urokinase regulates TLR2 mediated inflammatory responses in neutrophils.

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    Gang Liu

    Full Text Available The urokinase-type plasminogen activator receptor (uPAR, a glycosylphosphatidylinositol (GPI anchored membrane protein, regulates urokinase (uPA protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.

  6. The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

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    Enda Shevlin

    Full Text Available Toll-like receptor 7 (TLR7 plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM(-/- murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM(-/- cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

  7. Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-gamma and regulatory cytokine IL-10 expression in chicken monocytes

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    Toll-like receptors (TLRs) are the pattern recognition receptors of the innate immune system for various conserved pathogen-associated molecular motifs. The chicken TLR3 and TLR21 (avian equivalent to mammalian TLR9) recognize poly I:C (viral double-stranded RNA) and CpG-ODN (a CpG-motif containing...

  8. A 90-day toxicology study of meat from genetically modified sheep overexpressing TLR4 in Sprague-Dawley rats.

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    Hai Bai

    Full Text Available Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1 were confirmed using the polymerase chain reaction (PCR and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt of GM sheep meat, WT sheep meat or a commercial diet (CD. Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.

  9. Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

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    Ahmed R El-Awady

    2015-02-01

    Full Text Available Signaling via pattern recognition receptors (PRRs expressed on professional antigen presenting cells, such as dendritic cells (DCs, is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs. We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

  10. A 90-day toxicology study of meat from genetically modified sheep overexpressing TLR4 in Sprague-Dawley rats.

    Science.gov (United States)

    Bai, Hai; Wang, Zhixian; Hu, Rui; Kan, Tongtong; Li, Yan; Zhang, Xiaosheng; Zhang, Jinlong; Lian, Ling; Han, Hongbing; Lian, Zhengxing

    2015-01-01

    Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.

  11. A 90-Day Toxicology Study of Meat from Genetically Modified Sheep Overexpressing TLR4 in Sprague-Dawley Rats

    Science.gov (United States)

    Hu, Rui; Kan, Tongtong; Li, Yan; Zhang, Xiaosheng; Zhang, Jinlong; Lian, Ling; Han, Hongbing; Lian, Zhengxing

    2015-01-01

    Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals. PMID:25874566

  12. Human Intestinal Dendritic Cells Decrease Cytokine Release against Salmonella Infection in the Presence of Lactobacillus paracasei upon TLR Activation

    Science.gov (United States)

    Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, María J.; Romero, Fernando; Gil, Angel

    2012-01-01

    Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods. PMID:22905233

  13. Cloning and SNP screening of the TLR4 gene and the association ...

    African Journals Online (AJOL)

    Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Recognizing a broad class of pathogen-associated molecular patterns (PAMPs), TLR4 is implicated as a receptor mediating cellular activation in response to bacterial lipopolysaccharide (LPS), a cell-wall component ...

  14. Divergent effects of Tenofovir and Retrovir (AZT) on TLR-mediated cytokine production

    DEFF Research Database (Denmark)

    Melchjorsen, Jesper; Tolstrup, Martin; Paludan, Søren Riis

      Pathogen-recognizing Toll-like receptors 2 (TLR2) and TLR4 are known to recognize a number of pathogens, including E.Coli, S. Pneumoniae and N. Meningitidis. We have studied whether a number of HIV therapeutics affect immediate proinflammatory cytokine responses in cell cultures. Preliminary...

  15. TLR4 links podocytes with the innate immune system to mediate glomerular injury

    DEFF Research Database (Denmark)

    Banas, Miriam C; Banas, Bernhard; Hudkins, Kelly L

    2008-01-01

    profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered...... by the deposition of immune complexes....

  16. Porcine TLR3 characterization and expression in response to influenza virus and Bordetella bronchiseptica

    Science.gov (United States)

    We have provided a detailed structural analysis of porcine alveolar macrophage TLR3 extracellular domain (ECD). The pTLR3-ECD contains 18 leucine-rich repeat (LRRs) consisting of blocks of consensus motifs and non-consensus motifs containing insertions. Excluding the N-terminal and C-terminal LRRs, ...

  17. TLR3 Blockade in Rhinovirus-Induced Experimental Asthma Exacerbations: A Randomized Controlled Study

    NARCIS (Netherlands)

    Silkoff, Philip E.; Flavin, Susan; Gordon, Robert; Loza, Mathew J.; Sterk, Peter J.; Lutter, Rene; Diamant, Zuzana; Turner, Ronald B.; Lipworth, Brian J.; Proud, David; Singh, Dave; Eich, Andreas; Backer, Vibeke; Gern, James E.; Herzmann, Christian; Halperin, Scott A.; Mensinga, Tjeert T.; del Vecchio, Alfred M.; Branigan, Patrick; San Mateo, Lani; Baribaud, Frédéric; Barnathan, Elliot S.; Johnston, Sebastian L.

    2017-01-01

    Human rhinoviruses (HRV) commonly precipitate asthma exacerbations. TLR3, an innate pattern-recognition receptor, is triggered by HRV driving inflammation that may worsen asthma. To evaluate an inhibitory monoclonal antibody to TLR3, CNTO3157, on experimental HRV-16 inoculation in healthy and

  18. DMPD: LPS/TLR4 signal transduction pathway. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 2008 May;42(2):145-51. Epub 2008 Mar 4. (.png) (.svg) (.html) (.csml) Show LPS/TLR4 signal transduction path...way. PubmedID 18304834 Title LPS/TLR4 signal transduction pathway. Authors Lu YC, Yeh WC, Ohashi PS. Publica

  19. Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies.

    Science.gov (United States)

    Omotuyi, Olaposi; Matsunaga, Hayato; Ueda, Hiroshi

    2015-01-01

    During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF-IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied. Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics. Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 - 111) superficially interacts with similar TLR4/MD-2 (KD = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 - 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS. ProTα via its C-terminal peptide (aa 91 - 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

  20. TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy.

    Science.gov (United States)

    Ma, Jin; Chadban, Steven J; Zhao, Cathy Y; Chen, Xiaochen; Kwan, Tony; Panchapakesan, Usha; Pollock, Carol A; Wu, Huiling

    2014-01-01

    Toll like receptor (TLR) 4 has been reported to promote inflammation in diabetic nephropathy. However the role of TLR4 in the complicated pathophysiology of diabetic nephropathy is not understood. In this study, we report elevated expression of TLR4, its endogenous ligands and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ) diabetes. Subsequently, we demonstrated that TLR4-/- mice were protected against the development of diabetic nephropathy, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR4 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells in vitro, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. Our data indicate that TLR4 activation may promote inflammation, podocyte and tubular epithelial cell injury and interstitial fibrosis, suggesting TLR4 is a potential therapeutic target for diabetic nephropathy.

  1. TLR4 activation promotes podocyte injury and interstitial fibrosis in diabetic nephropathy.

    Directory of Open Access Journals (Sweden)

    Jin Ma

    Full Text Available Toll like receptor (TLR 4 has been reported to promote inflammation in diabetic nephropathy. However the role of TLR4 in the complicated pathophysiology of diabetic nephropathy is not understood. In this study, we report elevated expression of TLR4, its endogenous ligands and downstream cytokines, chemokines and fibrogenic genes in diabetic nephropathy in WT mice with streptozotocin (STZ diabetes. Subsequently, we demonstrated that TLR4-/- mice were protected against the development of diabetic nephropathy, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic wild-type controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA and expression of fibrogenic genes (TGF-β and fibronectin were also evident in TLR4 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR4 activation in podocytes and tubular epithelial cells in vitro, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses. Our data indicate that TLR4 activation may promote inflammation, podocyte and tubular epithelial cell injury and interstitial fibrosis, suggesting TLR4 is a potential therapeutic target for diabetic nephropathy.

  2. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

    Science.gov (United States)

    Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

    2009-08-14

    Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

  3. DMPD: Intracellular TLR signaling: a structural perspective on human disease. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16785490 Intracellular TLR signaling: a structural perspective on human disease. La...gnaling: a structural perspective on human disease. PubmedID 16785490 Title Intracellular TLR signaling: a s...tructural perspective on human disease. Authors Lasker MV, Nair SK. Publication J

  4. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

    Directory of Open Access Journals (Sweden)

    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  5. Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation

    National Research Council Canada - National Science Library

    Into, Takeshi; Kiura, Kazuto; Yasuda, Motoaki; Kataoka, Hideo; Inoue, Nobuo; Hasebe, Akira; Takeda, Kiyoshi; Akira, Shizuo; Shibata, Ken-ichiro

    2004-01-01

    .... The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid...

  6. Atopy and new-onset asthma in young Danish farmers and CD14, TLR2, and TLR4 genetic polymorphisms: a nested case-control study

    DEFF Research Database (Denmark)

    Smit, L A M; Bongers, S I M; Ruven, H J T

    2007-01-01

    genotyped 100 new-onset asthma cases and 88 control subjects for three CD14 SNPs, three TLR2 SNPs, and two TLR4 SNPs. Atopy at baseline (defined as a positive skin prick test to one or more common inhalant allergens) was found in 17 asthma cases (17.0%) and in 17 controls (19.3%). RESULTS: The CD14/-260T...... and atopy. OBJECTIVE: To study potential associations between single nucleotide polymorphisms (SNPs) in CD14, Toll-like receptor 2 (TLR2), and TLR4 genes, and atopy and new-onset asthma in young farmers. METHODS: A nested case-control study was conducted within a cohort of 1901 young Danish farmers. We...

  7. TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration in Acute Kidney Injury Induced by Sepsis

    Science.gov (United States)

    Castoldi, Angela; Braga, Tárcio Teodoro; Correa-Costa, Matheus; Aguiar, Cristhiane Fávero; Bassi, Ênio José; Correa-Silva, Reinaldo; Elias, Rosa Maria; Salvador, Fábia; Moraes-Vieira, Pedro Manoel; Cenedeze, Marcos Antônio; Reis, Marlene Antônia; Hiyane, Meire Ioshie; Pacheco-Silva, Álvaro; Gonçalves, Giselle Martins; Câmara, Niels Olsen Saraiva

    2012-01-01

    The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2−/−, TLR4−/− and MyD88−/− male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2−/−, TLR4−/− and MyD88−/− mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88−/− mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1+low cells migration compared with the knockout mice and decreased in GR1+high cells migration into the peritoneal cavity. The TLR2−/−, TLR4−/−, and MyD88−/− mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells. PMID:22655058

  8. TLR2, TLR4 and the MYD88 signaling pathway are crucial for neutrophil migration in acute kidney injury induced by sepsis.

    Directory of Open Access Journals (Sweden)

    Angela Castoldi

    Full Text Available The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-, TLR4(-/- and MyD88(-/- male mice were subjected to sepsis by cecal ligation and puncture (CLP. Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-, TLR4(-/- and MyD88(-/- mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT. MyD88(-/- mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low cells migration compared with the knockout mice and decreased in GR1(+high cells migration into the peritoneal cavity. The TLR2(-/-, TLR4(-/-, and MyD88(-/- mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.

  9. TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis.

    Science.gov (United States)

    Yin, Chaoran; Zhang, Ting; Qiao, Liangjun; Du, Jia; Li, Shuang; Zhao, Hengguang; Wang, Fangfang; Huang, Qiaorong; Meng, Wentong; Zhu, Hongyan; Bu, Hong; Li, Hui; Xu, Hong; Mo, Xianming

    2014-07-25

    Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.

  10. DMPD: TLR3: interferon induction by double-stranded RNA including poly(I:C). [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18262679 TLR3: interferon induction by double-stranded RNA including poly(I:C). Mat...l) Show TLR3: interferon induction by double-stranded RNA including poly(I:C). PubmedID 18262679 Title TLR3: interferon induction

  11. DMPD: Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation inrespiratory disease. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18031251 Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation...l) (.csml) Show Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation inrespiratory dis...ease. PubmedID 18031251 Title Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation

  12. Differential monocyte responses to TLR ligands in children with autism spectrum disorders.

    Science.gov (United States)

    Enstrom, Amanda M; Onore, Charity E; Van de Water, Judy A; Ashwood, Paul

    2010-01-01

    Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1beta, IL-6, IL-8, TNFalpha, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1beta, IL-6, and TNFalpha responses following TLR 2, and IL-1beta response following TLR 4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1beta, IL-6, GM-CSF, and TNFalpha responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

  13. Polymorphisms in the inflammatory pathway genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG are associated with susceptibility of inflammatory bowel disease in a Danish cohort

    DEFF Research Database (Denmark)

    Bank, Steffen; Skytt Andersen, Paal; Burisch, Johan

    2014-01-01

    associated with risk of CD and the polymorphisms TLR2 (rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs352139), LY96 (rs11465996), NFKBIA (rs696), TNFA (rs1800629), TNFRSF1A (rs4149570), IL10 (rs3024505), IL23R (rs11209026), PTPN22 (rs2476601) and PPARG (rs1801282) were associated with risk of UC. When...

  14. Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

    Directory of Open Access Journals (Sweden)

    Katherine L Irvine

    Full Text Available The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4 are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly

  15. Identification of key residues that confer Rhodobacter sphaeroides LPS activity at horse TLR4/MD-2.

    Science.gov (United States)

    Irvine, Katherine L; Gangloff, Monique; Walsh, Catherine M; Spring, David R; Gay, Nicholas J; Bryant, Clare E

    2014-01-01

    The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from Rhodobacter sphaeroides (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR

  16. Genetic and Pharmacologic Manipulation of TLR4 Has Minimal Impact on Ethanol Consumption in Rodents.

    Science.gov (United States)

    Harris, R Adron; Bajo, Michal; Bell, Richard L; Blednov, Yuri A; Varodayan, Florence P; Truitt, Jay M; de Guglielmo, Giordano; Lasek, Amy W; Logrip, Marian L; Vendruscolo, Leandro F; Roberts, Amanda J; Roberts, Edward; George, Olivier; Mayfield, Jody; Billiar, Timothy R; Hackam, David J; Mayfield, R Dayne; Koob, George F; Roberto, Marisa; Homanics, Gregg E

    2017-02-01

    Toll-like receptor 4 (TLR4) is a critical component of innate immune signaling and has been implicated in alcohol responses in preclinical and clinical models. Members of the Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune) consortium tested the hypothesis that TLR4 mediates excessive ethanol drinking using the following models: (1) Tlr4 knock-out (KO) rats, (2) selective knockdown of Tlr4 mRNA in mouse nucleus accumbens (NAc), and (3) injection of the TLR4 antagonist (+)-naloxone in mice. Lipopolysaccharide (LPS) decreased food/water intake and body weight in ethanol-naive and ethanol-trained wild-type (WT), but not Tlr4 KO rats. There were no consistent genotypic differences in two-bottle choice chronic ethanol intake or operant self-administration in rats before or after dependence. In mice, (+)-naloxone did not decrease drinking-in-the-dark and only modestly inhibited dependence-driven consumption at the highest dose. Tlr4 knockdown in mouse NAc did not decrease drinking in the two-bottle choice continuous or intermittent access tests. However, the latency to ethanol-induced loss of righting reflex increased and the duration decreased in KO versus WT rats. In rat central amygdala neurons, deletion of Tlr4 altered GABA A receptor function, but not GABA release. Although there were no genotype differences in acute ethanol effects before or after chronic intermittent ethanol exposure, genotype differences were observed after LPS exposure. Using different species and sexes, different methods to inhibit TLR4 signaling, and different ethanol consumption tests, our comprehensive studies indicate that TLR4 may play a role in ethanol-induced sedation and GABA A receptor function, but does not regulate excessive drinking directly and would not be an effective therapeutic target. Toll-like receptor 4 (TLR4) is a key mediator of innate immune signaling and has been implicated in alcohol responses in animal models and human alcoholics. Members of the

  17. Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.

    Directory of Open Access Journals (Sweden)

    Julien Pothlichet

    Full Text Available Influenza A virus (IAV triggers a contagious and potentially lethal respiratory disease. A protective IL-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58, TLR3, and NLRP3 in the IL-1β response to IAV in primary lung epithelial cells. To activate IL-1β secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet-dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1β response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1β responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

  18. Type I IFN triggers RIG-I/TLR3/NLRP3-dependent inflammasome activation in influenza A virus infected cells.

    Science.gov (United States)

    Pothlichet, Julien; Meunier, Isabelle; Davis, Beckley K; Ting, Jenny P-Y; Skamene, Emil; von Messling, Veronika; Vidal, Silvia M

    2013-01-01

    Influenza A virus (IAV) triggers a contagious and potentially lethal respiratory disease. A protective IL-1β response is mediated by innate receptors in macrophages and lung epithelial cells. NLRP3 is crucial in macrophages; however, which sensors elicit IL-1β secretion in lung epithelial cells remains undetermined. Here, we describe for the first time the relative roles of the host innate receptors RIG-I (DDX58), TLR3, and NLRP3 in the IL-1β response to IAV in primary lung epithelial cells. To activate IL-1β secretion, these cells employ partially redundant recognition mechanisms that differ from those described in macrophages. RIG-I had the strongest effect through a MAVS/TRIM25/Riplet-dependent type I IFN signaling pathway upstream of TLR3 and NLRP3. Notably, RIG-I also activated the inflammasome through interaction with caspase 1 and ASC in primary lung epithelial cells. Thus, NS1, an influenza virulence factor that inhibits the RIG-I/type I IFN pathway, strongly modulated the IL-1β response in lung epithelial cells and in ferrets. The NS1 protein derived from a highly pathogenic strain resulted in increased interaction with RIG-I and inhibited type I IFN and IL-1β responses compared to the least pathogenic virus strains. These findings demonstrate that in IAV-infected lung epithelial cells RIG-I activates the inflammasome both directly and through a type I IFN positive feedback loop.

  19. A Polysaccharide from the Culinary-Medicinal Mushroom Pholiota nameko (Agaricomycetes) Inhibits the NF-κB Pathway in Dendritic Cells Through the TLR2 Receptor.

    Science.gov (United States)

    Li, Haiping; Zhao, Pei; Wang, Fengling; Huai, Lihua; Zhu, Ruixuan; Li, Guoliang; Xu, Yufeng

    2016-01-01

    A polysaccharide purified from Pholiota nameko (PNPS-1) was found to have anticancer and anti-inflammatory activity. This study investigated the effect of PNPS-1 on the nuclear factor (NF)-κB signaling pathway of TLR2 small interfering RNA-silenced murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The expression of messenger RNA of 4 NF-κB-related genes, including MyD88, IKBKB, RelA(p65), and CCL2, was determined by real-time polymerase chain reaction; the expression of the phenotype molecule intercellular adhesion molecule-1 (ICAM-1) by flow cytometry; the protein expression of IKKβ and p65 by Western blot; the production of p65 by enzyme-linked immunosorbent assay; and the expression of p65 by immunocytochemistry. The results showed that TLR2-specific small interfering RNA could effectively inhibit the decrease in the expression of MyD88, IKBKB, CCL2, p65, and ICAM-1 in BMDCs induced by PNPS-1, and thus the transcription inactivation of NF-κB, which obviously suggests that PNPS-1 could downregulate the NF-κB signaling pathway via the TLR2 receptor.

  20. TGF-β1 Inhibits TLR-mediated Odontoblast Responses to Oral Bacteria

    Science.gov (United States)

    Horst, O.V.; Tompkins, K.A.; Coats, S.R.; Braham, P.H.; Darveau, R.P.; Dale, B.A.

    2009-01-01

    TGF-β1 exerts diverse functions in tooth development and tissue repair, but its role in microbial defenses of the tooth is not well-understood. Odontoblasts extending their cellular processes into the dentin are the first cells to recognize signals from TGF-β1 and bacteria in carious dentin. This study aimed to determine the role of TGF-β1 in modulating odontoblast responses to oral bacteria. We show that these responses depend upon the expression levels of microbial recognition receptors TLR2 and TLR4 on the cell surface. Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum activated both TLRs, but TLR4 played a greater role. Lack of cell-surface TLR2 was associated with poor response to Streptococcus mutans, Enterococcus faecalis, and Lactobacillus casei. TGF-β1 inhibited TLR2 and TLR4 expression and attenuated odontoblast responses. Our findings suggest that the balance between TLR-mediated inflammation and TGF-β1 anti-inflammatory activity plays an important role in pulpal inflammation. PMID:19407153

  1. TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C).

    Science.gov (United States)

    Park, Hong-Jai; Hong, Ju-ho; Kwon, Hyung-Joon; Kim, Youngchan; Lee, Kwan-Hee; Kim, Jong-Bae; Song, Seong K

    2010-06-04

    Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-alpha, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-alpha production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-alpha production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-alpha secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-alpha by interacting with the TLR4 molecule and activating its signaling pathways. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  2. [Cloning of mouse TLR-2 gene and its expression in Pichia pastroris].

    Science.gov (United States)

    Zhao, Wen-zhong; Jiang, Hai-yan; Liu, Yan-jun; Zhu, Ping; Fu, Ning

    2004-07-01

    To clone the mouse TLR-2 gene and to express it in Pichia pastroris. Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot. The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody. The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.

  3. Contrasting roles for TLR ligands in HIV-1 pathogenesis.

    Directory of Open Access Journals (Sweden)

    Beda Brichacek

    2010-09-01

    Full Text Available The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs. Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs 5 and 9, we examined their effect on human immunodeficiency virus (HIV-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist treatment enhanced replication of CC chemokine receptor 5 (CCR 5-tropic and CXC chemokine receptor 4 (CXCR4-tropic HIV-1, treatment with oligodeoxynucleotide (ODN M362 (TLR9 agonist suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention.

  4. Peculiairities of mRNA TLR-2, TLR-4 expression of the oral cavity epithelium in children under conditions of chronic catarrhal gingivitis and diabetes mellitus

    Directory of Open Access Journals (Sweden)

    А. V. Kotelban

    2017-06-01

    Full Text Available TLRs in Epithelium of the oral cavity can be effected as due to the dental as general somatic pathology. TLR availability in the external membrane of neutrophils, macrophages, keratocytes was found to be a starting point in triggering inflammation in the periodontal tissues ensuring molecular reception of a pathogen with further involvement of important components in the inherited immunity. These effectors possess phagocytic and killer activities, ensure a number of signals activating and directing antigen-specific response by the cells of the adaptive immune system. The aim of the study was to study mRNA TLR-2, TLR-4 in the epithelium of the oral cavity in children with chronic catarrhal gingivitis and diabetes mellitus taking to account revealed metabolic disorders. Materials and methods. 30 children with chronic catarrhal gingivitis and diabetes mellitus type I were included into Іst group, 30 children with chronic catarrhal gingivitis were included into the ІInd group and 30 absolutely healthy children -ІII group. Polymerase chain reaction with reverse transcription in the regime in real time (RT-RRT was used to analyze gene expression. Buccal epithelium was used as an object for molecular-genetic study. Results. Content of mRNA TLR-2 (90.0755 in diabetes mellitus patients was in 5.5 times higher as compared to children with CCG – 15.1505. Expression of mRNA TLR-4 in children of I group increased in 6 times as compared to the children from II group. Conclusion. Relative quantity of mRNA TLR-2 and TLR-4 in the epithelium of the oral cavity in children with diabetes mellitus was considerably higher than in patients without chronic inflammatory disease.

  5. The Fab Fragment of a Humanized Anti-Toll Like Receptor 4 (TLR4 Monoclonal Antibody Reduces the Lipopolysaccharide Response via TLR4 in Mouse Macrophage

    Directory of Open Access Journals (Sweden)

    Binggang Cai

    2015-10-01

    Full Text Available Lipopolysaccharides (LPS can induce acute inflammation, sepsis, or chronic inflammatory disorders through the Toll receptor 4 (TLR4 signaling pathway. The TLR4/MD2 (myeloid differentiation protein 2 complex plays a major role in the immune response to LPS. However, there is not a good method to suppress the immune response induced by LPS via this complex in macrophages. In this article, we aimed to evaluate the effects of humanized anti-TLR4 monoclonal antibodies on LPS-induced responses in mouse macrophages. The peritoneal macrophages of mice were incubated with anti-TLR4 monoclonal antibodies and stimulated with LPS. The expression levels of cytokines were analyzed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Additionally, activation of various signaling pathways was evaluated by Western blotting. The results showed that the humanized anti-TLR4 monoclonal antibody blocked the inflammatory cytokines expression at both the mRNA and protein level. We also found that the Fab fragment significantly inhibited the nuclear factor kappaB signaling pathway by reducing the phosphorylation of the inhibitor of kappaBalpha and decreasing the translocation of p65, resulting in the suppression of p38, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, and IFN-β regulatory factor 3 phosphorylation. Therefore, our study showed that this humanized anti-TLR4 monoclonal antibody could effectively protect against LPS-induced responses by blocking the TLR4 signaling pathway in mouse peritoneal macrophages.

  6. Monocyte-mediated inhibition of TLR9-dependent IFN-α induction in plasmacytoid dendritic cells questions bacterial DNA as the active ingredient of bacterial lysates.

    Science.gov (United States)

    Poth, Jens M; Coch, Christoph; Busch, Nicolas; Boehm, Olaf; Schlee, Martin; Janke, Markus; Zillinger, Thomas; Schildgen, Oliver; Barchet, Winfried; Hartmann, Gunther

    2010-12-15

    Bacterial DNA contains unmethylated CpG dinucleotides and is a potent ligand for TLR9. Bacterial DNA has been claimed the active ingredient in bacterial lysates used for immunotherapy. Whereas the detection of viral DNA by TLR9 expressed in plasmacytoid dendritic cells (PDCs) with subsequent IFN-α production is well defined, the role of bacterial DNA during microbial infection is less clear. In fact, IFN-α is not a hallmark of antibacterial immune responses. Unlike in mice, TLR9 expression in humans is restricted to PDCs and B cells; thus, conclusions from murine models of infection have limitations. In this study, we demonstrate that lysates of heat-killed Escherichia coli containing bacterial DNA induced IFN-α in isolated PDCs but not in the mixed cell populations of human PBMCs. Depletion of monocytes restored IFN-α secretion by PDCs within PBMCs. We found that monocyte-derived IL-10 and PGs contribute to monocyte-mediated inhibition of IFN-α release in PDCs. We conclude that human PDCs can be stimulated by bacterial DNA via TLR9; however, in the physiological context of mixed-cell populations, PDC activation is blocked by factors released from monocytes stimulated in parallel by other components of bacterial lysates such as LPS. This functional repression of PDCs by concomitantly stimulated monocytes avoids production of antiviral IFN-α during bacterial infection and thus explains how the innate immune system is enabled to distinguish bacterial from viral CpG DNA and thus to elicit the appropriate responses despite the presence of CpG DNA in both types of infection.

  7. Glaucocalyxin B Alleviates Lipopolysaccharide-Induced Parkinson’s Disease by Inhibiting TLR/NF-κB and Activating Nrf2/HO-1 Pathway

    Directory of Open Access Journals (Sweden)

    Wei Xu

    2017-12-01

    Full Text Available Background/Aims: Parkinson’s disease (PD is a common neurodegenerative disease in the old population, characterized by dopaminergic neuron loss, inflammation and oxidative stress injury in the substantia nigra. Glaucocalyxin B (GLB, an ent-kauranoid diterpenoid isolated from Rabdosia japonica, has anti-inflammation and anti-tumor effects. However, its effects on PD remain unclear. Methods: PD was introduced in rats via injection of lipopolysaccharide (LPS into cerebral corpus striatum, and GLB was given intracerebroventricularly to these rats. Their walking, climbing and sensory states were detected by Stepping, Whisker and Cylinder Tests. The expression of tyrosine hydroxylase (TH, glial fibrillary acidic protein (GFAP, CD11b and ionized calcium binding adaptor molecule (IBA-1 were detected by immunohischemical staining. The levels of a series of inflammatory factors, oxidative stress-related factors and apoptosis-related factors were measured by real-time PCR, immunoblotting and ELISA. In addition, Toll-like receptor (TLR/nuclear factor kappa B (NF-κB and nuclear factor erythroid 2-related factor 2 (Nrf2/heme oxygenase (HO-1 pathways were investigated to illustrate the underlying mechanism. In vitro, microglial cells exposed to LPS were treated with GLB. Results: The injection of LPS caused walking, climbing and sensory disturbances in rats, induced inflammation, oxidative stress response and apoptosis, and activated TLR/NF-κB and Nrf2/ HO-1 pathways in the cerebral tissue. GLB administration attenuated LPS-induced alterations. The TLR/NF-κB pathway was deactivated and Nrf2/HO-1 was activated after application of GLB. In vitro, cytotoxic effects induced by the conditioned medium derived from microglial cells exposed to LPS in PC12 cells were attenuated by GLB. Conclusion: GLB suppresses LPS-induced PD symptoms by modification of TLR/NF-κB and Nrf2/HO-1 pathways in vivo and in vitro.

  8. Evolution of the bovine TLR gene family and member associations with Mycobacterium avium subspecies paratuberculosis infection.

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    Colleen A Fisher

    Full Text Available Members of the Toll-like receptor (TLR gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50 between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to

  9. TLR4 Methylation Moderates the Relationship Between Alcohol Use Severity and Gray Matter Loss.

    Science.gov (United States)

    Karoly, Hollis C; Thayer, Rachel E; Hagerty, Sarah L; Hutchison, Kent E

    2017-09-01

    Alcohol use disorders (AUDs) are associated with decreased gray matter, and neuroinflammation is one mechanism through which alcohol may confer such damage, given that heavy alcohol use may promote neural damage via activation of toll-like receptor 4 (TLR4)-mediated inflammatory signaling cascades. We previously demonstrated that TLR4 is differentially methylated in AUD compared with control subjects, and the present study aims to extend this work by examining whether TLR4 methylation moderates the relationship between alcohol use and gray matter. We examined TLR4 methylation and gray matter thickness in a large sample (N = 707; 441 males) of adults (ages 18-56) reporting a range of AUD severity (mean Alcohol Use Disorders Identification Test score = 13.18; SD = 8.02). We used a series of ordinary least squares multiple regression equations to regress gray matter in four bilateral brain regions (precuneus, lateral orbitofrontal, inferior parietal, and superior temporal) on alcohol use, TLR4 methylation, and their interaction, controlling for demographic, psychological, and other substance use variables. After we corrected for multiple tests, a significant Alcohol × TLR4 Methylation interaction emerged in the equations modeling left precuneus and right inferior parietal gray matter. Follow-up analyses examining the nature of these interactions demonstrated a significant negative association between alcohol and precuneus and inferior parietal gray matter in individuals with low TLR4 methylation, but no relationship between alcohol and gray matter in the high methylation group. These findings suggest that TLR4 methylation may be protective against the damage conferred by alcohol on precuneus and inferior parietal gray matter, thereby implicating TLR4 for further investigation as a possible AUD treatment target.

  10. Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity.

    Science.gov (United States)

    Du, Yang; Yang, Marie; Lee, Syann; Behrendt, Cassie L; Hooper, Lora V; Saghatelian, Alan; Wan, Yihong

    2012-06-15

    For all newborn mammals, mother's milk is the perfect nourishment, crucial for their postnatal development. Here we report that, unexpectedly, maternal western diet consumption in mice causes the production of toxic milk that contains excessive long chain and saturated fatty acids, which triggers ceramide accumulation and inflammation in the nursing neonates, manifested as alopecia. This neonatal toxicity requires Toll-like-receptors (TLR), but not gut microbiota, because TLR2/4 deletion or TLR4 inhibition confers resistance, whereas germ-free mice remain sensitive. These findings unravel maternal western diet-induced inflammatory milk secretion as a novel aspect of the metabolic syndrome at the maternal offspring interface.

  11. TLR-mediated NF-kB-dependent cytokine production is differently affected by HIV therapeutics

    DEFF Research Database (Denmark)

    Melchjorsen, Jesper; Paludan, Søren Riis; Mogensen, Trine

      Pathogen-recognizing Toll-like receptors 2 (TLR2) and TLR4 are known to recognize a number of pathogens, including E.Coli, S. Pneumonia and N. Meningococcus. We have studied whether a number of HIV therapeutics affect immediate proinflammatory cytokine responses in cell cultures. Preliminary...... results suggest an opposing effect of the drugs Tenofovir and Retrovir (AZT) on TLR-mediated production of NF-kappaB-dependent proinflammatory cytokines. We present data on the mechanisms behind the drug-mediated remodeling of innate immune activation and how the drugs effect early host...

  12. Increased Expression Profile and Functionality of TLR6 in Peripheral Blood Mononuclear Cells and Hepatocytes of Morbidly Obese Patients with Non-Alcoholic Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    María Teresa Arias-Loste

    2016-11-01

    Full Text Available Current evidence suggests that gut dysbiosis drives obesity and non-alcoholic fatty liver disease (NAFLD pathogenesis. Toll-like receptor 2 (TLR2 and TLR6 specifically recognize components of Gram-positive bacteria. Despite the potential implications of TLR2 in NAFLD pathogenesis, the role of TLR6 has not been addressed. Our aim is to study a potential role of TLR6 in obesity-related NAFLD. Forty morbidly obese patients undergoing bariatric surgery were prospectively studied. Cell surface expression of TLR2 and TLR6 was assessed on peripheral blood mononuclear cells (PBMCs by flow cytometry. Freshly isolated monocytes were cultured with specific TLR2/TLR6 agonists and intracellular production of cytokines was determined by flow-cytometry. In liver biopsies, the expression of TLR2 and TLR6 was analyzed by immunohistochemistry and cytokine gene expression using RT-qPCR. TLR6 expression in PBMCs from non-alcoholic steatohepatitis (NASH patients was significantly higher when compared to those from simple steatosis. The production of pro-inflammatory cytokines in response to TLR2/TLR6 stimulation was also significantly higher in patients with lobular inflammation. Hepatocyte expression of TLR6 but not that of TLR2 was increased in NAFLD patients compared to normal liver histology. Deregulated expression and activity of peripheral TLR6 in morbidly obese patients can mirror the liver inflammatory events that are well known drivers of obesity-related NASH pathogenesis. Moreover, TLR6 is also significantly overexpressed in the hepatocytes of NAFLD patients compared to their normal counterparts. Thus, deregulated TLR6 expression may potentiate TLR2-mediated liver inflammation in NAFLD pathogenesis, and also serve as a potential peripheral biomarker of obesity-related NASH.

  13. Live Faecalibacterium prausnitzii induces greater TLR2 and TLR2/6 activation than the dead bacterium in an apical anaerobic co-culture system.

    Science.gov (United States)

    Maier, Eva; Anderson, Rachel C; Altermann, Eric; Roy, Nicole C

    2017-11-07

    Inappropriate activation of intestinal innate immune receptors, such as Toll-like receptors (TLRs), by pathogenic bacteria is linked to chronic inflammation. In contrast, a 'tonic' level of TLR activation by commensal bacteria is required for intestinal homeostasis. A technical challenge when studying this activation in vitro is the co-culturing of oxygen-requiring mammalian cells with obligate anaerobic commensal bacteria. To overcome this, we used a novel apical anaerobic co-culture system to successfully adapt a TLR activation assay to be conducted in conditions optimised for both cell types. Live Faecalibacterium prausnitzii, an abundant obligate anaerobe of the colonic microbiota, induced higher TLR2 and TLR2/6 activation than the dead bacterium. This enhanced TLR induction by live F. prausnitzii, which until now has not previously been described, may contribute to maintenance of gastrointestinal homeostasis. This highlights the importance of using physiologically relevant co-culture systems to decipher the mechanisms of action of live obligate anaerobes. This article is protected by copyright. All rights reserved.

  14. Vaccination inhibits TLR2 transcription via suppression of GR nuclear translocation and binding to TLR2 promoter in porcine lung infected with Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Sun, Zhiyuan; Liu, Maojun; Zou, Huafeng; Li, Xian; Shao, Guoqing; Zhao, Ruqian

    2013-12-27

    Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (Ppigs demonstrated significantly diminished nuclear GR content. TLRs 1-10 were all expressed in lung, among which TLR2 was the most abundant and was significantly up-regulated (Ppigs, but not in VI pigs. Accordingly, GR binding to the GR response element on TLR2 promoter was significantly increased (Ppigs, but not in VI pigs. These results suggest that the inhibition of GR nuclear translocation and binding to the TLR2 promoter, which results in diminished TLR2 expression, is associated with the protective effect of vaccination on M. hyopneumoniae-induced lung lesions in the pig. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Role of TLR4 gene polymorphisms in the colorectal cancer risk ...

    African Journals Online (AJOL)

    Role of TLR4 gene polymorphisms in the colorectal cancer risk modulation in ethnic Kashmiri population – A case–control study. Saniya Nissar, Aga Syed Sameer, Roohi Rasool, Qurteeba Qadri, Nissar A. Chowdri, Fouzia Rashid ...

  16. Paclitaxel therapy promotes breast cancer metastasis in a TLR4-dependent manner

    National Research Council Canada - National Science Library

    Volk-Draper, Lisa; Hall, Kelly; Griggs, Caitlin; Rajput, Sandeep; Kohio, Pascaline; DeNardo, David; Ran, Sophia

    2014-01-01

    .... Work in preclinical models of breast cancer has shown that acquired chemoresistance to the widely used drug paclitaxel can be mediated by activation of the Toll-like receptor TLR4 in cancer cells...

  17. TLR3 Mutations in Adult Patients With Herpes Simplex Virus and Varicella-Zoster Virus Encephalitis.

    Science.gov (United States)

    Sironi, Manuela; Peri, Anna Maria; Cagliani, Rachele; Forni, Diego; Riva, Stefania; Biasin, Mara; Clerici, Mario; Gori, Andrea

    2017-05-01

    Defects in genes of the Toll-like receptor 3 (TLR3) pathway are associated with susceptibility to herpes simplex virus type 1 encephalitis (HSE). We analyzed a cohort of 11 adult Italian patients in whom viral encephalitis developed. We detected 2 rare missense mutations in TLR3: 1 in a patient with HSE (p.Leu297Val) and 1 in a patient with varicella-zoster virus encephalitis (p.Leu199Phe). Both mutations are extremely rare in human populations and have pathogenicity scores highly suggestive of a functional effect. Data herein expand the phenotypic spectrum of TLR3 mutations to varicella-zoster virus encephalitis and support the role of TLR3 genetic defects as risk factors for HSE in adults. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  18. Lipid raft localization of TLR2 and its co-receptors is independent of membrane lipid composition

    Directory of Open Access Journals (Sweden)

    Christine Hellwing

    2018-01-01

    Full Text Available Background Toll like receptors (TLRs are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. Methods In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA or arachidonic acid (AA and analyzed for receptor expression and microdomain localization in context of TLR stimulation. Results and Conclusions Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

  19. TLR8 activation and inhibition by guanosine analogs in RNA: Importance of functional groups and chain length.

    Science.gov (United States)

    Hu, Tiannan; Suter, Scott R; Mumbleau, Madeline M; Beal, Peter A

    2018-01-01

    Toll-like receptor 8 (TLR8) is an important component of the human innate immune system that recognizes single stranded RNA (ssRNA). Recent X-ray crystal structures of TLR8 bound to ssRNA revealed a previously unrecognized binding site for a 5'-UpG-3' dinucleotide. Here we use an atomic mutagenesis strategy coupled with a cellular TLR8 activation assay to probe the importance of specific functional groups present on the guanine base in RNA-mediated receptor agonism and antagonism. Results from RNA analogs containing 7-deazaguanosine, 2-aminopurine and inosine confirm the importance of guanine N7, O6 and N2, respectively, in TLR8 activation. Nevertheless, these RNAs each retained TLR8 antagonism activity. RNA containing 7-deaza-8-azainosine (7d8aI) was prepared from a novel phosphoramidite and found to be a weaker TLR8 activator than guanosine-containing RNA. However, 7d8aI-containing RNA also retained TLR8 antagonism activity indicating that removal of multiple TLR8 H-bonding sites on guanine is insufficient for blocking TLR8 antagonism by guanine-containing RNA. We also identified an oligoribonucleotide length dependence on both TLR8 activation and antagonism. These studies extend our understanding of the effects of nucleobase modification on immune stimulation and will inform the design of novel RNA-based therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The role of TLR2 and bacterial lipoprotein in enhancing airway inflammation and immunity

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    Amit A Lugade

    2011-04-01

    Full Text Available Non-typeable Haemophilus influenzae (NTHI colonizes the lower respiratory tract of patients with chronic obstructive pulmonary disease (COPD and also causes exacerbations of the disease. The 16-kDa lipoprotein P6 has been widely studied as a potential vaccine antigen due to its highly conserved expression amongst NTHI strains. Although P6 is known to induce potent inflammatory responses, its role in the pathogenesis of NTHI infection in vivo has not been examined. Additionally, the presence of an amino-terminal lipid motif on P6 serves to activate host TLR2 signaling. The role of host Toll-like receptor 2 (TLR2 and NTHI expression of the lipoprotein P6 on the induction of airway inflammation and generation of adaptive immune responses following chronic NTHI stimulation was evaluated with TLR2-deficient mice and a P6-deficient NTHI strain. Absence of either host TLR2 or bacterial P6 resulted in diminished levels of immune cell infiltration within lungs of mice exposed to NTHI. Pro-inflammatory cytokine secretion was also reduced in lungs that did not express TLR2 or were exposed to NTHI devoid of P6. Induction of specific antibodies to P6 was severely limited in TLR2-deficient mice. Although mice exposed to the P6-deficient NTHI strain were capable of generating antibodies to other surface antigens of NTHI, these levels were lower compared to those observed in mice exposed to P6-expressing NTHI. Therefore, cognate interaction between host TLR2 and bacterial P6 serves to enhance lung inflammation and elicit robust adaptive immune responses during NTHI exposure. Strategies to limit NTHI inflammation while simultaneously promoting robust immune responses may benefit from targeting the TLR2:P6 signaling axis.

  1. TLR7 deficiency contributes to attenuated diabetic retinopathy via inhibition of inflammatory response.

    Science.gov (United States)

    Liao, Yun-Ru; Li, Zi-Jing; Zeng, Peng; Lan, Yu-Qing

    2017-11-18

    Diabetic retinopathy (DR) is a major microvascular complication of diabetes, resulting in neuronal dysfunction, retinal vascular leakage, and apoptosis within the retina. Innate immunity plays an important role in the pathogenesis of type 2 diabetes (T2D) and related complications. The toll-like receptors (TLRs), central to innate immunity, are essential participants in the progression and pathogenesis of the disease and its complications. In the study, streptozotocin (STZ) was combined with whole-body hypoxia for quicker induction of early-stage diabetic retinopathy (DR) in the wild type (WT) and TLR7-knockout (KO) C57BL/6 mice. The effects of TLR7 were also investigated in fructose-treated retinal pigment epithelial (RPE) cells. In the retinas of WT/DR mice, abnormal a-wave and b-wave activity, hyperfluorescence, and reduced retinal thickness were observed. DR development was associated with enhanced TLR7 expression, whose deletion dramatically reduced VEGF expression levels. And the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-18 and IL-12, was highly reduced by TLR7-deficiency in DR mice. Consistently, WT/DR mice exhibited higher phosphorylation of IκB kinase α (IKKα), inhibitor of NF-κB α (IκBα) and nuclear factor κB (NF-κB), which were found to be down-regulated in KO/DR mice. Similarly, DR-induced mitogen-activated protein kinases (MAPKs) activation was blocked by TLR7-knockout. In vitro, fructose incubation-triggered inflammation was reversed by TLR7 knockdown, accompanied with inactivated NF-κB and MAPKs pathways. And reduced reactive oxygen species (ROS) generation was observed in TLR7-knockdown cells with fructose treatment. Together, inhibiting TLR7 suppressed diabetic retinopathy by reducing inflammation and suggested a potential application in clinics. Copyright © 2017. Published by Elsevier Inc.

  2. TRIF is required for TLR4 mediated adjuvant effects on T cell clonal expansion.

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    Siva K Gandhapudi

    Full Text Available Toll like receptor 4 (TLR4 is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88 and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta. Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF (lps/lps mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4's adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response.

  3. The Importance of TLR2 and Macrophages in Modulating a Humoral Response after Encountering Streptococcus pneumoniae

    Science.gov (United States)

    2008-03-26

    350. Takeuchi, O., Hoshino, K., Akira, S. 2000. TLR2-Deficient and MyD88-Deficient Mice Are Highly Susceptible to Staphylococcus aureus Infection...Staphloccocus aureus , indicating a critical role for both MyD88 and TLR2 for defense against bacterial pathogen (350). Another group using the spirochete...monocytogenes to CD8+ T cells requires secretion of hemolysin and intracellular bacterial growth. J Immunol 145:3540-3546. 379. Kahlert, H., E. Grage

  4. Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries

    OpenAIRE

    Zhao, Alyssa; Blackburn, Corinne; Chin, Judith; Srinivasan, Mythily

    2014-01-01

    Background Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cario...

  5. TLR-4 and CD14 Polymorphisms in Respiratory Syncytial Virus Associated Disease

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    Beena Puthothu

    2006-01-01

    Full Text Available Respiratory syncytial virus (RSV is the most common viral respiratory pathogen during infancy world wide. It induces innate and adaptive immune response in host cells. The toll like receptor 4 (TLR4/CD14 complex is particularly important for the initiation of an innate immune response to RSV. Thus we were interested whether an association exists between severe RSV associated diseases and polymorphisms within TLR4 and CD14.

  6. Evaluation of TLR9 expression on PBMCs and CpG ODN-TLR9 ligation on IFN-α production in SLE patients.

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    Mortezagholi, Sahar; Babaloo, Zohreh; Rahimzadeh, Parisa; Namdari, Haideh; Ghaedi, Mojgan; Gharibdoost, Farhad; Mirzaei, Reza; Bidad, Katayoon; Salehi, Eisa

    2017-02-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoreactive antibodies. Recent findings revealed the importance of innate immune responses, especially Toll-like receptors (TLRs) in the pathogenesis of SLE. In this study, the level of TLR9 expression on peripheral blood mononuclear cells (PBMCs) was analyzed. The levels of produced IFN-α were also measured in supernatant of PBMCs from SLE patients and healthy controls after stimulation with CpG ODN2216 which is a plasmocytoid dendritic cell (pDC)-specific TLR9 ligand. TLR9 expression was analyzed by real-time polymerase chain reaction (PCR) and flow cytometry in 35 SLE patients and 38 healthy controls and IFN-α concentration was measured in supernatants using enzyme-linked immunosorbent assay (ELISA). The results showed that the TLR9 expression in the mRNA and the protein level was significantly higher in PBMCs from SLE patients. However, IFN-α concentration in patients and controls significantly increased in response to CpG stimulation but this increase was significantly higher in healthy controls compared with SLE patients. Our results do not show any association between taking hydroxychloroquine and reduction in IFN-α production in SLE patients. Regarding the findings of the study, there is the possibility that TLR9 has played a role in SLE pathogenesis, and consequently it implies that TLRs can be considered to be the therapeutic targets for systemic autoimmunity. We may conclude that PBMCs in patients are functionally impaired in response to TLR ligation via innate response stimulating pathogen-associated molecular patterns (PAMPs).

  7. TLR2 and TLR4 co-activation utilizes distinct signaling pathways for the production of Th1/Th2/Th17 cytokines in neonatal immune cells.

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    Sugitharini, V; Shahana, P; Prema, A; Berla Thangam, E

    2016-09-01

    Co-activation of TLR2 and TLR4 by gram negative and gram positive bacterial ligands induces a robust pro-inflammatory response in inflammatory cells. In order to understand the signaling mechanism, we aimed to delineate the signaling molecules involved in TLR2 and TLR4 co-activation in neonatal immune cells for the production of Th1/Th2/Th17 inflammatory cytokines. For this, we pretreated cord blood and peripheral blood mononuclear and human mast cells with specific signaling molecule inhibitors such as BAY117082, PD98059 and LY294002 and then stimulated with LPS and PGN and assayed for cytokines IL-6, IL-12/IL-23p40 (Th1), IL-13 (Th2), IL-23 (Th17) and RANTES secretion. We found that upon co-stimulation the phosphorylation of NFκBp65, ERK1/2 and Akt was found to be higher than when stimulated with individual ligands in CBMCs. Also, when compared to adult cells, neonatal cells were more potent in the activation of ERK and Akt through TLR2 and TLR4 co-activation. In addition, neonatal cells possess similar capacity to activate NFκB as that of adult cells for IL-6 secretion. Furthermore, all three signaling molecules were found to be involved in the production of Th17 cytokines which is detrimental during inflammation induced by infection in neonates whereas NFκB is mainly involved in the induction of pro-inflammatory response and Th2 cytokines production. In conclusion, different signaling molecules were utilized for the production of different cytokines in immune cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Systemic isotretinoin therapy normalizes exaggerated TLR-2-mediated innate immune responses in acne patients

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    Dispenza, Melanie C.; Wolpert, Ellen B.; Gilliland, Kathryn L.; Dai, Pingqi; Cong, Zhaoyuan; Nelson, Amanda M.; Thiboutot, Diane M.

    2012-01-01

    Retinoids are used in the treatment of inflammatory skin diseases and malignancies, but studies characterizing the in vivo actions of these drugs in humans are lacking. Isotretinoin is a pro-drug for all-trans retinoic acid that can induce long-term remissions of acne; however, its complete mechanism of action is unknown. We hypothesized that isotretinoin induces remission of acne by normalizing the innate immune response to the commensal bacterium P. acnes. Compared to normal subjects, peripheral blood monocytes from acne patients expressed significantly higher levels of TLR-2 and exhibited significantly greater induction of TLR-2 expression following P. acnes stimulation. Treatment of patients with isotretinoin significantly decreased monocyte TLR-2 expression and subsequent inflammatory cytokine response to P. acnes by one week of therapy. This effect was sustained six months following cessation of therapy, indicating that TLR-2 modulation may be involved in the durable therapeutic response to isotretinoin. This study demonstrates that isotretinoin exerts immunomodulatory effects in patients and sheds light on a potential mechanism for its long-term effects in acne. The modulation of TLR-2 expression on monocytes has important implications in other inflammatory disorders characterized by TLR-2 dysregulation. PMID:22513780

  9. TLR4 signaling is involved in brain vascular toxicity of PCB153 bound to nanoparticles.

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    Bei Zhang

    Full Text Available PCBs bind to environmental particles; however, potential toxicity exhibited by such complexes is not well understood. The aim of the present study is to study the hypothesis that assembling onto nanoparticles can influence the PCB153-induced brain endothelial toxicity via interaction with the toll-like receptor 4 (TLR4. To address this hypothesis, TLR4-deficient and wild type control mice (males, 10 week old were exposed to PCB153 (5 ng/g body weight bound to chemically inert silica nanoparticles (PCB153-NPs, PCB153 alone, silica nanoparticles (NPs; diameter, 20 nm, or vehicle. Selected animals were also subjected to 40 min ischemia, followed by a 24 h reperfusion. As compared to exposure to PCB153 alone, treatment with PCB153-NP potentiated the brain infarct volume in control mice. Importantly, this effect was attenuated in TLR4-deficient mice. Similarly, PCB153-NP-induced proinflammatory responses and disruption of tight junction integrity were less pronounced in TLR4-deficient mice as compared to control animals. Additional in vitro experiments revealed that TLR4 mediates toxicity of PCB153-NP via recruitment of tumor necrosis factor-associated factor 6 (TRAF6. The results of current study indicate that binding to seemingly inert nanoparticles increase cerebrovascular toxicity of PCBs and suggest that targeting the TLR4/TRAF6 signaling may protect against these effects.

  10. TLR4 signaling is involved in brain vascular toxicity of PCB153 bound to nanoparticles.

    Science.gov (United States)

    Zhang, Bei; Choi, Jeong June; Eum, Sung Yong; Daunert, Sylvia; Toborek, Michal

    2013-01-01

    PCBs bind to environmental particles; however, potential toxicity exhibited by such complexes is not well understood. The aim of the present study is to study the hypothesis that assembling onto nanoparticles can influence the PCB153-induced brain endothelial toxicity via interaction with the toll-like receptor 4 (TLR4). To address this hypothesis, TLR4-deficient and wild type control mice (males, 10 week old) were exposed to PCB153 (5 ng/g body weight) bound to chemically inert silica nanoparticles (PCB153-NPs), PCB153 alone, silica nanoparticles (NPs; diameter, 20 nm), or vehicle. Selected animals were also subjected to 40 min ischemia, followed by a 24 h reperfusion. As compared to exposure to PCB153 alone, treatment with PCB153-NP potentiated the brain infarct volume in control mice. Importantly, this effect was attenuated in TLR4-deficient mice. Similarly, PCB153-NP-induced proinflammatory responses and disruption of tight junction integrity were less pronounced in TLR4-deficient mice as compared to control animals. Additional in vitro experiments revealed that TLR4 mediates toxicity of PCB153-NP via recruitment of tumor necrosis factor-associated factor 6 (TRAF6). The results of current study indicate that binding to seemingly inert nanoparticles increase cerebrovascular toxicity of PCBs and suggest that targeting the TLR4/TRAF6 signaling may protect against these effects.

  11. TLR-dependent human mucosal epithelial cell responses to microbial pathogens.

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    Paola eMassari

    2014-08-01

    Full Text Available AbstractToll-Like Receptor (TLR signaling represents one of the best studied pathways to implement defense mechanisms against invading microbes in humans as well as in animals. TLRs respond to specific microbial ligands and to danger signals produced by the host during infection, and initiate downstream cascades that activate both innate and adaptive immunity. TLRs are expressed by professional immune cells and by the large majority of non-hematopoietic cells, including epithelial cells. In epithelial tissues, TLR functions are particularly important because these sites are constantly exposed to microorganisms, due to their location at the host interface with the environment. While at these sites, specific defense mechanisms and inflammatory responses are initiated via TLR signaling against pathogens, suppression or lack of TLR activation is also observed in response to the commensal microbiota. The mechanisms by which TLR signaling is regulated in mucosal epithelial cells include differential expression and levels of TLRs (and their signaling partners, their cellular localization and positioning within the tissue in a fashion that favors responses to pathogens while dampening responses to commensals and maintaining tissue homeostasis in physiologic conditions. In this review, the expression and activation of TLRs in mucosal epithelial cells of several sites of the human body are examined. Specifically, the oral cavity, the ear canal and eye, the airways, the gut and the reproductive tract are discussed, along with how site-specific host defense mechanisms are implemented via TLR signaling.

  12. S100A9 interaction with TLR4 promotes tumor growth.

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    Eva Källberg

    Full Text Available By breeding TRAMP mice with S100A9 knock-out (S100A9(-/- animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/- and TLR4(-/-, but not in RAGE(-/- animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b(+ cells. Lastly, treatment of mice with a small molecule (ABR-215050 that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

  13. Exacerbated Autoimmunity in the Absence of TLR9 in MRLFaslpr Mice Depends on Ifnar11

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    Nickerson, Kevin M.; Cullen, Jaime L.; Kashgarian, Michael; Shlomchik, Mark J.

    2013-01-01

    TLR9 suppresses TLR7-driven pathogenesis in the MRL.Faslpr murine model of systemic lupus erythematosus, but the mechanisms by which TLR7 promotes and TLR9 prevents disease in this and other lupus models remain unclear. Type I interferons have also been implicated in the pathogenesis of lupus both in patients and in several murine models of disease, but their role in MRL.Faslpr mice is controversial. Using MRL.Faslpr mice genetically deficient in a subunit of the receptor for type I interferon, Ifnar1, we show that type I interferons contribute significantly to renal disease in this model. Ifnar1 had no effect on anti-nucleosome or anti-Sm autoantibody titers, but instead regulated anti-cytoplasmic and anti-RNA specificities. Moreover, Ifnar1 deficiency prevented the exacerbation of clinical disease observed in Tlr9-deficient animals in this lupus model. Thus, type I interferon signaling is an important mediator of lupus pathogenesis and anti-RNA antibody production that is dysregulated in the absence of Tlr9. PMID:23467932

  14. Treatment of autoimmune inflammation by a TLR7 ligand regulating the innate immune system.

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    Tomoko Hayashi

    Full Text Available The Toll-like receptors (TLR have been advocated as attractive therapeutic targets because TLR signaling plays dual roles in initiating adaptive immune responses and perpetuating inflammation. Paradoxically, repeated stimulation of bone marrow mononuclear cells with a synthetic TLR7 ligand 9-benzyl-8-hydroxy-2-(2-methoxyethoxy adenine (called 1V136 leads to subsequent TLR hyporesponsiveness. Further studies on the mechanism of action of this pharmacologic agent demonstrated that the TLR7 ligand treatment depressed dendritic cell activation, but did not directly affect T cell function. To verify this mechanism, we utilized experimental allergic encephalitis (EAE as an in vivo T cell dependent autoimmune model. Drug treated SJL/J mice immunized with proteolipid protein (PLP(139-151 peptide had attenuated disease severity, reduced accumulation of mononuclear cells in the central nervous system (CNS, and limited demyelination, without any apparent systemic toxicity. Splenic T cells from treated mice produced less cytokines upon antigenic rechallenge. In the spinal cords of 1V136-treated EAE mice, the expression of chemoattractants was also reduced, suggesting innate immune cell hyposensitization in the CNS. Indeed, systemic 1V136 did penetrate the CNS. These experiments indicated that repeated doses of a TLR7 ligand may desensitize dendritic cells in lymphoid organs, leading to diminished T cell responses. This treatment strategy might be a new modality to treat T cell mediated autoimmune diseases.

  15. PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway

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    Dool-Ri Oh

    2014-01-01

    Full Text Available Toll-like receptor (TLR ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF-κB-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF-κB transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF-κB activation and IL-8 production in both TLR5− and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

  16. TLR4 ligation induces expression of APRIL molecule in human neutrophils — a preliminary study

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    Ewa Jabłońska

    2012-07-01

    Full Text Available In the present study we investigate the consequences of TLR4 activation by LPS for the synthesis of a proliferation-inducing ligand (APRIL by human neutrophils (PMNs, and the possible role of the ERK1/2 kinases signaling pathway. In order to make a comparison, the same examinations were carried out on autologous peripheral blood mononuclear cells (PBMCs. The levels of mRNA for APRIL and TLR4 were measured using the real-time PCR method. Western blot analysis was used to assay the expressions of APRIL and ERK1/2 in cell lysates. We discovered an increased expression of APRIL accompanying the increased expression of TLR4 in the LPS-stimulated PMNs and PBMCs. Furthermore, stimulation with LPS triggered similar changes in phospho-ERK1/2 proteins expression in those cells. The present study suggests that LPS plays a role in TLR4-ligation in APRIL induction through ERK1/2 pathway activation in human neutrophils and mononuclear cells of peripheral blood. The association between TLR4 activation and APRIL expression in examined leukocytes might have important implications for the  mmune response of the host exposed to TLR4 ligands such as LPS.

  17. Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries.

    Science.gov (United States)

    Zhao, Alyssa; Blackburn, Corinne; Chin, Judith; Srinivasan, Mythily

    2014-08-31

    Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cariogenic bacteria and modulate the disease process. The objective of this study is to determine the potential of salivary sTLR-2 and sCD14 as biomarkers of caries activity and indirect measures of the cariogenic bacterial burden. Unstimulated whole saliva was collected from twenty caries free and twenty caries active children between the ages of 5 and 13 years. The concentration of sCD14 and sTLR-2 together with that of the cytokine IL-8 reported to be increased in dental caries was assessed by the enzyme linked immunosorbent assay. While the level of sCD14 and that of IL-8 was equivocal between the two groups, the sTLR-2 concentration in caries active saliva was significantly higher than that in caries free saliva. The sTLR-2 in saliva could serve as a potential biomarker for caries activity.

  18. Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries

    Science.gov (United States)

    2014-01-01

    Background Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cariogenic bacteria and modulate the disease process. The objective of this study is to determine the potential of salivary sTLR-2 and sCD14 as biomarkers of caries activity and indirect measures of the cariogenic bacterial burden. Methods Unstimulated whole saliva was collected from twenty caries free and twenty caries active children between the ages of 5 and 13 years. The concentration of sCD14 and sTLR-2 together with that of the cytokine IL-8 reported to be increased in dental caries was assessed by the enzyme linked immunosorbent assay. Results While the level of sCD14 and that of IL-8 was equivocal between the two groups, the sTLR-2 concentration in caries active saliva was significantly higher than that in caries free saliva. Conclusions The sTLR-2 in saliva could serve as a potential biomarker for caries activity. PMID:25174416

  19. Tumor promotion by intratumoral plasmacytoid dendritic cells is reversed by TLR7 ligand treatment.

    Science.gov (United States)

    Le Mercier, Isabelle; Poujol, Dominique; Sanlaville, Amélien; Sisirak, Vanja; Gobert, Michael; Durand, Isabelle; Dubois, Bertrand; Treilleux, Isabelle; Marvel, Jacqueline; Vlach, Jaromir; Blay, Jean-Yves; Bendriss-Vermare, Nathalie; Caux, Christophe; Puisieux, Isabelle; Goutagny, Nadège

    2013-08-01

    Plasmacytoid dendritic cells (pDC) are key regulators of antiviral immunity. In previous studies, we reported that pDC-infiltrating human primary breast tumors represent an independent prognostic factor associated with poor outcome. To understand this negative impact of tumor-associated pDC (TApDC), we developed an orthotopic murine mammary tumor model that closely mimics the human pathology, including pDC and regulatory T cell (Treg) infiltration. We showed that TApDC are mostly immature and maintain their ability to internalize antigens in vivo and to activate CD4(+) T cells. Most importantly, TApDC were specifically altered for cytokine production in response to Toll-like receptor (TLR)-9 ligands in vitro while preserving unaltered response to TLR7 ligands (TLR7L). In vivo pDC depletion delayed tumor growth, showing that TApDC provide an immune-subversive environment, most likely through Treg activation, thus favoring tumor progression. However, in vivo intratumoral administration of TLR7L led to TApDC activation and displayed a potent curative effect. Depletion of pDC and type I IFN neutralization prevented TLR7L antitumoral effect. Our results establish a direct contribution of TApDC to primary breast tumor progression and rationalize the application of TLR7 ligands to restore TApDC activation in breast cancer. Cancer Res; 73(15); 4629-40. ©2013 AACR.

  20. Synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands as influenza virus vaccine adjuvants induce rapid, sustained, and broadly protective responses.

    Science.gov (United States)

    Goff, Peter H; Hayashi, Tomoko; Martínez-Gil, Luis; Corr, Maripat; Crain, Brian; Yao, Shiyin; Cottam, Howard B; Chan, Michael; Ramos, Irene; Eggink, Dirk; Heshmati, Mitra; Krammer, Florian; Messer, Karen; Pu, Minya; Fernandez-Sesma, Ana; Palese, Peter; Carson, Dennis A

    2015-03-01

    Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross-protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor 4 (TLR4) and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4-MD2 complex, and 1V270 is a phospholipid-conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2-associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous and heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that intramuscular (i.m.) administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long-lasting, and balanced Th1- and Th2-type immunity; demonstrates efficacy in a variety of murine influenza virus vaccine models assaying homologous, heterologous, and heterosubtypic challenge viruses; and has an excellent safety profile. Novel adjuvants are needed to enhance immunogenicity and increase the protective breadth of influenza virus vaccines to reduce the seasonal disease burden and ensure pandemic preparedness. We show

  1. Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.

    Science.gov (United States)

    Le Mandat Schultz, Aurelie; Bonnard, Arnaud; Barreau, Frédérick; Aigrain, Yves; Pierre-Louis, Coralie; Berrebi, Dominique; Peuchmaur, Michel

    2007-10-31

    The etiology of necrotizing enterocolitis (NEC) results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC. We specifically evaluated intestinal epithelial cell (IEC) expression of Toll-like receptor 2 (TLR-2), TLR-4, NOD2 and phosphorylated NF-kappaB (pNF-kappaB) after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1), neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2) or hand-gavaged with a protein rich formula after a hypoxia-reoxygenation procedure (group 3). Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-kappaB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia-reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-kappaB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged. These results show that this rat model of NEC induced mucosal injury, leading to a highly responsive IEC

  2. Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.

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    Aurelie Le Mandat Schultz

    Full Text Available BACKGROUND: The etiology of necrotizing enterocolitis (NEC results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC. METHODOLOGY/PRINCIPAL FINDINGS: We specifically evaluated intestinal epithelial cell (IEC expression of Toll-like receptor 2 (TLR-2, TLR-4, NOD2 and phosphorylated NF-kappaB (pNF-kappaB after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1, neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2 or hand-gavaged with a protein rich formula after a hypoxia-reoxygenation procedure (group 3. Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-kappaB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia-reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-kappaB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged. CONCLUSIONS/SIGNIFICANCE: These results show that this

  3. Expression of TLR-2, TLR-4, NOD2 and pNF-κB in a Neonatal Rat Model of Necrotizing Enterocolitis

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    Barreau, Frédérick; Aigrain, Yves; Pierre-Louis, Coralie

    2007-01-01

    Background The etiology of necrotizing enterocolitis (NEC) results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC. Methodology/Principal Findings We specifically evaluated intestinal epithelial cell (IEC) expression of Toll-like receptor 2 (TLR-2), TLR-4, NOD2 and phosphorylated NF-κB (pNF-κB) after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1), neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2) or hand-gavaged with a protein rich formula after a hypoxia–reoxygenation procedure (group 3). Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-κB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia–reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-κB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged. Conclusions/Significance These results show that this rat model of NEC induced

  4. A New Immunomodulatory Role for Peroxisomes in Macrophages Activated by the TLR4 Ligand Lipopolysaccharide.

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    Vijayan, Vijith; Srinu, Tumpara; Karnati, Srikanth; Garikapati, Vannuruswamy; Linke, Monika; Kamalyan, Lilit; Mali, Srihari Reddy; Sudan, Kritika; Kollas, Andreas; Schmid, Tobias; Schulz, Sabine; Spengler, Bernhard; Weichhart, Thomas; Immenschuh, Stephan; Baumgart-Vogt, Eveline

    2017-03-15

    Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation. Copyright © 2017 by The American Association of Immunologists, Inc.

  5. First evidence of comparative responses of Toll-like receptor 22 (TLR22) to relatively resistant and susceptible Indian farmed carps to Argulus siamensis infection.

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    Panda, Rudra Prasanna; Chakrapani, V; Patra, Swagat Kumar; Saha, Jatindra Nath; Jayasankar, Pallipuram; Kar, Banya; Sahoo, Pramoda Kumar; Barman, Hirak Kumar

    2014-11-01

    Toll-like receptor 22 (TLR22) is present in teleost but not in mammals. Among Indian farmed carps, Catla catla is relatively more resistant than Labeo rohita to Argulus siamensis lice infection. TLR22 is believed to be associated with innate immunity against ectoparasite infection. To investigate the TLR22 mediated immunity against argulosis, we have cloned and characterized TLR22 genes of L. rohita (rTLR22) and C. catla (cTLR22). The full-length cDNAs of rTLR22 and cTLR22 contained an open reading frame of 2838 and 2841 nucleotides, respectively; bearing the typical structural features. Phylogenetically rTLR22/cTLR22 was most closely related to Cyprinus carpio (common carp) counterpart, having highest sequence identity of 86.0%. The TIR domain remained highly conserved with 90% identity within freshwater fishes. The sequence information of cDNA and genomic DNA together revealed that the rTLR22/cTLR22 genes are encoded by uninterrupted exons. The co-habitation challenge study with A. siamensis infection confirmed that C. catla is comparatively more resistant than L. rohita. Further, comparative mRNA expression profile in immune relevant tissues also suggested about the participatory role of TLR22 during lice infection. However, TLR22 might not solely be involved in conferring relative resistance among carp species against argulosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Intrinsic renal cell and leukocyte-derived TLR4 aggravate experimental anti-MPO glomerulonephritis

    NARCIS (Netherlands)

    Summers, Shaun A.; van der Veen, Betty S.; O'Sullivan, Kim M.; Gan, Poh-Yi; Ooi, Joshua D.; Heeringa, Peter; Satchell, Simon C.; Mathieson, Peter W.; Saleem, Moin A.; Visvanathan, Kumar; Holdsworth, Stephen R.; Kitching, A. Richard

    2010-01-01

    Antimyeloperoxidase antibodies can cause crescentic glomerulonephritis and pulmonary hemorrhage. Toll-like receptors (TLRs) respond to infectious agents activating host defenses, whereas infections potentially initiate disease and provoke relapses. Neutrophils were found to be key effector cells of

  7. The role of CNS TLR2 activation in mediating innate versus adaptive neuroinflammation.

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    Luz, Avital; Fainstein, Nina; Einstein, Ofira; Ben-Hur, Tamir

    2015-11-01

    Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates both innate and adaptive immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis (EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and myelination. We investigated the roles of CNS TLR2 activation in mediating neuro-inflammatory responses in intact versus EAE animals. We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymosan, a TLR2 agonist, on naive versus EAE animals. The neuro-inflammatory response was characterized by immune-fluorescent staining for IBA-1+ microglia/macrophages and CD3+ T cells, and by semi-quantitative real time PCR for TLR2 and immune cytokines. The nature of the immune cells isolated from EAE brain tissue was assessed by their proliferative response to the PLP peptide autoantigen. Survival and clinical scores were monitored; demyelination and axonal loss were quantified by Gold-Black and Bielschowsky stains. Our findings showed that Zymosan injection in naïve mice induced a massive neuro-inflammatory response without any clinical manifestations. In EAE mice, ICV Zymosan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease was not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymosan injection. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide suggested that TLR2 activation induces innate but not adaptive immune response. We conclude that EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. As CNS TLR2 activation

  8. Increased expression of TLR9 associated with pro-inflammatory S100A8 and IL-8 in diabetic wounds could lead to unresolved inflammation in type 2 diabetes mellitus (T2DM) cases with impaired wound healing.

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    Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Sinha, Pratima; Singh, Kiran

    2016-01-01

    Type 2 diabetes mellitus (T2DM) is characterized by persistent hyperglycemia which causes a chain of abrupt biochemical and physiological changes. Immune dys-regulation is the hallmark of T2DM that could contribute to prolonged inflammation causing transformation of wounds into non-healing chronic ulcers. Toll like receptor -9 (TLR9) is a major receptor involved in innate immune regulation. TLR9 activation induces release of pro-inflammatory molecules like S100A8 and interleukin-8 (IL-8) by myeloid cells causing migration of myeloid cells to the site of inflammation. We hypothesized that pro-inflammatory S100A8 and IL-8 proteins could cause persistent inflammation in chronic wounds like diabetic foot ulcer (DFU) and may contribute to impaired wound healing in T2DM patients. Expression of TLR9 and its downstream effector molecules S100A8, and IL-8 were analyzed in chronic diabetic wound and non-diabetic control wound tissue samples by semiquantitative reverse transcriptase - polymerase chain reaction (RT-PCR), quantitative RT-PCR, western blot and immunofluorescence. CD11b(+)CD33(+) myeloid cells were analyzed by flow cytometry. TLR9 message and protein were higher in diabetic wounds compared to control wounds (p=0.03, t=2.21 for TLR9 mRNA; p=21 for TLR9 protein). TLR9 down-stream effector molecules S100A8 and IL-8 were also increased in diabetic wounds (p=0.003, t=3.1 for S100A8 mRNA; p=0.04, t=2.04 for IL-8). CD11b(+) CD33(+) myeloid cells were decreased in T2DM as compared to non-diabetic controls (p=0.001, t=3.6). DFU subjects had higher levels of CD11b(+) CD33(+) myeloid cells as compared to non-DFU T2DM control (p=0.003, t=2.8). Infection in the wound microenvironment could be the cause of increase in CD11b(+)CD33(+) myeloid cells in DFU (p=0.03, t=2.5). The up-regulation of myeloid cell-derived pro-inflammatory molecules S100A8 and IL-8 in combination with lower levels of CD11b(+) CD33(+) myeloid cells may cause the impairment of wound healing in T2DM

  9. Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages

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    Elisa Araldi

    2017-06-01

    Full Text Available Macrophages perform critical functions in both innate immunity and cholesterol metabolism. Here, we report that activation of Toll-like receptor 4 (TLR4 in macrophages causes lanosterol, the first sterol intermediate in the cholesterol biosynthetic pathway, to accumulate. This effect is due to type I interferon (IFN-dependent histone deacetylase 1 (HDAC1 transcriptional repression of lanosterol-14α-demethylase, the gene product of Cyp51A1. Lanosterol accumulation in macrophages, because of either treatment with ketoconazole or induced conditional disruption of Cyp51A1 in mouse macrophages in vitro, decreases IFNβ-mediated signal transducer and activator of transcription (STAT1-STAT2 activation and IFNβ-stimulated gene expression. These effects translate into increased survival to endotoxemic shock by reducing cytokine secretion. In addition, lanosterol accumulation increases membrane fluidity and ROS production, thus potentiating phagocytosis and the ability to kill bacteria. This improves resistance of mice to Listeria monocytogenes infection by increasing bacterial clearance in the spleen and liver. Overall, our data indicate that lanosterol is an endogenous selective regulator of macrophage immunity.

  10. HIV-1 Structural Proteins Serve as PAMPs for TLR2 Heterodimers Significantly Increasing Infection and Innate Immune Activation.

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    Henrick, Bethany M; Yao, Xiao-Dan; Rosenthal, Kenneth Lee

    2015-01-01

    Immune activation is critical to HIV infection and pathogenesis; however, our understanding of HIV innate immune activation remains incomplete. Recently we demonstrated that soluble TLR2 (sTLR2) physically inhibited HIV-induced NFκB activation and inflammation, as well as HIV-1 infection. In light of these findings, we hypothesized that HIV-1 structural proteins may serve as pathogen-associated molecular patterns (PAMPs) for cellular TLR2 heterodimers. These studies made use of primary human T cells and TZMbl cells stably transformed to express TLR2 (TZMbl-2). Our results demonstrated that cells expressing TLR2 showed significantly increased proviral DNA compared to cells lacking TLR2, and mechanistically this may be due to a TLR2-mediated increased CCR5 expression. Importantly, we show that HIV-1 structural proteins, p17, p24, and gp41, act as viral PAMPs signaling through TLR2 and its heterodimers leading to significantly increased immune activation via the NFκB signaling pathway. Using co-immunoprecipitation and a dot blot method, we demonstrated direct protein interactions between these viral PAMPs and TLR2, while only p17 and gp41 bound to TLR1. Specifically, TLR2/1 heterodimer recognized p17 and gp41, while p24 lead to immune activation through TLR2/6. These results were confirmed using TLR2/1 siRNA knock down assays which ablated p17 and gp41-induced cellular activation and through studies of HEK293 cells expressing selected TLRs. Interestingly, our results show in the absence of TLR6, p24 bound to TLR2 and blocked p17 and gp41-induced activation, thus providing a novel mechanism by which HIV-1 can manipulate innate sensing. Taken together, our results identified, for the first time, novel HIV-1 PAMPs that play a role in TLR2-mediated cellular activation and increased proviral DNA. These findings have important implications for our fundamental understanding of HIV-1 immune activation and pathogenesis, as well as HIV-1 vaccine development.

  11. Molecular cloning of orange-spotted grouper (Epinephelus coioides) TLR21 and expression analysis post Cryptocaryon irritans infection.

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    Li, Yan-Wei; Luo, Xiao-Chun; Dan, Xue-Ming; Qiao, Wei; Huang, Xia-Zi; Li, An-Xing

    2012-03-01

    TLR21, a non-mammalian Toll like receptor, was recently identified in chicken as a pattern recognition receptor of unmethyl-CpG ODN, functionally similar to that of mammalian TLR9. Its role in fish immune defense and whether it is involved in anti-parasite immunity has not yet been proven. In this study, we identified a cDNA sequence encoding orange-spotted grouper Toll-like receptor 21 (EcTLR21), the open reading frame (ORF) was 2937 bp encoding a putative polypeptide of 979 amino acid residues. Some conserved motifs in mammalian TLR9 were also conserved in grouper and other fish species' TLR9 and TLR21. Tissue distribution analysis indicated that EcTLR21 is broadly expressed in all the tissue we tested except muscle. High expression levels were found in the head kidney, trunk kidney, spleen and heart. Post Cryptocaryon irritans infection, TLR21 and TLR9 transcripts were induced at the local infection sites (skin and gill), while suppressed in systemic immune organs (spleen and head kidney), indicating that these two receptors may play a role in host anti-parasitic immune responses. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis.

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    Reithmeier-Rost, Dagmar; Hill, Jim; Elvin, Stephen J; Williamson, Diane; Dittmann, Svea; Schmid, Annika; Wilharm, Gottfried; Sing, Andreas

    2007-07-01

    Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.

  13. Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

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    Adeline M Hajjar

    Full Text Available Although lipopolysaccharide (LPS stimulation through the Toll-like receptor (TLR-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

  14. Hepatic TLR4 signaling is activated by LPS from digestive tract during SARA, and epigenetic mechanisms contribute to enforced TLR4 expression

    Science.gov (United States)

    Chang, Guangjun; Zhuang, Su; Seyfert, Hans-Martin; Zhang, Kai; Xu, Tianle; Jin, Di; Guo, Junfei; Shen, Xiangzhen

    2015-01-01

    Subacute ruminal acidosis (SARA) is known to trigger a systemic inflammatory response that is possibly caused by the translocationof lipopolysaccharides (LPS) from the gastrointestinal tract into the bloodstream. The aim of this study is to investigate this causal relationship between the increases of circulating LPS and liver inflammation. Here we found that SARA goats exhibited significantly increased LPS concentrations in both the rumen and portal vein. The livers of these goats exhibited increased mRNA concentrations of pro-inflammatory genes that indicated inflammation. Meanwhile, the occurrence of liver inflammation was further validated by the enhanced protein expression of those cytokines in the livers of SARA goats. These increased expressions of detected pro-inflammatory genes were likely mediated by enforced TLR4 signaling because SARA increased the concentrations of TLR4 mRNA and protein in the liver and the abundance of both the NF-kB-p65 factor and its active phosphorylated variant. We also verified that the enhanced TLR4 expression was accompanied by chromatin decompaction and demethylation of the proximal TLR4 promoter. Hence, epigenetic mechanisms are involved in the enforced expression of immune genes during SARA, and these findings open innovative routes for interventions via the modulation of these epigenetic mechanisms. PMID:26498350

  15. Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.

    Science.gov (United States)

    Li, Di; Liu, Yao; Yang, Ya; Chen, Jian-hong; Yang, Jie; Zou, Lin-yun; Tian, Zhi-qiang; Lv, Jun; Xia, Pei-yuan

    2013-06-15

    The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and β-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and β-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.

  16. Higher activation of TLR9 in plasmacytoid dendritic cells by microbial DNA compared with self-DNA based on CpG-specific recognition of phosphodiester DNA.

    Science.gov (United States)

    Coch, Christoph; Busch, Nicolas; Wimmenauer, Vera; Hartmann, Evelyn; Janke, Markus; Abdel-Mottaleb, Mona Mohamed Ahmed; Lamprecht, Alf; Ludwig, Janos; Barchet, Winfried; Schlee, Martin; Hartmann, Gunther

    2009-09-01

    TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.

  17. Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

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    Nives Hörmann

    Full Text Available The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs. Here, we report that colonization of germ-free (GF Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2 and protein-kinase B (AKT induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

  18. Cutting edge: natural DNA repetitive extragenic sequences from gram-negative pathogens strongly stimulate TLR9.

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    Magnusson, Mattias; Tobes, Raquel; Sancho, Jaime; Pareja, Eduardo

    2007-07-01

    Bacterial DNA exerts immunostimulatory effects on mammalian cells via the intracellular TLR9. Although broad analysis of TLR9-mediated immunostimulatory potential of synthetic oligonucleotides has been developed, which kinds of natural bacterial DNA sequences are responsible for immunostimulation are not known. This work provides evidence that the natural DNA sequences named repetitive extragenic palindromic (REPs) sequences present in Gram-negative bacteria are able to produce innate immune system stimulation via TLR9. A strong induction of IFN-alpha production by REPs from Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Neisseria meningitidis was detected in splenocytes from 129 mice. In addition, the involvement of TLR9 in immune stimulation by REPs was confirmed using B6.129P2-Tlr9(tm1Aki) knockout mice. Considering the involvement of TLRs in Gram-negative septic shock, it is conceivable that REPs play a role in its pathogenesis. This study highlights REPs as a potential novel target in septic shock treatment.

  19. Allelic Variants of NRF2 and TLR9 Genes in Critical Illness

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    A. G. Chumachenko

    2016-01-01

    Full Text Available Aim of the study. To elucidate the association of allelic variants of single nucleotide polymorphism in NRF2 (rs6726395, 177238501A>G and TLR9 (rs352162, 52218953T>C genes, each gene separately and in their combination, with peculiarities of the course of critical conditions during lung infection. Materials and methods. DNA from 86 post#operative patients and oncologic patients was genotyped in an allelespecific fashion using tetra#primer polymerase chain reaction followed by gel electrophoresis analysis of products.Results. It has been found that septic shock patients with NRF2 177238501A>G GG genotype had increased mortality and higher APACHE II score and developed non#responsive edema more frequently. Patients with NRF2 177238501A>G GG/TLR9 52218953T>C CC genotype combination developed septic shock and nosocomialpneumonia more rarely.Conclusion. The homozygous NRF2 177238501A>G (GG allele combination is unfavorable for the course and outcome of critical conditions only in combination with TLR9 52218953T>C СT or TLR9 52218953T>C TT alleles in septic shock patients. At the same time, the combination of TLR9 52218953T>C СС alleles in the same patients with 'unfavorable' NRF2 177238501A>G GG protects against development of septic shock and nosocomial pneumonia.

  20. TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis

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    Nikolay N. Kuzmich

    2017-10-01

    Full Text Available Toll-Like Receptor 4 (TLR4 signal pathway plays an important role in initiating the innate immune response and its activation by bacterial endotoxin is responsible for chronic and acute inflammatory disorders that are becoming more and more frequent in developed countries. Modulation of the TLR4 pathway is a potential strategy to specifically target these pathologies. Among the diseases caused by TLR4 abnormal activation by bacterial endotoxin, sepsis is the most dangerous one because it is a life-threatening acute system inflammatory condition that still lacks specific pharmacological treatment. Here, we review molecules at a preclinical or clinical phase of development, that are active in inhibiting the TLR4-MyD88 and TLR4-TRIF pathways in animal models. These are low-molecular weight compounds of natural and synthetic origin that can be considered leads for drug development. The results of in vivo studies in the sepsis model and the mechanisms of action of drug leads are presented and critically discussed, evidencing the differences in treatment results from rodents to humans.

  1. TLR2-induced IL-10 production impairs neutrophil recruitment to infected tissues during neonatal bacterial sepsis.

    Science.gov (United States)

    Andrade, Elva B; Alves, Joana; Madureira, Pedro; Oliveira, Liliana; Ribeiro, Adília; Cordeiro-da-Silva, Anabela; Correia-Neves, Margarida; Trieu-Cuot, Patrick; Ferreira, Paula

    2013-11-01

    Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti-IL-10R mAb treatment or by using IL-10-deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis.

  2. Universal vaccine against influenza virus: linking TLR signaling to anti-viral protection.

    Science.gov (United States)

    Schmitz, Nicole; Beerli, Roger R; Bauer, Monika; Jegerlehner, Andrea; Dietmeier, Klaus; Maudrich, Melanie; Pumpens, Paul; Saudan, Philippe; Bachmann, Martin F

    2012-04-01

    A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis.

    Directory of Open Access Journals (Sweden)

    T Eoin West

    2014-09-01

    Full Text Available Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice and type III secretion system components (in mice and humans. In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4(-/- mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.

  4. Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation

    Science.gov (United States)

    Schmidt, Nathan W.; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Lee, Calvin; Frasca, Loredana; Frenkel, Daan; Dobnikar, Jure; Gilliet, Michel; Wong, Gerard C. L.

    2015-07-01

    Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs , , , , ). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

  5. Activation of TLR4 signaling promotes gastric cancer progression by inducing mitochondrial ROS production.

    Science.gov (United States)

    Yuan, X; Zhou, Y; Wang, W; Li, J; Xie, G; Zhao, Y; Xu, D; Shen, L

    2013-09-12

    Chronic infection, such as Helicobacter pylori infection, has been associated with the development of gastric cancer (GC). Pathogen-associated molecular patterns can trigger inflammatory responses via Toll-like receptors (TLRs) in GC. Here we showed that Toll-like receptor 4 (TLR4) was highly expressed in GC cells and was associated with the aggressiveness of GC. The binding of lipopolysaccharide (LPS) to TLR4 on GC cells enhanced proliferation without affecting apoptosis. Higher level of reactive oxygen species (ROS) was induced after activation of TLR4 signaling in GC. Using oxidase inhibitors and antioxidants, we found that mitochondrial ROS (mROS) was major source of TLR4-stimulated ROS generation. This elevated mROS production can be inhibited by diphenylene iodonium (DPI), and the blocking of the mROS production rather than ROS neutralization resulted in cell cycle arrest and the loss of mitochondrial potential, which were plausible reason for decreased cell viability. Furthermore, the increased mROS owing to TLR4 signaling resulted in the activation of Akt phosphorylation and NF-κB p65 nuclear translocation. Altogether, these results reveal a novel pathway linking innate immune signaling to GC cell proliferation, implicate mROS as an important component of cell survival signals and further establish mitochondria as hubs for GC therapies.

  6. Relationship of intestinal flora disorder with TLR/NK-kB and inflammatory mediator expression in patients with ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Ma Tao

    2017-07-01

    Full Text Available Objective: To study the relationship of intestinal flora disorder with TLR/NK-kB and inflammatory mediator expression in patients with ulcerative colitis (UC. Methods: Patients who were diagnosed with UC in Yan’an People’s Hospital between May 2015 and February 2017 were selected, patients with active UC and patients with remission UC were included in the AUC and RUC group; patients who were diagnosed with colonic polyps in Yan’an People’s Hospital through physical examination during the same period were selected as the control group. Feces was collected to test the flora number, mucosa lesion was collected to determine the expression of TLR/NK-kB and inflammatory mediators, and serum was collected to detect the levels of inflammatory mediators. Results: Bifidobacterium, lactobacillus acidophilus and bacteroides number in feces of AUC group and RUC group were significantly lower than those of control group while escherichia coli and enterococcus number in feces, TLR2, TLR4, TLR5, TLR9, NK-kB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were significantly higher than those of control group; bifidobacterium, lactobacillus acidophilus and bacteroides number in feces of AUC group were significantly lower than those of RUC group while escherichia coli and enterococcus number in feces, TLR2, TLR4, TLR5, TLR9, NKkB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were significantly higher than those of RUC group; TLR2, TLR4, TLR5, TLR9, NK-kB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were negatively correlated with bifidobacteria, lactobacillus acidophilus and bacteroides number, and positively correlated with escherichia coli and enterococcus number. Conclusion: The intestinal flora

  7. TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Song Min

    2011-08-01

    Full Text Available Abstract Background Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD, are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs, a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start. Methods Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. Results While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg was less than that in a TLR4 wild-type AD model (TLR4W Tg. At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes. Conclusion This is the first demonstration of TLR4

  8. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling.

    Science.gov (United States)

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.

  9. TLR3 is required for survival following Coxsackievirus B3 infection by driving T lymphocyte activation and polarization: The role of dendritic cells

    National Research Council Canada - National Science Library

    Renata Sesti-Costa; Marcela Cristina Santiago Françozo; Grace Kelly Silva; José Luiz Proenca-Modena; João Santana Silva

    2017-01-01

    .... In the current study, we found that TLR3 expression in dendritic cells plays a role in their activation upon CVB3 infection in vitro, as TLR3-deficient dendritic cells up-regulate CD80 and CD86...

  10. TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro.

    Science.gov (United States)

    Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

    2014-09-01

    Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4(+) T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2(-/-) and TLR4(-/-) mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2(-/-) and TLR4(-/-) mice. In addition, CD4(+) T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4(+) T cells from TLR2(-/-) and TLR4(-/-) mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2(-/-) and TLR4(-/-) mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2(-/-) and TLR4(-/-) mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.

  11. The helminth Trichuris suis suppresses TLR4-induced inflammatory responses in human macrophages

    DEFF Research Database (Denmark)

    Ottow, M. K.; Klaver, E. J.; van der Pouw Kraan, T. C. T. M.

    2014-01-01

    include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM......-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM......-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation...

  12. TLR9 agonists: immune mechanisms and therapeutic potential in domestic animals.

    Science.gov (United States)

    Mutwiri, George

    2012-07-15

    Toll like receptors (TLRs) are transmembrane glycoproteins that recognize conserved microbial molecules. Engagement of TLRs activates innate and adaptive immunity. TLR-mediated activation of immune cells results in upregulation of cytokines, chemokines and costimulatory molecules. These early innate responses control pathogen spread and initiates adaptive immune responses. Synthetic CpG oligodeoxynucleotides (ODN), agonists for TLR9, had shown great promise as immunotherapeutic agents and vaccine adjuvants in laboratory animal models of infectious disease, allergy and cancer. However, it has become apparent that CpG ODN are less potent immune activators in domestic animals and humans. The disparity in immune responses between rodents and mammals has been mainly attributed to differences in cellular expression of TLR9 in the various species. In this article, our current understanding of the immune mechanisms, as well as the potential applications of CpG ODN will be reviewed, with particular emphasis on domestic animals. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. DMPD: Transcriptional signaling by double-stranded RNA: role of TLR3. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15733829 Transcriptional signaling by double-stranded RNA: role of TLR3. Sen GC, Sa...rkar SN. Cytokine Growth Factor Rev. 2005 Feb;16(1):1-14. (.png) (.svg) (.html) (.csml) Show Transcriptional... signaling by double-stranded RNA: role of TLR3. PubmedID 15733829 Title Transcriptional signaling by double

  14. Respiratory virus-induced TLR7 activation controls IL-17-associated increased mucus via IL-23 regulation

    NARCIS (Netherlands)

    Lukacs, Nicholas W; Smit, Joost J; Mukherjee, Sumanta; Morris, Susan B; Nunez, Gabriel; Lindell, Dennis M

    2010-01-01

    The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen-associated targets. In the current study, the role of TLR7 that recognizes ssRNA was examined. Using TLR7(-/-) mice, we found that the response to RSV infection in

  15. Inducible Lentivirus-Mediated siRNA against TLR4 Reduces Nociception in a Rat Model of Bone Cancer Pain

    Directory of Open Access Journals (Sweden)

    Ruirui Pan

    2015-01-01

    Full Text Available Although bone cancer pain is still not fully understood by scientists and clinicians alike, studies suggest that toll like receptor 4 (TLR4 plays an important role in the initiation and/or maintenance of pathological pain state in bone cancer pain. A promising treatment for bone cancer pain is the downregulation of TLR4 by RNA interference; however, naked siRNA (small interference RNA is not effective in long-term treatments. In order to concoct a viable prolonged treatment for bone cancer pain, an inducible lentivirus LvOn-siTLR4 (tetracycline inducible lentivirus carrying siRNA targeting TLR4 was prepared and the antinociception effects were observed in bone cancer pain rats induced by Walker 256 cells injection in left leg. Results showed that LvOn-siTLR4 intrathecal injection with doxycycline (Dox oral administration effectively reduced the nociception induced by Walker 256 cells while inhibiting the mRNA and protein expression of TLR4. Proinflammatory cytokines as TNF-α and IL-1β in spinal cord were also decreased. These findings suggest that TLR4 could be a target for bone cancer pain treatment and tetracycline inducible lentivirus LvOn-siTLR4 represents a new potential option for long-term treatment of bone cancer pain.

  16. DMPD: Pellino proteins: novel players in TLR and IL-1R signalling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17635639 Pellino proteins: novel players in TLR and IL-1R signalling. Schauvliege R..., Janssens S, Beyaert R. J Cell Mol Med. 2007 May-Jun;11(3):453-61. (.png) (.svg) (.html) (.csml) Show Pellino proteins...: novel players in TLR and IL-1R signalling. PubmedID 17635639 Title Pellino proteins: novel play

  17. Influence of age, past smoking, and disease severity on TLR2, neutrophilic inflammation, and MMP-9 levels in COPD.

    Science.gov (United States)

    Simpson, Jodie L; McDonald, Vanessa M; Baines, Katherine J; Oreo, Kevin M; Wang, Fang; Hansbro, Philip M; Gibson, Peter G

    2013-01-01

    Chronic obstructive pulmonary disease (COPD) is a common and serious respiratory disease, particularly in older individuals, characterised by fixed airway obstruction and persistent airway neutrophilia. The mechanisms that lead to these features are not well established. We investigated the contribution of age, prior smoking, and fixed airflow obstruction on sputum neutrophils, TLR2 expression, and markers of neutrophilic inflammation. Induced sputum from adults with COPD (n = 69) and healthy controls (n = 51) was examined. A sputum portion was dispersed, total, differential cell count and viability recorded, and supernatant assayed for CXCL8, matrix metalloproteinase- (MMP-) 9, neutrophil elastase, and soluble TLR2. Peripheral blood cells (n = 7) were stimulated and TLR2 activation examined. TLR2 levels were increased with ageing, while sputum neutrophils and total sputum MMP-9 levels increased with age, previous smoking, and COPD. In multivariate regression, TLR2 gene expression and MMP-9 levels were significant independent contributors to the proportion of sputum neutrophils after adjustment for age, prior smoking, and the presence of airflow obstruction. TLR2 stimulation led to enhanced release of MMP-9 from peripheral blood granulocytes. TLR2 stimulation activates neutrophils for MMP-9 release. Efforts to understand the mechanisms of TLR2 signalling and subsequent MMP-9 production in COPD may assist in understanding neutrophilic inflammation in COPD.

  18. Molecular modeling-based evaluation of hTLR10 and identification of potential ligands in Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Rajiv Gandhi Govindaraj

    Full Text Available Toll-like receptors (TLRs are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (hTLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D structure of hTLR10 and refined the model through molecular dynamics (MD simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1. The docked complexes were then used for interaction with ligands (Pam(3CSK(4 and PamCysPamSK(4 using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam(3CSK(4 might be the ligand for the hTLR10/2 complex and PamCysPamSK(4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.

  19. Ozone therapy ameliorates tubulointerstitial inflammation by regulating TLR4 in adenine-induced CKD rats.

    Science.gov (United States)

    Chen, Zhiyuan; Liu, Xiuheng; Yu, Gang; Chen, Hui; Wang, Lei; Wang, Zhishun; Qiu, Tao; Weng, Xiaodong

    2016-06-01

    Tubulointerstitium inflammation is a common pathway aggravating chronic kidney disease (CKD) progression and the mechanism is partly associated with excessive activation of toll-like receptor 4 (TLR4) in tubulointerstitium. Ozone therapy is demonstrated to alleviate inflammation in some experiments. The aim of this study is to examine whether ozone therapy could ameliorate chronic tubulointerstitium inflammation by suppressing TLR4 in adenine-induced CKD rats. Sprague-Dawley rats were fed with 0.75% adenine-containing diet to induce CKD and tubulointerstitium inflammation injury. Ozone therapy (1.1 mg/kg) was simultaneously administrated by rectal insufflations (i.r.). After 4 weeks, serum and kidney samples were collected for detection. Renal function and systemic electrolyte were detected. Renal pathological changes were assessed by hematoxylin-eosin (H&E) staining and Masson trichrome (MT) staining. Immunohistochemistry, Western blot and Real-time PCR were applied to evaluate tubulointerstitium inflammation as well as the expression of TLR4 and phosphorylated nuclear factor kappa B P65 (p-NF-κB P65) in rats. The results showed ozone therapy improved serious renal insufficiency, systemic electrolyte disorder and tubulointerstitium morphology damages in adenine-induced CKD rats. In addition, ozone therapy suppressed excessive activation of TLR4 and p-NF-κB P65 in the tubulointerstitium of adenine-induced CKD rats, accompanied by the reduction of inflammation-related cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). The protein expression of TLR4 was positively correlated with the protein expression levels of MCP-1 (r = 0.7863, p ozone therapy could attenuate tubulointerstitium inflammation injury in adenine-induced CKD rats and the mechanism might associate with the mediation of TLR4.

  20. S100A9 Interaction with TLR4 Promotes Tumor Growth

    Science.gov (United States)

    Källberg, Eva; Vogl, Thomas; Liberg, David; Olsson, Anders; Björk, Per; Wikström, Pernilla; Bergh, Anders; Roth, Johannes; Ivars, Fredrik; Leanderson, Tomas

    2012-01-01

    By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies. PMID:22470535

  1. TLR4 Signaling in MPP+-Induced Activation of BV-2 Cells

    Directory of Open Access Journals (Sweden)

    Peng Zhou

    2016-01-01

    Full Text Available Aims. This work was conducted to establish an in vitro Parkinson’s disease (PD model by exposing BV-2 cells to 1-methyl-4-phenylpyridinium (MPP+ and exploring the roles of TLR2/TLR4/TLR9 in inflammatory responses to MPP+. Methods/Results. MTT assay showed that cell viability of BV-2 cells was 84.78 ± 0.86% and 81.18 ± 0.99% of the control after incubation with 0.1 mM MPP+ for 12 hours and 24 hours, respectively. Viability was not significantly different from the control group. With immunofluorescence technique, we found that MPP+ incubation at 0.1 mM for 12 hours was the best condition to activate BV-2 cells. In this condition, the levels of TNF-α, IL-1β, and iNOS protein were statistically increased compared to the control according to ELISA tests. Real time RT-PCR and western blot measurements showed that TLR4 was statistically increased after 0.1 mM MPP+ incubation for 12 hours. Furthermore, after siRNA interference of TLR4 mRNA, NF-κB activation and the levels of TNF-α, IL-1β, and iNOS were all statistically decreased in this cell model. Conclusion. MPP+ incubation at the concentration of 0.1 mM for 12 hours is the best condition to activate BV-2 cells for mimicking PD inflammation in BV-2 cells. TLR4 signalling plays a critical role in the activation of BV-2 cells and the induction of inflammation in this cell model.

  2. Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage.

    Science.gov (United States)

    Pascual, María; Baliño, Pablo; Alfonso-Loeches, Silvia; Aragón, Carlos M G; Guerri, Consuelo

    2011-06-01

    Toll-like receptors (TLRs) play an important role in the innate immune response, and emerging evidence indicates their role in brain injury and neurodegeneration. Our recent results have demonstrated that ethanol is capable of activating glial TLR4 receptors and that the elimination of these receptors in mice protects against ethanol-induced glial activation, induction of inflammatory mediators and apoptosis. This study was designed to assess whether ethanol-induced inflammatory damage causes behavioral and cognitive consequences, and if behavioral alterations are dependent of TLR4 functions. Here we show in mice drinking alcohol for 5months, followed by a 15-day withdrawal period, that activation of the astroglial and microglial cells in frontal cortex and striatum is maintained and that these events are associated with cognitive and anxiety-related behavioral impairments in wild-type (WT) mice, as demonstrated by testing the animals with object memory recognition, conditioned taste aversion and dark and light box anxiety tasks. Mice lacking TLR4 receptors are protected against ethanol-induced inflammatory damage, and behavioral associated effects. We further assess the possibility of the epigenetic modifications participating in short- or long-term behavioral effects associated with neuroinflammatory damage. We show that chronic alcohol treatment decreases H4 histone acetylation and histone acetyltransferases activity in frontal cortex, striatum and hippocampus of WT mice. Alterations in chromatin structure were not observed in TLR4(-/-) mice. These results provide the first evidence of the role that TLR4 functions play in the behavioral consequences of alcohol-induced inflammatory damage and suggest that the epigenetic modifications mediated by TLR4 could contribute to short- or long-term alcohol-induced behavioral or cognitive dysfunctions. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response.

    Science.gov (United States)

    Aldinucci, Alessandra; Bonechi, Elena; Manuelli, Cinzia; Nosi, Daniele; Masini, Emanuela; Passani, Maria Beatrice; Ballerini, Clara

    2016-07-08

    Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1-4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Histamine Regulates Actin Cytoskeleton in Human Toll-like Receptor 4-activated Monocyte-derived Dendritic Cells Tuning CD4+ T Lymphocyte Response*

    Science.gov (United States)

    Bonechi, Elena; Manuelli, Cinzia

    2016-01-01

    Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1–4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance. PMID:27226579

  5. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function.

    Science.gov (United States)

    Krug, Anne; French, Anthony R; Barchet, Winfried; Fischer, Jens A A; Dzionek, Andrzej; Pingel, Jeanette T; Orihuela, Michael M; Akira, Shizuo; Yokoyama, Wayne M; Colonna, Marco

    2004-07-01

    Natural interferon-producing cells (IPC) respond to viruses by secreting type I interferon (IFN) and interleukin-12 (IL-12). Toll-like receptor (TLR) 9 mediates IPC recognition of some of these viruses in vitro. However, whether TLR9-induced activation of IPC is necessary for an effective antiviral response in vivo is not clear. Here, we demonstrate that IPC and dendritic cells (DC) recognize murine cytomegalovirus (MCMV) through TLR9. TLR9-mediated cytokine secretion promotes viral clearance by NK cells that express the MCMV-specific receptor Ly49H. Although depletion of IPC leads to a drastic reduction of the IFN-alpha response, this allows other cell types to secrete IL-12, ensuring normal IFN-gamma and NK cell responses to MCMV. We conclude that the TLR9/MyD88 pathway mediates antiviral cytokine responses by IPC, DC, and possibly other cell types, which are coordinated to promote effective NK cell function and MCMV clearance.

  6. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  7. Prothymosin-alpha preconditioning activates TLR4-TRIF signaling to induce protection of ischemic retina.

    Science.gov (United States)

    Halder, Sebok Kumar; Matsunaga, Hayato; Ishii, Ken J; Ueda, Hiroshi

    2015-12-01

    Prothymosin-alpha protects the brain and retina from ischemic damage. Although prothymosin-alpha contributes to toll-like receptor (TLR4)-mediated immnunopotentiation against viral infection, the beneficial effects of prothymosin-alpha-TLR4 signaling in protecting against ischemia remain to be elucidated. In this study, intravitreal administration of prothymosin-alpha 48 h before induction of retinal ischemia prevented retinal cellular damage as evaluated by histology, and retinal functional deficits as evaluated by electroretinography. Prothymosin-alpha preconditioning completely prevented the ischemia-induced loss of ganglion cells with partial survival of bipolar and photoreceptor cells, but not amacrine cells, in immunohistochemistry experiments. Prothymosin-alpha treatment in the absence of ischemia caused mild activation, proliferation, and migration of retinal microglia, whereas the ischemia-induced microglial activation was inhibited by prothymosin-alpha preconditioning. All these preventive effects of prothymosin-alpha preconditioning were abolished in TLR4 knock-out mice and by pre-treatments with anti-TLR4 antibodies or minocycline, a microglial inhibitor. Prothymosin-alpha preconditioning inhibited the retinal ischemia-induced up-regulation of TLR4-related injury genes, and increased expression of TLR4-related protective genes. Furthermore, the prothymosin-alpha preconditioning-induced prevention of retinal ischemic damage was abolished in TIR-domain-containing adapter-inducing interferon-β knock-out mice, but not in myeloid differentiation primary response gene 88 knock-out mice. Taken together, the results of this study suggest that prothymosin-alpha preconditioning selectively drives TLR4-TIR-domain-containing adapter-inducing interferon-β signaling and microglia in the prevention of retinal ischemic damage. We propose the following mechanism for prothymosin-alpha (ProTα) preconditioning-induced retinal prevention against ischemia: Pro

  8. DMPD: Regulation of TLR4 signaling and the host interface with pathogens and danger:the role of RP105. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17470533 Regulation of TLR4 signaling and the host interface with pathogens and dan...tion of TLR4 signaling and the host interface with pathogens and danger:the role ...of RP105. PubmedID 17470533 Title Regulation of TLR4 signaling and the host interface with pathogens and dan

  9. Delivery of TLR7 agonist to monocytes and dendritic cells by DCIR targeted liposomes induces robust production of anti-cancer cytokines

    DEFF Research Database (Denmark)

    Klauber, Thomas Christopher Bogh; Laursen, Janne Marie; Zucker, Daniel

    2017-01-01

    as their immune activating potential in blood-derived monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). Monocytes and mDCs were targeted with high specificity over lymphocytes, and exhibited potent TLR7-specific secretion of the anti-cancer cytokines IL-12p70, IFN-α 2a, and IFN-γ. This delivery system...... targeted by the tumor escape mechanisms to develop immunosuppressive phenotypes. Providing activated monocytes and DCs to the tumor tissue is therefore an attractive way to break the tumor-derived immune suppression and reinstate cancer immune surveillance. To activate monocytes and DCs with high...... could be a way to improve cancer treatment either in the form of a vaccine with co-formulated antigen or as an immunotherapeutic vector to boost monocyte and DC activity in combination with other treatment protocols such as chemotherapy or radiotherapy. Cancer immunotherapy is a powerful new tool...

  10. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    Directory of Open Access Journals (Sweden)

    Marion eDuriez

    2014-07-01

    Full Text Available Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis, where maternal and fetal cells are in close contact. Toll-like receptors (TLRs may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs and NK cells (dNKs, the major decidual immune cell populations.We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

  11. Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

    Directory of Open Access Journals (Sweden)

    Yan Tan

    Full Text Available Stiffening of large arteries is increasingly used as an independent predictor of risk and therapeutic outcome for small artery dysfunction in many diseases including pulmonary hypertension. The molecular mechanisms mediating downstream vascular cell responses to large artery stiffening remain unclear. We hypothesize that high pulsatility flow, induced by large artery stiffening, causes inflammatory responses in downstream pulmonary artery endothelial cells (PAECs through toll-like receptor (TLR pathways. To recapitulate the stiffening effect of large pulmonary arteries that occurs in pulmonary hypertension, ultrathin silicone tubes of variable mechanical stiffness were formulated and were placed in a flow circulatory system. These tubes modulated the simulated cardiac output into pulsatile flows with different pulsatility indices, 0.5 (normal or 1.5 (high. PAECs placed downstream of the tubes were evaluated for their expression of proinflammatory molecules (ICAM-1, VCAM-1, E-selectin and MCP-1, TLR receptors and intracellular NF-κB following flow exposure. Results showed that compared to flow with normal pulsatility, high pulsatility flow induced proinflammatory responses in PAECs, enhanced TLR2 expression but not TLR4, and caused NF-κB activation. Pharmacologic (OxPAPC and siRNA inhibition of TLR2 attenuated high pulsatility flow-induced pro-inflammatory responses and NF-κB activation in PAECs. We also observed that PAECs isolated from small pulmonary arteries of hypertensive animals exhibiting proximal vascular stiffening demonstrated a durable ex-vivo proinflammatory phenotype (increased TLR2, TLR4 and MCP-1 expression. Intralobar PAECs isolated from vessels of IPAH patients also showed increased TLR2. In conclusion, this study demonstrates for the first time that TLR2/NF-κB signaling mediates endothelial inflammation under high pulsatility flow caused by upstream stiffening, but the role of TLR4 in flow pulsatility-mediated endothelial

  12. TLR2, TLR4 and the MyD88 signaling are crucial for the in vivo generation and the longevity of long-lived antibody-secreting cells.

    Directory of Open Access Journals (Sweden)

    Evilin Naname Komegae

    Full Text Available This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent modulated the migration of innate-like B cells (B1a and B2 out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent modulated the emigration from the spleen of Bmem as well as ASC B220(pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent and ASC B220(pos (MyD88-independent. We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent and in BM (MyD88-independent as well as of ASC B220(neg in the spleen (MyD88-independent. TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent and ASC B220(pos (MyD88-dependent in BM. Finally, TLR4 signals sustained the longevity of ASC B220(pos (MyD88-independent and ASC B220(neg into the peritoneum (MyD88-dependent and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220(neg in peritoneum and BM. Terminally differentiated ASC B220(neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220(pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220(pos and ASC B220(neg.

  13. Aging and contribution of MyD88 and TRIF to expression of TLR pathway-associated genes following stimulation with Porphyromonas gingivalis.

    Science.gov (United States)

    Shaik-Dasthagirisaheb, Y B; Huang, N; Weinberg, E O; Shen, S S; Genco, C A; Gibson, F C

    2015-02-01

    Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease, including socio-economic status, genetics and age; however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft- and hard-tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in the host response to P. gingivalis. The focus of the current study was to use microarray analysis to define the contributions of the TLR adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), and aging, on the expression of TLR pathway-associated mRNAs in response to P. gingivalis. Bone marrow-derived macrophages (BMØ) from wild-type (Wt), MyD88 knockout (MyD88-KO) and Trif(Lps2) [i.e. containing a point mutation in the lipopolysaccharide 2 (Lps2) gene rendering the Toll/interleukin (IL)-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) protein nonfunctional] mice, at 2-and 12-mo of age, were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison with expression of genes in BMØ cultured in medium only. Using, as criteria, a twofold increase or decrease in mRNA expression, differential expression of 32 genes was observed when Wt BMØ from 2-mo-old mice were cultured with P. gingivalis compared with the medium-only control. When compared with 2-mo-old Wt mice, 21 and 12 genes were differentially expressed (p gingivalis. Age also influenced the expression of genes in MyD88-KO and Trif(Lps2) mice challenged with P. gingivalis. Our results indicate that P. gingivalis induces differential expression of TLR pathway-associated genes, and both MyD88 and TRIF play roles in the expression of these genes

  14. TLR9 gene polymorphism -1486T/C (rs187084) is associated with uterine cervical neoplasm in Mexican female population.

    Science.gov (United States)

    Martínez-Campos, Cecilia; Bahena-Román, Margarita; Torres-Poveda, Kirvis; Burguete-García, Ana I; Madrid-Marina, Vicente

    2017-12-01

    The aim of this work was to evaluate the association of single nucleotide polymorphisms in TLR9 (-1486 T/C [rs187084], -1237T/C [rs5743836] and G2848A [rs352140]) with HPV infection, squamous intraepithelial lesions, and uterine cervical neoplasm in a Mexican population. Additionally, the peripheral expression of TLR9 was evaluated to evaluate the differences in the TLR9 expression associated with every genotype in the locus -1486 of the TLR9 gene. The serum concentration of TLR9 was evaluated in a randomly selected subsample. Genotyping was performed using predesigned 5' endonuc lease assays and the association of the polymorphisms with the diagnosis groups were assessed by performing multinomial regression models. The relative expression of TLR9 in peripheral blood mononuclear cells was evaluated by real-time polymerase chain reaction and the association of the level of TLR9 expression with the diagnosis was evaluated by performing multinomial regression models. The serum concentration of TLR9 was evaluated in a subsample of patients diagnosed with uterine cervical neoplasm by ELISA. The results showed that genotype TT in the -1486 locus of TLR9 was significantly associated with HPV infection (OR = 3.25, 95% CI 1.12-9.46), squamous intraepithelial cervical lesion (OR = 3.76, 95% CI 1.36-10.41), and uterine cervical neoplasm (OR = 5.30, 95% CI 1.81-15.55). Moreover, the highest level of TLR9 expression was significantly associated with a greater risk for developing squamous intraepithelial cervical lesion and uterine cervical neoplasm. The serum TLR9 concentration was higher in patients with uterine cervical cancer than in controls. Our findings indicate that genotype TT in the -1486 locus of the TLR9 gene could comprise a risk genotype for HPV infection, squamous intraepithelial cervical lesion, and uterine cervical neoplasm in Mexican female population. Further studies with larger samples are needed to evaluate if the peripheral expression of TLR9 could be

  15. Should a Toll-like receptor 4 (TLR-4 agonist or antagonist be designed to treat cancer? TLR-4: its expression and effects in the ten most common cancers

    Directory of Open Access Journals (Sweden)

    Mai CW

    2013-11-01

    Full Text Available Chun Wai Mai, Yew Beng Kang, Mallikarjuna Rao PichikaDepartment of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, Kuala Lumpur, MalaysiaAbstract: Toll-like receptor 4 (TLR-4 is well known for its host innate immunity. Despite the fact that TLR-4 activation confers antitumor responses; emerging evidence suggests that TLR-4 is associated with tumor development and progression. It is now clear that overactivation of TLR-4, through various immune mediators, may cause immune response dysfunction, resulting in tumorigenesis. Different cancers could have different extents of TLR-4 involvement during tumorigenesis or tumor progression. In this review, we focus on infection- and inflammation-related TLR-4 activation in noncancer and cancer cells, as well as on the current evidence about the role of TLR-4 in ten of the most common cancers, viz, head and neck cancer, lung cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, skin cancer, breast cancer, ovarian cancer, cervical cancer, and prostate cancer.Keywords: drug design, cancer treatment, myeloid differentiation factor 2, MD-2, tumor progression, pathogen-associated molecular patterns, PAMPs

  16. Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway

    Directory of Open Access Journals (Sweden)

    Xiaoxia Li

    2016-04-01

    Full Text Available Abstract Background In tumor microenvironment, a continuous cross-talk between cancer cells and other cellular components is required to sustain tumor progression. Accumulating evidence suggests that exosomes, a novel way of cell communication, play an important role in such cross-talk. Exosomes could facilitate the direct intercellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells. Since mesenchymal stem cells (MSCs can be attracted to tumor sites and become an important component of the tumor microenvironment, there is an urgent need to reveal the effect of tumor exosomes on MSCs and to further explore the underlying molecular mechanisms. Methods Exosomes were harvested from lung cancer cell line A549 and added to MSCs. Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA. The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model. Signaling pathway involved in exosomes-treated MSCs was detected by PCR array of human toll-like receptor signaling pathway, RT-PCR, and Western blot. Results Data showed that lung tumor cell A549-derived exosomes could induce a pro-inflammatory phenotype in MSCs named P-MSCs, which have significantly elevated secretion of IL-6, IL-8, and MCP-1. P-MSCs possess a greatly enhanced ability in promoting lung tumor growth in mouse xenograft model. Analysis of the signaling pathways in P-MSCs revealed a fast triggering of NF-κB. Genetic ablation of Toll-like receptor 2 (TLR2 by siRNA and TLR2-neutralizing antibody could block NF-κB activation by exosomes. We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes. Conclusions Our studies suggest a novel mechanism by which lung tumor cell-derived exosomes induce pro-inflammatory activity of MSCs which in turn get tumor supportive characteristics.

  17. Analysis of the Toll-Like Receptor 2-2 (TLR2-2 and TLR4 mRNA Expression in the Intestinal Mucosal Immunity of Broilers Fed on Diets Supplemented with Nickel Chloride

    Directory of Open Access Journals (Sweden)

    Bangyuan Wu

    2014-01-01

    Full Text Available Toll-like receptor (TLRs are important innate immune receptors, and TLR2 and TLR4 play an important role in intestinal mucosal innate immunity. It has been found that nickel (Ni can affect the immune system in broilers. The purpose of this study was to analyze changes in TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosal immunity system of broilers induced by dietary nickel chloride (NiCl2 using quantitative real-time polymerase chain reaction (qRT-PCR assays. Two hundred and forty one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet or the same basal diet supplemented with 300, 600 and 900 mg/kg of NiCl2 for 42 days. Results showed that the TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosa and the cecal tonsil were lower (p < 0.05 or p < 0.01 in the 300, 600 and 900 mg/kg groups than those in the control group. It was concluded that dietary NiCl2 in excess of 300 mg/kg could reduce TLR2-2 and TLR4 mRNA expression levels in the intestinal mucosa and cecal tonsil in broilers, implying that the innate immunity in intestinal mucosal immune system could be impaired by pathways involving injured surface epithelium cells or/and the inhibition of the TLR signal transduction.

  18. DMPD: PI3K and negative regulation of TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available mmunol. 2003 Jul;24(7):358-63. (.png) (.svg) (.html) (.csml) Show PI3K and negative regulation of TLR signal...cation Trends Immunol. 2003 Jul;24(7):358-63. Pathway - PNG File (.png) SVG File (.svg) HTML File (.html

  19. Polymorphism of the CD14 and TLR4 genes and post-treatment apical periodontitis.

    Science.gov (United States)

    Rôças, Isabela N; Siqueira, José F; Del Aguila, Camila A; Provenzano, José C; Guilherme, Bianca P S; Gonçalves, Lucio S

    2014-02-01

    This study investigated the association of CD14 -260C>T and TLR4 +896A>G gene polymorphisms with post-treatment apical periodontitis in Brazilian individuals. The study population consisted of 41 patients with post-treatment apical periodontitis and 42 individuals with root canal-treated teeth exhibiting healed/healing periradicular tissues (controls). All teeth had apical periodontitis lesions at the time of treatment, which was completed at least 1 year previously. Saliva was collected from the participants; DNA was extracted and used for CD14 and TLR4 genotyping using the polymerase chain reaction-restriction fragment length polymorphism approach and a real-time polymerase chain reaction TaqMan assay (Applied Biosystems, Foster City, CA), respectively. No specific genotype or allele of the CD14 and TLR4 genes or any combination thereof was positively associated with post-treatment apical periodontitis (P > .05). Data from the present study suggest that polymorphisms in the CD14 and TLR4 genes do not influence the response to endodontic treatment of teeth with apical periodontitis. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  20. Genetic variation at Exon2 of TLR4 gene and its association with ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... role as a key linkage between innate immunity and specific immunity. But to date, no mutation has been detected in exon 2 in chicken. The correlation between polymorphism of TLR4 gene and its resistance to disease have rarely been reported in china. This study analyzed the polymorphism of chicken ...

  1. Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist

    DEFF Research Database (Denmark)

    Johansen, Pia Thermann; Zucker, Daniel; Parhamifar, Ladan

    2015-01-01

    adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of nnonocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged...... cytokines. We envision this technology as a promising tool in future cancer immunotherapy....

  2. TLR2 Controls Intestinal Carcinogen Detoxication by CYP1A1

    DEFF Research Database (Denmark)

    Do, Khoa; Fink, Lisbeth Nielsen; Jensen, Thomas Elbenhardt

    2012-01-01

    of ligands for TLR2 of bacterial origin seems to be crucial for detoxication of luminal carcinogens by CYP1A1 in the intestine. This unprecedented finding indicates a complex interplay between the immune system of the host and intestinal bacteria with detoxication mechanisms. This highlights the relevance...

  3. Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis

    Science.gov (United States)

    Maekawa, Tomoki; Krauss, Jennifer L.; Abe, Toshiharu; Jotwani, Ravi; Triantafilou, Martha; Triantafilou, Kathy; Hashim, Ahmed; Hoch, Shifra; Curtis, Michael A.; Nussbaum, Gabriel; Lambris, John D.; Hajishengallis, George

    2014-01-01

    SUMMARY Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides ‘bystander’ protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR, and can contribute to the persistence of microbial communities that drive dysbiotic diseases. PMID:24922578

  4. TLR4 genotype and environmental LPS mediate RSV bronchiolitis through Th2 polarization

    Science.gov (United States)

    Caballero, Mauricio T.; Serra, M. Elina; Acosta, Patricio L.; Marzec, Jacqui; Gibbons, Luz; Salim, Maximiliano; Rodriguez, Andrea; Reynaldi, Andrea; Garcia, Alejandro; Bado, Daniela; Buchholz, Ursula J.; Hijano, Diego R.; Coviello, Silvina; Newcomb, Dawn; Bellabarba, Miguel; Ferolla, Fausto M.; Libster, Romina; Berenstein, Ada; Siniawaski, Susana; Blumetti, Valeria; Echavarria, Marcela; Pinto, Leonardo; Lawrence, Andrea; Ossorio, M. Fabiana; Grosman, Arnoldo; Mateu, Cecilia G.; Bayle, Carola; Dericco, Alejandra; Pellegrini, Mariana; Igarza, Ignacio; Repetto, Horacio A.; Grimaldi, Luciano Alva; Gudapati, Prathyusha; Polack, Norberto R.; Althabe, Fernando; Shi, Min; Ferrero, Fernando; Bergel, Eduardo; Stein, Renato T.; Peebles, R. Stokes; Boothby, Mark; Kleeberger, Steven R.; Polack, Fernando P.

    2015-01-01

    While 30%–70% of RSV-infected infants develop bronchiolitis, 2% require hospitalization. It is not clear why disease severity differs among healthy, full-term infants; however, virus titers, inflammation, and Th2 bias are proposed explanations. While TLR4 is associated with these disease phenotypes, the role of this receptor in respiratory syncytial virus (RSV) pathogenesis is controversial. Here, we evaluated the interaction between TLR4 and environmental factors in RSV disease and defined the immune mediators associated with severe illness. Two independent populations of infants with RSV bronchiolitis revealed that the severity of RSV infection is determined by the TLR4 genotype of the individual and by environmental exposure to LPS. RSV-infected infants with severe disease exhibited a high GATA3/T-bet ratio, which manifested as a high IL-4/IFN-γ ratio in respiratory secretions. The IL-4/IFN-γ ratio present in infants with severe RSV is indicative of Th2 polarization. Murine models of RSV infection confirmed that LPS exposure, Tlr4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-γ ratio is associated with severe disease. Moreover, these molecules should be explored as potential targets for therapeutic intervention. PMID:25555213

  5. Immunologic activity of schistosomal and bacterial TLR2 ligands in Gabonese children

    NARCIS (Netherlands)

    Retra, K.; Riet, E. van; Adegnika, A.A.; Everts, B.; Geest, S. van; Kremsner, P.G.; Hellemond, J.J. van; Kleij, D. van der; Tielens, A.G.M.; Yazdanbakhsh, M.

    2008-01-01

    Schistosomes carry lipid moieties that interact with the immune system. To understand the consequence of interactions in terms of polarizing the cytokine profiles, the effect of two Toll-like receptor-2 (TLR2) activating schistosomal lipid fractions was studied on whole blood from Gabonese children

  6. Snapin promotes HIV-1 transmission from dendritic cells by dampening TLR8 signaling.

    Science.gov (United States)

    Khatamzas, Elham; Hipp, Madeleine Maria; Gaughan, Daniel; Pichulik, Tica; Leslie, Alasdair; Fernandes, Ricardo A; Muraro, Daniele; Booth, Sarah; Zausmer, Kieran; Sun, Mei-Yi; Kessler, Benedikt; Rowland-Jones, Sarah; Cerundolo, Vincenzo; Simmons, Alison

    2017-10-16

    HIV-1 traffics through dendritic cells (DCs) en route to establishing a productive infection in T lymphocytes but fails to induce an innate immune response. Within DC endosomes, HIV-1 somehow evades detection by the pattern-recognition receptor (PRR) Toll-like receptor 8 (TLR8). Using a phosphoproteomic approach, we identified a robust and diverse signaling cascade triggered by HIV-1 upon entry into human DCs. A secondary siRNA screen of the identified signaling factors revealed several new mediators of HIV-1 trans-infection of CD4(+) T cells in DCs, including the dynein motor protein Snapin. Inhibition of Snapin enhanced localization of HIV-1 with TLR8(+) early endosomes, triggered a pro-inflammatory response, and inhibited trans-infection of CD4(+) T cells. Snapin inhibited TLR8 signaling in the absence of HIV-1 and is a general regulator of endosomal maturation. Thus, we identify a new mechanism of innate immune sensing by TLR8 in DCs, which is exploited by HIV-1 to promote transmission. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  7. Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

    Directory of Open Access Journals (Sweden)

    Guoku Hu

    Full Text Available Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

  8. Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.

    Science.gov (United States)

    Abhyankar, Mayuresh M; Noor, Zannatun; Tomai, Mark A; Elvecrog, James; Fox, Christopher B; Petri, William A

    2017-02-07

    Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. 2'-O-methyl-modified RNAs act as TLR7 antagonists.

    Science.gov (United States)

    Robbins, Marjorie; Judge, Adam; Liang, Lisa; McClintock, Kevin; Yaworski, Ed; MacLachlan, Ian

    2007-09-01

    RNA molecules such as single-stranded RNA (ssRNA) and small interfering RNA (siRNA) duplexes induce Toll-like receptor (TLR)-mediated immune stimulation after intracellular delivery. We have previously shown that selective incorporation of 2'-O-methyl (2'OMe) residues into siRNA abrogates cytokine production without reduction of gene silencing activity. Here we show that 2'OMe-modified RNA acts as a potent inhibitor of RNA-mediated cytokine induction in both human and murine systems. This activity does not require the direct incorporation of 2'OMe nucleotides into the immunostimulatory RNA or that the 2'OMe nucleotide-containing RNA be annealed as a complementary strand to form a duplex. Our results indicate that 2'OMe RNA acts as a potent antagonist of immunostimulatory RNA. We further show that 2'OMe RNA is able significantly to reduce both interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) induction by the small-molecule TLR7 agonist loxoribine in human peripheral blood mononuclear cells (human PBMCs), in murine Flt3L dendritic cells (Flt3L DCs), and in vivo in mice. These results indicate that 2'OMe-modified RNA may have utility as an inhibitor of TLR7 with potential applications in the treatment of inflammatory and autoimmune diseases that involve TLR7-mediated immune stimulation.

  10. [Role of several periodontopathogenic microorganisms and tlr4 gene Asp299Gly polymorphism in atherosclerosis pathogenesis].

    Science.gov (United States)

    Skochko, O V; Bobrova, N A; Izmaylova, O V; Kaĭdashev, I P

    2011-01-01

    Establishment of presence of periodonto-pathogenic microorganisms in atherosclerosis plaque and surrounding tissues, and possible relation of development of atherosclerosis and TLR4 gene Asp299Gly polymorphism in ischemic heart disease patients (IHD). Samples of coronary vessels obtained during autopsy of 31 individuals deceased from IHD and 5 individuals deceased due to reasons not related with IHD were studied. PCR was used to determine DNA of the microorganisms. TLR4 gene polymorphic segment was amplified by using specific primers. Analysis of coronary vessel atherosclerotic plaques revealed presence of the studied periodontopathogenic microorganisms in 83.9% of cases. The most frequently detected were Porphyromonas gingivalis (64.5%), Treponema denticola (41.9%), Actinobacillus actinomycetemcomitans (32.3%), less frequently--Bacteroides forsythus and Prevotella intermedia (12.9% and 6.5% respectively). In 51.6% of cases 2 or more microorganisms were detected. Only in 11.1% ofcoronary artery samples, with plaques containing microorganisms, the microorganisms were detected in undamaged tissues. Patients deceased from IHD had TLR4 gene 299Gly allele significantly more frequently. The studied periodontopathogenic microorganisms can play an important role in the pathogenesis of atherosclerotic injury of coronary arteries in IHD. The presence of TLR4 gene allele 299Gly significantly contributes to these processes.

  11. TLR2 Signaling Pathway in Alveolar Bone Osteogenesis Induced by Aloe vera and Xenograft (XCB).

    Science.gov (United States)

    Kresnoadi, Utari; Rahayu, Retno Pudji; Rubianto, M; Sudarmo, Subijanto Marto; Budi, Hendrik Setia

    2017-01-01

    The aim of this study was to find the role of TLR2 signaling pathway in reducing osteoclast activity and promoting osteoblast growth by inducing a combination of Aloe vera and cancellous bovine xenograft (XCB) into dental extraction socket. Forty-eight Cavia cobayas were used. They were divided into eight groups (n=6). For control group, their mandibular incisors were extracted and filled with PEG. For treatment groups, they were extracted and filled with XCB, Aloe vera and the combination of Aloe vera and XCB. The first four groups were sacrificed after 7 days and the other groups after 30 days. Immunohistochemistry and histopathology examination were conducted to examine TLR2, TNFa, OPG, collagen-1, and the osteoblast and osteoclast expressions. The expressions of TLR2, OPG and Collagen-1, as well as the number of osteoblast were increased. Meanwhile, the expressions of TNFa and osteoclast were decreased. The study finding was that TLR2 signaling pathway influenced alveolar bone osteogenesis process by reducing osteoclast activity and stimulating osteoblast growth induced by the combination of Aloe vera and XCB.

  12. Irinotecan-Induced Gastrointestinal Dysfunction and Pain Are Mediated by Common TLR4-Dependent Mechanisms.

    Science.gov (United States)

    Wardill, Hannah R; Gibson, Rachel J; Van Sebille, Ysabella Z A; Secombe, Kate R; Coller, Janet K; White, Imogen A; Manavis, Jim; Hutchinson, Mark R; Staikopoulos, Vasiliki; Logan, Richard M; Bowen, Joanne M

    2016-06-01

    Strong epidemiological data indicate that chemotherapy-induced gut toxicity and pain occur in parallel, indicating common underlying mechanisms. We have recently outlined evidence suggesting that TLR4 signaling may contribute to both side effects. We therefore aimed to determine if genetic deletion of TLR4 improves chemotherapy-induced gut toxicity and pain. Forty-two female wild-type (WT) and 42 Tlr4 null (-/-) BALB/c mice weighing between 18 and 25 g (10-13 weeks) received a single 270 mg/kg (i.p.) dose of irinotecan hydrochloride or vehicle control and were killed at 6, 24, 48, 72, and 96 hours. Bacterial sequencing was conducted on cecal samples of control animals to determine the gut microbiome profile. Gut toxicity was assessed using validated clinical and histopathologic markers, permeability assays, and inflammatory markers. Chemotherapy-induced pain was assessed using the validated rodent facial grimace criteria, as well as immunologic markers of glial activation in the lumbar spinal cord. TLR4 deletion attenuated irinotecan-induced gut toxicity, with improvements in weight loss (P = 0.0003) and diarrhea (P induced gut toxicity and pain, highlighting the possibility of a targetable gut/CNS axis for improved toxicity outcomes. Mol Cancer Ther; 15(6); 1376-86. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Chemical inhomogeneity in the ultrafast dynamics of the DXCF cyanobacteriochrome Tlr0924.

    Science.gov (United States)

    Freer, Lucy H; Kim, Peter W; Corley, Scott C; Rockwell, Nathan C; Zhao, Lu; Thibert, Arthur J; Lagarias, J Clark; Larsen, Delmar S

    2012-09-06

    Cyanobacteriochromes (CBCRs) are diverse biliprotein photosensors distantly related to the red/far-red photoreceptors of the phytochrome family. There are several subfamilies of CBCRs, displaying varied spectral responses spanning the entire visible region. Tlr0924 belongs to the DXCF subfamily that utilizes the Cys residue in a conserved Asp-Xaa-Cys-Phe (DXCF) motif to form a second covalent linkage to the chromophore, resulting in a blue-absorbing dark state. Photoconversion leads to elimination of this linkage, resulting in a green-absorbing photoproduct. Tlr0924 initially incorporates phycocyanobilin (PCB) as a chromophore, exhibiting a blue/orange photocycle, but slowly isomerizes PCB to phycoviolobilin (PVB) to yield a blue/green photocycle. Ultrafast transient absorption spectroscopy was used to study both forward and reverse reaction photodynamics of the recombinant GAF domain of Tlr0924. Primary photoproducts were identified, as were subsequent intermediates at 1 ms. PCB and PVB population photodynamics were decomposed using global target analysis. PCB and PVB populations exhibit similar and parallel photocycles in Tlr0924, but the PVB population exhibits faster excited-state decay in both reaction directions. On the basis of longer time analysis, we show that the photochemical coordinate (15,16-isomerization) and second-linkage coordinate (elimination or bond formation at C10) are separate processes in both directions.

  14. A role for intestinal TLR4-driven inflammatory response during activity-based anorexia.

    Science.gov (United States)

    Belmonte, Liliana; Achamrah, Najate; Nobis, Séverine; Guérin, Charlène; Riou, Gaëtan; Bôle-Feysot, Christine; Boyer, Olivier; Richard, Vincent; Rego, Jean Claude Do; Déchelotte, Pierre; Goichon, Alexis; Coëffier, Moïse

    2016-10-25

    Anorexia nervosa (AN) is associated with low-grade systemic inflammation and altered gut microbiota. However, the molecular origin of the inflammation remains unknown. Toll-like receptors are key regulators of innate immune response and their activation seems also to be involved in the control of food intake. We used activity-based anorexia (ABA) model to investigate the role of TLR4 and its contribution in anorexia-associated low-grade inflammation. Here, we found that ABA affected early the intestinal inflammatory status and the hypothalamic response. Indeed, TLR4 was upregulated both on colonic epithelial cells and intestinal macrophages, leading to elevated downstream mucosal cytokine production. These mucosal changes occurred earlier than hypothalamic changes driving to increased levels of IL-1β and IL-1R1 as well as increased levels of plasma corticosterone. Paradoxically, TLR4-deficient mice exhibited greater vulnerability to ABA with increased mortality rate, suggesting a major contribution of TLR4-mediated responses during ABA-induced weight loss.

  15. Role of TLR4 gene polymorphisms in the colorectal cancer risk ...

    African Journals Online (AJOL)

    Saniya Nissar

    2016-05-26

    May 26, 2016 ... morphic status of Toll-like receptor 4 gene in various populations of the world. Aim: The aim of the study was to determine the genetic association of TLR4 gene polymor- phisms (Asp299Gly and Thr399Ile) with disease susceptibility and risk development in colorectal cancer (CRC) patients of Kashmir, India ...

  16. DMPD: TLR3 in antiviral immunity: key player or bystander? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available Trends Immunol. 2005 Sep;26(9):462-8. (.png) (.svg) (.html) (.csml) Show TLR3 in antiviral immunity: key pl... File (.png) SVG File (.svg) HTML File (.html) CSML File (.csml) Open .csml file with CIOPlayer Open .csml f

  17. CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

    NARCIS (Netherlands)

    Klein, D.C.; Skjesol, A.; Kers-Rebel, E.D.; Sherstova, T.; Sporsheim, B.; Egeberg, K.W.; Stokke, B.T.; Espevik, T.; Husebye, H.

    2015-01-01

    Toll-like receptor 4 (TLR4) is responsible for the immediate response to Gram-negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal-MyD88 [Mal (MyD88-adaptor-like) - MYD88 (Myeloid differentiation primary response gene (88))] is recruited to

  18. Evolutionary analysis of TLR9 genes reveals the positive selection of extant teleosts in Perciformes.

    Science.gov (United States)

    Zhu, Zhihuang; Sun, Yuena; Wang, Rixin; Xu, Tianjun

    2013-08-01

    The innate immune system can recognize non-self through pattern recognition receptors. Toll-like receptors were the best-known members of these receptors, and they could sense, recognize, and bind pathogen-associated molecular patterns. TLRs played an important role in innate immune system and were conserved in both invertebrate and vertebrate lineages. Thereinto, TLR9 could detect unmethylated CpG motifs in dsDNA and was expected to undergo coevolution with its microbial ligands. It was known that aquatic and terrestrial organisms dwelled in different environments which contained different pathogens, and they had to adapt to their local environmental conditions. Therefore, we collected TLR9 genes from invertebrate to vertebrate to further explore whether the huge differences between aquatic and terrestrial environments affected the TLR9s evolution between aquatic and terrestrial organisms. Molecular evolution analysis detected positively selected sites in the ancestral lineages of vertebrates, teleosts, and Perciformes but not in the ancestral lineage of mammals. In PAML, site model revealed that extant mammalian TLR9 genes underwent positive selection. However, the positive selection of extant teleosts appeared primarily in Perciformes in which there were 14 positively selected sites. Among these sites, two of them were located on the amino acid insertions of the leucine-rich repeats which could create DNA binding sites, three were found on the convex surface which might possibly affect the flexibility of the TLR solenoids, and six were located on the β-face of concave surface which contained the ligand-binding sites of the TLR solenoids. In other ML methods, we also found three sites under selection that coincided with the codons identified by M8 and these sites were all located in LRRs. The diverse aquatic and terrestrial environments might possess different pathogens to make the living organisms adapt to their local environmental conditions. The positive

  19. Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Koji; Komohara, Yoshihiro; Fujiwara, Yukio; Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Lei, XiaoFeng [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Nakagawa, Takenobu [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Department of Human Pathology, Institute of Health Biosciences, The University of Tokushima, Tokushima (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan)

    2011-08-05

    Highlights: {yields} We focused on the interaction between SR-A and TLR4 signaling in this study. {yields} SR-A deletion promoted NF{kappa}B activation in macrophages in septic model mouse. {yields} SR-A suppresses both MyD88-dependent and -independent TLR4 signaling in vitro. {yields} SR-A clears LPS binding to TLR4 which resulting in the suppression of TLR4 signals. -- Abstract: The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A{sup -/-}) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6 and interferon (IFN)-{beta} were significantly increased in SR-A{sup -/-} mice compared to wild-type mice, and elevated nuclear factor kappa B (NF{kappa}B) activation was detected in SR-A{sup -/-} macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NF{kappa}B in vitro. SR-A deletion also promoted the nuclear translocation of NF{kappa}B and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A{sup -/-} macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.

  20. The activation of CD14, TLR4, and TLR2 by mmLDL induces IL-1β, IL-6, and IL-10 secretion in human monocytes and macrophages

    Directory of Open Access Journals (Sweden)

    Blanco-Favela Francisco

    2010-10-01

    Full Text Available Abstract Atherosclerosis is considered a chronic inflammatory disease in which monocytes and macrophages are critical. These cells express CD14, toll-like receptor (TLR 2, and TLR4 on their surfaces, are activated by minimally modified low-density lipoprotein (mmLDL and are capable of secreting pro-inflammatory cytokines. The aim of this research was thus to demonstrate that the activation of CD14, TLR2, and TLR4 by mmLDL induces the secretion of cytokines. Methods Human monocytes and macrophages were incubated with monoclonal antibodies specific for CD14, TLR4, and TLR2 prior to stimulation with mmLDL. Cytokine secretion was then compared to that observed upon mmLDL stimulation in untreated cells. Results Stimulation with mmLDL induced the secretion of pro-inflammatory cytokines. Blocking CD14 in monocytes inhibited secretion of interleukin (IL-1β (72%, IL-6 (58% and IL-10 (63%, and blocking TLR4 inhibited secretion of IL-1β by 67%, IL-6 by 63% and IL-10 by 60%. Blocking both receptors inhibited secretion of IL-1β by 73%, IL-6 by 69% and IL-10 by 63%. Furthermore, blocking TLR2 inhibited secretion of IL-1β by 65%, IL-6 by 62% and IL-10 by 75%. In macrophages, we found similar results: blocking CD14 inhibited secretion of IL-1β by 59%, IL-6 by 52% and IL-10 by 65%; blocking TLR4 inhibited secretion of IL-1β by 53%, IL-6 by 63% and IL-10 by 61%; and blocking both receptors inhibited secretion of IL-1β by 69%, IL-6 by 67% and IL-10 by 65%. Blocking TLR2 in macrophages inhibited secretion of IL-1β by 57%, IL-6 by 40% and IL-10 by 72%. Conclusion Our study demonstrates that CD14, TLR4, and TLR2 participate in the immune response against mmLDL by inducing the production of pro-inflammatory cytokines in both monocytes and macrophages. These findings suggest that the activation of these receptors by mmLDL contributes to the inflammatory process of atherosclerosis.

  1. TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

    Science.gov (United States)

    Tomlinson, Gillian; Chimalapati, Suneeta; Pollard, Tracey; Lapp, Thabo; Cohen, Jonathan; Camberlein, Emilie; Stafford, Sian; Periselneris, Jimstan; Aldridge, Christine; Vollmer, Waldemar; Picard, Capucine; Casanova, Jean-Laurent; Noursadeghi, Mahdad; Brown, Jeremy

    2014-10-01

    Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae. Copyright © 2014 The Authors.

  2. A member of the Tlr family is involved in dsRNA innate immune response in Paracentrotus lividus sea urchin.

    Science.gov (United States)

    Russo, Roberta; Chiaramonte, Marco; Matranga, Valeria; Arizza, Vincenzo

    2015-08-01

    The innate immune response involves proteins such as the membrane receptors of the Toll-like family (TLRs), which trigger different intracellular signalling pathways that are dependent on specific stimulating molecules. In sea urchins, TLR proteins are encoded by members of a large multigenic family composed of 60-250 genes in different species. Here, we report a newly identified mRNA sequence encoding a TLR protein (referred to as Pl-Tlr) isolated from Paracentrotus lividus immune cells. The partial protein sequence contained the conserved Toll/IL-1 receptor (TIR) domain, the transmembrane domain and part of the leucine repeats. Phylogenetic analysis of the Pl-Tlr protein was accomplished by comparing its sequence with those of TLRs from different classes of vertebrates and invertebrates. This analysis was suggestive of an evolutionary path that most likely represented the course of millions of years, starting from simple organisms and extending to humans. Challenge of the sea urchin immune system with poly-I:C, a chemical compound that mimics dsRNA, caused time-dependent Pl-Tlr mRNA up-regulation that was detected by QPCR. In contrast, bacterial LPS injury did not affect Pl-Tlr transcription. The study of the Tlr genes in the sea urchin model system may provide new perspectives on the role of Tlrs in the invertebrate immune response and clues concerning their evolution in a changing world. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Determination of Genetic Variations of Toll-Like Receptor (TLR 2, 4, and 6 with Next-Generation Sequencing in Native Cattle Breeds of Anatolia and Holstein Friesian

    Directory of Open Access Journals (Sweden)

    Nuket Bilgen

    2016-11-01

    Full Text Available In recent years, the focus of disease resistance and susceptibility studies in cattle have been on determining patterns in the innate immune response of key proteins, such as Toll-like receptors (TLR. In the bovine genome, there are 10 TLR family members and, of these, TLR2, TLR4, and TLR6 are specialized in the recognition of bacterial ligands. Indigenous cattle breeds of Anatolia have been reported to show fewer signs of clinical bacterial infections, such as bovine tuberculosis and mastitis, and it is hypothesized that this might be due to a less stringent genetic selection during breeding. In contrast, Holstein-Friesian cattle have been under strong selection for milk production, which may have resulted in greater susceptibility to diseases. To test this hypothesis, we have compared the TLR2, TLR4, and TLR6 genes of Anatolian Black (AB, East Anatolian Red (EAR, South Anatolian Red (SAR, Turkish Grey (TG, and Holstein (HOL cattle using next-generation sequencing. The SAR breed had the most variations overall, followed by EAR, AB, TG, and HOL. TG had the most variations for TLR2, whereas SAR had the most variations in TLR4 and TLR6. We compared these variants with those associated with disease and susceptibility traits. We used exon variants to construct haplotypes, investigated shared haplotypes within breeds, and proposed candidate haplotypes for a disease resistance phenotype in Anatolian cattle breeds.