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Sample records for natural tlr response

  1. 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

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

    DEFF Research Database (Denmark)

    Holm, Thomas H; 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...

  3. Structure-based discovery of an immunomodulatory inhibitor of TLR1-TLR2 heterodimerization from a natural product-like database.

    Science.gov (United States)

    Zhong, Zhangfeng; Liu, Li-Juan; Dong, Zhi-Qiang; Lu, Lihua; Wang, Modi; Leung, Chung-Hang; Ma, Dik-Lung; Wang, Yitao

    2015-06-30

    We report herein the identification of an immunomodulatory natural product-like compound as a direct inhibitor of TLR1-TLR2 heterodimerization. Compound suppressed TNF-α and IL-6 secretion in Pam3CSK4-induced macrophages. Moreover, compound inhibited the phagocytic activity of macrophages, presumably through modulation of TLR1-TLR2 signaling and inactivation of NF-κB. Molecular docking revealed that compound bound to the interface region of TLR1-TLR2 by forming two hydrogen bonds with residues lining the binding site. To our knowledge, compound has been only the second inhibitor overall of TLR1-TLR2 heterodimerization reported to date.

  4. 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.

  5. 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

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

    Science.gov (United States)

    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.

  7. 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.

  8. Expression of TLR 2, TLR 4 and iNOS in cervical monocytes of Chlamydia trachomatis-infected women and their role in host immune response.

    Science.gov (United States)

    Agrawal, Tanvi; Bhengraj, Apurb R; Vats, Vikas; Salhan, Sudha; Mittal, Aruna

    2011-12-01

    To study the innate immune response -TLR2 TLR 4 and iNOS expression in female genital Chlamydia trachomatis infection. TLR 2, TLR 4, and iNOS expression was evaluated by real-time PCR in C. trachomatis-infected asymptomatic, mucopurulent cervicitis (MPC), and fertility disorders (FD) women. Expression of TLR signaling pathway genes was checked in vivo in C. trachomatis-infected cervical monocytes. Further, inos gene expression and nitric oxide release was assessed in vitro in THP-1 cell line upon chlamydial infection. TLR2, TLR4, and iNOS expression was significantly (P cervical monocytes upon chlamydial infection. © 2011 John Wiley & Sons A/S.

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

    Science.gov (United States)

    Alizadeh, Hassan; Tripathi, Trivendra; Abdi, Mahshid; Smith, Ashley Dawn

    2014-01-01

    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 (PAcanthamoeba 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.

  10. 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.

  11. Pathogenic Strains of Acanthamoeba Are Recognized by TLR4 and Initiated Inflammatory Responses in the Cornea

    Science.gov (United States)

    Alizadeh, Hassan; Tripathi, Trivendra; Abdi, Mahshid; Smith, Ashley Dawn

    2014-01-01

    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 (PAcanthamoeba 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. PMID:24633052

  12. TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

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    Chassin, Cécilia; Picardeau, Mathieu; Goujon, Jean-Michel; Bourhy, Pascale; Quellard, Nathalie; Darche, Sylvie; Badell, Edgar; d'Andon, Martine Fanton; Winter, Nathalie; Lacroix-Lamandé, Sonia; Buzoni-Gatel, Dominique; Vandewalle, Alain; Werts, Catherine

    2009-08-15

    Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

  13. Purinergic signaling to terminate TLR responses in macrophages

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    Kajal eHamidzadeh

    2016-03-01

    Full Text Available Macrophages undergo profound physiological alterations when they encounter pathogen associated molecular patterns (PAMPs. These alterations can result in the elaboration of cytokines and mediators that promote immune responses and contribute to the clearance of pathogens. These innate immune responses by myeloid cells are transient. The termination of these secretory responses is not due to the dilution of stimuli, but rather to the active down-regulation of innate responses induced by the very PAMPs that initiated them. Here we describe a purinergic autoregulatory program whereby TLR-stimulated macrophages control their activation state. In this program, TLR stimulated macrophages undergo metabolic alterations that result in the production of ATP and its release through membrane pannexin channels. This purine nucleotide is rapidly hydrolyzed to adenosine by ectoenzymes on the macrophage surface, CD39 and CD73. Adenosine then signals through the P1 class of seven transmembrane receptors to induce a regulatory state that is characterized by the down-regulation of inflammatory cytokines and the production of anti-inflammatory cytokines and growth factors. This purinergic autoregulatory system mitigates the collateral damage that would be caused by the prolonged activation of macrophages, and rather allows the macrophage to maintain homeostasis. The transient activation of macrophages can be prolonged by treating macrophages with IFN-γ. IFN-γ treated macrophages become less sensitive to the regulatory effects of adenosine, allowing them to sustain macrophage activation for the duration of an adaptive immune response.

  14. Impaired TLR9 responses in B cells from patients with systemic lupus erythematosus.

    Science.gov (United States)

    Gies, Vincent; Schickel, Jean-Nicolas; Jung, Sophie; Joublin, Aurélie; Glauzy, Salomé; Knapp, Anne-Marie; Soley, Anne; Poindron, Vincent; Guffroy, Aurélien; Choi, Jin-Young; Gottenberg, Jacques-Eric; Anolik, Jennifer H; Martin, Thierry; Soulas-Sprauel, Pauline; Meffre, Eric; Korganow, Anne-Sophie

    2018-03-08

    B cells play a central role in systemic lupus erythematosus (SLE) pathophysiology but dysregulated pathways leading to a break in B cell tolerance remain unclear. Since Toll-like receptor 9 (TLR9) favors the elimination of autoreactive B cells in the periphery, we assessed TLR9 function in SLE by analyzing the responses of B cells and plasmacytoid dendritic cells (pDCs) isolated from healthy donors and patients after stimulation with CpG, a TLR9 agonist. We found that SLE B cells from patients without hydroxychloroquine treatment displayed defective in vitro TLR9 responses, as illustrated by the impaired upregulation of B cell activation molecules and the diminished production of various cytokines including antiinflammatory IL-10. In agreement with CD19 controlling TLR9 responses in B cells, decreased expression of the CD19/CD21 complex on SLE B cells was detected as early as the transitional B cell stage. In contrast, TLR7 function was preserved in SLE B cells, whereas pDCs from SLE patients properly responded to TLR9 stimulation, thereby revealing that impaired TLR9 function in SLE was restricted to B cells. We conclude that abnormal CD19 expression and TLR9 tolerogenic function in SLE B cells may contribute to the break of B cell tolerance in these patients.

  15. TLR-2 is involved in airway epithelial cell response to air pollution particles

    International Nuclear Information System (INIS)

    Becker, Susanne; Dailey, Lisa; Soukup, Joleen M.; Silbajoris, Robert; Devlin, Robert B.

    2005-01-01

    Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of several oxidant stress response genes. Components of ambient air PM responsible for stimulating epithelial cells have not been conclusively identified, although metal contaminants, benzo[a]pyrene and biological matter have been implicated. Stimulation of IL-8 release from NHBE with coarse (PM 2.5-10 ), fine (PM 2.5 ), and UF particle fractions has shown that the coarse particle fraction has the greatest effect on the epithelial cells as well as alveolar macrophages (AM). Since this fraction concentrates fugitive dusts and particle-associated microbial matter, it was hypothesized that NHBE may recognize PM through microbial pattern recognition receptors TLR2 and TLR4, as has been previously shown with AM. NHBE were shown to release IL-8 when exposed to a Gram-positive environmental isolate of Staphylococcus lentus, and lower levels when exposed to Gram-negative Pseudomonas spp. Comparison of TLR2 and TLR4 mRNA expression in NHBE and AM showed that NHBE express similar levels of TLR2 mRNA as the AM, but expressed very low levels of TLR4. When NHBE were stimulated with PM 2.5-10 , PM 2.5 , and UF PM, in the presence or absence of inhibitors of TLR2 and TLR4 activation, a blocking antibody to TLR2 inhibited production of IL-8, while TLR4 antagonist E5531 or the LPS inhibitor Polymixin B had no effect. Furthermore, effects on expression of TLR2 and TLR4 mRNA, as well as the stress protein HSP70 was assessed in NHBE exposed to PM. TLR4 expression was increased in these cells while TLR2 mRNA levels were unchanged. Hsp70 was increased by PM 2.5-10 > PM 2.5 > UF PM suggesting the possibility of indirect activation of TLR pathway by this endogenous TLR2/4 agonist

  16. Ginkgolide B Suppresses TLR4-Mediated Inflammatory Response by Inhibiting the Phosphorylation of JAK2/STAT3 and p38 MAPK in High Glucose-Treated HUVECs

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    Kun Chen

    2017-01-01

    Full Text Available Aim. Ginkgolide B is a Ginkgo biloba leaf extract that has been identified as a natural platelet-activating factor receptor (PAFR antagonist. We investigated the effect of ginkgolide B on high glucose-induced TLR4 activation in human umbilical vein endothelial cells (HUVECs. Methods. Protein expression was analyzed by immunoblotting. Small-interfering RNA (siRNA was used to knock down PAFR and TLR4 expression. Results. Ginkgolide B suppressed the expression of TLR4 and MyD88 that was induced by high glucose. Ginkgolide B also reduced the levels of platelet endothelial cell adhesion molecule-1, interleukin-6, and monocyte chemotactic protein 1. Further, we examined the association between PAFR and TLR4 by coimmunoprecipitation. The result showed that high glucose treatment caused the binding of PAFR and TLR4, whereas ginkgolide B abolished this binding. The functional analysis indicated that PAFR siRNA treatment reduced TLR4 expression, and TLR4 siRNA treatment decreased PAFR expression in high glucose-treated HUVECs, further supporting the coimmunoprecipitation data. Ginkgolide B inhibited the phosphorylation of Janus kinase 2 (JAK2/signal transducer and activator of transcription 3 (STAT3 and p38 mitogen-activated protein kinase (MAPK. Conclusion. Ginkgolide B exerted protective effects by inhibiting the TLR4-mediated inflammatory response in high glucose-treated endothelial cells. The mechanism of action of ginkgolide B might be associated with inhibition of the JAK2/STAT3 and p38 MAPK phosphorylation.

  17. Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

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    Laure Escoubet-Lozach

    2011-12-01

    Full Text Available Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4-dependent activation of nearly all immediate/early- (I/E and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.

  18. Active Hexose Correlated Compound (AHCC) promotes an intestinal immune response in BALB/c mice and in primary intestinal epithelial cell culture involving toll-like receptors TLR-2 and TLR-4.

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    Mallet, Jean-François; Graham, Émilie; Ritz, Barry W; Homma, Kohei; Matar, Chantal

    2016-02-01

    Active Hexose Correlated Compound (AHCC(®)) is a cultured mushroom extract that is commercially available and promoted for immune support. Available data suggest that AHCC supplementation affects immune cell populations and immune outcomes, including natural killer cell response to infection. The mechanism by which AHCC exerts its effects is not well understood. The present work aimed to characterize the immunomodulatory activity of AHCC in the gut and to study the effects of AHCC on toll-like receptor (TLR) signaling in intestinal epithelial cells (IECs). BALB/c mice were fed AHCC by gavage. In vivo activities were assessed by immunohistochemistry and cytokine production. The effects of AHCC on ex vivo primary cell culture from IECs were examined after challenge with LPS or E. coli alone or in the presence of anti-TLR-2 and TLR-4 blocking antibodies. Feeding AHCC resulted in increased IgA+ cells in the intestine and increased sIgA, IL-10, and IFN-γ in the intestinal fluid. In IECs, contact with AHCC increased IL-6 production but not to the pro-inflammatory level of positive controls, LPS and E. coli. Blocking TLR-2 and TLR-4 reduced the induction of IL-6 by AHCC, suggesting that these innate receptors are involved in generating the immune response of IECs to AHCC. AHCC may play a role in the orchestration of immune response and the maintenance of immune homeostasis in part by priming the TLR-2 and TLR-4 gate at the intestinal epithelium. Such a response is likely due to the recognition of non-pathogenic food-associated molecular patterns (FAMPs) such as those found associated with other mushroom or yeast-derived compounds.

  19. The TLR5 ligand flagellin promotes asthma by priming allergic responses to indoor allergens

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    Wilson, Rhonda H.; Maruoka, Shuichiro; Whitehead, Gregory S.; Foley, Julie F.; Flake, Gordon P.; Sever, Michelle L.; Zeldin, Darryl C.; Kraft, Monica; Garantziotis, Stavros; Nakano, Hideki; Cook, Donald N.

    2012-01-01

    Allergic asthma is a complex disease characterized by eosinophilic pulmonary inflammation, mucus production and reversible airway obstruction1. Exposure to indoor allergens is a clear risk factor for asthma, but this disease is also associated with high household levels of total and Gram-negative bacteria2. The ability of bacterial products to act as adjuvants3 suggests they might promote asthma by priming allergic sensitization to inhaled allergens. In support of this idea, house dust extracts (HDEs) can activate antigen presenting dendritic cells (DC) in vitro and promote allergic sensitization to inhaled innocuous proteinsin vivo4. It is unknown which microbial products provide most of the adjuvant activity in HDEs. A screen of microbial products for their adjuvant activity in the airway revealed that the bacterial protein, flagellin (FLA) stimulated strong allergic responses to an innocuous inhaled protein. Moreover, toll-like receptor (TLR)5, the mammalian receptor for FLA5,6, was required for priming strong allergic responses to natural indoor allergens present in HDEs. In addition, the incidence of human asthma was associated with high serum levels of FLA-specific antibodies. Together, these findings suggest that household FLA promotes the development of allergic asthma by TLR5-dependent priming of allergic responses to indoor allergens. PMID:23064463

  20. Identification of the key differential transcriptional responses of human whole blood following TLR2 or TLR4 ligation in-vitro.

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    Simon Blankley

    Full Text Available The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4 and the TLR4 ligand (LPS, human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.

  1. Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses

    DEFF Research Database (Denmark)

    Ugolini, Matteo; Gerhard, Jenny; Burkert, Sanne

    2018-01-01

    Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation...... and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific...... cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism...

  2. 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.

  3. 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.

  4. TLR9 activation suppresses inflammation in response to Helicobacter pylori infection.

    Science.gov (United States)

    Varga, Matthew G; Piazuelo, M Blanca; Romero-Gallo, Judith; Delgado, Alberto G; Suarez, Giovanni; Whitaker, Morgan E; Krishna, Uma S; Patel, Rachna V; Skaar, Eric P; Wilson, Keith T; Algood, Holly M S; Peek, Richard M

    2016-11-01

    Helicobacter pylori (H. pylori) induces chronic gastritis in humans, and infection can persist for decades. One H. pylori strain-specific constituent that augments disease risk is the cag pathogenicity island. The cag island encodes a type IV secretion system (T4SS) that translocates DNA into host cells. Toll-like receptor 9 (TLR9) is an innate immune receptor that detects hypo-methylated CpG DNA motifs. In this study, we sought to define the role of the H. pylori cag T4SS on TLR9-mediated responses in vivo. H. pylori strain PMSS1 or its cagE - mutant, which fails to assemble a T4SS, were used to infect wild-type or Tlr9 -/- C57BL/6 mice. PMSS1-infected Tlr9 -/- mice developed significantly higher levels of inflammation, despite similar levels of colonization density, compared with PMSS1-infected wild-type mice. These changes were cag dependent, as both mouse genotypes infected with the cagE - mutant only developed minimal inflammation. Tlr9 -/- genotypes did not alter the microbial phenotypes of in vivo-adapted H. pylori strains; therefore, we examined host immunological responses. There were no differences in levels of T H 1 or T H 2 cytokines in infected mice when stratified by host genotype. However, gastric mucosal levels of IL-17 were significantly increased in infected Tlr9 -/- mice compared with infected wild-type mice, and H. pylori infection of IL-17A -/- mice concordantly led to significantly decreased levels of gastritis. Thus loss of Tlr9 selectively augments the intensity of IL-17-driven immune responses to H. pylori in a cag T4SS-dependent manner. These results suggest that H. pylori utilizes the cag T4SS to manipulate the intensity of the host immune response. Copyright © 2016 the American Physiological Society.

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

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    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.

  6. Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages

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    Kirill V. Korneev

    2018-02-01

    Full Text Available Toll-like receptor 4 (TLR4 initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS, the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

  7. Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

    Science.gov (United States)

    Korneev, Kirill V; Kondakova, Anna N; Sviriaeva, Ekaterina N; Mitkin, Nikita A; Palmigiano, Angelo; Kruglov, Andrey A; Telegin, Georgy B; Drutskaya, Marina S; Sturiale, Luisa; Garozzo, Domenico; Nedospasov, Sergei A; Knirel, Yuriy A; Kuprash, Dmitry V

    2018-01-01

    Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni , the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

  8. 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

  9. Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

    OpenAIRE

    Zakharova, Ekaterina; Grandhi, Jaykumar; Wewers, Mark D.; Gavrilin, Mikhail A.

    2010-01-01

    Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 beta was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respective...

  10. 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.

  11. FimH adhesin of type 1 fimbriae is a potent inducer of innate antimicrobial responses which requires TLR4 and type 1 interferon signalling.

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    Ali A Ashkar

    2008-12-01

    Full Text Available Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs. We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-beta production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88(-/-, Trif(-/-, IFN-alpha/betaR(-/- or IRF3(-/- mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88(-/-, IFN-alpha/betaR(-/-, IRF3(-/- or TLR4(-/- mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-beta, but not IFN-alpha or IFN-gamma. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6 mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4(-/- mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.

  12. microRNA-124 negatively regulates TLR signaling in alveolar macrophages in response to mycobacterial infection.

    Science.gov (United States)

    Ma, Chunyan; Li, Yong; Li, Min; Deng, Guangcun; Wu, Xiaoling; Zeng, Jin; Hao, Xiujing; Wang, Xiaoping; Liu, Jing; Cho, William C S; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years; and the alveolar macrophages (AMs) are the main targets of mycobacterial infection, which play a pivotal role in the pathogenesis of Mycobacterium tuberculosis infection. However, the immunoregulatory role of miRNAs in AMs has not been fully demonstrated. In this study, we find that miR-124 is up-regulated in the peripheral leukocytes of patients with pulmonary tuberculosis; furthermore, the expression miR-124 can be induced upon Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection in both RAW264.7 AM cells in vitro and murine AMs in vivo. Mechanistically, miR-124 is able to modulate toll-like receptor (TLR) signaling activity in RAW264.7 cells in response to BCG infection. In this regard, multiple components of TLR signaling cascade, including the TLR6, myeloid differentiation factor 88 (MyD88), TNFR-associated factor 6 and tumor necrosis factor-α are directly targeted by miR-124. In addition, both overexpression of TLR signaling adaptor MyD88 and BCG infection are able to augment miR-124 transcription, while MyD88 expression silenced by small interfering RNA dramatically suppresses miR-124 expression in AMs in vitro. Moreover, the abundance of miR-124 transcript in murine AMs of MyD88 deficient mice is significantly less than that of their wild-type or heterozygous littermates; and the BCG infection fails to induce miR-124 expression in the lung of MyD88 deficient mouse. These results indicate a negative regulatory role of miR-124 in fine-tuning inflammatory response in AMs upon mycobacterial infection, in part through a mechanism by directly targeting TLR signaling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Mycoplasma suppression of THP-1 Cell TLR responses is corrected with antibiotics.

    Science.gov (United States)

    Zakharova, Ekaterina; Grandhi, Jaykumar; Wewers, Mark D; Gavrilin, Mikhail A

    2010-03-25

    Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 beta was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1beta and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.

  14. Mycoplasma suppression of THP-1 Cell TLR responses is corrected with antibiotics.

    Directory of Open Access Journals (Sweden)

    Ekaterina Zakharova

    2010-03-01

    Full Text Available Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 beta was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1beta and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003. Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.

  15. 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.

  16. Differential host response to LPS variants in amniochorion and the TLR4/MD-2 system in Macaca nemestrina

    Science.gov (United States)

    Chang, Justine; Jain, Sumita; Carl, David J.; Paolella, Louis; Darveau, Richard P.; Gravett, Michael G.; Waldorf, Kristina M. Adams

    2010-01-01

    OBJECTIVES Microbial-specific factors are likely critical in determining whether bacteria trigger preterm labor. Structural variations in lipopolysaccharide (LPS), a component of gram-negative bacteria, can determine whether LPS has an inflammatory (agonist) or anti-inflammatory (antagonist) effect through Toll-like receptor 4 (TLR4). Our objective was to determine whether amniochorion can discriminate between LPS variants in a nonhuman primate model. We also cloned Macaca nemestrina TLR4 and MD-2 and compared this complex functionally to the human homologue to establish whether nonhuman primates could be used to study TLR4 signaling in preterm birth. STUDY DESIGN Amniochorion explants from M. nemestrina were stimulated with a panel of LPS variants for 24 hours. Supernatants were analyzed for IL-1β, TNF-α, IL-6, IL-8 and prostaglandins E2 and F2α. Tissue expression of TLR1, 2, 4, 6, MyD88 and NF-kB was studied by RT-PCR. M. nemestrina TLR4 and MD2 genes were cloned and compared with their human counterparts in a recombinant TLR4 signaling system to determine LPS sensitivity. RESULTS LPS variants differentially stimulated cytokines and prostaglandins, which was not related to transcriptional changes of TLR4 or other TLRs. Nearly all elements of LPS binding and TLR4 leucine-rich repeats were conserved between humans and M. nemestrina. TLR4/MD-2 signaling complexes from both species were equally sensitive to LPS variants. CONCLUSIONS LPS variants elicit a hierarchical inflammatory response within amniochorion that may contribute to preterm birth. LPS sensitivity is similar between M. nemestrina and humans, validating M. nemestrina as an appropriate model to study TLR4 signaling in preterm birth. PMID:20619890

  17. Subcellular Localization of Large Yellow Croaker ( Larimichthys crocea) TLR21 and Expression Profiling of Its Gene in Immune Response

    Science.gov (United States)

    Sun, Qingxue; Fan, Zejun; Yao, Cuiluan

    2018-04-01

    Toll-like receptor 21 (TLR21) is a non-mammalian type TLR, and plays an important role in innate immune response in fish. In this paper, the full-length cDNA sequence of TLR21 gene was identified and characterized from large yellow croaker, Larimichthys crocea and was termed as LcTLR21. It consists of 3365 bp, including a 5'-terminal untranslated region (UTR) of 97 bp, a 3'-terminal UTR of 331 bp, and an open reading frame (ORF) of 2937 bp encoding a polypeptide of 978 amino acid residues. The deduced LcTLR21 contains a signal peptide domain at N-terminal, 12 leucine-rich repeats (LRRs) at the extracellular region, a transmembrane domain and a cytoplasmic toll-interleukin-1 receptor (TIR) domain at the C-terminal. Subcellular localization analysis revealed that the LcTLR21-GFP was constitutively expressed in cytoplasm. Tissue expression analysis indicated that LcTLR21 gene broadly expressed in most of the examined tissues, with the most predominant abundance in spleen, followed by head-kidney and liver, while the weakest expression was detected in brain. The expression level of LcTLR21 after LPS, poly I:C and Vibrio parahaemolyticus challenges was investigated in spleen, head-kidney and liver. LcTLR21 gene transcripts increased significantly in all examined tissues after the challenges, and the highest expression level was detected in liver at 24 h after poly I:C stimulation ( P < 0.05), suggesting that LcTLR21 might play a crucial role in fish resistance to viral and bacterial infections.

  18. Enhancement of LPS-Induced Microglial Inflammation Response via TLR4 Under High Glucose Conditions

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    Xiang Zhang

    2015-03-01

    Full Text Available Background: Microglia activation mediated by toll-like receptor 4 (TLR4 plays an important role in neuroinflammation and postoperative cognitive dysfunction (POCD. Diabetes mellitus (DM has been recently suggested as an independent risk factor for POCD. In this study, we investigate the potential exacerbation of the inflammatory response in primary microglia due to high glucose conditions. Methods: Primary microglial cells were exposed to normal glucose (25 mmol/L and high glucose (35 mmol/L levels alone or with lipopolyscaccharide (LPS 0, 2, 5, 10 ng/mL. The pro-inflammatory response of the cells was assessed by measuring changes in cytokine levels and the evaluation of associated signaling pathways. Results: Neither high glucose nor low LPS (≤5ng/ml alone had an effect on TNF-a and IL-6 levels, but the combination of low LPS and high glucose stimulated the inflammatory response. Analyses of the associated signaling pathways demonstrated that high glucose enhanced the LPS-induced microglial activation via the TLR4/JAK2/STAT3 pathway. Conclusion: This study demonstrates that high glucose, one of the key abnormalities characteristic of DM, can augment LPS-induced microglial activation and inflammatory cytokine levels through the TLR4/JAK2/STAT3 pathway, offering new insight into the pathophysiological relationship between DM and POCD.

  19. 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.

  20. The scavenger receptor MARCO modulates TLR-induced responses in dendritic cells.

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    Haydn T Kissick

    Full Text Available The scavenger receptor MARCO mediates macrophage recognition and clearance of pathogens and their polyanionic ligands. However, recent studies demonstrate MARCO expression and function in dendritic cells, suggesting MARCO might serve to bridge innate and adaptive immunity. To gain additional insight into the role of MARCO in dendritic cell activation and function, we profiled transcriptomes of mouse splenic dendritic cells obtained from MARCO deficient mice and their wild type counterparts under resting and activating conditions. In silico analysis uncovered major alterations in gene expression in MARCO deficient dendritic cells resulting in dramatic alterations in key dendritic cell-specific pathways and functions. Specifically, changes in CD209, FCGR4 and Complement factors can have major consequences on DC-mediated innate responses. Notably, these perturbations were magnified following activation with the TLR-4 agonist lipopolysaccharide. To validate our in silico data, we challenged DC's with various agonists that recognize all mouse TLRs and assessed expression of a set of immune and inflammatory marker genes. This approach identified a differential contribution of MARCO to TLR activation and validated a major role for MARCO in mounting an inflammatory response. Together, our data demonstrate that MARCO differentially affects TLR-induced DC activation and suggest targeting of MARCO could lead to different outcomes that depend on the inflammatory context encountered by DC.

  1. Role of TLR4 polymorphisms in inflammatory responses: implications for unsuccessful aging.

    Science.gov (United States)

    Balistreri, Carmela Rita; Candore, Giuseppina; Listì, Florinda; Fazio, Teresa; Gangi, Simona; Incalcaterra, Egle; Caruso, Marco; Vecchi, Maurizio Li; Lio, Domenico; Caruso, Calogero

    2007-11-01

    The total burden of infection at various sites may affect the progression of atherosclerosis and Alzheimer's disease (AD), the risk being modulated by host genotype. The role of lipopolysaccharide (LPS) receptor TLR4 is paradigmatic. It initiates the innate immune response against gram-negative bacteria, and TLR4 single nucleotide polymorphisms (SNPs), such as +896A/G, known to attenuate receptor signaling, have been described. This SNP shows a significantly lower frequency in patients affected by myocardial infarction or AD. Thus, people genetically predisposed to developing lower inflammatory activity seem to have less chance of developing cardiovascular disease (CVD) or AD. In the present report, to validate this hypothesis, the levels of the eicosanoids, leukotriene B4 (LTB4) and prostaglandin E2 (PGE2), known to be involved as mediators in age-related diseases, were determined by an enzyme-linked immunosorbent assay in supernatants from a whole blood assay, after stimulation with subliminal doses of LPS from Escherichia coli. The samples, genotyped for the +896A/G SNP, were challenged with LPS for 4, 24, and 48 h. Both LTB4 and PGE2 values were significantly lower in carriers bearing the TLR4 mutation. Therefore, the pathogen burden, by interacting with the host genotype, determines the type and intensity of the inflammatory responses accountable for proinflammatory status, CVD, AD, and unsuccessful aging (i.e., age-related inflammatory diseases).

  2. Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

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    Yun Jeong Kim

    Full Text Available Botulinum neurotoxin type A (BoNT/A is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO and tumor necrosis factor alpha (TNFα were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2 and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK, extracellular signal-regulated kinase (ERK, and p38 mitogen-activated protein kinase (MAPK. BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK.

  3. Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung.

    Science.gov (United States)

    Fonceca, Angela M; Zosky, Graeme R; Bozanich, Elizabeth M; Sutanto, Erika N; Kicic, Anthony; McNamara, Paul S; Knight, Darryl A; Sly, Peter D; Turner, Debra J; Stick, Stephen M

    2018-01-22

    Accumulation mode particles (AMP) are formed from engine combustion and make up the inhalable vapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposure far from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung when inhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negative bacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS in ambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure. This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airways disease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor 4 (TLR-4). The effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatory infiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4. The presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity via TLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independent of TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those induced by LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression. These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.

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

    OpenAIRE

    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 TLR...

  5. 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.

  6. CpG oligodeoxynucleotide-specific goose TLR21 initiates an anti-viral immune response against NGVEV but not AIV strain H9N2 infection.

    Science.gov (United States)

    Qi, Yulin; Yan, Bing; Chen, Shun; Chen, Hongjun; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Yang, Qiao; Sun, Kunfeng; Wu, Ying; Chen, Xiaoyue; Jing, Bo; Cheng, Anchun

    2016-03-01

    Toll-like receptors (TLRs) recognize components of pathogens and mediate the host innate immune response. TLR21 is a TLR that specifically recognizes exogenous double-stranded DNA and rapidly signals to downstream innate immune factors. This study reports the cDNA of goose TLR21 and identifies its immune characteristics. The goose TLR21 is 3161 base pairs and encodes a 975 amino acid protein. As predicted, the goose transmembrane protein TLR21 has a signal peptide, leucine-rich repeat regions, a transmembrane domain, and a Toll/interleukin-1 receptor domain. Multiple sequence alignments and phylogenetic analyses showed that goose TLR21 has homology to chicken TLR21. The tissue distribution of TLR21 suggested that it has high transcript levels in immune-associated tissues, especially in the bursa of Fabricius, the Hadrian gland, and the thymus. After challenge with agonist ODN2006 and new type gosling viral enteritis virus (NGVEV), significant induction of TLR21 production, pro-inflammatory cytokines IL-1β and IL-6, and interferons were observed in peripheral blood mononuclear cells. Both synthetic DNA (ODN2006) and viral DNA (NGVEV) can be recognized by goose TLR21, which leads to a rapid up-regulation of pro-inflammatory cytokines and anti-viral molecules. In vivo, avian influenza A virus H9N2 and NGVEV were used to infect goslings, which was followed by a significant up-regulation of TLR21 mRNA transcripts in multiple tissues of NGVEV-infected geese. In general, goose TLR21 plays an important role in binding invading pathogenic DNA viruses, which subsequently triggers an innate immune response; furthermore, it acts as a functional homologue of mammalian TLR9, as TLR21 recognizes a mammalian TLR9 agonist. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. 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.

  8. Retinal photoreceptor expresses toll-like receptors (TLRs and elicits innate responses following TLR ligand and bacterial challenge.

    Directory of Open Access Journals (Sweden)

    Pawan Kumar Singh

    Full Text Available Toll-like receptors (TLRs play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W; and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1β, MIP-2, and KC in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus. Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity.

  9. Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway.

    Science.gov (United States)

    Marshall, Nikki B; Lukomska, Ewa; Nayak, Ajay P; Long, Carrie M; Hettick, Justin M; Anderson, Stacey E

    2017-12-01

    The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfα, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (T H 2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy.

  10. Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

    Science.gov (United States)

    Cheng, Liang; Zhang, Zheng; Li, Guangming; Li, Feng; Wang, Li; Zhang, Liguo; Zurawski, Sandra M; Zurawski, Gerard; Levy, Yves; Su, Lishan

    2017-10-27

    TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4 + T cell response, while only R848 and Poly I:C induced CD8 + cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Lipid motif of a bacterial antigen mediates immune responses via TLR2 signaling.

    Directory of Open Access Journals (Sweden)

    Amit A Lugade

    Full Text Available The cross-talk between the innate and the adaptive immune system is facilitated by the initial interaction of antigen with dendritic cells. As DCs express a large array of TLRs, evidence has accumulated that engagement of these molecules contributes to the activation of adaptive immunity. We have evaluated the immunostimulatory role of the highly-conserved outer membrane lipoprotein P6 from non-typeable Haemophilus influenzae (NTHI to determine whether the presence of the lipid motif plays a critical role on its immunogenicity. We undertook a systematic analysis of the role that the lipid motif plays in the activation of DCs and the subsequent stimulation of antigen-specific T and B cells. To facilitate our studies, recombinant P6 protein that lacked the lipid motif was generated. Mice immunized with non-lipidated rP6 were unable to elicit high titers of anti-P6 Ig. Expression of the lipid motif on P6 was also required for proliferation and cytokine secretion by antigen-specific T cells. Upregulation of T cell costimulatory molecules was abrogated in DCs exposed to non-lipidated rP6 and in TLR2(-/- DCs exposed to native P6, thereby resulting in diminished adaptive immune responses. Absence of either the lipid motif on the antigen or TLR2 expression resulted in diminished cytokine production from stimulated DCs. Collectively, our data suggest that the lipid motif of the lipoprotein antigen is essential for triggering TLR2 signaling and effective stimulation of APCs. Our studies establish the pivotal role of a bacterial lipid motif on activating both innate and adaptive immune responses to an otherwise poorly immunogenic protein antigen.

  12. RO 90-7501 enhances TLR3 and RLR agonist induced antiviral response.

    Directory of Open Access Journals (Sweden)

    Fang Guo

    Full Text Available Recognition of virus infection by innate pattern recognition receptors (PRRs, including membrane-associated toll-like receptors (TLR and cytoplasmic RIG-I-like receptors (RLR, activates cascades of signal transduction pathways leading to production of type I interferons (IFN and proinflammatory cytokines that orchestrate the elimination of the viruses. Although it has been demonstrated that PRR-mediated innate immunity plays an essential role in defending virus from infection, it also occasionally results in overwhelming production of proinflammatory cytokines that cause severe inflammation, blood vessel leakage and tissue damage. In our efforts to identify small molecules that selectively enhance PRR-mediated antiviral, but not the detrimental inflammatory response, we discovered a compound, RO 90-7501 ('2'-(4-Aminophenyl-[2,5'-bi-1H-benzimidazol]-5-amine, that significantly promoted both TLR3 and RLR ligand-induced IFN-β gene expression and antiviral response, most likely via selective activation of p38 mitogen-activated protein kinase (MAPK pathway. Our results thus imply that pharmacological modulation of PRR signal transduction pathways in favor of the induction of a beneficial antiviral response can be a novel therapeutic strategy.

  13. 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.

  14. Synergic activation of toll-like receptor (TLR 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Martha Triantafilou

    Full Text Available Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM, preterm labour (PL pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR. In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA, trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

  15. 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

  16. 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.

  17. Rhinovirus attenuates non-typeable Hemophilus influenzae-stimulated IL-8 responses via TLR2-dependent degradation of IRAK-1.

    Directory of Open Access Journals (Sweden)

    Benjamin L Unger

    Full Text Available Bacterial infections following rhinovirus (RV, a common cold virus, are well documented, but pathogenic mechanisms are poorly understood. We developed animal and cell culture models to examine the effects of RV on subsequent infection with non-typeable Hemophilus influenzae (NTHi. We focused on NTHI-induced neutrophil chemoattractants expression that is essential for bacterial clearance. Mice infected with RV1B were superinfected with NTHi and lung bacterial density, chemokines and neutrophil counts determined. Human bronchial epithelial cells (BEAS-2B or mouse alveolar macrophages (MH-S were infected with RV and challenged with NHTi, TLR2 or TLR5 agonists. Chemokine levels were measured by ELISA and expression of IRAK-1, a component of MyD88-dependent TLR signaling, assessed by immunoblotting. While sham-infected mice cleared all NTHi from the lungs, RV-infected mice showed bacteria up to 72 h post-infection. However, animals in RV/NTHi cleared bacteria by day 7. Delayed bacterial clearance in RV/NTHi animals was associated with suppressed chemokine levels and neutrophil recruitment. RV-infected BEAS-2B and MH-S cells showed attenuated chemokine production after challenge with either NTHi or TLR agonists. Attenuated chemokine responses were associated with IRAK-1 protein degradation. Inhibition of RV-induced IRAK-1 degradation restored NTHi-stimulated IL-8 expression. Knockdown of TLR2, but not other MyD88-dependent TLRs, also restored IRAK-1, suggesting that TLR2 is required for RV-induced IRAK-1 degradation.In conclusion, we demonstrate for the first time that RV infection delays bacterial clearance in vivo and suppresses NTHi-stimulated chemokine responses via degradation of IRAK-1. Based on these observations, we speculate that modulation of TLR-dependent innate immune responses by RV may predispose the host to secondary bacterial infection, particularly in patients with underlying chronic respiratory disorders.

  18. CETP Lowers TLR4 Expression Which Attenuates the Inflammatory Response Induced by LPS and Polymicrobial Sepsis

    Directory of Open Access Journals (Sweden)

    Tatiana Martins Venancio

    2016-01-01

    Full Text Available Sepsis is a systemic inflammatory response to infection eliciting high mortality rate which is a serious health problem. Despite numerous studies seeking for therapeutic alternatives, the mechanisms involved in this disease remain elusive. In this study we evaluated the influence of cholesteryl ester transfer protein (CETP, a glycoprotein that promotes the transfer of lipids between lipoproteins, on the inflammatory response in mice. Human CETP transgenic mice were compared to control mice (wild type, WT after polymicrobial sepsis induced by cecal ligation and puncture (CLP, aiming at investigating their survival rate and inflammatory profiles. Macrophages from the peritoneal cavity were stimulated with LPS in the presence or absence of recombinant CETP for phenotypic and functional studies. In comparison to WT mice, CETP mice showed higher survival rate, lower IL-6 plasma concentration, and decreased liver toll-like receptor 4 (TLR4 and acyloxyacyl hydrolase (AOAH protein. Moreover, macrophages from WT mice to which recombinant human CETP was added decreased LPS uptake, TLR4 expression, NF-κB activation and IL-6 secretion. This raises the possibility for new therapeutic tools in sepsis while suggesting that lowering CETP by pharmacological inhibitors should be inconvenient in the context of sepsis and infectious diseases.

  19. Molecular characterization and expression analysis of three TLR genes in yellow catfish (Pelteobagrus fulvidraco): Responses to stimulation of Aeromonas hydrophila and TLR ligands.

    Science.gov (United States)

    Wang, Kai-Lun; Ji, Wei; Zhang, Gui-Rong; Wei, Kai-Jian; Shi, Ze-Chao; Zhang, Xiao-Ting; Zheng, Huan; Fan, Qi-Xue

    2017-07-01

    Toll-like receptors (TLRs) are one of the most extensively researched pattern recognition receptors (PRRs) and play an important role in the innate immune system. In this study, partial cDNA sequences of the Pf_TLR18 and Pf_TLR19 genes and complete cDNA sequence of the Pf_TLR21 gene were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORFs) of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes were 1956 bp, 2262 bp and 2949 bp in length, encoding 651, 753 and 982 amino acids, respectively. The Pf_TLR18 and Pf_TLR19 consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor domain, and the Pf_TLR21 only has LRRs and TIR domain. Homologous identity revealed that the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes have high nucleotide and protein sequence similarity with channel catfish, especially the TIR domains that exhibited the greatest conservation compared to channel catfish. Ontogenetic expression analyses indicated that the mRNA expressions of the Pf_TLR18, Pf_TLR19 and Pf_TLR21 genes could be detected from fertilized eggs to 30 day post-hatching and they exhibited different variation trends after hatching. The three TLR genes were expressed in various tissues, but they were mostly highly expressed in the spleen. The mRNA expression levels of the three genes were up-regulated in the spleen, head kidney, trunk kidney, liver and blood after challenge of killed Aeromonas hydrophila. In addition, the expressions of the three TLR genes were induced to up-regulate in isolated peripheral blood lymphocytes of yellow catfish after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). Our findings indicate that the three TLR genes may play a potential role in the host defense against pathogenic microbes. These results will provide valuable information to better understand the function of TLR genes in the innate immune system of yellow catfish. Copyright © 2017

  20. 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

  1. Engagement of Toll-like receptors by mycoplasmal superantigen: downregulation of TLR2 by MAM/TLR4 interaction.

    Science.gov (United States)

    Mu, H-H; Pennock, N D; Humphreys, J; Kirschning, C J; Cole, B C

    2005-06-01

    Mycoplasma arthritidis mitogen (MAM) is a superantigen (SAg) from M. arthritidis, an agent of murine toxic shock syndrome and arthritis. We previously demonstrated that C3H/HeJ and C3H/HeSnJ mice that differ in expression of TLR4 differed in immune reactivity to MAM. We show here that MAM directly interacts with TLR2 and TLR4 by using monoclonal antibodies to TLR2 and TLR4 which inhibit cytokine responses of THP-1 cells to MAM. Also, using macrophages from C3H substrains and TLR2-deficient mice, we confirmed that both TLR2 and TLR4 are used by MAM. Levels of IL-6 in supernatants of MAM-challenged macrophages were higher in mice which expressed only TLR2, lesser with both TLR2 and TLR4, and absent in mice lacking both TLR2 and TLR4. In addition, expression of TLR2 and TLR4 was moderately upregulated in wild-type cells but cells lacking TLR4 showed a fivefold increase in TLR2 expression. Further, blockade of TLR4 on macrophages of C3H/HeN mice with antibody greatly increased expression of TLR2 and release of IL-12p40 in response to MAM. These results indicate that the SAg, MAM, interacts with both TLR2 and TLR4 and that TLR4 signalling might downregulate the MAM/TLR2 inflammatory response.

  2. HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

    Directory of Open Access Journals (Sweden)

    Olga Moreno-Gonzalo

    2017-12-01

    Full Text Available Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR. Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88, and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.

  3. Increased responsiveness of peripheral blood mononuclear cells to in vitro TLR 2, 4 and 7 ligand stimulation in chronic pain patients.

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    Yuen H Kwok

    Full Text Available Glial activation via Toll-like receptor (TLR signaling has been shown in animals to play an important role in the initiation and establishment of chronic pain. However, our ability to assess this central immune reactivity in clinical pain populations is currently lacking. Peripheral blood mononuclear cells (PBMCs are an accessible source of TLR expressing cells that may mirror similarities in TLR responsiveness of the central nervous system. The aim of this study was to characterize the IL-1β response to various TLR agonists in isolated PBMCs from chronic pain sufferers (on and not on opioids and pain-free controls. Venous blood was collected from 11 chronic pain sufferers on opioids (≥ 20 mg of morphine / day, 8 chronic pain sufferers not on opioids and 11 pain-free controls. PBMCs were isolated and stimulated in vitro with a TLR2 (Pam3CSK4, TLR4 (LPS or TLR7 (imiquimod agonist. IL-1β released into the supernatant was measured with ELISA. Significantly increased IL-1β expression was found in PBMCs from chronic pain sufferers (on and not on opioids compared with pain-free controls for TLR2 (F((6, 277 = 15, P<0.0001, TLR4 (F((8, 263 = 3, P = 0.002 and TLR7 (F((2,201 = 5, P = 0.005 agonists. These data demonstrate that PBMCs from chronic pain sufferers were more responsive to TLR agonists compared with controls, suggesting peripheral cells may have the potential to become a source of biomarkers for chronic pain.

  4. CNS cell-type localization and LPS response of TLR signaling pathways [version 1; referees: 2 approved

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    Gizelle M. McCarthy

    2017-07-01

    Full Text Available Background: Innate immune signaling in the brain has emerged as a contributor to many central nervous system (CNS pathologies, including mood disorders, neurodegenerative disorders, neurodevelopmental disorders, and addiction. Toll-like receptors (TLRs, a key component of the innate immune response, are particularly implicated in neuroimmune dysfunction. However, most of our understanding about TLR signaling comes from the peripheral immune response, and it is becoming clear that the CNS immune response is unique. One controversial aspect of neuroimmune signaling is which CNS cell types are involved. While microglia are the CNS cell-type derived from a myeloid lineage, studies suggest that other glial cell types and even neurons express TLRs, although this idea is controversial. Furthermore, recent work suggests a discrepancy between RNA and protein expression within the CNS. Methods: To elucidate the CNS cell-type localization of TLRs and their downstream signaling molecules, we isolated microglia and astrocytes from the brain of adult mice treated with saline or the TLR4 ligand lipopolysaccharide (LPS. Glial mRNA and protein expression was compared to a cellular-admixture to determine cell-type enrichment. Results: Enrichment analysis revealed that most of the TLR pathway genes are localized in microglia and changed in microglia following immune challenge. However, expression of Tlr3 was enriched in astrocytes, where it increased in response to LPS. Furthermore, attempts to determine protein cell-type localization revealed that many antibodies are non-specific and that antibody differences are contributing to conflicting localization results. Conclusions: Together these results highlight the cell types that should be looked at when studying TLR signaling gene expression and suggest that non-antibody approaches need to be used to accurately evaluate protein expression.

  5. The natural product phyllanthusmin C enhances IFN-γ production by human NK cells through upregulation of TLR-mediated NF-κB signaling.

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    Deng, Youcai; Chu, Jianhong; Ren, Yulin; Fan, Zhijin; Ji, Xiaotian; Mundy-Bosse, Bethany; Yuan, Shunzong; Hughes, Tiffany; Zhang, Jianying; Cheema, Baljash; Camardo, Andrew T; Xia, Yong; Wu, Lai-Chu; Wang, Li-Shu; He, Xiaoming; Kinghorn, A Douglas; Li, Xiaohui; Caligiuri, Michael A; Yu, Jianhua

    2014-09-15

    Natural products are a major source for cancer drug development. NK cells are a critical component of innate immunity with the capacity to destroy cancer cells, cancer-initiating cells, and clear viral infections. However, few reports describe a natural product that stimulates NK cell IFN-γ production and unravel a mechanism of action. In this study, through screening, we found that a natural product, phyllanthusmin C (PL-C), alone enhanced IFN-γ production by human NK cells. PL-C also synergized with IL-12, even at the low cytokine concentration of 0.1 ng/ml, and stimulated IFN-γ production in both human CD56(bright) and CD56(dim) NK cell subsets. Mechanistically, TLR1 and/or TLR6 mediated PL-C's activation of the NF-κB p65 subunit that in turn bound to the proximal promoter of IFNG and subsequently resulted in increased IFN-γ production in NK cells. However, IL-12 and IL-15Rs and their related STAT signaling pathways were not responsible for the enhanced IFN-γ secretion by PL-C. PL-C induced little or no T cell IFN-γ production or NK cell cytotoxicity. Collectively, we identify a natural product with the capacity to selectively enhance human NK cell IFN-γ production. Given the role of IFN-γ in immune surveillance, additional studies to understand the role of this natural product in prevention of cancer or infection in select populations are warranted. Copyright © 2014 by The American Association of Immunologists, Inc.

  6. Increased TLR responses in dendritic cells lacking the ITAM-containing adapters DAP12 and FcRγ

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    Chu, Ching-Liang; Yu, Yen-Ling; Shen, Kuan-Yin; Lowell, Clifford A.; Lanier, Lewis L.; Hamerman, Jessica A.

    2008-01-01

    The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12-deficient mice. In this report, we demonstrate that both DAP12 and the FcεRIγ-chain (FcRγ) are required for negative regulation of TLR responses in bone marrow-derived dendritic cells (DC). Loss of both DAP12 and FcRγ enhanced the pro-inflammatory cytokine production and maturation of DC after TLR stimulation, resulting in a greater percentage of DC that produced IL-12 p40, TNF, and IL-6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DC lacking only DAP12 showed some increased TLR responses, those lacking only FcRγ had a greater enhancement of maturation and cytokine production, though to a lesser extent than DC lacking both DAP12 and FcRγ. Additionally, antigen-specific T cell proliferation was enhanced by DAP12−/−FcRγ−/− DC relative to wild-type DC after maturation. Similar to DAP12−/−FcRγ−/− DC, Syk-deficient DC also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of immunoreceptor tyrosine-based activation motif (ITAM) signaling in myeloid cells and show that DC and macrophages differ in their dependence on the ITAM-containing adapters DAP12 and FcRγ for negative regulation of TLR signaling. PMID:18081038

  7. TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders.

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    Pascual, María; Montesinos, Jorge; Montagud-Romero, Sandra; Forteza, Jerónimo; Rodríguez-Arias, Marta; Miñarro, José; Guerri, Consuelo

    2017-07-24

    Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal sera and brains of fetuses/offspring, which impairs brain ontogeny and causes cognitive dysfunction. WT and TLR4-KO female mice treated with or without 10% ethanol in the drinking water during gestation and lactation were used. Cytokine/chemokine levels were determined by ELISA in the amniotic fluid, maternal serum, and cerebral cortex, as well as in the offspring cerebral cortex. Microglial and neuronal markers (evaluated by western blotting), myelin proteins (immunohistochemical and western blotting) and synaptic parameters (western blotting and electron microscopy) were assessed in the cortices of the WT and TLR4-KO pups on PND 0, 20, and 66. Behavioral tests (elevated plus maze and passive avoidance) were performed in the WT and TLR4-KO mice on PND 66 exposed or not to ethanol. We show that alcohol intake during gestation and lactation increases the levels of several cytokines/chemokines (IL-1β, IL-17, MIP-1α, and fractalkine) in the maternal sera, amniotic fluid, and brains of fetuses and offspring. The upregulation of cytokines/chemokines is associated with an increase in activated microglia markers (CD11b and MHC-II), and with a reduction in some synaptic (synaptotagmin, synapsin IIa) and myelin (MBP, PLP) proteins in the brains of offspring on days 0, 20, and 66 (long-term effects

  8. Chicken Immune Response after In Ovo Immunization with Chimeric TLR5 Activating Flagellin of Campylobacter jejuni.

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    Katarzyna A Radomska

    Full Text Available Campylobacter jejuni is the main cause of bacterial food-borne diseases in developed countries. Chickens are the most important source of human infection. Vaccination of poultry is an attractive strategy to reduce the number of C. jejuni in the intestinal tract of chickens. We investigated the immunogenicity and protective efficacy of a recombinant C. jejuni flagellin-based subunit vaccine with intrinsic adjuvant activity. Toll-like receptor activation assays demonstrated the purity and TLR5 stimulating (adjuvant activity of the vaccine. The antigen (20-40 μg was administered in ovo to 18 day-old chicken embryos. Serum samples and intestinal content were assessed for antigen-specific systemic and mucosal humoral immune responses. In ovo vaccination resulted in the successful generation of IgY and IgM serum antibodies against the flagellin-based subunit vaccine as determined by ELISA and Western blotting. Vaccination did not induce significant amounts of flagellin-specific secretory IgA in the chicken intestine. Challenge of chickens with C. jejuni yielded similar intestinal colonization levels for vaccinated and control animals. Our results indicate that in ovo delivery of recombinant C. jejuni flagellin subunit vaccine is a feasible approach to yield a systemic humoral immune response in chickens but that a mucosal immune response may be needed to reduce C. jejuni colonization.

  9. TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis.

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    Mu, Hong-Hua; Humphreys, Jennifer; Chan, Fok Vun; Cole, Barry C

    2006-03-01

    Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.

  10. Genetic drift outweighs natural selection at toll-like receptor (TLR) immunity loci in a re-introduced population of a threatened species.

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    Grueber, Catherine E; Wallis, Graham P; Jamieson, Ian G

    2013-09-01

    During population establishment, genetic drift can be the key driver of changes in genetic diversity, particularly while the population is small. However, natural selection can also play a role in shaping diversity at functionally important loci. We used a well-studied, re-introduced population of the threatened Stewart Island robin (N = 722 pedigreed individuals) to determine whether selection shaped genetic diversity at innate immunity toll-like receptor (TLR) genes, over a 9-year period of population growth following establishment with 12 genetic founders. We found no evidence for selection operating with respect to TLR diversity on first-year overwinter survival for the majority of loci, genotypes and alleles studied. However, survival of individuals with TLR4BE genotype was significantly improved: these birds were less than half as likely to die prior to maturity compared with all other TLR4 genotypes. Furthermore, the population frequency of this genotype, at a two-fold excess over Hardy-Weinberg expectation, was increased by nonrandom mating. Near-complete sampling and full pedigree and reproductive data enabled us to eliminate other potential causes of these patterns including inbreeding, year effects, density dependence, selection on animals at earlier life history stages or genome-level association of the TLR4E allele with 'good genes'. However, comparison of observed levels of gene diversity to predictions under simulated genetic drift revealed results consistent with neutral expectations for all loci, including TLR4. Although selection favoured TLR4BE heterozygotes in this population, these effects were insufficient to outweigh genetic drift. This is the first empirical study to show that genetic drift can overwhelm natural selection in a wild population immediately following establishment. © 2013 John Wiley & Sons Ltd.

  11. Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors

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    Belderbos, Mirjam E.; Levy, Ofer; Stalpers, Femke; Kimpen, Jan L.; Meyaard, Linde; Bont, Louis

    2012-01-01

    Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection. PMID:22442690

  12. Neonatal plasma polarizes TLR4-mediated cytokine responses towards low IL-12p70 and high IL-10 production via distinct factors.

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    Mirjam E Belderbos

    Full Text Available Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP or soluble CD14 (sCD14. The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection.

  13. Proteomic Profiling of Iron Overload-Induced Human Hepatic Cells Reveals Activation of TLR2-Mediated Inflammatory Response

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    Xiang Li

    2016-03-01

    Full Text Available Background: Hepatic iron overload is common in patients who have undergone hematopoietic cell transplantation (HCT and may predispose to peri- and post-HCT toxicity. To better reveal more molecules that might be involved in iron overload-induced liver injury, we utilized proteomics to investigate differentially expressed proteins in iron overload-induced hepatocytes vs. untreated hepatocytes. Methods and Results: HH4 hepatocytes were exposed to ferric ammonium citrate (FAC to establish an in vitro iron overload model. Differentially expressed proteins initiated by the iron overload were studied by two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS analysis. We identified 93 proteins whose quantity statistically significantly changes under excess hepatocyte iron conditions. Gene Ontology (GO analysis showed that these differentially expressed proteins in HH4 cells are involved in various biological process including endocytosis, response to wounding, di-, trivalent inorganic cation homeostasis, inflammatory response, positive regulation of cytokine production, and etc. Meanwhile, proteomics data revealed protein level of TLR2 and IL6ST significantly increased 7 times and 2.9 times, respectively, in iron overloaded HH4 cells. Our subsequent experiments detected that FAC-treated HH4 cells can activate IL6 expression through TLR2-mediated inflammatory responses via the NF-κB pathway. Conclusions: In this study, we demonstrated that iron overload induced hepatocytes triggering TLR2-mediated inflammatory response via NF-κB signaling pathway in HH4 cells.

  14. Importance of TLR2 on hepatic immune and non-immune cells to attenuate the strong inflammatory liver response during Trypanosoma cruzi acute infection.

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    Eugenio Antonio Carrera-Silva

    Full Text Available BACKGROUND: Toll-like receptors (TLR and cytokines play a central role in the pathogen clearance as well as in pathological processes. Recently, we reported that TLR2, TLR4 and TLR9 are differentially modulated in injured livers from BALB/c and C57BL/6 (B6 mice during Trypanosoma cruzi infection. However, the molecular and cellular mechanisms involved in local immune response remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate that hepatic leukocytes from infected B6 mice produced higher amounts of pro-inflammatory cytokines than BALB/c mice, whereas IL10 and TGFβ were only released by hepatic leukocytes from BALB/c. Strikingly, a higher expression of TLR2 and TLR4 was observed in hepatocytes of infected BALB/c mice. However, in infected B6 mice, the strong pro-inflammatory response was associated with a high and sustained expression of TLR9 and iNOS in leukocytes and hepatic tissue respectively. Additionally, co-expression of gp91- and p47-phox NADPH oxidase subunits were detected in liver tissue of infected B6 mice. Notably, the pre-treatment previous to infection with Pam3CSK4, TLR2-agonist, induced a significant reduction of transaminase activity levels and inflammatory foci number in livers of infected B6 mice. Moreover, lower pro-inflammatory cytokines and increased TGFβ levels were detected in purified hepatic leukocytes from TLR2-agonist pre-treated B6 mice. CONCLUSIONS/SIGNIFICANCE: Our results describe some of the main injurious signals involved in liver immune response during the T. cruzi acute infection. Additionally we show that the administration of Pam3CSk4, previous to infection, can attenuate the exacerbated inflammatory response of livers in B6 mice. These results could be useful to understand and design novel immune strategies in controlling liver pathologies.

  15. Chronic lymphocytic leukemia cells acquire regulatory B-cell properties in response to TLR9 and CD40 activation.

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    Ringelstein-Harlev, Shimrit; Avivi, Irit; Fanadka, Mona; Horowitz, Netanel A; Katz, Tami

    2018-02-15

    Circulating chronic lymphocytic leukemia (CLL) cells share phenotypic features with certain subsets of regulatory B-cells (Bregs). The latter cells have been reported to negatively regulate immune cell responses, mostly by provision of IL-10. The purpose of the current study was to identify and delineate Breg properties of CLL cells. B-cells and T-cells were obtained from the peripheral blood of untreated CLL patients diagnosed according to the 2008 Guidelines of the International Workshop on Chronic Lymphocytic Leukemia. Co-culture assays were used to examine the ability of CLL cells to suppress autologous T-cell immune responses. IL-10 potency of CLL cells was assessed following stimulation with activators of the toll-like receptor 9 (TLR9) or CD40 and was correlated with the inhibitory activity of the cells. TLR9-activated CLL cells were found to increase the frequency of CD4 + CD25 hi FOXp3 + regulatory T-cells (Tregs) and to inhibit autologous CD4 + T-cell proliferation. This signaling cascade proved to control IL-10 generation in CLL cells, which in turn promoted the inhibition of T-cell proliferation by CLL cells. However, CD40 activation of CLL cells, while exhibiting a similar ability to augment Treg frequency, did not either affect IL-10 generation or T-cell proliferation. In conclusion, CLL cells demonstrate a unique clonal quality of adopting Breg properties which promote modulation of T-cell characteristics. TLR9 appears to be a potent activator of regulatory abilities in CLL cells, possibly contributing to preferential immune escape of TLR9-responsive cells.

  16. Roles of TLR3 and RIG-I in Mediating the Inflammatory Response in Mouse Microglia following Japanese Encephalitis Virus Infection

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    Rong Jiang

    2014-01-01

    Full Text Available Japanese encephalitis virus (JEV infection can cause central nervous system disease with irreversible neurological damage in humans and animals. Evidence suggests that overactivation of microglia leads to greatly increased neuronal damage during JEV infection. However, the mechanism by which JEV induces the activation of microglia remains unclear. Toll-like receptor 3 (TLR3 and retinoic acid-inducible gene I (RIG-I can recognize double-stranded RNA, and their downstream signaling results in production of proinflammatory mediators. In this study, we investigated the roles of TLR3 and RIG-I in the inflammatory response caused by JEV infection in the mouse microglial cell line. JEV infection induced the expression of TLR3 and RIG-I and the activation of extracellular signal-regulated kinase (ERK and p38 mitogen-activated protein kinase (p38MAPK. Knockdown of TLR3 and RIG-I attenuated activation of ERK, p38MAPK, activator protein 1 (AP-1, and nuclear factor κB (NF-κB. Secretion of TNF-α, IL-6, and CCL-2, which was induced by JEV, was reduced by TLR3 and RIG-I knockdown and inhibitors of phosphorylated ERK and p38MAPK. Furthermore, viral proliferation was increased following knockdown of TLR3 and RIG-I. Our findings suggest that the signaling pathways of TLR3 and RIG-I play important roles in the JEV-induced inflammatory response of microglia.

  17. Infant anemia is associated with reduced TLR-stimulated cytokine responses and increased nasopharyngeal colonization with Moxarella catarrhalis.

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    Liao, Sui-Ling; Hsu, Shih-Yun; Lai, Shen-Hao; Chen, Shih-Hsiang; Hua, Man-Chin; Yao, Tsung-Chieh; Chen, Li-Chen; Tsai, Ming-Han; Huang, Jing-Long

    2018-03-20

    Anemia is a major public health problem in young children. Reports on the role of anemia on infectious diseases remained controversial. We aim to investigate the effect of anemia on innate immunity, nasopharyngeal bacterial colonization, and subsequent infectious outcome. Blood tests were examined at the age of 12 months. TLR-induced cytokine production was assessed by ELISA. Bacteria from nasopharyngeal specimens were identified with traditional culture. Clinical infectious diseases were followed yearly until 3 years of age. Result showed that of the 423 infants, 72 had hemoglobin level ≤ 11 g/dL, among which 55% had normal iron level. There was significant association between hemoglobin level and TLR1-2, and 4 induced IL-6 (p = 0.04, 0.02) and that of TLR4 stimulated TNF-α response (p = 0.04). Children with anemia had higher nasopharyngeal colonization with Moxarella catarrhalis. Clinical analysis did not show anemia to be associated with infectious morbidity. However, children who developed LRTIs had mean lower ferritin levels. We speculated that iron might be the key factor related to infectious morbidity. Thus, to investigate the role of anemia in infectious diseases, it is important to first consider the prevalence of iron deficit, since the incidence of iron deficiency-induced anemia may vary among different regions.

  18. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

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    Baroni, Adone; Orlando, Manuela; Donnarumma, Giovanna; Farro, Pietro; Iovene, Maria Rosaria; Tufano, Maria Antonietta; Buommino, Elisabetta

    2006-01-01

    Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

  19. Responses to natural disasters

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    Maggs, William Ward

    Since 1964, natural disasters caused by earthquakes, volcanic eruptions, or extreme weather in the form of floods, droughts, or hurricanes, have been responsible for more than 2,756,000 deaths worldwide in nations other than the United States, the Soviet Union, and the Eastern European Bloc, according to figures tabulated by the Office of Foreign Disaster Assistance (OFDA) of the Agency for International Development (AID). Over 95% of these fatalities occurred in developing or third world countries. Damage resulting from these calamities has been severe but extremely difficult to estimate in monetary terms. In 1986, U.S. government and voluntary agencies spent $303 million on natural disaster assistance around the world, 79% of total world assistance. In 1985 the U.S. total was nearly $900 million, 48% of the $1.84 billion world total.

  20. HMGB1/TLR4 signaling induces an inflammatory response following high-pressure renal pelvic perfusion in a porcine model.

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    Shao, Yi; Sha, Minglei; Chen, Lei; Li, Deng; Lu, Jun; Xia, Shujie

    2016-11-01

    Percutaneous nephrolithotomy (PCNL) causes a rapid increase in renal pelvic pressure in the kidney, which induces an inflammatory response. High-mobility group box-1 (HMGB1) is known to trigger the recruitment of inflammatory cells and the release of proinflammatory cytokines following ischemia reperfusion injury in the kidney, but the contribution of HMGB1 to the inflammatory response following high-pressure renal pelvic perfusion has not been investigated. In this study, high-pressure renal pelvic perfusion was induced in anesthetized pigs to examine the effect of HMGB1 on the inflammatory response. HMGB1 levels in the kidney increased following high-pressure renal pelvic perfusion, together with elevated levels of inflammatory cytokines in the plasma and kidney and an accumulation of neutrophils and macrophages. Inhibition of HMGB1 alleviated this inflammatory response while perfusion with recombinant HMGB1 had an augmentative effect, confirming the involvement of HMGB1 in the inflammatory response to high-pressure renal pelvic perfusion. HMGB1 regulated the inflammatory response by activating Toll-like receptor 4 (TLR4) signaling. In conclusion, this study has demonstrated that HMGB1/TLR4 signaling contributes to the inflammatory response following high-pressure renal pelvic perfusion in a porcine model and has implications for the management of inflammation after PCNL. Copyright © 2016 the American Physiological Society.

  1. Rapid loss of dendritic cell and monocyte responses to TLR ligands following venipuncture.

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    Meier, Angela; Fisher, Amelia; Sidhu, Harlyn K; Chang, Judy J; Wen, Tom F; Streeck, Hendrik; Alter, Galit; Silvestri, Guido; Altfeld, Marcus

    2008-12-31

    Blood samples from multiple sites are collected in multicenter trials, and frequently shipped to centralized laboratories for processing and comparable experimental evaluation. It is therefore of crucial interest to assess the preservation of immune cell functions after overnight shipment of whole blood. Here we evaluated the ability of pDCs, mDCs and monocytes to respond to TLR ligands at multiple timepoints following venipuncture as compared to immediate processing. Our results demonstrate a profound impairment of APC function, in particular of IFN-alpha production of pDCs, if whole blood was processed later than 6 h after venipuncture. Overnight shipment or extended rest of whole blood before processing therefore severely compromises the ability of APCs to respond to TLR ligands, and this has to be taken into consideration when designing multicenter trials.

  2. 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.

  3. The Effect of TLR9 Agonist CpG Oligodeoxynucleotides on the Intestinal Immune Response of Cobia (Rachycentron canadum

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    Omkar Byadgi

    2014-01-01

    Full Text Available Cytosine-guanine oligodeoxynucleotide (CpG ODN motifs of bacterial DNA are recognized through toll-like receptor 9 (TLR9 and are potent activators of innate immunity. However, the interaction between TLR9 and CpG ODN in aquatic species has not been well characterized. Hence, cobia TLR9 isoform B (RCTLR9B was cloned and its expression and induction in intestine were investigated. RCTLR9B cDNA consists of 3113bp encoding 1009 amino acids containing three regions, leucine rich repeats, transmembrane domain, and toll/interleukin-1 receptor (TIR domain. Intraperitoneal injection of CpG ODN 2395 upregulated RCTLR9 A and B and MyD88 and also induced the expressions of Mx, chemokine CC, and interleukin IL-1β. Cobia intraperitoneally injected with CpG ODN 1668 and 2395 had increased survival rates after challenge with Photobacterium damselae subsp. piscicida. In addition, formulation of CpG ODN with formalin-killed bacteria (FKB and aluminum hydroxide gel significantly increased expressions of RCTLR9 A (50 folds and B (30 folds isoforms at 10 dpi (CpG ODN 1668 and MyD88 (21 folds at 6 dpv (CpG ODN 2395. Subsequently, IL-1β increased at 6 dpv in 1668 group. No histopathological damage and inflammatory responses were observed in the injected cobia. Altogether, these results facilitate CpG ODNs as an adjuvant to increase bacterial disease resistance and efficacy of vaccines in cobia.

  4. Genomic evidence of gene duplication and adaptive evolution of Toll like receptors (TLR2 and TLR4) in reptiles.

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    Shang, Shuai; Zhong, Huaming; Wu, Xiaoyang; Wei, Qinguo; Zhang, Huanxin; Chen, Jun; Chen, Yao; Tang, Xuexi; Zhang, Honghai

    2018-04-01

    Toll-like receptors (TLRs) encoded by the TLR multigene family play an important role in initial pathogen recognition in vertebrates. Among the TLRs, TLR2 and TLR4 may be of particular importance to reptiles. In order to study the evolutionary patterns and structural characteristics of TLRs, we explored the available genomes of several representative members of reptiles. 25 TLR2 genes and 19 TLR4 genes from reptiles were obtained in this study. Phylogenetic results showed that the TLR2 gene duplication occurred in several species. Evolutionary analysis by at least two methods identified 30 and 13 common positively selected codons in TLR2 and TLR4, respectively. Most positively selected sites of TLR2 and TLR4 were located in the Leucine-rich repeat (LRRs). Branch model analysis showed that TLR2 genes were under different evolutionary forces in reptiles, while the TLR4 genes showed no significant selection pressure. The different evolutionary adaptation of TLR2 and TLR4 among the reptiles might be due to their different function in recognizing bacteria. Overall, we explored the structure and evolution of TLR2 and TLR4 genes in reptiles for the first time. Our study revealed valuable information regarding TLR2 and TLR4 in reptiles, and provided novel insights into the conservation concern of natural populations. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL.

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    Sengupta, Dola; Koblansky, Alicia; Gaines, Jennifer; Brown, Tim; West, A Phillip; Zhang, Dekai; Nishikawa, Tak; Park, Sung-Gyoo; Roop, R Martin; Ghosh, Sankar

    2010-01-15

    Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter-like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNbeta. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiquitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

  6. 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.

  7. Targeting Deficiencies in the TLR5 Mediated Vaginal Response to Treat Female Recurrent Urinary Tract Infection.

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    Ali, Ased S M; Mowbray, Catherine; Lanz, Marcelo; Stanton, Anna; Bowen, Samantha; Varley, Claire L; Hilton, Paul; Brown, Karen; Robson, Wendy; Southgate, Jennifer; Aldridge, Phillip D; Tyson-Capper, Alison; Abraham, Soman; Pickard, Robert S; Hall, Judith

    2017-09-08

    The identification of the host defence peptides as target effectors in the innate defence of the uro-genital tract creates new translational possibilities for immunomodulatory therapies, specifically vaginal therapies to treat women suffering from rUTI, particularly those carrying the TLR5_C1174T SNP. Urinary tract infections (UTIs) are a microbial disease reported worldwide. Women are particularly susceptible with many suffering debilitating recurrent (r) infections. Treatment is by antibiotics, but such therapy is linked to antibiotic resistance and re-infection. This study explored the innate protective mechanisms of the urogenital tract with the aim of boosting such defences therapeutically. Modelling UTIs in vitro, human vaginal and bladder epithelial cells were challenged with uropathogenic Escherichia coli (CFT073) and microbial PAMPs including flagellin, LPS and peptidoglycan. Flagellin functioning via the TLR5/NFκB pathway was identified as the key UPEC virulence factor causing a significant increase (P < 0.05) in the production of the host-defence peptide (HDP), BD2. BD2-depleted urine samples from bladder infected mice supported increased UPEC growth, strengthening the significance of the HDPs in protecting the urogenital tissues from infection. Clinically, vaginal-douche BD2 concentrations were reduced (p < 0.05) in women suffering rUTIs, compared to age-matched healthy controls with concentrations further decreased (p < 0.05) in a TLR5 392Stop SNP rUTI subgroup. Topical vaginal estrogen treatment increased (p < 0.001) BD2 concentrations in all women, including those carrying the SNP. These data identify therapeutic and antibiotic sparing roles for vaginal immunomodulatory agents that specifically target HDP induction, facilitate bacterial killing and disrupt the UPEC infection cycle.

  8. Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-beta.

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    Juan C Salazar

    2009-05-01

    Full Text Available It is widely believed that innate immune responses to Borrelia burgdorferi (Bb are primarily triggered by the spirochete's outer membrane lipoproteins signaling through cell surface TLR1/2. We recently challenged this notion by demonstrating that phagocytosis of live Bb by peripheral blood mononuclear cells (PBMCs elicited greater production of proinflammatory cytokines than did equivalent bacterial lysates. Using whole genome microarrays, we show herein that, compared to lysates, live spirochetes elicited a more intense and much broader transcriptional response involving genes associated with diverse cellular processes; among these were IFN-beta and a number of interferon-stimulated genes (ISGs, which are not known to result from TLR2 signaling. Using isolated monocytes, we demonstrated that cell activation signals elicited by live Bb result from cell surface interactions and uptake and degradation of organisms within phagosomes. As with PBCMs, live Bb induced markedly greater transcription and secretion of TNF-alpha, IL-6, IL-10 and IL-1beta in monocytes than did lysates. Secreted IL-18, which, like IL-1beta, also requires cleavage by activated caspase-1, was generated only in response to live Bb. Pro-inflammatory cytokine production by TLR2-deficient murine macrophages was only moderately diminished in response to live Bb but was drastically impaired against lysates; TLR2 deficiency had no significant effect on uptake and degradation of spirochetes. As with PBMCs, live Bb was a much more potent inducer of IFN-beta and ISGs in isolated monocytes than were lysates or a synthetic TLR2 agonist. Collectively, our results indicate that the enhanced innate immune responses of monocytes following phagocytosis of live Bb have both TLR2-dependent and -independent components and that the latter induce transcription of type I IFNs and ISGs.

  9. 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.

  10. Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis.

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    Tania Køllgaard

    Full Text Available Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD. Epidemiological evidence has linked periodontal disease (PD with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL-1β, tumor necrosis factor (TNF-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1β by monocytes stimulated with the toll-like receptor (TLR 4 agonist Escherichia coli lipopolysaccharide (LPS, and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by P. gingivalis LPS and IL-1β secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.

  11. Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis.

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    Køllgaard, Tania; Enevold, Christian; Bendtzen, Klaus; Hansen, Peter R; Givskov, Michael; Holmstrup, Palle; Nielsen, Claus H

    2017-01-01

    Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD). Epidemiological evidence has linked periodontal disease (PD) with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs) and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1β by monocytes stimulated with the toll-like receptor (TLR) 4 agonist Escherichia coli lipopolysaccharide (LPS), and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by P. gingivalis LPS and IL-1β secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.

  12. MyD88 and TLR9 dependent immune responses mediate resistance to Leishmania guyanensis infections, irrespective of Leishmania RNA virus burden.

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    Ives, Annette; Masina, Slavica; Castiglioni, Patrik; Prével, Florence; Revaz-Breton, Mélanie; Hartley, Mary-Anne; Launois, Pascal; Fasel, Nicolas; Ronet, Catherine

    2014-01-01

    Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB-/- mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.

  13. MyD88 and TLR9 dependent immune responses mediate resistance to Leishmania guyanensis infections, irrespective of Leishmania RNA virus burden.

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    Annette Ives

    Full Text Available Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL, and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL and mutocutaneous (MCL leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13, which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB-/- mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.

  14. Neonatal immune responses to TLR2 stimulation: Influence of maternal atopy on Foxp3 and IL-10 expression

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    Gold Diane R

    2006-03-01

    Full Text Available Abstract Background Maternal atopic background and stimulation of the adaptive immune system with allergen interact in the development of allergic disease. Stimulation of the innate immune system through microbial exposure, such as activation of the innate Toll-like-receptor 2 (TLR2, may reduce the development of allergy in childhood. However, little is known about the immunological effects of microbial stimulation on early immune responses and in association with maternal atopy. Methods We analyzed immune responses of cord blood mononuclear cells (CBMC from 50 healthy neonates (31 non-atopic and 19 atopic mothers. Cells were stimulated with the TLR2 agonist peptidoglycan (Ppg or the allergen house dust mite Dermatophagoides farinae (Derf1, and results compared to unstimulated cells. We analyzed lymphocyte proliferation and cytokine secretion of CBMC. In addition, we assessed gene expression associated with T regulatory cells including the transcription factor Foxp3, the glucocorticoid-induced TNF receptor (GITR, and the cytotoxic lymphocyte antigen 4 (CTLA4. Lymphocyte proliferation was measured by 3H-Thymidine uptake, cytokine concentrations determined by ELISA, mRNA expression of T cell markers by real-time RT-PCR. Results Ppg stimulation induced primarily IL-10 cytokine production, in addition to IFN-γ, IL-13 and TNF-α secretion. GITR was increased following Ppg stimulation (p = 0.07. Ppg-induced IL-10 production and induction of Foxp3 were higher in CBMC without, than with maternal atopy (p = 0.04, p = 0.049. IL-10 production was highly correlated with increased expression of Foxp3 (r = 0.53, p = 0.001, GITR (r = 0.47, p = 0.004 and CTLA4 (r = 0.49, p = 0.003, independent of maternal atopy. Conclusion TLR2 stimulation with Ppg induces IL-10 and genes associated with T regulatory cells, influenced by maternal atopy. Increased IL-10 and Foxp3 induction in CBMC of non-atopic compared to atopic mothers, may indicate an increased capacity to

  15. TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

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    Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Shi, Yun-Liang; Wan, Xiao-Ling; Yang, Yi-Chao

    2017-08-07

    Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5)  = 2.595, P = 0.049), 60 (t (5)  = 7.838, P = 0.001) and 90 dpi (t (5)  = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5)  = 5.139, P = 0.004), 60 (t (5)  = 6.138, P = 0.002) and 90 dpi (t (5)  = 6

  16. The Fas-Associated Death Domain Protein (FADD) is Required in Apoptosis and TLR-induced Proliferative Responses in B Cells1

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    Imtiyaz, Hongxia Z.; Rosenberg, Stephen; Zhang, Yuhang; Rahman, Ziaur S. M.; Hou, Ying-Ju; Manser, Tim L.; Zhang, Jianke

    2011-01-01

    The Fas-associated death domain protein FADD/Mort1 is a signaling adaptor protein which mediates the activation of caspase 8 during death receptor-induced apoptosis. Disruption of FADD in germ cells results in death receptor-independent embryonic lethality in mice. Previous studies indicated that in addition to its function in apoptosis, FADD is also required in peripheral T cell homeostasis and TCR-induced proliferative responses. In this report, we generated B cell-specific FADD-deficient mice and showed that deletion of FADD at the pro-B cell stage had minor effects on B cell development in the bone marrow, and resulted in increased splenic and lymph node B cell numbers and decreased peritoneal B1 cell numbers. As in T cells, a FADD deficiency inhibited Fas-induced apoptosis in B cells. However, B cell proliferative responses induced by stimulation of the BCR and CD40 using anti-IgM or anti-CD40 antibodies were unaffected by the absence of FADD. Further analyses revealed that FADD-deficient B cells were defective in proliferative responses induced by treatments with dsRNA and LPS which stimulate TLR3 and TLR4 respectively. Therefore, in addition to its apoptotic function, FADD also plays a role in TLR3- and TLR4-induced proliferative responses in B cells. PMID:16709845

  17. Expression of Toll-like receptors (TLR, in lymphoid organ of black tiger shrimp (Penaeus monodon in response to Vibrio harveyi infection

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    Mundanda Muthappa Dechamma

    2015-05-01

    Full Text Available The Toll-like receptors (TLR, being pattern recognition molecules, are a powerful first line of defense in response to pathogen invasion. They are known to play a crucial role in detecting and binding to the microbial molecule and triggering a non-specific immune response. Quantitative real time PCR (qPCR expression of the TLR gene was studied in healthy and Vibrio harveyi infected black tiger shrimp (Penaeus monodon. Lymphoid tissue expression of TLR in V. harveyi infected animals 24 h post injection showed statistically significant up regulation of the gene as compared to the control animals sham injected with phosphate buffered saline (PBS. The qPCR expression pattern of TLR at different time points in shrimp administered with the immunostimulant glucan for 6 days by oral feeding followed by challenge with V. harveyi showed statistically significant level at 48 h post bacterial challenge as compared to the control (immunostimulant treated animals sham injected with PBS. The novelty of the study is that it elicits the role of TLRs as important response proteins of the innate immune system in the shrimp.

  18. Systemic immune activation in HIV infection is associated with decreased MDC responsiveness to TLR ligand and inability to activate naive CD4 T-cells.

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    Nicole L Yonkers

    Full Text Available HIV infection is characterized by ineffective anti-viral T-cell responses and impaired dendritic cell (DC functions, including response to Toll-Like Receptor (TLR ligands. Because TLR responsiveness may affect a host's response to virus, we examined TLR ligand induced Myeloid and Plasmacytoid DC (MDC and PDC activation of naïve T-cells in HIV+ subjects.Freshly purified MDC and PDC obtained from HIV+ subjects and healthy controls were cultured in the presence and absence of TLR ligands (poly I∶C or R-848. We evaluated indices of maturation/activation (CD83, CD86, and HLA-DR expression, cytokine secretion (IFN-alpha and IL-6, and ability to activate allogeneic naïve CD4 T-cells to secrete IFN-gamma and IL-2.MDC from HIV+ subjects had increased spontaneous IL-6 production and increased CD83 and CD86 expression when compared to MDC of controls. MDC IL-6 expression was associated with plasma HIV level. At the same time, poly I∶C induced HLA-DR up-regulation on MDC was reduced in HIV+ persons when compared to controls. The latter finding was associated with impaired ability of MDC from HIV+ subjects to activate allogeneic naïve CD4 T-cells. PDC from HIV+ persons had increased spontaneous and TLR ligand induced IL-6 expression, and increased HLA-DR expression at baseline. The latter was associated with an intact ability of HIV PDC to activate allogeneic naïve CD4 T-cells.These results have implications for the ability of the HIV+ host to form innate and adaptive responses to HIV and other pathogens.

  19. Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice

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    Jonathan Laiño

    2017-01-01

    Full Text Available Background. TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim. To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods. Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand and Pam2CysK4 (TLR2-ligand, were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. Results. CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions. Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.

  20. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

    Science.gov (United States)

    Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

    2015-01-01

    Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

  1. Cigarette smoke amplifies inflammatory response and atherosclerosis progression through activation of the H1R-TLR2/4-COX2 axis

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    Rajat S Barua

    2015-11-01

    Full Text Available Emerging evidence suggest that infection and persistent inflammation are key players in the pathogenesis of atherosclerotic cardiovascular disease (CVD. Although it is well established that cigarette smoke (CS promotes atherosclerotic CVD, very little is known about the potential impact of the collective effects of CS and intermittent or chronic subclinical infection on atherosclerosis. Our previous studies demonstrated that mast cell-derived histamine and lipopolysaccharide (LPS synergistically enhance endothelial cell inflammatory response. We further noted that the synergy between histamine and LPS was due to reciprocal upregulation of histamine receptor (H1R and Toll-like receptor 4 (TLR4 expression and functions. These results suggest that the combined and persistent effects of mast cell mediators and bacterial agents on the vasculature are risk factors of CVD. Our recent data demonstrated that CS extract enhances histamine- and LPS-induced expression of cyclooxygenase-2 (COX-2 in endothelial cells suggesting that CS and mast cell mediators may collectively amplify inflammatory response in the vessel wall. We hypothesize that CS enhances histamine-mediated upregulation of TLR2/TLR4 signaling in the endothelium and promotes progression of atherosclerosis. This article presents our perspective on the modulatory effects of CS and nicotine on the ‘histamine-TLR-COX-2 axis’.

  2. Cigarette Smoke Amplifies Inflammatory Response and Atherosclerosis Progression Through Activation of the H1R-TLR2/4-COX2 Axis.

    Science.gov (United States)

    Barua, Rajat S; Sharma, Mukut; Dileepan, Kottarappat N

    2015-01-01

    Emerging evidence suggests that infection and persistent inflammation are key players in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Although it is well established that cigarette smoke (CS) promotes atherosclerotic CVD, very little is known about the potential impact of the collective effects of CS and intermittent or chronic subclinical infection on atherosclerosis. Our previous studies demonstrated that mast cell-derived histamine and lipopolysaccharide (LPS) synergistically enhance endothelial cell inflammatory response. We further noted that the synergy between histamine and LPS was due to reciprocal upregulation of histamine receptor and Toll-like receptor 4 (TLR4) expression and functions. These results suggest that the combined and persistent effects of mast cell mediators and bacterial agents on the vasculature are risk factors of CVD. Our recent data demonstrated that CS extract enhances histamine- and LPS-induced expression of cyclooxygenase-2 (COX-2) in endothelial cells, suggesting that CS and mast cell mediators may collectively amplify inflammatory response in the vessel wall. We hypothesize that CS enhances histamine-mediated upregulation of TLR2/TLR4 signaling in the endothelium and promotes progression of atherosclerosis. This article presents our perspective on the modulatory effects of CS and nicotine on the "histamine-TLR-COX-2 axis."

  3. Biophysical Attributes of CpG Presentation Control TLR9 Signaling to Differentially Polarize Systemic Immune Responses

    Directory of Open Access Journals (Sweden)

    Jardin A. Leleux

    2017-01-01

    Full Text Available It is currently unknown whether and how mammalian pathogen recognition receptors (PRRs respond to biophysical patterns of pathogen-associated molecular danger signals. Using synthetic pathogen-like particles (PLPs that mimic physical properties of bacteria or large viruses, we have discovered that the quality and quantity of Toll-like receptor 9 (TLR9 signaling by CpG in mouse dendritic cells (mDCs are uniquely dependent on biophysical attributes; specifically, the surface density of CpG and size of the presenting PLP. These physical patterns control DC programming by regulating the kinetics and magnitude of MyD88-IRAK4 signaling, NF-κB-driven responses, and STAT3 phosphorylation, which, in turn, controls differential T cell responses and in vivo immune polarization, especially T helper 1 (Th1 versus T helper 2 (Th2 antibody responses. Our findings suggest that innate immune cells can sense and respond not only to molecular but also pathogen-associated physical patterns (PAPPs, broadening the tools for modulating immunity and helping to better understand innate response mechanisms to pathogens and develop improved vaccines.

  4. A non-synonymous coding variant (L616F in the TLR5 gene is potentially associated with Crohn's disease and influences responses to bacterial flagellin.

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    Jared Sheridan

    Full Text Available Although numerous studies have implicated TLR5, or its ligands, bacterial flagellins, in the pathogenesis of Crohn's disease (CD, genome-wide association studies (GWAS have not reported associations with the TLR5 gene. We aimed to examine potential CD-associated TLR5 variants and assess whether they modified inflammatory responses to bacterial flagellins.A two-stage study was carried out. In stage 1, we genotyped tagging single-nucleotide polymorphisms (tag-SNPs in the TLR5 gene in a sample of CD cases (<20 years of age, N = 566 and controls (N = 536. Single SNP and haplotype analysis was carried out. In Stage 2, we assessed the functional significance of potential CD-associated variant(s vis-à-vis effects on the inflammatory response to bacterial flagellin using HEK293T cells. We observed marginal association between a non-synonymous coding SNP rs5744174 (p = 0.05 and CD. Associations between SNP rs851139 that is in high linkage disequilibrium (LD with SNP rs5744174 were also suggested (p = 0.07. Haplotype analysis revealed that a 3 marker haplotype was significantly associated with CD (p = 0.01. Functional studies showed that the risk allele (616F (corresponding to the C allele of SNP rs5744174 conferred significantly greater production of CCL20 in response to a range of flagellin doses than the comparator allele (616L.Our findings suggest that a non-synonymous coding variation in the TLR5 gene may confer modest susceptibility for CD.

  5. 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

    studies have defined essential roles for single TLRs in innate immune defense in vivo. This could suggest that PRRs act in concert to mount the first line of defense against virus infections. To test this hypothesis we have examined the host response of C57BL/6, TLR2(-/-), TLR9(-/-), and TLR2/9(-/-) mice......9 in a cytokine- and cell type-dependent manner. With respect to the cellular response to infection, we found that recruitment but not activation of NK cells was impaired in TLR2/9(-/-) mice. Importantly, the viral load in the brain, but not liver, was significantly higher in the brain of TLR2......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...

  6. Synthetic TLR4 agonists enhance functional antibodies and CD4+ T-cell responses against the Plasmodium falciparum GMZ2.6C multi-stage vaccine antigen

    DEFF Research Database (Denmark)

    Baldwin, Susan L; Roeffen, Will; Singh, Susheel K

    2016-01-01

    , liposomes, and alum) in C57BL/6 mice. Some, but not all, formulations containing either the synthetic TLR4 agonist GLA or SLA elicited the highest parasite-specific antibody titers, the greatest IFN-γ responses in CD4+ TH1 cells, and the highest percentage of multifunctional CD4+ T cells expressing IFN......-γ and TNF in response to GMZ2.6C. Both of these agonists have good safety records in humans....

  7. Inactivated influenza vaccine adjuvanted with bacterium-like particles induce systemic and mucosal influenza A virus specific T-cell and B-cell responses after nasal administration in a TLR2 dependent fashion.

    Science.gov (United States)

    Keijzer, C; Haijema, B J; Meijerhof, T; Voorn, P; de Haan, A; Leenhouts, K; van Roosmalen, M L; van Eden, W; Broere, F

    2014-05-19

    Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies showed that non-living bacterium-like particles (BLPs) from the food-grade bacterium Lactococcus lactis are effective stimulators of local and systemic immune responses when administered intranasally. Moreover, in vitro, BLPs specifically interact with human Toll-like receptor 2 (TLR2), suggestive of a role for TLR2 dependent immune activation by BLPs. In the present study, we examined the role of TLR2 in vivo in immune activation after nasal administration of BLP mixed with split influenza vaccine (BLP-SV) of influenza A virus (IAV) using TLR2 knockout mice. The systemic Th1 cell and subsequent B-cell responses induced after intranasal BLP-SV vaccination depended on the interaction of BLPs with TLR2. Notably, the BLP-SV-induced class switch to IgG2c depended on the interaction of BLP with TLR2. Local induced IAV-specific Th1 cell responses and the mucosal B-cell responses also depended on interaction of BLP with TLR2. Strongly reduced SIgA levels were observed in TLR2 knockout mice both in the nasal and vaginal lavages. In addition, detailed analysis of the T-cell response revealed that nasal BLP-SV vaccination promoted Th1/Th17 immune responses that coincided with increased IAV-specific IgG2c antibody production. Altogether these results indicate that nasal BLP-SV vaccination induces IAV-specific T-cell and B-cell responses, both systemically and at the site of virus entry in a TLR2-dependent manner. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients

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    David Heftrig

    2017-01-01

    Full Text Available Objective. Trauma patients (TP frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro “hit” have been reported. Methods/Results. Ex vivo-in vitro lipopolysaccharide- (LPS- stimulated blood from TP showed diminished interleukin- (IL- 1β-release in TP for five postinjury days compared to healthy volunteers (HV. The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV. Conclusion. Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

  9. Impaired Surface Expression of HLA-DR, TLR2, TLR4, and TLR9 in Ex Vivo-In Vitro Stimulated Monocytes from Severely Injured Trauma Patients.

    Science.gov (United States)

    Heftrig, David; Sturm, Ramona; Oppermann, Elsie; Kontradowitz, Kerstin; Jurida, Katrin; Schimunek, Lukas; Woschek, Mathias; Marzi, Ingo; Relja, Borna

    2017-01-01

    Objective . Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro "hit" have been reported. Methods/Results . Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1 β -release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV. Conclusion . Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.

  10. TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

    Science.gov (United States)

    Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

    2016-05-01

    Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9. © Society for Leukocyte Biology.

  11. Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

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    Weith Andreas

    2005-11-01

    Full Text Available Abstract Background Bacterial and viral exacerbations play a crucial role in a variety of lung diseases including COPD or asthma. Since the lung epithelium is a major source of various inflammatory mediators that affect the immune response, we analyzed the inflammatory reaction of primary lung epithelial cells to different microbial molecules that are recognized by Toll-like receptors (TLR. Methods The effects of TLR ligands on primary small airway epithelial cells were analyzed in detail with respect to cytokine, chemokine and matrix metalloproteinase secretion. In addition, the regulation of the expression of TLRs and their adaptor proteins in small airway epithelial cells was investigated. Results Our data demonstrate that poly(I:C, a synthetic analog of viral dsRNA, mediated the strongest proinflammatory effects among the tested ligands, including an increased secretion of IL-6, IL-8, TNF-α, GM-CSF, GRO-α, TARC, MCP-1, MIP-3α, RANTES, IFN-β, IP-10 and ITAC as well as an increased release of MMP-1, MMP-8, MMP-9, MMP-10 and MMP-13. Furthermore, our data show that poly(I:C as well as type-1 and type-2 cytokines have a pronounced effect on the expression of TLRs and molecules involved in TLR signaling in small airway epithelial cells. Poly(I:C induced an elevated expression of TLR1, TLR2 and TLR3 and increased the gene expression of the general TLR adaptor MyD88 and IRAK-2. Simultaneously, poly(I:C decreased the expression of TLR5, TLR6 and TOLLIP. Conclusion Poly(I:C, an analog of viral dsRNA and a TLR3 ligand, triggers a strong inflammatory response in small airway epithelial cells that is likely to contribute to viral exacerbations of pulmonary diseases like asthma or COPD. The pronounced effects of poly(I:C on the expression of Toll-like receptors and molecules involved in TLR signaling is assumed to influence the immune response of the lung epithelium to viral and bacterial infections. Likewise, the regulation of TLR expression by type

  12. TLR3 signaling in macrophages is indispensable for the protective immunity of invariant natural killer T cells against enterovirus 71 infection.

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    Kai Zhu

    2015-01-01

    Full Text Available Enterovirus 71 (EV71 is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs. Mechanistically, interleukin (IL-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.

  13. TLR3 signaling in macrophages is indispensable for the protective immunity of invariant natural killer T cells against enterovirus 71 infection.

    Science.gov (United States)

    Zhu, Kai; Yang, Juhao; Luo, Kaiming; Yang, Chunhui; Zhang, Na; Xu, Ruifeng; Chen, Jianxia; Jin, Mingfei; Xu, Bin; Guo, Nining; Wang, Jianrong; Chen, Zuolong; Cui, Ying; Zhao, Hui; Wang, Yan; Deng, Chaoyang; Bai, Li; Ge, Baoxue; Qin, Cheng-Feng; Shen, Hao; Yang, Chun-Fu; Leng, Qibin

    2015-01-01

    Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.

  14. The proteoglycan biglycan mediates inflammatory response by activating TLR-4 in human chondrocytes: Inhibition by specific siRNA and high polymerized Hyaluronan.

    Science.gov (United States)

    Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Mandraffino, Giuseppe; Calatroni, Alberto; Saitta, Antonino; Campo, Salvatore; Campo, Giuseppe M

    2018-02-15

    Cartilage degeneration are hallmarks of wear, tear, mechanical and inflammatory damage of the joint cartilage. Tissue degradation as well as compromising the integrity and function of the organ, produces different intermediates, directly able to stimulate further inflammatory effect, therefore, amplifying the inflammation response. Biglycan is a soluble component of the extracellular matrix that is released during tissue injury. It has been reported that released biglycan is an endogenous ligand for TLR-2/4 in some cell type. We studied the role of biglycan in an experimental model of biglycan-induced inflammatory response in human chondrocytes and the effect of high polymerized HA on reducing its activity. Exposition of chondrocytes to LPS generated cell injury, including high levels of biglycan. Chondrocyte treatment with biglycan produces a high mRNA expression of several detrimental inflammation mediators such as IL-1β, IL-6, MMP-13, and IL-17, as well as NF-kB and TLR-4 activation. Administration of high polymerized HA to chondrocytes exposed to biglycan was able to attenuate the inflammatory response by decreasing the expression of the inflammatory mediators. Involvement of the TLR-4 in the mediation of the biglycan action was confirmed using a specific silent agent (siRNA). Taken together, these data could be used to develop new anti-inflammatory approaches. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Role of human Toll-like receptors in naturally occurring influenza A infections.

    Science.gov (United States)

    Lee, Nelson; Wong, Chun Kwok; Hui, David S C; Lee, Sharon K W; Wong, Rity Y K; Ngai, Karry L K; Chan, Martin C W; Chu, Yi Jun; Ho, Amy W Y; Lui, Grace C Y; Wong, Bonnie C K; Wong, Sunny H; Yip, Shea Ping; Chan, Paul K S

    2013-09-01

    We investigated the roles of Toll-like receptors (TLRs) in naturally occurring influenza. A prospective, case - control study was conducted. Adults hospitalized with virologically confirmed influenza A infections (onset respiratory/cardiovascular complications. There were increased cellular expressions of TLR9, TLR8, TLR3, and TLR7 during influenza; TLR2 and TLR4 were suppressed. Results were similar for both virus strains. Higher TLR expression levels at presentation significantly correlated with lower viral loads (Spearman's rho: -0.46 to -0.69 for TLR9, TLR8, and TLR3; P-values <0.05). Multivariate regression models (adjusted for age, comorbidity, disease severity, time from onset) confirmed their independent associations. Increased signaling molecules (phospho-MAPKs, IκB) and inflammatory cytokines (IL-6, sTNFR-1, CCL2/MCP-1; CXCL10/IP-10, IFN-γ) correlated with increased TLR expression. RLRs were upregulated simultaneously. PBMCs of patients with influenza showed significant, dynamic changes in their cytokine responses upon TLR stimulation, compared with controls. Our results suggest that TLRs play an important role in early, innate viral inhibition in naturally occurring influenza. Inflammatory cytokine responses are concomitantly induced. These findings support investigation of TLR targeting as a novel intervention approach for prophylaxis against influenza. © 2013 John Wiley & Sons Ltd.

  16. TLR2 Expression in Peripheral CD4+ T Cells Promotes Th17 Response and Is Associated with Disease Aggravation of Hepatitis B Virus-Related Acute-On-Chronic Liver Failure

    Directory of Open Access Journals (Sweden)

    Chunli Xu

    2017-11-01

    Full Text Available Th17 responses have been shown to play crucial roles in the pathogenesis of hepatitis B virus (HBV-associated acute-on-chronic liver failure (ACLF. The mechanism underlying the enhanced Th17 responses in these patients remains largely unclear. Here we investigated toll-like receptors (TLRs expression in peripheral T cells and their roles in Th17 cell differentiation and disease aggravation in ACLF patients. 18 healthy subjects (HS, 20 chronic HBV-infected (CHB patients, and 26 ACLF patients were enrolled and examined for TLRs expression in peripheral blood mononuclear cells (PBMCs. The correlations of T cell TLR2 expression with the antigen non-specific Th17 responses and disease aggravation, as well as the Th17 response to TLR2 ligand stimulation were evaluated in ACLF patients. Compared to HS and CHB patients, ACLF patients showed a distinct TLRs expression pattern in PBMCs. Significantly increased TLR2 expression in T cells was observed in ACLF patients. The TLR2 expression in CD4+ T cells was correlated with the Th17 responses and the clinical markers for disease aggravation in ACLF patients. Moreover, TLR2 ligands stimulation promoted Th17 cell differentiation and response in PBMCs of ACLF patients. These findings implicate that TLR2 signaling plays critical roles in Th17 cell differentiation and disease aggravation of HBV-related ACLF.

  17. A key role for the endothelium in NOD1 mediated vascular inflammation: comparison to TLR4 responses.

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    Timothy Gatheral

    Full Text Available Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of gram negative and some gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.

  18. Evaluation of an intranasal virosomal vaccine against respiratory syncytial virus in mice: effect of TLR2 and NOD2 ligands on induction of systemic and mucosal immune responses.

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    Muhammad Shafique

    Full Text Available INTRODUCTION: RSV infection remains a serious threat to newborns and the elderly. Currently, there is no vaccine available to prevent RSV infection. A mucosal RSV vaccine would be attractive as it could induce mucosal as well as systemic antibodies, capable of protecting both the upper and lower respiratory tract. Previously, we reported on a virosomal RSV vaccine for intramuscular injection with intrinsic adjuvant properties mediated by an incorporated lipophilic Toll-like receptor 2 (TLR2 ligand. However, it has not been investigated whether this virosomal RSV vaccine candidate would be suitable for use in mucosal immunization strategies and if additional incorporation of other innate receptor ligands, like NOD2-ligand, could further enhance the immunogenicity and protective efficacy of the vaccine. OBJECTIVE: To explore if intranasal (IN immunization with a virosomal RSV vaccine, supplemented with TLR2 and/or NOD2-ligands, is an effective strategy to induce RSV-specific immunity. METHODS: We produced RSV-virosomes carrying TLR2 (Pam3CSK4 and/or NOD2 (L18-MDP ligands. We tested the immunopotentiating properties of these virosomes in vitro, using TLR2- and/or NOD2-ligand-responsive murine and human cell lines, and in vivo by assessing induction of protective antibody and cellular responses upon IN immunization of BALB/c mice. RESULTS: Incorporation of Pam3CSK4 and/or L18-MDP potentiates the capacity of virosomes to activate (antigen-presenting cells in vitro, as demonstrated by NF-κB induction. In vivo, incorporation of Pam3CSK4 in virosomes boosted serum IgG antibody responses and mucosal antibody responses after IN immunization. While L18-MDP alone was ineffective, incorporation of L18-MDP in Pam3CSK4-carrying virosomes further boosted mucosal antibody responses. Finally, IN immunization with adjuvanted virosomes, particularly Pam3CSK4/L18-MDP-adjuvanted-virosomes, protected mice against infection with RSV, without priming for enhanced

  19. The TLR9 agonist MGN1703 triggers a potent type I interferon response in the sigmoid colon

    DEFF Research Database (Denmark)

    Krarup, A R; Abdel-Mohsen, M; Schleimann, M H

    2017-01-01

    Toll-like receptor 9 (TLR9) agonists are being developed for treatment of colorectal and other cancers, yet the impact of these drugs on human intestines remains unknown. This, together with the fact that there are additional potential indications for TLR9 agonist therapy (e.g., autoimmune...... (60 mg s.c.) twice weekly for 4 weeks in a single-arm, phase 1b/2a study. Within sigmoid mucosa, global transcriptomic analyses revealed 248 modulated genes (false discovery rate...=0.001) and ISG15 (P=0.014) protein expression. No changes were observed in neutrophil infiltration (myeloperoxidase; P=0.97). No systematic effect on fecal microbiota structure was observed (analysis of similarity Global R=-0.105; P=0.929). TLR9 expression at baseline was inversely proportional...

  20. Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response.

    Science.gov (United States)

    Sartorius, Rossella; D'Apice, Luciana; Trovato, Maria; Cuccaro, Fausta; Costa, Valerio; De Leo, Maria Giovanna; Marzullo, Vincenzo Manuel; Biondo, Carmelo; D'Auria, Sabato; De Matteis, Maria Antonietta; Ciccodicola, Alfredo; De Berardinis, Piergiuseppe

    2015-07-01

    Filamentous bacteriophage fd particles delivering antigenic determinants via DEC-205 (fdsc-αDEC) represent a powerful delivery system that induces CD8(+) T-cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA-Sequencing of fd-pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co-stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc-αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc-αDEC is delivered into LAMP-1-positive compartments and co-localizes with TLR9. Thus, phage particles containing a single-strand DNA genome rich in CpG motifs delivered via DEC-205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  1. The TLR13-MyD88-NF-κB signalling pathway of Cyclina sinensis plays vital roles in innate immune responses.

    Science.gov (United States)

    Ren, Yipeng; Ding, Dan; Pan, Baoping; Bu, Wenjun

    2017-11-01

    Toll-like receptors, the best known pattern recognition receptors, play important roles in recognizing non-self molecules and binding pathogen-associated molecular patterns in the innate immune system. In the present research, the cDNA and protein characterization of the TLR signalling pathway genes including IRAK4, TRAK6 and IKKα (named CsIRAK4, CsTRAF6 and CsIKKα, respectively) with the typical motifs from Cyclina sinensis showed significant similarity with their homologues from other shellfish. Furthermore, the mRNA transcripts of these three genes are ubiquitously expressed in all tissues tested and are dominantly expressed in C. sinensis haemocytes (P sinensis by RNA interference and immune challenges. The results suggested the mRNA expression patterns of CsMyD88, CsIRAK4, CsTRAF6, CsIKKα, CsIκB, CsNF-κB, CsC-LYZ and CsAMP were all down-regulated (P sinensis haemocytes, revealing the involvement of the TLR13-MyD88-NF-κB signalling pathway in innate immunity by positively adjusting internal signalling factors and immune-related genes. In summary, a TLR13-MyD88-NF-κB signalling pathway exists and plays vital roles in innate immune responses in C. sinensis. These findings collectively lay the foundation for studying the functional characterization of internal signalling factors and establishing a regulatory network for the TLR signalling pathway in molluscs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Addition of a TLR7 agonist to an acellular pertussis vaccine enhances Th1 and Th17 responses and protective immunity in a mouse model.

    Science.gov (United States)

    Misiak, Alicja; Leuzzi, Rosanna; Allen, Aideen C; Galletti, Bruno; Baudner, Barbara C; D'Oro, Ugo; O'Hagan, Derek T; Pizza, Mariagrazia; Seubert, Anja; Mills, Kingston H G

    2017-09-18

    A resurgence of whooping cough (pertussis) has been observed in recent years in a number of developed countries, despite widespread vaccine coverage. Although the exact reasons of the recurrence of pertussis are not clear, there are a number of potential causes, like antigenic variation in the circulating strains of Bordetella pertussis, changes in surveillance and diagnostic tools, and potential differences in protection afforded by current acellular pertussis (aP) vaccines compared to more reactogenic whole cell (wP) vaccines, which they replaced. Studies in animal models have shown that induction of cellular as well as humoral immune responses are key to conferring effective and long lasting protection against B. pertussis. wP vaccines induce robust Th1/Th17 responses, which are associated with good protection against lung infection. In contrast, aP vaccines induce mixed Th2/Th17 responses. One research option is to modify current aP vaccines with the intention of inducing protective T cell responses, without compromising on their low reactogenicity profile. Here we found that formulation of an aP vaccine with a novel adjuvant based on a Toll-like receptor 7 agonist (TLR7a) adsorbed to aluminum hydroxide (alum) enhanced B. pertussis-specific Th1 and Th17 responses and serum IgG2a/b antibodies, which had greater functional capacity than those induced by aP formulated with alum alone. Furthermore, addition of a TLR7a enhanced the protective efficacy of the aP vaccine against B. pertussis aerosol challenge; protection was comparable to that of a wP vaccine. These findings suggest that alum-TLR7a is a promising adjuvant for clinical development of next generation pertussis vaccines. Copyright © 2017. Published by Elsevier Ltd.

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

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

    2010-01-01

    levels of pro-inflammatory and type I interferon cytokines mRNA in response to stimulation with the human TLR7/8 agonist R848 or the TLR3 agonist poly I:C. Only poly I:C-induced IFN2 transcription was significantly suppressed in the presence of chloroquine, a compound known to block endosomal acidification and inhibit endosomal maturation. The effect of chloroquine on R848-induced cytokine expression was equivocal and so it remains questionable whether rainbow trout recognition of R848 requires endosomal maturation. TLR7 and TLR8a1 expression levels in rainbow trout anterior kidney leukocytes were not affected by poly I:C or R848 treatments, but surprisingly, TLR8a2 expression was moderately down-regulated by R848. The down-regulation of omTLR8a2 may imply that this gene has evolved to a new or altered function in rainbow trout, as often occurs when the two duplicated genes remain active.

  4. Nogo-B Facilitates LPS-Mediated Immune Responses by Up-Regulation of TLR4-Signaling in Macrophage RAW264.7

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    Ying Zhu

    2017-01-01

    Full Text Available Background/Aims: Nogo-B, a member of the reticulon family of proteins, is mainly located in the endoplasmic reticulum (ER. Here, we investigate the function and mechanism of Nogo-B in the regulation of TLR4-associated immune responses in the macrophage cell line of RAW264.7. Methods: Nogo-B was up- and down-regulated through the use of appropriate adenoviral vectors or siRNA, and the effects of Nogo-B on macrophages under liposaccharide (LPS stimulation were evaluated via western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA, flow cytometric analysis, and transwell assay. Results: Our data indicates that the protein of Nogo-B was down-regulated in a time- and dose-dependent manner following LPS administration in the macrophage. Nogo-B overexpression increased the production of inflammatory cytokines (MCP-1, TNF-α, IL-1β, and TGF-β, enhanced macrophage migration activities, activated major histocompatibility complex II (MHC II, and elevated the expression of macrophage scavenger receptor 1(MSR1, all of which suggest that Nogo-B is necessary for immune responses and plays an important role in regulating macrophage recruitment. Mechanistically, Nogo-B may enhance TLR4 expression in macrophage surfaces, activate mitogen-activated protein kinase (MAPK pathways, and initiate inflammatory responses. Conclusion: These findings illustrate the key regulatory functions of Nogo-B in facilitating LPS-mediated immune responses through promoting the phosphorylation of MAP kinase.

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

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    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

  6. A pseudopterane diterpene isolated from the octocoral Pseudopterogorgia acerosa inhibits the inflammatory response mediated by TLR-ligands and TNF-alpha in macrophages.

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    Yisett González

    Full Text Available Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1 isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL-6, IL-1β, nitric oxide (NO, interferon gamma-induced protein 10 (IP-10, ciclooxygenase (COX-2, inducible nitric oxide synthase (iNOS and monocyte chemoattractant protein-1 (MCP-1 induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation.

  7. A Pseudopterane Diterpene Isolated From the Octocoral Pseudopterogorgia acerosa Inhibits the Inflammatory Response Mediated by TLR-Ligands and TNF-Alpha in Macrophages

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    González, Yisett; Doens, Deborah; Santamaría, Ricardo; Ramos, Marla; Restrepo, Carlos M.; Barros de Arruda, Luciana; Lleonart, Ricardo; Gutiérrez, Marcelino; Fernández, Patricia L.

    2013-01-01

    Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1) isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α) and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), interferon gamma-induced protein 10 (IP-10), ciclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation. PMID:24358331

  8. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

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    Ganguli, Kriston; Collado, Maria Carmen; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M.; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W. Allan; Rautava, Samuli

    2015-01-01

    Background Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Methods TNF-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Results Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. Conclusion The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human intestinal epithelial cells and directly modulates intestinal epithelial cell innate immune gene expression. PMID:25580735

  9. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

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    Ganguli, Kriston; Collado, Maria C; Rautava, Jaana; Lu, Lei; Satokari, Reetta; von Ossowski, Ingemar; Reunanen, Justus; de Vos, Willem M; Palva, Airi; Isolauri, Erika; Salminen, Seppo; Walker, W Allan; Rautava, Samuli

    2015-04-01

    Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

  10. Targeting the TLR4 signaling pathway by polyphenols: A novel therapeutic strategy for neuroinflammation.

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    Rahimifard, Mahban; Maqbool, Faheem; Moeini-Nodeh, Shermineh; Niaz, Kamal; Abdollahi, Mohammad; Braidy, Nady; Nabavi, Seyed Mohammad; Nabavi, Seyed Fazel

    2017-07-01

    A wide array of cell signaling mediators and their interactions play vital roles in neuroinflammation associated with ischemia, brain trauma, developmental disorders and age-related neurodegeneration. Along with neurons, microglia and astrocytes are also affected by the inflammatory cascade by releasing pro-inflammatory cytokines, chemokines and reactive oxygen species. The release of pro-inflammatory mediators in response to neural dysfunction may be helpful, neutral or even deleterious to normal cellular survival. Moreover, the important role of NF-κB factors in the central nervous system (CNS) through toll-like receptor (TLR) activation has been well established. This review demonstrates recent findings regarding therapeutic aspects of polyphenolic compounds for the treatment of neuroinflammation, with the aim of regulating TLR4. Polyphenols including flavonoids, phenolic acids, phenolic alcohols, stilbenes and lignans, can target TLR4 signaling pathways in multiple ways. Toll interacting protein expression could be modulated by epigallocatechin-3-gallate. Resveratrol may also exert neuroprotective effects via the TLR4/NF-κB/STAT signaling cascade. Its role in activation of cascade via interfering with TLR4 oligomerization upon receptor stimulation has also been reported. Curcumin, another polyphenol, can suppress overexpression of inflammatory mediators via inhibiting the TLR4-MAPK/NF-κB pathway. It can also reduce neuronal apoptosis via a mechanism concerning the TLR4/MyD88/NF-κB signaling pathway in microglia/macrophages. Despite a symphony of in vivo and in vitro studies, many molecular and pharmacological aspects of neuroinflammation remain unclear. It is proposed that natural compounds targeting TLR4 may serve as important pharmacophores for the development of potent drugs for the treatment of neurological disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Poly-thymidine oligonucleotides mediate activation of murine glial cells primarily through TLR7, not TLR8.

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    Min Du

    Full Text Available The functional role of murine TLR8 in the inflammatory response of the central nervous system (CNS remains unclear. Murine TLR8 does not appear to respond to human TLR7/8 agonists, due to a five amino acid deletion in the ectodomain. However, recent studies have suggested that murine TLR8 may be stimulated by alternate ligands, which include vaccinia virus DNA, phosphothioate oligodeoxynucleotides (ODNs or the combination of phosphothioate poly-thymidine oligonucleotides (pT-ODNs with TLR7/8 agonists. In the current study, we analyzed the ability of pT-ODNs to induce activation of murine glial cells in the presence or absence of TLR7/8 agonists. We found that TLR7/8 agonists induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines in mixed glial cultures. In contrast, pT-ODNs alone induced only low level expression of two cytokines, CCL2 and CXCL10. The combination of pT-ODNs along with TLR7/8 agonists induced a synergistic response with substantially higher levels of proinflammatory cytokines and chemokines compared to CL075. This enhancement was not due to cellular uptake of the agonist, indicating that the pT-ODN enhancement of cytokine responses was due to effects on an intracellular process. Interestingly, this response was also not due to synergistic stimulation of both TLR7 and TLR8, as the loss of TLR7 abolished the activation of glial cells and cytokine production. Thus, pT-ODNs act in synergy with TLR7/8 agonists to induce strong TLR7-dependent cytokine production in glial cells, suggesting that the combination of pT-ODNs with TLR7 agonists may be a useful mechanism to induce pronounced glial activation in the CNS.

  12. IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals.

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    Le, Hongnga T; Tran, Vuvi G; Kim, Wonyoung; Kim, Juyang; Cho, Hong R; Kwon, Byungsuk

    2012-07-01

    IL-33 is known to play an important role in Th2 immunity. In this study, we investigated the effect of IL-33 pretreatment on anti-fungal response using an acute Candida albicans peritoneal infection model. IL-33 pretreatment induced a rapid fungal clearance and markedly reduced the C. albicans infection-associated mortality. The priming effect of IL-33 occurred during multiple steps of the neutrophil-mediated anti-fungal response. First, the anti-fungal effect occurred due to the rapid and massive recruitment of neutrophils to the site of infection as a result of the release of CXCR2 chemokines by peritoneal macrophages and by reversal of the TLR-induced reduction of CXCR2 expression in neutrophils during IL-33 priming. Second, conditioning of neutrophils by IL-33 activated the TLR and dectin-1 signaling pathways, leading to the upregulation of complement receptor 3 expression induced by C. albicans. Upregulated CR3 in turn increased the phagocytosis of opsonized C. albicans and resulted in the production of high levels of reactive oxygen species and the subsequent enhanced killing activity of neutrophils. Taken together, our results suggest that IL-33 can regulate the anti-fungal activity of neutrophils by collaborative modulation of the signaling pathways of different classes of innate immune receptors.

  13. TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA enhances CD8+ T Cell responses providing protection against Leishmania (Viannia.

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    Asha Jayakumar

    2011-06-01

    Full Text Available Leishmania (Viannia parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V. panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that

  14. Targeting TLR2 for Vaccine Development

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    Afonso P. Basto

    2014-01-01

    Full Text Available Novel and more effective immunization strategies against many animal diseases may profit from the current knowledge on the modulation of specific immunity through stimulation of innate immune receptors. Toll-like receptor (TLR2-targeting formulations, such as synthetic lipopeptides and antigens expressed in fusion with lipoproteins, have been shown to have built-in adjuvant properties and to be effective at inducing cellular and humoral immune mechanisms in different animal species. However, contradictory data has arisen concerning the profile of the immune response elicited. The benefits of targeting TLR2 for vaccine development are thus still debatable and more studies are needed to rationally explore its characteristics. Here, we resume the main features of TLR2 and TLR2-induced immune responses, focusing on what has been reported for veterinary animals.

  15. Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-κB signaling pathway in lipopolysaccharide-induced mastitis in mice.

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    Fu, Yunhe; Gao, Ruifeng; Cao, Yongguo; Guo, Mengyao; Wei, Zhengkai; Zhou, Ershun; Li, Yimeng; Yao, Minjun; Yang, Zhengtao; Zhang, Naisheng

    2014-05-01

    Curcumin, the main constituent of the spice turmeric, has been reported to have potent anti-inflammatory properties. However, the effect of curcumin on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The aim of this study was to investigate whether curcumin could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of the mammary gland. Curcumin was applied 1h before and 12h after LPS treatment. The results showed that curcumin attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in a dose-dependent manner. Additionally, Western blotting results showed that curcumin inhibited the phosphorylation of IκB-α and NF-κB p65 and the expression of TLR4. These results indicated that curcumin has protective effect on mice mastitis and the anti-inflammatory mechanism of curcumin on LPS-induced mastitis in mice may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathways. Curcumin may be a potential therapeutic agent against mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Genetic Variability as a Regulator of TLR4 and NOD Signaling in Response to Bacterial Driven DNA Damage Response (DDR and Inflammation: Focus on the Gastrointestinal (GI Tract

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    Evagelia Spanou

    2017-05-01

    Full Text Available The fundamental role of human Toll-like receptors (TLRs and NOD-like receptors (NLRs, the two most studied pathogen recognition receptors (PRRs, is the protection against pathogens and excessive tissue injury. Recent evidence supports the association between TLR/NLR gene mutations and susceptibility to inflammatory, autoimmune, and malignant diseases. PRRs also interfere with several cellular processes, such as cell growth, apoptosis, cell proliferation, differentiation, autophagy, angiogenesis, cell motility and migration, and DNA repair mechanisms. We briefly review the impact of TLR4 and NOD1/NOD2 and their genetic variability in the process of inflammation, tumorigenesis and DNA repair, focusing in the gastrointestinal tract. We also review the available data on new therapeutic strategies utilizing TLR/NLR agonists and antagonists for cancer, allergic diseases, viral infections and vaccine development against both infectious diseases and cancer.

  17. Genetic Variability as a Regulator of TLR4 and NOD Signaling in Response to Bacterial Driven DNA Damage Response (DDR) and Inflammation: Focus on the Gastrointestinal (GI) Tract.

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    Spanou, Evagelia; Kalisperati, Polyxeni; Pateras, Ioannis S; Papalampros, Alexandros; Barbouti, Alexandra; Tzioufas, Athanasios G; Kotsinas, Athanassios; Sougioultzis, Stavros

    2017-01-01

    The fundamental role of human Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the two most studied pathogen recognition receptors (PRRs), is the protection against pathogens and excessive tissue injury. Recent evidence supports the association between TLR/NLR gene mutations and susceptibility to inflammatory, autoimmune, and malignant diseases. PRRs also interfere with several cellular processes, such as cell growth, apoptosis, cell proliferation, differentiation, autophagy, angiogenesis, cell motility and migration, and DNA repair mechanisms. We briefly review the impact of TLR4 and NOD1/NOD2 and their genetic variability in the process of inflammation, tumorigenesis and DNA repair, focusing in the gastrointestinal tract. We also review the available data on new therapeutic strategies utilizing TLR/NLR agonists and antagonists for cancer, allergic diseases, viral infections and vaccine development against both infectious diseases and cancer.

  18. Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation.

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    Katja Derkow

    Full Text Available Interleukin-17 (IL-17 acts as a key regulator in central nervous system (CNS inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear.In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays.We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88 expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell contact between T

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

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    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.

  20. 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.

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

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    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.

  2. 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.

  3. Genetically determined high activity of IL-12 and IL-18 in ulcerative colitis and TLR5 in Crohns disease were associated with non-response to anti-TNF therapy

    DEFF Research Database (Denmark)

    Bank, S.; Andersen, P. S.; Burisch, J.

    2018-01-01

    and the IFNG pathways were assessed in 482 and 256 prior anti-TNF naïve Danish patients with CD and UC, respectively. The results were analysed using logistic regression (adjusted for age and gender). Eight functional SNPs were associated with anti-TNF response either among patients with CD (TLR5 (rs5744174...

  4. Human Response to Natural Disasters

    Directory of Open Access Journals (Sweden)

    Dara Nix-Stevenson

    2013-07-01

    Full Text Available This study elaborates on the connection between socioeconomic status, education, and the ability to respond to natural disasters. Using the aftermath of Hurricane Katrina and other natural disasters as teachable moments, I foreground how uneven access to resources and capital leave some people more vulnerable than others to natural disasters and how marginal communities inevitably bear the accompanying repercussions of who gets what, when, and how much in the postdisaster emergency relief and reconstruction phase. This occurs not necessarily and merely through a “natural” disaster, as the Boxer Day Tsunami or Hurricane Katrina, but through processes of social, political, and economic disempowerment associated with prior racialized histories and inequitable access to cultural capital.

  5. Primary development and participation in a foreign antigen-driven immune response of a chromatin-reactive B cell clonotype are not influenced by TLR9 or other MyD88-dependent TLRs.

    Science.gov (United States)

    Coffey, Francis; Liu, Xiaohe; Manser, Tim

    2007-11-15

    Recent findings support a central role for TLRs in both foreign Ag-driven immune responses and systemic autoimmune diseases mediated by B lymphocytes. In vitro studies have shown that the Ag receptors (BCRs) on B cells specific for nuclear autoantigens can facilitate the delivery of these autoantigens to the endocytic compartment, resulting in activation of the nucleic acid-specific TLRs present in this subcellular locale. If this pathway is operative in vivo it might promote the development, survival, or activation of such autoreactive B cells. To test this idea, we evaluated the influence of a deficiency in the CpG DNA-specific TLR, TLR9, or all MyD88-dependent TLRs on the primary development and foreign Ag-driven immune response of B cells in a line of V(H) knockin mice that contains a high frequency of "dual reactive" B cells specific for DNA-based autoantigens such as chromatin, as well as the hapten arsonate. We found that although development and activation of these B cells in vitro are clearly influenced by DNA-based autoantigens, TLR9 or MyD88 deficiencies had no apparent effect on the primary development and participation in the anti-arsonate response of these B cells in vivo. We discuss these results in the context of previous models for the role of TLR9 and other TLRs in the regulation of antinuclear Ag B cell development and activity.

  6. Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells.

    Science.gov (United States)

    Coelho-Dos-Reis, Jordana G; Huang, Jing; Tsao, Tiffany; Pereira, Felipe V; Funakoshi, Ryota; Nakajima, Hiroko; Sugiyama, Haruo; Tsuji, Moriya

    2016-07-01

    In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8+ T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells

    Science.gov (United States)

    Coelho-dos-Reis, Jordana G.; Huang, Jing; Tsao, Tiffany; Pereira, Felipe V.; Funakoshi, Ryota; Nakajima, Hiroko; Sugiyama, Haruo; Tsuji, Moriya

    2016-01-01

    In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8+ T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8+ T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8+ T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44+CD62L−NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation. PMID:27132023

  8. Measurement of TLR-induced macrophage spreading by automated image analysis: differential role of Myd88 and MAPK in early and late responses

    Directory of Open Access Journals (Sweden)

    Jens eWenzel

    2011-10-01

    Full Text Available Sensing of infectious danger by Toll-like receptors (TLR on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluorescence microscopy pictures is very time consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deificiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the MAPK ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8 – 24 h after stimulation. The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger through-put analysis of macrophage spreading, e.g. in siRNA knockdown screens.

  9. Histones from Dying Renal Cells Aggravate Kidney Injury via TLR2 and TLR4

    Science.gov (United States)

    Allam, Ramanjaneyulu; Scherbaum, Christina Rebecca; Darisipudi, Murthy Narayana; Mulay, Shrikant R.; Hägele, Holger; Lichtnekert, Julia; Hagemann, Jan Henrik; Rupanagudi, Khader Valli; Ryu, Mi; Schwarzenberger, Claudia; Hohenstein, Bernd; Hugo, Christian; Uhl, Bernd; Reichel, Christoph A.; Krombach, Fritz; Monestier, Marc; Liapis, Helen; Moreth, Kristin; Schaefer, Liliana

    2012-01-01

    In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI. PMID:22677551

  10. Genetic Variability as a Regulator of TLR4 and NOD Signaling in Response to Bacterial Driven DNA Damage Response (DDR) and Inflammation: Focus on the Gastrointestinal (GI) Tract

    OpenAIRE

    Spanou, Evagelia; Kalisperati, Polyxeni; Pateras, Ioannis S.; Papalampros, Alexandros; Barbouti, Alexandra; Tzioufas, Athanasios G.; Kotsinas, Athanassios; Sougioultzis, Stavros

    2017-01-01

    The fundamental role of human Toll-like receptors (TLRs) and NOD-like receptors (NLRs), the two most studied pathogen recognition receptors (PRRs), is the protection against pathogens and excessive tissue injury. Recent evidence supports the association between TLR/NLR gene mutations and susceptibility to inflammatory, autoimmune, and malignant diseases. PRRs also interfere with several cellular processes, such as cell growth, apoptosis, cell proliferation, differentiation, autophagy, angioge...

  11. TOLL-LIKE RECEPTORS (TLR 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

    Directory of Open Access Journals (Sweden)

    Cristiane Beatriz de Oliveira

    2015-02-01

    Full Text Available There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs 2 and 4 in localized (LD and disseminated (DD dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.

  12. TLR4 (not TLR2) dominate cognate TLR activity associated with CoCrMo implant particles.

    Science.gov (United States)

    Samelko, Lauryn; Landgraeber, Stefan; McAllister, Kyron; Jacobs, Joshua; Hallab, Nadim J

    2017-05-01

    Innate immune reactions to orthopedic implant debris are the primary cause of total joint replacement (TJR) failure over the long term (15-20 years). The role of pathogen associated pattern recognition receptors (i.e., TLRs) in regulating immune reactivity to metal implant particles remains controversial. Do different TLRs (i.e., TLR2 vs. TLR4) activated by their respective ligands in concert with metal implant debris elicit equivalent innate immune responses? In this investigation, our in vitro and in vivo data indicate that Gram-negative PAMPs are more pro-inflammatory than Gram-positive PAMPs. In vitro results indicated TLR4 activation in concert with CoCrMo orthopedic implant debris (CoCrMo/LPS+) challenged primary macrophages resulted in significantly greater inflammatory responses than CoCrMo/PAM3CSK+ (TLR2). Similarly, in vivo results indicated CoCrMo/LPS+ TLR4 challenge induced a twofold increase in inflammation-induced bone resorption (osteolysis) than CoCrMo/PAM3CSK+ (p alloy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1007-1017, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Antigen presentation by B cells guides programing of memory CD4+T-cell responses to a TLR4-agonist containing vaccine in mice.

    Science.gov (United States)

    Dubois Cauwelaert, Natasha; Baldwin, Susan L; Orr, Mark T; Desbien, Anthony L; Gage, Emily; Hofmeyer, Kimberly A; Coler, Rhea N

    2016-12-01

    The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T-helper 1 (T H 1) CD4 + T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B-cell deficient mice (μMT -/- ), tetramer-positive CD4 + T cells, markers of memory "precursor" effector cells (MPECs), and T-cell adoptive transfers and demonstrated that the early antigen-specific cytokine-producing T H 1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory T H 1 response in μMT -/- mice. We further show that antigen-presentation by B cells is necessary for their role in MPEC generation using B-cell adoptive transfers from wt or MHC class II knock-out mice into μMT -/- mice. Our study challenges the view that B-cell deficiency exclusively alters the T H 1 response at memory time-points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring T H 1 cell-mediated immunity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

    Science.gov (United States)

    Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa

    2015-01-01

    Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis. PMID:26720149

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

    Directory of Open Access Journals (Sweden)

    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.

  16. 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.

  17. TLR-4/miRNA-32-5p/FSTL1 signaling regulates mycobacterial survival and inflammatory responses in Mycobacterium tuberculosis-infected macrophages.

    Science.gov (United States)

    Zhang, Zhi-Min; Zhang, Ai-Rong; Xu, Min; Lou, Jun; Qiu, Wei-Qiang

    2017-03-15

    Macrophages play a pivotal role in host immune response against mycobacterial infection, which is tightly modulated by multiple factors, including microRNAs. The purpose of the present study was to investigate the biological function and potential mechanism of miR-32-5p in human macrophages during Mycobacterium tuberculosis (M.tb) infection. The results demonstrated that miR-32-5p was robustly enhanced in THP-1 and U937 cells in response to M.tb infection. TLR-4 signaling was required for upregulation of miR-32-5p induced by M.tb infection. Additionally, the introduction of miR-32-5p strongly increased the survival rate of intracellular mycobacteria, whereas inhibition of miR-32-5p suppressed intracellular growth of mycobacteria during M.tb challenged. Furthermore, forced expression of miR-32-5p dramatically attenuated the accumulation of inflammatory cytokines IL-1β, IL-6 and TNF-α induced by M.tb infection. Conversely, downregulated expression of miR-32-5p led to enhancement in these inflammatory cytokines. More importantly, our study explored that Follistatin-like protein 1 (FSTL1) was a direct and functional target of miR-32-5p. qRT-PCR and western blot analysis further validated that miR-32-5p negatively regulated the expression of FSTL1. Mechanistically, re-expression of FSTL1 attenuated the ability of miR-32-5p to promote mycobacterial survival. Meanwhile, miR-32-5p-mediated inhibition of the inflammatory cytokine production were completely reversed by overexpression of FSTL1. Collectively, our findings demonstrated a novel role of TLR-4/miRNA-32-5p/FSTL1 in the modulation of host defense against mycobacterial infection, which may provide a better understanding of the pathogenesis of tuberculosis and useful information for developing potential therapeutic interventions against the disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The GITRL-GITR system alters TLR-4 expression on DC during fungal infection.

    Science.gov (United States)

    Vecchiarelli, Anna; Pericolini, Eva; Gabrielli, Elena; Agostini, Massimiliano; Bistoni, Francesco; Nocentini, Giuseppe; Cenci, Elio; Riccardi, Carlo

    2009-01-01

    The glucocorticoid-induced TNFR-related (GITR) protein is a member of the tumor necrosis factor receptor superfamily influencing natural and acquired immune response. GITR is activated by its ligand, GITRL, mainly expressed on antigen presenting cells. Previously, we demonstrated that GITR plays a role in regulating immune response to Candida albicans. Here we analyzed whether GITRL-GITR interaction influences the recognition of C. albicans by regulating the expression of pattern recognition receptors on splenic dendritic cells. Our report demonstrates that under physiological conditions and during candidiasis the GITRL-GITR system affects TLR-2 and TLR-4 expression on DC. These changes correlate with decrease in: MyD88 activation; CD80 and CD40 expression on DC; T cell activation response, including CD28 expression, IL-2 and IFN-gamma production. Our results point out that, during fungal infection, GITRL-GITR interaction modulates TLR-4 and TLR-2 expression, thereby altering the antigen presentation process, and suggesting a role of GITRL-GITR interaction in resistance against infectious diseases.

  19. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

    International Nuclear Information System (INIS)

    González-Guerrero, Cristian; Ocaña-Salceda, Carlos; Berzal, Sergio; Carrasco, Susana; Fernández-Fernández, Beatriz

    2013-01-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory

  20. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-κB-mediated inflammatory responses in kidney tubular cells

    Energy Technology Data Exchange (ETDEWEB)

    González-Guerrero, Cristian, E-mail: cristian.gonzalez@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Ocaña-Salceda, Carlos, E-mail: carlos.ocana@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Berzal, Sergio, E-mail: sberzal@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Carrasco, Susana, E-mail: scarrasco@fjd.es [Renal and Vascular Pathology Laboratory, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); Fernández-Fernández, Beatriz, E-mail: bfernandez@fjd.es [Nephrology, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Av. Reyes Católicos 2, 28040 Madrid (Spain); and others

    2013-11-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-κB activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-κB, STAT3 and IRE1α activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-κB in CNIs-treated tubular cells. • A NF-κB-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory

  1. Helminth-excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway.

    Science.gov (United States)

    Terrazas, César A; Alcántara-Hernández, Marcela; Bonifaz, Laura; Terrazas, Luis I; Satoskar, Abhay R

    2013-11-01

    Dendritic cells (DCs) recognize pathogens and initiate the T-cell response. The DC-helminth interaction induces an immature phenotype in DCs; as a result, these DCs display impaired responses to TLR stimulation and prime Th2-type responses. However, the DC receptors and intracellular pathways targeted by helminth molecules and their importance in the initiation of the Th2 response are poorly understood. In this report, we found that products excreted/secreted by Taenia crassiceps (TcES) triggered cRAF phosphorylation through MGL, MR, and TLR2. TcES interfered with the LPS-induced NFκB p65 and p38 MAPK signaling pathways. In addition, TcES-induced cRAF signaling pathway was critical for down-regulation of the TLR-mediated DC maturation and secretion of IL-12 and TNF-α. Finally, we show for the first time that blocking cRAF in DCs abolishes their ability to induce Th2 polarization in vitro after TcES exposure. Our data demonstrate a new mechanism by which helminths target intracellular pathways to block DC maturation and efficiently program Th2 polarization.

  2. Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

    Science.gov (United States)

    Wong, Eric B; Mallet, Jean-François; Duarte, Jairo; Matar, Chantal; Ritz, Barry W

    2014-04-01

    Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. An unusual dimeric structure and assembly for TLR4 regulator RP105-MD-1

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    Yoon, Sung-il; Hong, Minsun; Wilson, Ian A [Scripps

    2011-11-16

    RP105-MD-1 modulates the TLR4-MD-2-mediated, innate immune response against bacterial lipopolysaccharide (LPS). The crystal structure of the bovine 1:1 RP105-MD-1 complex bound to a putative endogenous lipid at 2.9 Å resolution shares a similar overall architecture to its homolog TLR4-MD-2 but assembles into an unusual 2:2 homodimer that differs from any other known TLR-ligand assembly. The homodimer is assembled in a head-to-head orientation that juxtaposes the N-terminal leucine-rich repeats (LRRs) of the two RP105 chains, rather than the usual tail-to-tail configuration of C-terminal LRRs in ligand-activated TLR dimers, such as TLR1-TRL2, TLR2-TLR6, TLR3-TLR3 and TLR4-TLR4. Another unusual interaction is mediated by an RP105-specific asparagine-linked glycan, which wedges MD-1 into the co-receptor binding concavity on RP105. This unique mode of assembly represents a new paradigm for TLR complexes and suggests a molecular mechanism for regulating LPS responses.

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

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    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

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

    Science.gov (United States)

    Kirschning, Carsten J; Dreher, Stefan; Maass, Björn; Fichte, Sylvia; Schade, Jutta; Köster, Mario; Noack, Andreas; Lindenmaier, Werner; Wagner, Hermann; Böldicke, Thomas

    2010-04-13

    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 (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib 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 (Gly4Ser)3 amino acid sequence. Coexpression of alphaT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with alphaT2ib indicated interaction of alphaT2ib with its cognate antigen within cells. alphaT2ib inhibited NF-kappaB 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 alphaT2ib into HEK293 cells demonstrated high efficiency of the TLR2-alphaT2ib interaction. The alphaT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-alphaT2ib. Transduction with AdValphaT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFalpha mRNA accumulation, as

  6. Inflammatory response of TLR4 deficient spleen macrophages (CRL 2471) to Brucella abortus S19 and an isogenic ΔmglA deletion mutant.

    Science.gov (United States)

    Jacob, Jens; Makou, Patricia; Finke, Antje; Mielke, Martin

    2016-05-01

    Brucellosis is a worldwide distributed zoonosis caused by members of the genus Brucella. One of them, Brucella abortus, is the etiological agent of bovine brucellosis. With the attenuated strain B. abortus S19 a vaccine is available. However, both, virulence (safety) and the ability to induce a protective B and T cell response (efficacy) have to be tested in suitable assays before successful use in the field. For this purpose, several macrophage cell lines of various origins have been used while splenic macrophages are the preferred host cells in vivo. We here characterized the in vitro response of the murine splenic macrophage cell line CRL 2471(I-13.35) to B. abortus. This cell line still depends on the presence of colony-stimulating factor 1 (CSF1) and is derived from LPS resistant (TLR4 deficient) C3H/HeJ mice. For infection the vaccine strain B. abortus S19A as well as the formerly described isogenic deletion mutant B. abortus S19A ΔmglA 3.14 were used. While numbers of viable bacteria did not differ significantly between the vaccine strain and the deletion mutant at 6h post infection, a higher bacterial load was measured in case of the mutant at 24h and 48h after infection. This was also true, when IFNγ was used for macrophage activation. A comprehensive gene expression profile of macrophages was analysed 6 and 24h after infection by means of an RT-PCR based gene expression array. The mutant strain B. abortus S19A ΔmglA 3.14 elicited a stronger cellular response of the splenic macrophages as compared to the parental vaccine strain. This was most prominent for the pro-inflammatory cytokines IL-1α, IL-1β, TNF-α and IL6 as well as for the chemokine ligands CXCL1, CXCL2, CXCL10, CCL2, CCL5, CCL7, CCL17 and the co-stimulatory molecules CD40 and ICAM1. While these differences were also present in IFNγ-stimulated macrophages, an addition of IFNγ after infection not only resulted in a dramatic increase of the translation of the afore mentioned genes but also

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

    Science.gov (United States)

    Redondo, Ana C C; Ceccon, Maria E J R; Silveira-Lessa, Ana L; Quinello, Camila; Palmeira, Patrícia; Carvalho, Werther B; Carneiro-Sampaio, Magda

    2014-01-01

    To analyze toll-like receptor (TLR)-2 and TLR-4 expression in monocytes of newborns with late-onset sepsis. This prospective study included 27 full-term newborns aged 8 to 29 days, with clinical and laboratory diagnosis of late-onset sepsis. Ten newborns (37%) had positive cultures. Cytokines were measured by cytometric bead array in peripheral blood, while TLR-2, TLR-4 expression, and median fluorescence intensity (MFI) were determined by immunophenotyping peripheral whole blood monocytes, and were analyzed with a BD FACSDiva flow cytometer (Becton, Dickinson and Company, USA). A comparison was performed with healthy adults. Microorganisms were identified in 37% of these septic newborns, and all of them had high levels of pro-inflammatory cytokines (IL-8, IL-6, IL-1β) and anti-inflammatory cytokine (IL-10) corroborating the inflammatory/septic process. In monocytes, the frequency of TLR-4 expression was higher in infected newborns (p = 0.01). This study investigated the innate immune response in septic newborns. Septic newborns that relied almost exclusively on the innate immune system showed little in vivo response at monocyte activation, suggesting impaired immune response and increased susceptibility to infection. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  8. Human dendritic cell DC-SIGN and TLR-2 mediate complementary immune regulatory activities in response to Lactobacillus rhamnosus JB-1.

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    Patrycja Konieczna

    Full Text Available The microbiota is required for optimal host development and ongoing immune homeostasis. Lactobacilli are common inhabitants of the mammalian large intestine and immunoregulatory effects have been described for certain, but not all, strains. The mechanisms underpinning these protective effects are beginning to be elucidated. One such protective organism is Lactobacillus rhamnosus JB-1 (Lb. rhamnosus JB-1. Lb. murinus has no such anti-inflammatory protective effects and was used as a comparator organism. Human monocyte-derived dendritic cells (MDDCs were co-incubated with bacteria and analysed over time for bacterial adhesion and intracellular processing, costimulatory molecule expression, cytokine secretion and induction of lymphocyte polarization. Neutralising antibodies were utilized to identify the responsible MDDC receptors. Lb. rhamnosus JB-1 adhered to MDDCs, but internalization and intracellular processing was significantly delayed, compared to Lb. murinus which was rapidly internalized and processed. Lb. murinus induced CD80 and CD86 expression, accompanied by high levels of cytokine secretion, while Lb. rhamnosus JB-1 was a poor inducer of costimulatory molecule expression and cytokine secretion. Lb. rhamnosus JB-1 primed MDDCs induced Foxp3 expression in autologous lymphocytes, while Lb. murinus primed MDDCs induced Foxp3, T-bet and Ror-γt expression. DC-SIGN was required for Lb. rhamnosus JB-1 adhesion and influenced IL-12 secretion, while TLR-2 influenced IL-10 and IL-12 secretion. Here we demonstrate that the delayed kinetics of bacterial processing by MDDCs correlates with MDDC activation and stimulation of lymphocytes. Thus, inhibition or delay of intracellular processing may be a novel strategy by which certain commensals may avoid the induction of proinflammatory responses.

  9. 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.

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

    Science.gov (United States)

    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

  11. IN VITRO INTERACTION OF INFLUENZA VIRUS A(H1N1pdm09 WITH MONOCYTIC MACROPHAGES: INDIVIDUAL RESPONSES OF TLR7 AND RIG1 RECEPTOR GENES

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    T. M. Sokolova

    2017-01-01

    Full Text Available In vitro differentiation of donor blood monocytes to macrophages (Mph following GM-CSF treatment was accompanied by a significant increase in the levels of gene transcription signaling receptors TLR7 or RIG1. The levels of intracellular viral RNA (M1 gene in Mph remained high upon infection by influenza virus A H1N1pdm (Moscow 2009 for 24-96 hours. The innate immunity reactions caused by influenza virus show individual features: they are decreased in Mph from donor 1 which had initially high level of endosomal TLR7 gene activity, and it increased by influenza virus in MPh from donor 2 who had a very low level of TLR7 gene expression. The influenza H1N1pdm virus weakly stimulated expression of gene RIG1 and production of inflammatory cytokines in Mf in donor 1. The differences may be connected with individual sensitivity of the donors to influenza infection.

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

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    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.

  13. Identification and Immune Functional Characterization of Pigeon TLR7

    Science.gov (United States)

    Xiong, Dan; Song, Li; Pan, Zhiming; Chen, Xiang; Geng, Shizhong; Jiao, Xinan

    2015-01-01

    Toll-like receptor 7 (TLR7) is activated by single-stranded RNA and synthetic imidazoquinoline components, and induces interferon production. In this study, we cloned the TLR7 gene from King pigeon (Columba livia). The TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with 15 leucine-rich repeats (LRRs). Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. The tissue distribution of pigeon TLR7 suggests that immune-associated tissues, especially the spleen and liver, have high TLR7 expression. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Following R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at three day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups. Triggering pigeon TLR7 leads to robust up-regulation of inflammatory cytokines and chemokines, suggesting an important role in the innate immune response. PMID:25874762

  14. Inhibitory effect of BMAP-28 on Leptospiral Lipopolysaccharide-Induced TLR2-Dependent Immune Response in Bovine Cells

    OpenAIRE

    GUO, Yijie; Ding, Cuiping; Zhang, Bo; XU, Jun; XUN, Meng; XU, Jiru

    2016-01-01

    Background Bovine leptospirosis is a widespread zoonotic disease, leading to serious economic losses in animal production and causing potential hazards to human health. Leptospiral lipopolysaccharide (L-LPS) plays an important role in leptospirosis pathogenicity. Objectives With respect to L-LPS endotoxin-like activity, we examined bovine immune response to L-LPS and the inhibitory ability of bovine myeloid antimicrobial peptide-28 (BMAP-28) against L-LPS-induced immune activation in bovine c...

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

    OpenAIRE

    Kirschning, Carsten J; Dreher, Stefan; Maa?, Bj?rn; Fichte, Sylvia; Schade, Jutta; K?ster, Mario; Noack, Andreas; Lindenmaier, Werner; Wagner, Hermann; B?ldicke, Thomas

    2010-01-01

    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 blo...

  16. Selective Inhibitors of Kv11.1 Regulate IL-6 Expression by Macrophages in Response to TLR/IL-1R Ligands

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    Cheryl Hunter

    2010-01-01

    Full Text Available The mechanism by which the platelet-endothelial cell adhesion molecule PECAM-1 regulates leukodiapedesis, vascular endothelial integrity, and proinflammatory cytokine expression in vivo is not known. We recently identified PECAM-1 as a negative regulator of Kv11.1, a specific voltage-gated potassium channel that functioned in human macrophages to reset a resting membrane potential following depolarization. We demonstrate here that dofetilide (DOF, a selective inhibitor of the Kv11.1 current, had a profound inhibitory effect on neutrophil recruitment in mice following TLR/IL-1R–elicited peritonitis or intrascrotal injection of IL-1β, but had no effect on responses seen with TNFα. Furthermore, inhibitors of Kv11.1 (DOF, E4031, and astemizole, but not Kv1.3 (margatoxin, suppressed the expression of IL-6 and MCP-1 cytokines by murine resident peritoneal macrophages, while again having no effect on TNFα. In contrast, IL-6 expression by peritoneal mesothelial cells was unaffected. Using murine P388 cells, which lack endogenous C/EBPβexpression and are unresponsive to LPS for the expression of both IL-6 and MCP-1, we observed that DOF inhibited LPS-induced expression of IL-6 mRNA following ectopic expression of wild-type C/EBPβ, but not a serine-64 point mutant. Finally, DOF inhibited the constitutive activation of cdk2 in murine peritoneal macrophages; cdk2 is known to phosphorylate C/EBPβ at serine-64. Taken together, our results implicate a potential role for Kv11.1 in regulating cdk2 and C/EBPβ activity, where robust transactivation of both IL-6 and MCP-1 transcription is known to be dependent on serine-64 of C/EBPβ. Our data might also explain the altered phenotypes displayed by PECAM-1 knockout mice in several disease models.

  17. Meat and fiber intake and interaction with pattern recognition receptors (TLR1, TLR2, TLR4, and TLR10) in relation to colorectal cancer in a Danish prospective, case-cohort study.

    Science.gov (United States)

    Kopp, Tine Iskov; Vogel, Ulla; Tjonneland, Anne; Andersen, Vibeke

    2018-03-01

    Meat and dietary fiber are associated with increased and decreased risk of colorectal cancer (CRC), respectively. Toll-like receptors (TLRs) regulate the intestinal immune response in a complex interplay between the mucosal epithelium and the microbiota and may therefore be important modulators of diet-induced CRC together with other inflammatory mediators. Our aim was to investigate the association between functional TLR polymorphisms and risk of CRC and the interaction with dietary factors. Additionally, interactions with previously studied polymorphisms in IL10, IL1B, PTGS2, and NFKB1 were assessed in order to examine possible biological pathways in meat-induced CRC. A nested case-cohort study of 897 CRC cases and 1689 randomly selected participants from the Danish prospective "Diet, Cancer and Health" study encompassing 57,053 persons was performed using Cox proportional hazard models and the likelihood ratio test. We found associations between polymorphisms in TLR2 (P = 0.018) and TLR4 (P = 0.044) and risk of CRC per se, interactions between intake of red and processed meat (10 g/d) and polymorphisms in TLR1 (P-interaction = 0.032) and TLR10 (P-interaction = 0.026 and 0.036), and intake of cereals (50 g/d) and TLR4 (P-interaction = 0.044) in relation to risk of CRC. Intake of red and processed meat also interacted with combinations of polymorphisms in TLR1 and TLR10 and polymorphisms in NFKB1, IL10, IL1B, and PTGS2 (P-interaction; TLR1/rs4833095 × PTGS2/rs20417 = 0.021, TLR10/rs11096955 × IL10/rs3024505 = 0.047, TLR10/rs11096955 × PTGS2/rs20417 = 0.017, TLR10/rs4129009 × NFKB1/rs28362491 = 0.027, TLR10/rs4129009 × IL1B/rs4848306 = 0.020, TLR10/rs4129009 × IL1B/rs1143623 = 0.021, TLR10/rs4129009 × PTGS2/rs20417 = 0.027), whereas intake of dietary fiber (10 g/d) interacted with combinations of polymorphisms in TLR4, IL10, and PTGS2 (P-interaction; TLR4/rs1554973 × IL10/rs3024505 = 0.0012, TLR4/rs1554973 × PTGS2/rs20417 = 0.0041, TLR4/rs1554973 × PTGS

  18. Modulation of cell proliferation, survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

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    Marcell Costa de MEDEIROS

    2014-06-01

    Full Text Available Objective: The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response. Material and Methods: T lymphocyte (JM and monocyte (U937 cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR. Results: RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively. Conclusions: There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types.

  19. Similar structures but different roles - An updated perspective on TLR structures

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    Balachandran eManavalan

    2011-07-01

    Full Text Available Toll-like receptors (TLRs are pattern recognition receptors (PRRs that recognize conserved structures in pathogens, trigger innate immune responses and prime antigen-specific adaptive immunity. Elucidation of crystal structures of TLRs interacting with their ligands such as TLR1-2 with triacylated lipopeptide, TLR2-6 with diacylated lipopeptide, TLR4-MD-2 with LPS and TLR3 with dsRNA have enabled an understanding of the initiation of TLR signaling. Agonistic ligands such as LPS, dsRNA and lipopeptides induce ‘m’ shaped TLR dimers in which C-termini converge at the center. Such central convergence is necessary to bring the two intracellular receptor TIR domains closer together and promote their dimerization, which serves as an essential step in downstream signaling. In this review, we summarize TLR ECD structures that have been reported to date with special emphasis on ligand recognition and activation mechanism.

  20. N(6)-(2-Hydroxyethyl)adenosine in the Medicinal Mushroom Cordyceps cicadae Attenuates Lipopolysaccharide-Stimulated Pro-inflammatory Responses by Suppressing TLR4-Mediated NF-κB Signaling Pathways.

    Science.gov (United States)

    Lu, Meng-Ying; Chen, Chin-Chu; Lee, Li-Ya; Lin, Ting-Wei; Kuo, Chia-Feng

    2015-10-23

    Natural products play an important role in promoting health with relation to the prevention of chronic inflammation. N(6)-(2-Hydroxyethyl)adenosine (HEA), a physiologically active compound in the medicinal mushroom Cordyceps cicadae, has been identified as a Ca(2+) antagonist and shown to control circulation and possess sedative activity in pharmacological tests. The fruiting body of C. cicadae has been widely applied in Chinese medicine. However, neither the anti-inflammatory activities of HEA nor the fruiting bodies of C. cicadae have been carefully examined. In this study, we first cultured the fruiting bodies of C. cicadae and then investigated the anti-inflammatory activities of water and methanol extracts of wild and artificially cultured C. cicadae fruiting bodies. Next, we determined the amount of three bioactive compounds, adenosine, cordycepin, and HEA, in the extracts and evaluated their synergistic anti-inflammatory effects. Moreover, the possible mechanism involved in anti-inflammatory action of HEA isolated from C. cicadae was investigated. The results indicate that cordycepin is more potent than adenosine and HEA in suppressing the lipopolysaccharide (LPS)-stimulated release of pro-inflammatory cytokines by RAW 264.7 macrophages; however, no synergistic effect was observed with these three compounds. HEA attenuated the LPS-induced pro-inflammatory responses by suppressing the toll-like receptor (TLR)4-mediated nuclear factor-κB (NF-κB) signaling pathway. This result will support the use of HEA as an anti-inflammatory agent and C. cicadae fruiting bodies as an anti-inflammatory mushroom.

  1. Primate immune responses to HIV-1 Env formulated in the saponin-based adjuvant AbISCO-100 in the presence or absence of TLR9 co-stimulation.

    Science.gov (United States)

    Martinez, Paola; Sundling, Christopher; O'Dell, Sijy; Mascola, John R; Wyatt, Richard T; Karlsson Hedestam, Gunilla B

    2015-03-12

    Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines.

  2. A triacylated lipoprotein from Mycoplasma genitalium activates NF-kappaB through Toll-like receptor 1 (TLR1) and TLR2.

    Science.gov (United States)

    Shimizu, Takashi; Kida, Yutaka; Kuwano, Koichi

    2008-08-01

    Mycoplasma genitalium is a sexually transmitted bacterial pathogen that causes nongonococcal chlamydia-negative urethritis, mucopurulent cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility in humans. However, pathogenic agents that induce inflammatory responses have not been identified in M. genitalium. In this study, we examined the involvement of Toll-like receptors (TLRs) in activation of the immune response by a lipoprotein from M. genitalium and their active component responsible for NF-kappaB activation. The Triton X-114 detergent phase of M. genitalium was found to induce NF-kappaB through TLR2. The active component of the Triton X-114 detergent phase was a lipoprotein precursor, MG149. The activation of NF-kappaB by MG149 was inhibited by a dominant negative (DN) construct of TLR1 but not by a DN construct of TLR6. These results indicate that the activation of NF-kappaB by MG149 is dependent on TLR1 and TLR2. A synthetic lipopeptide derived from MG149 containing three acyl chains also induced NF-kappaB through TLR1 and TLR2. Thus, the results show that MG149, a triacylated lipoprotein from M. genitalium, activates NF-kappaB through TLR1 and TLR2.

  3. Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8+ T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells

    OpenAIRE

    Coelho-dos-Reis, Jordana G.; Huang, Jing; Tsao, Tiffany; Pereira, Felipe V.; Funakoshi, Ryota; Nakajima, Hiroko; Sugiyama, Haruo; Tsuji, Moriya

    2016-01-01

    In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8+ T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8+ T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immuniza...

  4. TLR ligands, but not modulators of histone modifiers, can induce the complex immune response pattern of endotoxin tolerance in mammary epithelial cells

    Science.gov (United States)

    Günther, Juliane; Petzl, Wolfram; Zerbe, Holm; Schuberth, Hans-Joachim

    2016-01-01

    Excessive stimulation of the TLR4 axis through LPS reduces the expression of some cytokine genes in immune cells, while stimulating the expression of immune defense genes during a subsequent bacterial infection. This endotoxin tolerance (ET) is mediated via epigenetic mechanisms. Priming the udder of cows with LPS was shown to induce ET in mammary epithelial cells (MEC), thereby protecting the udder against reinfection for some time. Seeking alternatives to LPS priming we tried to elicit ET by priming MEC with either lipopeptide (Pam2CSK4) via the TLR2/6 axis or inhibitors of histone-modifying enzymes. Pre-incubation of MEC with Pam2CSK4 enhanced baseline and induced expression of bactericidal (β-defensin; SLPI) and membrane protecting factors (SAA3, TGM3), while reducing the expression of cytokine- and chemokine-encoding genes (TNF, IL1β) after a subsequent pathogen challenge, the latter, however, not as efficiently as after LPS priming. Pre-treating MEC with various inhibitors of histone H3 modifiers (for demethylation, acetylation or deacetylation) all failed to induce any of the protective factors and only resulted in some dampening of cytokine gene expression after the re-challenge. Hence, triggering immune functions via the TLR axis, but not through those histone modifiers, induced the beneficial phenomenon of ET in MEC. PMID:27913794

  5. TL response of a natural fluorite

    CERN Document Server

    Balogun, F A; Ogundare, F O; Fasasi, M K; Hussein, L A

    1999-01-01

    A batch of a naturally occurring fluorite (CaF sub 2) from the Middle Benue Valley region of Nigeria has been studied in some detail for its thermoluminescence (TL) properties. TL glow peaks are observed at 119, 144 and 224 deg. C at a heating rate of 10 deg. C s sup - sup 1. The TL response is observed to increase with increasing dose, as expected, over the dose range examined. Variations are observed in the decay curves of the various glow peaks with storage at room temperature. While the lower temperature peaks are observed to decay, enhancement of the TL signal is observed for the 224 deg. C glow peak when stored for four weeks. A low-level radioactivity measurement showed no evidence of self-irradiation from naturally occurring radionuclides. UV exposure was suppressed by storage in a black sealed container to exclude sunlight contribution to the observed TL response. A scheme involving the formation of large defect complexes, from smaller ones, during storage, as possible route leading to loss of signal...

  6. Radiation response of Philippine natural rubber latex

    International Nuclear Information System (INIS)

    Dela Rosa, A.M.; Abad, L.V.; Ana-Relleve, L.S.; Tranquilan-Aranilla, C.; Pascual, C.L.

    1998-01-01

    Our earlier work has shown that the natural rubber latex (NRL) produced and processed in the Philippines is suited for radiation vulcanization. The cast films from NRL with 50% TSC exhibited maximum tensile strengths of 25-32 MPa at 15 kGy, which is the vulcanization dose or Dv. In the manufacture of dipped NRL products, certain specifications such as %TSC, protein content and tensile properties, must be met to ensure an acceptable product. For radiation vulcanization of natural rubber latex (RVNRL) to be accepted as an alternative process, it must also meet the requirements. Thus, this paper presents additional data on the radiation response of local NRL at different total solids contents (TSC), leachable proteins from NRL films as a function of dose, and the thermal activities of irradiated natural rubber latex (INRL). Different formulations of NRL showed varying tolerances to nBA. Data showed that as %TSC increases, the maximum concentration of nBA that can be added without affecting the stability of the latex decreases. The Dv increases as the %TSC increases and the nBA content decreases. This difference in response may be attributed to a lower concentration of nBA in formulations with higher %TSC. These data indicate that the parameters in the radiation treatment will be dictated by the intended applications of INRL. The thermogravimetric data showed greater stability of INRL to thermal oxidation relative to the unirradiated NRL, which correlates directly with the tensile properties of the INRL. A radiation dose of 10 kGy increased the amount of proteins leached from cast latex films. The amount of extractable proteins did not increase significantly at higher doses. The SDS PAGE analysis of the extractable proteins from unirradiated latex film showed distinct bands. An additional band at 60 Kda appeared at 10 kGy. All these bands became diffuse at higher doses, indicating the radiolysis of the proteins

  7. The nucleosome (histone-DNA complex) is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs.

    Science.gov (United States)

    Gowda, Nagaraj M; Wu, Xianzhu; Gowda, D Channe

    2011-01-01

    The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.

  8. The nucleosome (histone-DNA complex is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs.

    Directory of Open Access Journals (Sweden)

    Nagaraj M Gowda

    Full Text Available The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-, TLR9(-/- and MyD88(-/- mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.

  9. Increased Expression Profile and Functionality of TLR6 in Peripheral Blood Mononuclear Cells and Hepatocytes of Morbidly Obese Patients with Non-Alcoholic Fatty Liver Disease.

    Science.gov (United States)

    Arias-Loste, María Teresa; Iruzubieta, Paula; Puente, Ángela; Ramos, David; Santa Cruz, Carolina; Estébanez, Ángel; Llerena, Susana; Alonso-Martín, Carmen; San Segundo, David; Álvarez, Lorena; López Useros, Antonio; Fábrega, Emilio; López-Hoyos, Marcos; Crespo, Javier

    2016-11-10

    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.

  10. 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

    2011-03-01

    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.

  11. CXC195 suppresses proliferation and inflammatory response in LPS-induced human hepatocellular carcinoma cells via regulating TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway

    International Nuclear Information System (INIS)

    Wang, Yiting; Tu, Qunfei; Yan, Wei; Xiao, Dan; Zeng, Zhimin; Ouyang, Yuming; Huang, Long; Cai, Jing; Zeng, Xiaoli; Chen, Ya-Jie; Liu, Anwen

    2015-01-01

    Highlights: • CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. • CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells. • CXC195 regulated TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway in LPS-induced HepG2 cells. - Abstract: CXC195 showed strong protective effects in neuronal apoptosis by exerting its antioxidant activity. However, the anti-cancer effects of CXC195 is still with limited acquaintance. Here, we investigated the role of CXC195 in lipopolysaccharide (LPS)-induced human hepatocellular carcinoma (HCC) cells lines (HepG2) and the possible signaling pathways. CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. In addition, CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells, including TNF-α, iNOS, IL-1β, IL-6, CC chemokine ligand (CCL)-2, CCL-22 and epidermal growth factor receptor (EGFR). Moreover, CXC195 inhibited the expressions and interactions of TLR4, MyD88 and TAK1, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of ERK1/2, p38 and JNK. Our results suggested that treatment with CXC195 could attenuate the TLR4-mediated proliferation and inflammatory response in LPS-induced HepG2 cells, thus might be beneficial for the treatment of HCC

  12. Man's nature: innate determinants of response to natural environments

    Science.gov (United States)

    B. L. Driver; Peter Greene

    1977-01-01

    Man's sensory mechanisms evolved by natural selection in natural settings and humans survived as a species not so much by the "club in the hand" but by the "plan in the head." That plan or ability enabled man to remember, interpret, and predict environmental events. Humans have an innate capacity (but not necessarily a developed ability) to...

  13. Mycobacterium tuberculosis and TLR2 agonists inhibit induction of type I IFN and class I MHC antigen cross processing by TLR9.

    Science.gov (United States)

    Simmons, Daimon P; Canaday, David H; Liu, Yi; Li, Qing; Huang, Alex; Boom, W Henry; Harding, Clifford V

    2010-08-15

    Dendritic cells (DCs) cross process exogenous Ags and present them by class I MHC (MHC-I) molecules to CD8(+) T cells specific for Ags from viruses and bacteria such as Mycobacterium tuberculosis. Unmethylated CpG DNA signals through TLR9 to induce type I IFN (IFN-alpha/beta), which enhances MHC-I Ag cross processing, but lipoproteins that signal through TLR2 do not induce IFN-alpha/beta. In these studies we observed that M. tuberculosis, which expresses agonists of both TLR9 and TLR2, did not induce production of IFN-alpha/beta or cross processing by murine DCs. Furthermore, M. tuberculosis and TLR2 agonists inhibited induction of IFN-alpha/beta and DC cross processing by CpG DNA. Exogenous IFN-alpha/beta effectively enhanced cross processing of M. bovis bacillus Calmette-Guérin expressing OVA, bypassing the inhibition of induction of endogenous IFN-alpha/beta. In addition, inhibition of TLR9-induced cross processing of M. bovis bacillus Calmette-Guérin expressing OVA could be circumvented by pretreating cells with CpG DNA to induce IFN-alpha/beta and MHC-I cross processing before inhibitory mycobacterial TLR2 agonists were present. Inhibition of the response to one TLR by another may affect the ultimate response to pathogens like M. tuberculosis that express agonists of multiple TLRs, including TLR2 and TLR9. This mechanism may contribute to immune evasion and explain why IFN-alpha/beta provides little contribution to host immunity to M. tuberculosis. However, downregulation of certain TLR responses may benefit the host by preventing detrimental excessive inflammation that may occur in the presence of persistent infection.

  14. IL-21 enhances the activity of the TLR-MyD88-STAT3 pathway but not the classical TLR-MyD88-NF-κB pathway in human B cells to boost antibody production.

    Science.gov (United States)

    Liu, Bi-Sheng; Stoop, Jeroen N; Huizinga, Tom W; Toes, Rene E M

    2013-10-15

    Both IL-21 and TLR agonists are important regulators of B cell responses, and the combination of IL-21 and TLR stimulation results in increased Ab production. However, it is not clear yet how IL-21 interacts with TLR signaling in B cells. In this study, we show that IL-21 enhances TLR-induced IgG production, whereas it has no effect on TLR-induced IL-6 production by human B cell cultures. These observations are explained by the finding that IL-21 augments TLR-induced IgG production via the TLR-MyD88-STAT3 pathway but not the classical TLR-MyD88-NF-κB pathway. We further demonstrate that stimulation of human B cells with IL-21 and TLR7/8 or TLR9 agonists increases the phosphorylation of STAT3, whereas the activation of NF-κB is not affected. Interestingly, like IL-21, IL-10 in combination with TLR signaling also enhances phosphorylation of STAT3, resulting in an increase of IgG production. Hence, IL-21 and IL-10 increase the activity of the TLR-MyD88-STAT3 pathway in human B cells via enhancing the phosphorylation of STAT3 for Ab production.

  15. Respiratory Influenza A Virus Infection Triggers Local and Systemic Natural Killer Cell Activation via Toll-Like Receptor 7

    Science.gov (United States)

    Stegemann-Koniszewski, Sabine; Behrens, Sarah; Boehme, Julia D.; Hochnadel, Inga; Riese, Peggy; Guzmán, Carlos A.; Kröger, Andrea; Schreiber, Jens; Gunzer, Matthias; Bruder, Dunja

    2018-01-01

    The innate immune system senses influenza A virus (IAV) through different pathogen-recognition receptors including Toll-like receptor 7 (TLR7). Downstream of viral recognition natural killer (NK) cells are activated as part of the anti-IAV immune response. Despite the known decisive role of TLR7 for NK cell activation by therapeutic immunostimulatory RNAs, the contribution of TLR7 to the NK cell response following IAV infection has not been addressed. We have analyzed lung cytokine responses as well as the activation, interferon (IFN)-γ production, and cytotoxicity of lung and splenic NK cells following sublethal respiratory IAV infection in wild-type and TLR7ko mice. Early airway IFN-γ levels as well as the induction of lung NK cell CD69 expression and IFN-γ production in response to IAV infection were significantly attenuated in TLR7-deficient hosts. Strikingly, respiratory IAV infection also primed splenic NK cells for IFN-γ production, degranulation, and target cell lysis, all of which were fully dependent on TLR7. At the same time, lung type I IFN levels were significantly reduced in TLR7ko mice early following IAV infection, displaying a potential upstream mechanism of the attenuated NK cell activation observed. Taken together, our data clearly demonstrate a specific role for TLR7 signaling in local and systemic NK cell activation following respiratory IAV infection despite the presence of redundant innate IAV-recognition pathways. PMID:29497422

  16. Cytomegalovirus evasion of natural killer cell responses.

    Science.gov (United States)

    Farrell, H E; Degli-Esposti, M A; Davis-Poynter, N J

    1999-04-01

    Natural killer (NK) cells are an important component of the innate cellular immune system. They are particularly important during the early immune responses following virus infection, prior to the induction of cytotoxic T cells (CTL). Unlike CTL, which recognize specific peptides displayed on the surface of cells by class I MHC, NK cells respond to aberrant expression of cell surface molecules, in particular class I MHC, in a non-specific manner. Thus, cells expressing low levels of surface class I MHC are susceptible to recognition by NK cells, with concomitant triggering of cytolytic and cytokine-mediated responses. Many viruses, including the cytomegaloviruses, downregulate cell surface MHC class I: this is likely to provide protection against CTL-mediated clearance of infected cells, but may also render infected cells sensitive to NK-cell attack. This review focuses upon cytomegalovirus-encoded proteins that are believed to promote evasion of NK-cell-mediated immunity. The class I MHC homologues, encoded by all cytomegaloviruses characterised to date, have been implicated as molecular 'decoys', which may mimic the ability of cellular MHC class I to inhibit NK-cell functions. Results from studies in vitro are not uniform, but in general they support the proposal that the class I homologues engage inhibitory receptors from NK cells and other cell types that normally interact with cellular class I. Consistent with this, in vivo studies of murine cytomegalovirus indicate that the class I homologue is required for efficient evasion of NK-cell-mediated clearance. Recently a second murine cytomegalovirus protein, a C-C chemokine homologue, has been implicated as promoting evasion of NK and T-cell-mediated clearance in vivo.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer.

    Science.gov (United States)

    Kim, Tae Woo; Lee, Seon-Jin; Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-26

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells.

  19. Genetically determined high activity of IL-12 and IL-18 in ulcerative colitis and TLR5 in Crohns disease were associated with non-response to anti-TNF therapy.

    Science.gov (United States)

    Bank, S; Andersen, P S; Burisch, J; Pedersen, N; Roug, S; Galsgaard, J; Turino, S Y; Brodersen, J B; Rashid, S; Rasmussen, B K; Avlund, S; Olesen, T B; Hoffmann, H J; Nexø, B A; Sode, J; Vogel, U; Andersen, V

    2018-01-01

    Anti-tumour necrosis factor-α (TNF-α) is used for treatment of severe cases of inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). However, one-third of the patients do not respond to the treatment. A recent study indicated that genetically determined high activity of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6 and interferon gamma (IFN-γ), are associated with non-response to anti-TNF therapy. Using a candidate gene approach, 21 functional single-nucleotide polymorphisms (SNPs) in 14 genes in the Toll-like receptors, the inflammasome and the IFNG pathways were assessed in 482 and 256 prior anti-TNF naïve Danish patients with CD and UC, respectively. The results were analysed using logistic regression (adjusted for age and gender). Eight functional SNPs were associated with anti-TNF response either among patients with CD (TLR5 (rs5744174) and IFNGR2 (rs8126756)), UC (IL12B (rs3212217), IL18 (rs1946518), IFNGR1 (rs2234711), TBX21 (rs17250932) and JAK2 (rs12343867)) or in the combined cohort of patient with CD and UC (IBD) (NLRP3 (rs10754558), IL12B (rs3212217) and IFNGR1 (rs2234711)) (P<0.05). Only the association with heterozygous genotype of IL12B (rs3212217) (OR: 0.24, 95% CI: 0.11-0.53, P=0.008) among patients with UC withstood Bonferroni correction for multiple testing. In conclusion, Our results suggest that SNPs associated with genetically determined high activity of TLR5 among patients with CD and genetically determined high IL-12 and IL-18 levels among patients with UC were associated with non-response. Further studies will evaluate whether these genes may help stratifying patients according to the expected response to anti-TNF treatment.

  20. Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications

    NARCIS (Netherlands)

    Malgorzata-Miller, G.; Heinbockel, L.; Brandenburg, K.; Meer, J.W.M. van der; Netea, M.G.; Joosten, L.A.B.

    2016-01-01

    The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic

  1. Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique

    Science.gov (United States)

    Wilson, Ian A.

    2013-01-01

    Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1–D2–D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins. PMID:22989748

  2. Recognition of Candida albicans by gingival fibroblasts: The role of TLR2, TLR4/CD14, and MyD88.

    Science.gov (United States)

    Pinheiro, Claudia Ramos; Coelho, Ana Lúcia; de Oliveira, Carine Ervolino; Gasparoto, Thaís Helena; Garlet, Gustavo Pompermaier; Silva, João Santana; Santos, Carlos Ferreira; Cavassani, Karen Angélica; Hogaboam, Cory M; Campanelli, Ana Paula

    2017-11-08

    Recent evidence indicates that nonprofessional immune cells such as epithelial cells, endothelial cells, and fibroblasts also contribute to innate immunity via secretion of cytokines. Fibroblasts are the principal type of cell found in the periodontal connective tissues and they are involved in the immune response during periodontal disease. The role of fibroblasts in the recognition of pathogens via Toll-like receptors (TLRs) has been established; however, few studies have been conducted concerning the involvement of innate immune receptors in the recognition of Candida albicans by gingival fibroblast. In the current study, we investigate the functional activity of TLR2, cluster of differentiation 14 (CD14), and myeloid differentiation primary response gene 88 (MyD88) molecules in the recognition of C. albicans by gingival fibroblast. First, we identified that gingival fibroblasts expressed TLR2, TLR3, and TLR4. Our results showed that TLR agonists had no effect on these receptors' expression by TLR2, MyD88, and CD14-deficient cells. Notably, C. albicans and a synthetic triacylated lipoprotein (Pam3CSK4) induced a remarkable increase of TLR3 expression on MyD88-deficient gingival fibroblasts. TLR4 expression levels were lower than TLR2 and TLR3 levels and remained unchanged after TLR agonist stimulation. Gingival fibroblasts presented morphological similarities; however, TLR2 deficiency on these cells leads to a lower proliferative response, whereas the deficiency on CD14 expression resulted in lower levels of type I collagen by these cells. In addition, the recognition of C. albicans by gingival fibroblasts had an effect on the secretion of cytokines and it was dependent on a specific recognition molecule. Specifically, tumor necrosis factor-α (TNF-α) production after the recognition of C. albicans was dependent on MyD88, CD14, and TLR2 molecules, whereas the production of interleukin-1β (IL-1β) and IL-13 was dependent on TLR2. These findings are the first to

  3. 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.

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

    Science.gov (United States)

    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

  5. Differential gene expression following TLR stimulation in rag1-/- mutant zebrafish tissues and morphological descriptions of lymphocyte-like cell populations.

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    Preeti J Muire

    Full Text Available In the absence of lymphocytes, rag1-/- mutant zebrafish develop protective immunity to bacteria. In mammals, induction of protection by innate immunity can be mediated by macrophages or natural killer (NK cells. To elucidate potential responsive cell populations, we morphologically characterized lymphocyte-like cells (LLCs from liver, spleen and kidney hematopoietic tissues. In fish, these cells include NK cells and Non-specific cytotoxic cells (NCCs. We also evaluated the transcriptional expression response of select genes that are important indicators of NK and macrophage activation after exposure to specific TLR ligands. The LLC cell populations could be discriminated by size and further discriminated by the presence of cytoplasmic granules. Expression levels of mx, tnfα, ifnγ, t-bet and nitr9 demonstrated dynamic changes in response to intra-coelomically administered β glucan (a TLR2/6 ligand, Poly I:C (a TLR3 ligand and resiquimod (R848 (a TLR7/8 ligand. Following TLR 2/6 stimulation, there was a greater than 100 fold increase in ifnγ in liver, kidney and spleen and moderate increases in tnfα in liver and kidney. TLR3 stimulation caused broad up regulation of mx, down-regulation of tnfα in kidney and spleen tissues and up regulation of nitr9 in the kidney. Following TLR 7/8 stimulation, there was a greater than 100 fold increase in ifnγ in liver and kidney and t-bet in liver. Our gene expression findings suggest that LLCs and macrophages are stimulated following β glucan exposure. Poly I:C causes type I interferon response and mild induction of LLC in the kidney and R-848 exposure causes the strongest LLC stimulation. Overall, the strongest NK like gene expression occurred in the liver. These differential effects of TLR ligands in rag1-/- mutant zebrafish shows strong NK cell-like gene expression responses, especially in the liver, and provides tools to evaluate the basis for protective immunity mediated by the innate immune cells

  6. 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...

  7. 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...

  8. Differential expression of Toll-like receptor 4 (TLR4) in healthy and infected canine endometrium.

    Science.gov (United States)

    Chotimanukul, S; Sirivaidyapong, S

    2011-10-01

    This study provides the first report into immunohistochemical localization of Toll-like receptor (TLR) in the canine reproductive tract. TLR4 was investigated in endometrium during the estrous cycle and in pyometra. Pyometra is the most important pathological condition of the uterus due to bacterial infection in dogs. To protect against invading pathogens, the female reproductive tract has evolved immune mechanisms. TLRs are the cellular components of the afferent arm of the innate immune system. The expression of TLR4 was significantly higher in the endometrial stroma compared to the endometrial surface epithelium and glandular epithelium in proestrus. The glandular epithelium and stroma at the diestrous stage expressed TLR4 significantly higher than surface epithelium. Furthermore, when compared to other healthy groups, the glandular epithelium at diestrus also higher expressed TLR4 than other stages. The expression of TLR4 in the surface epithelium was higher in dogs with pyometra compared with all other groups. And, the surface epithelium of dogs suffering from pyometra also expressed TLR4 more intensely than the glandular epithelium. The innate immunity of infected canine endometrium response to bacterial infection is intensely extremely increased by the expression of TLR4. Furthermore, the different levels of TLR4 expression seems related to physiological changes in distinct cell types of endometrium, leukocytes populations, cytokines and sex hormones. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Toll-like receptors-2 and -9 (TLR2 and TLR9) gene polymorphism in patients with type 2 diabetes and diabetic foot.

    Science.gov (United States)

    Wifi, Mohamed-Naguib Abdalla; Assem, Maha; Elsherif, Rasha Hamed; El-Azab, Hameda Abdel-Fattah; Saif, Aasem

    2017-04-01

    Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response in diabetes mellitus (DM). The aim of this study is to evaluate the association of TLR2 and TLR9 gene polymorphism in patients with type 2 DM (T2DM) and diabetic foot (DF).The study included 90 subjects divided into group I (30 patients with T2DM and DF), group II (30 patients with T2DM and no evidence of DF), and group III (normal control subjects). TLR2 (1350 T/C, rs3804100) and TLR9 (1237 T/C, rs5743836) genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for all subjects.There was a statistically significant difference in the distribution of TLR9-1237 T/C genotypes between groups I and II (P < .029) as well as between groups I and III (P < .001). Calculated risk estimation revealed that TLR9-1237 polymorphism conferred almost 20 times increased risk of DF disorders in T2DM (OR = 20, 95% CI = 5.38-74.30). There was no statistical difference in the distribution of TLR2-1350T/C genotypes between the 3 groups.TLR9-1237 T/C gene polymorphism may be considered as a molecular risk for DF among patients with T2DM.

  10. TLR3 Signaling in Macrophages Is Indispensable for the Protective Immunity of Invariant Natural Killer T Cells against Enterovirus 71 Infection

    OpenAIRE

    Zhu, Kai; Yang, Juhao; Luo, Kaiming; Yang, Chunhui; Zhang, Na; Xu, Ruifeng; Chen, Jianxia; Jin, Mingfei; Xu, Bin; Guo, Nining; Wang, Jianrong; Chen, Zuolong; Cui, Ying; Zhao, Hui; Wang, Yan

    2015-01-01

    Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice fr...

  11. HOUSEHOLD EXPENDITURE IN RESPONSE TO NATURAL DISASTERS

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    Eny Sulistyaningrum

    2015-09-01

    Full Text Available Natural disasters have increased in their frequency, and the intensity of their destruction over the last ten years in Indonesia. Households usually respond to these difficulties by cutting their consump-tion, especially for non-essential goods. Arguably natural disasters are exogenous events, so this paper uses the exogenous variation from natural disasters as a natural experiment design to estimate the effect of disasters on household expenditure. When a certain group is exposed to the causal variable of interest, such as a disaster, and other groups are not, the Difference In Difference model (DID can be used for estimation. Using a micro level survey data set from the Indonesian Family Life Survey (IFLS which covers approximately 83 percent of the Indonesian population within the survey area, this paper examines the effects of natural disasters on household expenditure. This paper also examines whether there are any different impacts from different types of disasters. The finding is there are no significant effects of disasters on total household expenditure for households living in disaster regions, whether they are affected directly or not by the disaster.

  12. 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.

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

    Science.gov (United States)

    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.

  14. Contrasting roles for TLR ligands in HIV-1 pathogenesis.

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    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.

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

    Science.gov (United States)

    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.

  16. Berberine inhibits the LPS-induced proliferation and inflammatory response of stromal cells of adenomyosis tissues mediated by the LPS/TLR4 signaling pathway.

    Science.gov (United States)

    Liu, Li; Chen, Li; Jiang, Caixia; Guo, Jing; Xie, Yan; Kang, Le; Cheng, Zhongping

    2017-12-01

    A previous study by our group has demonstrated that lipopolysaccharide (LPS) induces adenomyosis through stimulating inflammatory cell proliferation and invasive growth of stromal cells via Toll-like receptor 4 (TLR4) signaling. The present study aimed to investigate the effects of berberine (BBR) on LPS-induced ectopic endometrial stromal cells (EESCs) isolated from patients with adenomyosis. The viability of EESCs treated with LPS or LPS plus BBR was detected by a cell counting kit-8 assay, and the cell cycle distribution and apoptosis were evaluated by flow cytometry. The effect of BBR on the expression of key molecules of inflammatory proliferation and invasive growth of LPS-induced EESCs was also evaluated. BBR significantly inhibited the LPS-induced proliferation of EESCs in a dose- and time-dependent manner. BBR induced cell cycle arrest in G0/G1 phase and enhanced apoptosis of LPS-induced EESCs. Furthermore, BBR inhibited the expression of interleukin (IL)-6, IL-8, transforming growth factor-β, epithelial growth factor, vascular endothelial growth factor and matrix metalloproteinase 2 in LPS-induced EESCs. To the best of our knowledge, the present study was the first to demonstrate that BBR has a protective effect on ameliorating the LPS-induced progression of adenomyosis. This result may provide a novel therapeutic strategy for the clinical treatment of the disease.

  17. Baicalein attenuates inflammatory responses by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice.

    Science.gov (United States)

    He, Xuexiu; Wei, Zhengkai; Zhou, Ershun; Chen, Libin; Kou, Jinhua; Wang, Jingjing; Yang, Zhengtao

    2015-09-01

    Baicalein is a phenolic flavonoid presented in the dry roots of Scutellaria baicalensis Georgi. It has been reported that baicalein possesses a number of biological properties, such as antiviral, antioxidative, anti-inflammatory, antithrombotic, and anticancer properties. However, the effect of baicalein on mastitis has not yet been reported. This research aims to detect the effect of baicalein on lipopolysaccharide (LPS)-induced mastitis in mice and to investigate the molecular mechanisms. Baicalein was administered intraperitoneally 1h before and 12h after LPS treatment. The results indicated that baicalein treatment markedly attenuated the damage of the mammary gland induced by LPS, suppressed the activity of myeloperoxidase (MPO) and the levels of tumor necrosis factor (TNF-α) and interleukin (IL-1β) in mice with LPS-induced mastitis. Besides, baicalein blocked the expression of Toll-like receptor 4 (TLR4) and then suppressed the phosphorylation of nuclear transcription factor-kappaB (NF-κB) p65 and degradation inhibitor of NF-κBα (IκBα) and, and inhibited the phosphorylation of p38, extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) in mitogen-activated protein kinase (MAPK) signal pathway. These findings suggested that baicalein may have a potential prospect against mastitis. Copyright © 2015. Published by Elsevier B.V.

  18. CD14 and TLR4 mediate cytokine release promoted by electronegative LDL in monocytes.

    Science.gov (United States)

    Estruch, Montserrat; Bancells, Cristina; Beloki, Lorea; Sanchez-Quesada, Jose Luis; Ordóñez-Llanos, Jordi; Benitez, Sonia

    2013-08-01

    Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes. Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells. CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

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    Johanna Poecheim

    2015-12-01

    Full Text Available Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA. The formulations included (1 trimethyl chitosan (TMC nanoparticles, (2 a squalene-in-water nanoemulsion, and (3 a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9. In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2 was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

  2. HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia

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    Nabanita Das

    2016-10-01

    Full Text Available Infectious and sterile inflammatory diseases are correlated with increased levels of high mobility group box 1 (HMGB1 in tissues and serum. Extracellular HMGB1 is known to activate Toll-like receptors (TLRs 2 and 4 and RAGE (receptor for advanced glycation endproducts in inflammatory conditions. Here, we find that TLR5 is also an HMGB1 receptor that was previously overlooked due to lack of functional expression in the cell lines usually used for studying TLR signaling. HMGB1 binding to TLR5 initiates the activation of NF-κB signaling pathway in a MyD88-dependent manner, resulting in proinflammatory cytokine production and pain enhancement in vivo. Biophysical and in vitro results highlight an essential role for the C-terminal tail region of HMGB1 in facilitating interactions with TLR5. These results suggest that HMGB1-modulated TLR5 signaling is responsible for pain hypersensitivity.

  3. Hyperspectral Cubesat Constellation for Natural Hazard Response

    Science.gov (United States)

    Mandl, Daniel; Crum, Gary; Ly, Vuong; Handy, Matthew; Huemmrich, Karl F.; Ong, Lawrence; Holt, Ben; Maharaja, Rishabh

    2016-01-01

    The authors on this paper are team members of the Earth Observing 1 (E0-1) mission which has flown an imaging spectrometer (hyperspectral) instrument called Hyperion for the past 15+ years. The satellite is able to image any spot on Earth in the nadir looking direction every 16 days and with slewing, of the satellite for up to a 23 degree view angle, any spot on the Earth can be imaged approximately every 2 to 3 days. EO-1 has been used to track many natural hazards such as wildfires, volcanoes and floods. An enhanced capability that has been sought is the ability to image natural hazards in a daily time series for space-based imaging spectrometers. The Hyperion cannot provide this capability on EO-1 with the present polar orbit. However, a constellation of cubesats, each with the same imaging spectrometer, positioned strategically can be used to provide daily coverage or even diurnal coverage, cost-effectively. This paper sought to design a cubesat constellation mission that would accomplish this goal and then to articulate the key tradeoffs.

  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. Mycobacterium tuberculosis Latent Antigen Rv2029c from the Multistage DNA Vaccine A39 Drives TH1 Responses via TLR-mediated Macrophage Activation

    Directory of Open Access Journals (Sweden)

    Haibo Su

    2017-11-01

    Full Text Available Targeting of Mycobacterium tuberculosis (MTB latent antigens comprises a crucial strategy for the development of alternative tuberculosis (TB vaccine(s that protects against TB reactivation. Here, we generated a multistage DNA vaccine, A39, containing the early antigens Ag85A and Rv3425 as well as the latency-associated protein Rv2029c, which conferred protective immunity in a pre-exposure mouse model. Moreover, administration of the A39 vaccination after MTB exposure inhibited reactivation and resulted in significantly lower bacterial loads in the lungs and spleen of mice, compared to those in the control population. Subsequently, we investigated the effect of Rv2029c on innate immunity and characterized the molecular details of the interaction of this protein with the host via iTRAQ proteomic and biochemical assay analyses. Rv2029c activated macrophages, triggered the production of pro-inflammatory cytokines, and promoted toll-like receptor/mitogen-activated protein kinase (TLR/MAPK-dependent macrophage apoptosis. Furthermore, Rv2029c treatment enhanced the ability of Mycobacterium bovis Bacillus Calmette-Guérin (BCG-infected macrophages to present antigens to CD4+ T cells in vitro, which correlated with an increase in MHC-II expression. Lastly, Rv2029c-treated macrophages activated T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, and specifically expanded a population of CD44highCD62LlowCD4+/CD8+ effector/memory cells, indicating that Rv2029c, as a specific recall antigen, contributes to Th1 polarization in T cell immunity. These results suggest that Rv2029c and A39 comprise promising targets for the development of next-generation clinical TB therapeutic vaccines.

  6. 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.

  7. 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

    2017-01-01

    ABSTRACT 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. PMID:28630066

  8. Brand logo design: examining consumer response to naturalness

    OpenAIRE

    Machado, Joana Machado; Vacas-de-Carvalho, Leonor; Torres, Anna; Costa, Patrício

    2015-01-01

    Purpose – This paper aims to study how logo design characteristics influence consumer response. Based on an in-depth literature review on consumer responses to logo design, the authors included in this research one fundamental dimension of logo design, namely, naturalness and investigated the influence of the different types of natural logo designs on affective response. Design/methodology/approach – In total, 96 logos were selected as design stimuli. The logos were previously classi...

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

    Science.gov (United States)

    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.

  10. An accelerated rabies vaccine schedule based on toll-like receptor 3 (TLR3) agonist PIKA adjuvant augments rabies virus specific antibody and T cell response in healthy adult volunteers.

    Science.gov (United States)

    Wijaya, Limin; Tham, Christine Y L; Chan, Yvonne F Z; Wong, Abigail W L; Li, L T; Wang, Lin-Fa; Bertoletti, Antonio; Low, Jenny G

    2017-02-22

    Rabies is a fatal disease where post-exposure prophylaxis (PEP) is crucial in preventing infection. However, deaths even after appropriate PEP, have been reported. The PIKA Rabies vaccine adjuvant is a TLR3 agonist that activates B and T cells leading to a robust immune response. We conducted a phase I, open label, randomized study in healthy adults to assess the safety and immunogenicity of the PIKA Rabies vaccine and an accelerated vaccine regimen. Thirty-seven subjects were randomized into 3 groups: control vaccine classic regimen, PIKA vaccine classic regimen and PIKA vaccine accelerated regimen. Subjects were followed up for safety, rabies virus neutralizing antibodies (RVNA) and T cell responses. Both the control and PIKA Rabies vaccine were well tolerated. All adverse events (AEs) were mild and self-limiting. Seventy-five percent of subjects in the PIKA accelerated regimen achieved a RVNA titer ⩾0.5IU/mL on day 7, compared to 53.9% in the PIKA classic regimen (p=0.411) and 16.7% in control vaccine classic regimen (p=0.012). The PIKA rabies vaccine elicited multi-specific rabies CD4 mediated T cell response already detectable ex vivo at day 7 after vaccination and that was maintained at day 42. The investigational PIKA rabies vaccine was well tolerated and more immunogenic than the commercially available vaccine in healthy adults. Clinical trial registry: The study was registered with clinicaltrials.gov NCT02657161. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. 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...... induction of IL-6 and IL-12p40, and differentiation into CD14+ and DC-SIGN+ DCs.Conclusion: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory...

  12. Plastic Surgery Response in Natural Disasters.

    Science.gov (United States)

    Chung, Susan; Zimmerman, Amanda; Gaviria, Andres; Dayicioglu, Deniz

    2015-06-01

    Disasters cause untold damage and are often unpredictable; however, with proper preparation, these events can be better managed. The initial response has the greatest impact on the overall success of the relief effort. A well-trained multidisciplinary network of providers is necessary to ensure coordinated care for the victims of these mass casualty disasters. As members of this network of providers, plastic surgeons have the ability to efficiently address injuries sustained in mass casualty disasters and are a valuable member of the relief effort. The skill set of plastic surgeons includes techniques that can address injuries sustained in large-scale emergencies, such as the management of soft-tissue injury, tissue viability, facial fractures, and extremity salvage. An approach to disaster relief, the types of disasters encountered, the management of injuries related to mass casualty disasters, the role of plastic surgeons in the relief effort, and resource management are discussed. In order to improve preparedness in future mass casualty disasters, plastic surgeons should receive training during residency regarding the utilization of plastic surgery knowledge in the disaster setting.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

    Directory of Open Access Journals (Sweden)

    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.

  15. Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

    Science.gov (United States)

    Hajjar, Adeline M; Ernst, Robert K; Fortuno, Edgardo S; Brasfield, Alicia S; Yam, Cathy S; Newlon, Lindsay A; Kollmann, Tobias R; Miller, Samuel I; Wilson, Christopher B

    2012-01-01

    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.

  16. Natural semantic networks in the Social Representations of Responsibility

    Directory of Open Access Journals (Sweden)

    Humberto Emilio Aguilera Arévalo

    2010-07-01

    Full Text Available The study of social representations of responsibility is a fundamental construct of the present democratic societies. Different empirical techniques such as natural semantic networks can significantly improve the approach to the object of study than the traditional associationist techniques. The present study examines natural semantic networks of six stimulus words with respect to responsibility and irresponsibility at the individual, in group and out group level in a sample of Guatemalan students.

  17. Utilizing a TLR5-Adjuvanted Cytomegalovirus as a Lentiviral Vaccine in the Nonhuman Primate Model for AIDS.

    Directory of Open Access Journals (Sweden)

    Jesse D Deere

    Full Text Available Despite tremendous progress in our understanding of human immunodeficiency virus (HIV natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV and transiently expressed in telomerized rhesus fibroblast (TeloRF cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α, in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.

  18. Characteristic and functional analysis of toll-like receptors (TLRs in the lophotrocozoan, Crassostrea gigas, reveals ancient origin of TLR-mediated innate immunity.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    Full Text Available The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostreagigas (CgTLRs. All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C. gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.

  19. The combination of maltose-binding protein and BCG-induced Th1 activation is involved in TLR2/9-mediated upregulation of MyD88-TRAF6 and TLR4-mediated downregulation of TRIF-TRAF3.

    Science.gov (United States)

    Liu, Guomu; Zhai, Xiaoyu; Zhou, Hongyue; Yang, Xiaoyu; Zhang, Nannan; Tai, Guixiang; Ni, Weihua

    2018-03-01

    Our previous study demonstrated that maltose-binding protein (MBP) activated Th1 through the TLR2-mediated MyD88-dependent pathway and the TLR4-mediated TRIF-dependent pathway. The combination of MBP and BCG synergistically induced Th1 activation, and the TLR2/9-mediated MyD88-dependent pathway is involved in this process. To further explore this mechanism, we stimulated purified mouse CD4 + T cells with MBP and BCG in vitro. The results demonstrated that MBP combined with BCG synergistically increased IFN-γ production and TLR2/4/9 expression, suggesting the involvement of TLR2/4/9 in the combination-induced Th1 activation. Next, TLRs 2/4/9 were blocked to analyze the effects of TLRs on Th1 activation. The results demonstrated that MBP induced a low level of Th1 activation by upregulating TLR2-mediated MyD88-TRAF6 and TLR4-mediated TRIF-TRAF3 expression, whereas MBP combined with BCG induced synergistic Th1 activation, which was not only triggered by strong upregulation of TLR2/9-mediated MyD88-TRAF6 expression but also by shifting TLR4-mediated TRIF-TRAF3 into the TRIF-TRAF6 pathway. Moreover, we observed that a TLR4 antibody upregulated MyD88 expression and a TLR9 inhibitor downregulated TRIF expression, indicating that there was cross-talk between TLRs 2/4/9 in MBP combined with BCG-induced Th1 activation. Our findings may expand the knowledge regarding TLR cross-talk involved in regulating the Th1 response. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation.

    Directory of Open Access Journals (Sweden)

    Lauryn Samelko

    Full Text Available Cobalt alloy debris has been implicated as causative in the early failure of some designs of current total joint implants. The ability of implant debris to cause excessive inflammation via danger signaling (NLRP3 inflammasome vs. pathogen associated pattern recognition receptors (e.g. Toll-like receptors; TLRs remains controversial. Recently, specific non-conserved histidines on human TLR4 have been shown activated by cobalt and nickel ions in solution. However, whether this TLR activation is directly or indirectly an effect of metals or secondary endogenous alarmins (danger-associated molecular patterns, DAMPs elicited by danger signaling, remains unknown and contentious. Our study indicates that in both a human macrophage cell line (THP-1 and primary human macrophages, as well as an in vivo murine model of inflammatory osteolysis, that Cobalt-alloy particle induced NLRP3 inflammasome danger signaling inflammatory responses were highly dominant relative to TLR4 activation, as measured respectively by IL-1β or TNF-α, IL-6, IL-10, tissue histology and quantitative bone loss measurement. Despite the lack of metal binding histidines H456 and H458 in murine TLR4, murine calvaria challenge with Cobalt alloy particles induced significant macrophage driven in vivo inflammation and bone loss inflammatory osteolysis, whereas LPS calvaria challenge alone did not. Additionally, no significant increase (p500pg/mL. Therefore, not only do the results of this investigation support Cobalt alloy danger signaling induced inflammation, but under normal homeostasis low levels of hematogenous PAMPs (<2pg/mL from Gram-negative bacteria, seem to have negligible contribution to the danger signaling responses elicited by Cobalt alloy metal implant debris. This suggests the unique nature of Cobalt alloy particle bioreactivity is strong enough to illicit danger signaling that secondarily activate concomitant TLR activation, and may in part explain Cobalt particulate

  1. Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation

    Science.gov (United States)

    Samelko, Lauryn; Landgraeber, Stefan; McAllister, Kyron; Jacobs, Joshua; Hallab, Nadim James

    2016-01-01

    Cobalt alloy debris has been implicated as causative in the early failure of some designs of current total joint implants. The ability of implant debris to cause excessive inflammation via danger signaling (NLRP3 inflammasome) vs. pathogen associated pattern recognition receptors (e.g. Toll-like receptors; TLRs) remains controversial. Recently, specific non-conserved histidines on human TLR4 have been shown activated by cobalt and nickel ions in solution. However, whether this TLR activation is directly or indirectly an effect of metals or secondary endogenous alarmins (danger-associated molecular patterns, DAMPs) elicited by danger signaling, remains unknown and contentious. Our study indicates that in both a human macrophage cell line (THP-1) and primary human macrophages, as well as an in vivo murine model of inflammatory osteolysis, that Cobalt-alloy particle induced NLRP3 inflammasome danger signaling inflammatory responses were highly dominant relative to TLR4 activation, as measured respectively by IL-1β or TNF-α, IL-6, IL-10, tissue histology and quantitative bone loss measurement. Despite the lack of metal binding histidines H456 and H458 in murine TLR4, murine calvaria challenge with Cobalt alloy particles induced significant macrophage driven in vivo inflammation and bone loss inflammatory osteolysis, whereas LPS calvaria challenge alone did not. Additionally, no significant increase (p500pg/mL). Therefore, not only do the results of this investigation support Cobalt alloy danger signaling induced inflammation, but under normal homeostasis low levels of hematogenous PAMPs (<2pg/mL) from Gram-negative bacteria, seem to have negligible contribution to the danger signaling responses elicited by Cobalt alloy metal implant debris. This suggests the unique nature of Cobalt alloy particle bioreactivity is strong enough to illicit danger signaling that secondarily activate concomitant TLR activation, and may in part explain Cobalt particulate associated

  2. TLR2, TLR4 and Dectin-1 signalling in hematopoietic stem and progenitor cells determines the antifungal phenotype of the macrophages they produce.

    Science.gov (United States)

    Megías, Javier; Martínez, Alba; Yáñez, Alberto; Goodridge, Helen S; Gozalbo, Daniel; Gil, M Luisa

    2016-05-01

    TLRs represent an attractive target for the stimulation of myeloid cell production by HSPCs. We have previously demonstrated that HSPCs use TLR2 to sense Candida albicans in vivo and induce the production of macrophages. In this work, we used an in vitro model of HSPCs differentiation to investigate the functional consequences for macrophages of exposure of HSPCs to various PAMPs and C. albicans cells. Mouse HSPCs (Lin(-) cells) were cultured with M-CSF to induce macrophage differentiation, in the presence or absence of the following PRR agonists: Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand), depleted zymosan (which only activates Dectin-1), or C. albicans yeasts (which activate several PRRs, but principally TLR2 and Dectin-1). Our data show that these PAMPs differentially impact the anti-microbial function of the macrophages produced by the exposed HSPCs. Pure TLR2 and TLR4 ligands generate macrophages with a diminished ability to produce inflammatory cytokines. In contrast, HSPCs activation in response to C. albicans leads to the generation of macrophages that are better prepared to deal with the infection, as they produce higher amounts of inflammatory cytokines and have higher fungicidal capacity than control macrophages. Therefore, the tailored manipulation of the differentiation process may help to boost the innate immune response to infection. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. 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):11...1-3. (.png) (.svg) (.html) (.csml) Show TLR ignores methylated RNA? PubmedID 16111629 Title TLR ignores methylated RNA

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

    Lifescience Database Archive (English)

    Full Text Available 17275323 TLR signaling. Kawai T, Akira S. Semin Immunol. 2007 Feb;19(1):24-32. Epub... 2007 Feb 1. (.png) (.svg) (.html) (.csml) Show TLR signaling. PubmedID 17275323 Title TLR signaling. Author

  5. 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) (.csml) Show TLR signaling. PubmedID 16410796 Title TLR signaling. Authors Kawai T, A

  6. Deep Learning Models of the Retinal Response to Natural Scenes.

    Science.gov (United States)

    McIntosh, Lane T; Maheswaranathan, Niru; Nayebi, Aran; Ganguli, Surya; Baccus, Stephen A

    2016-01-01

    A central challenge in sensory neuroscience is to understand neural computations and circuit mechanisms that underlie the encoding of ethologically relevant, natural stimuli. In multilayered neural circuits, nonlinear processes such as synaptic transmission and spiking dynamics present a significant obstacle to the creation of accurate computational models of responses to natural stimuli. Here we demonstrate that deep convolutional neural networks (CNNs) capture retinal responses to natural scenes nearly to within the variability of a cell's response, and are markedly more accurate than linear-nonlinear (LN) models and Generalized Linear Models (GLMs). Moreover, we find two additional surprising properties of CNNs: they are less susceptible to overfitting than their LN counterparts when trained on small amounts of data, and generalize better when tested on stimuli drawn from a different distribution (e.g. between natural scenes and white noise). An examination of the learned CNNs reveals several properties. First, a richer set of feature maps is necessary for predicting the responses to natural scenes compared to white noise. Second, temporally precise responses to slowly varying inputs originate from feedforward inhibition, similar to known retinal mechanisms. Third, the injection of latent noise sources in intermediate layers enables our model to capture the sub-Poisson spiking variability observed in retinal ganglion cells. Fourth, augmenting our CNNs with recurrent lateral connections enables them to capture contrast adaptation as an emergent property of accurately describing retinal responses to natural scenes. These methods can be readily generalized to other sensory modalities and stimulus ensembles. Overall, this work demonstrates that CNNs not only accurately capture sensory circuit responses to natural scenes, but also can yield information about the circuit's internal structure and function.

  7. Distinct evolution of TLR-mediated dendritic cell cytokine secretion in patients with limited and diffuse cutaneous systemic sclerosis.

    NARCIS (Netherlands)

    Bon, L. van; Popa, C.; Huibens, R.J.F.; Vonk, M.C.; York, M.; Simms, R.; Hesselstrand, R.; Wuttge, D.M.; Lafyatis, R.; Radstake, T.R.D.J.

    2010-01-01

    BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease and accumulating evidence suggests a role for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). OBJECTIVE: To map TLR-mediated cytokine responses of DCs from patients with SSc. METHODS: 45 patients with SSc were

  8. Molecular cloning of orange-spotted grouper (Epinephelus coioides) TLR21 and expression analysis post Cryptocaryon irritans infection.

    Science.gov (United States)

    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.

  9. Temporal response of laser power standards with natural convective cooling.

    Science.gov (United States)

    Xu, Tao; Gan, Haiyong; Yu, Jing; Zang, Erjun

    2016-01-25

    Laser power detectors with natural convective cooling are convenient to use and hence widely applicable in a power range below 150 W. However, the temporal response characteristics of the laser power detectors need to be studied in detail for accurate measurement. The temporal response based on the absolute laser power standards with natural convective cooling is studied through theoretical analysis, numerical simulations, and experimental verifications. Our results show that the response deviates from a single exponential function and that an ultimate response balance is difficult to achieve because the temperature rise of the heat sink leads to continuous increase of the response. To determine the measurement values, an equal time reading method is proposed and validated by the laser power calibrations.

  10. Bartonella quintana lipopolysaccharide (LPS): structure and characteristics of a potent TLR4 antagonist for in-vitro and in-vivo applications

    Science.gov (United States)

    Malgorzata-Miller, Gosia; Heinbockel, Lena; Brandenburg, Klaus; van der Meer, Jos W. M.; Netea, Mihai G.; Joosten, Leo A. B.

    2016-01-01

    The pattern recognition receptor TLR4 is well known as a crucial receptor during infection and inflammation. Several TLR4 antagonists have been reported to inhibit the function of TLR4. Both natural occurring antagonists, lipopolysaccharide (LPS) from Gram-negative bacteria as well as synthetic compounds based on the lipid A structure of LPS have been described as potent inhibitors of TLR4. Here, we have examined the characteristics of a natural TLR4 antagonist, isolated from Bartonella quintana bacterium by elucidating its chemical primary structure. We have found that this TLR4 antagonist is actually a lipooligosaccharide (LOS) instead of a LPS, and that it acts very effective, with a high inhibitory activity against triggering by the LPS-TLR4 system in the presence of a potent TLR4 agonist (E. coli LPS). Furthermore, we demonstrate that B. quintana LPS is not inactivated by polymyxin B, a classical cyclic cationic polypeptide antibiotic that bind the lipid A part of LPS, such as E. coli LPS. Using a murine LPS/D-galactosamine endotoxaemia model we showed that treatment with B. quintana LPS could improve the survival rate significantly. Since endogenous TLR4 ligands have been associated with several inflammatory- and immune-diseases, B. quintana LPS might be a novel therapeutic strategy for TLR4-driven pathologies. PMID:27670746

  11. The natural environment as an area of Corporate Social Responsibility

    Directory of Open Access Journals (Sweden)

    Wolak-Tuzimek Anna

    2017-09-01

    Full Text Available Areas of Corporate Social Responsibility (CSR have been defined in ISO 26000. Guidelines of the International Standardisation Organisation distinguish seven areas: corporate governance, human rights, labour practices, natural environment, fair operating practices, consumer issues, social commitment and development of local communities. This article presents good practices implemented by enterprises in the individual areas, in particular, actions in the area of the natural environment. Two research hypotheses are posited concerning the rate of implementing good CSR practices and the number of actions in the natural environment area. National Responsible Business Forum research and a survey of a group of enterprises in the Mazovian region, conducted by the authors in 2014–2016, served to verify the hypotheses. The results imply that the number of good practices realised in CSR areas tends to grow. In addition, actions in the area of the natural environment rank third with regard to good practices implemented.

  12. Fungal Chitin Dampens Inflammation through IL-10 Induction Mediated by NOD2 and TLR9 Activation

    Science.gov (United States)

    Wagener, Jeanette; Malireddi, R. K. Subbarao; Lenardon, Megan D.; Köberle, Martin; Vautier, Simon; MacCallum, Donna M.; Biedermann, Tilo; Schaller, Martin; Netea, Mihai G.; Kanneganti, Thirumala-Devi; Brown, Gordon D.; Brown, Alistair J. P.; Gow, Neil A. R.

    2014-01-01

    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. Authors Summary Chitin is the second most abundant polysaccharide in nature after cellulose and an essential component of the cell wall of all fungal pathogens. The discovery of human chitinases and chitinase-like binding proteins indicates that fungal chitin is recognised by cells of the human immune system, shaping the immune response towards the invading pathogen. We show that three immune cell receptors– the mannose receptor, NOD2 and TLR9 recognise chitin and act together to mediate an anti-inflammatory response via secretion of the cytokine IL-10. This mechanism may prevent inflammation-based damage

  13. A new non-phagocytic TLR6 with broad recognition ligands from Pacific oyster Crassostrea gigas.

    Science.gov (United States)

    Wang, Weilin; Zhang, Tao; Wang, Lingling; Xu, Jiachao; Li, Meijia; Zhang, Anguo; Qiu, Limei; Song, Linsheng

    2016-12-01

    Toll like receptors (TLRs) are evolutionarily prevalent recognition molecules in the Animalia and Plantae kingdom, which play vital roles in immune defense and homeostasis maintenance. Recently, the expansion of TLRs has been reported in invertebrate genomes, but the characters and immune functions of these expanded TLRs were still not well known. In the present study, a new member of TLR family with five LRR domains was identified in Crassostrea gigas (designated CgTLR6). It shared homology with TLRs from other organisms with the closest phylogenic relationship with molluscan TLRs. The recombinant protein of CgTLR6 (rCgTLR6) displayed direct bind activity to gram-negative bacteria Vibrio anguillarum and Vibrio splendidus, gram-positive bacteria Staphylococci aureus and Micrococcus luteus, and fungi Pichia pastoris, but not to fungi Yarrowia lipolytica. It also exhibited affinity to lipopolysaccharide (LPS) and peptidoglycan (PGN), while no affinity to mannan (MAN). The mRNA of CgTLR6 was mainly detected in hemocytes and hepatopancreas, and was significantly induced (p < 0.01) in hemocytes after the oyster was stimulated with LPS, PGN or bacteria V. splendidus. Immunofluorescence analysis indicated that CgTLR6 was mainly located at the membrane of hemocytes. The blockage of CgTLR6 by anti-rCgTLR6 antibody did not significantly inhibit the phagocytic rates of hemocytes toward recognized gram-negative bacteria V. anguillarum and V. splendidus, and unrecognized fungi Y. lipolytica. These results collectively implied that CgTLR6 was a novel non-phagocytic receptor of C. gigas to mediate humoral immune response by recognizing pathogen-associated molecular patterns on the invaders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Evolution of the bovine TLR gene family and member associations with Mycobacterium avium subspecies paratuberculosis infection.

    Directory of Open Access Journals (Sweden)

    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

  15. Cobalt Alloy Implant Debris Induces Inflammation and Bone Loss Primarily through Danger Signaling, Not TLR4 Activation: Implications for DAMP-ening Implant Related Inflammation.

    Science.gov (United States)

    Samelko, Lauryn; Landgraeber, Stefan; McAllister, Kyron; Jacobs, Joshua; Hallab, Nadim James

    2016-01-01

    Cobalt alloy debris has been implicated as causative in the early failure of some designs of current total joint implants. The ability of implant debris to cause excessive inflammation via danger signaling (NLRP3 inflammasome) vs. pathogen associated pattern recognition receptors (e.g. Toll-like receptors; TLRs) remains controversial. Recently, specific non-conserved histidines on human TLR4 have been shown activated by cobalt and nickel ions in solution. However, whether this TLR activation is directly or indirectly an effect of metals or secondary endogenous alarmins (danger-associated molecular patterns, DAMPs) elicited by danger signaling, remains unknown and contentious. Our study indicates that in both a human macrophage cell line (THP-1) and primary human macrophages, as well as an in vivo murine model of inflammatory osteolysis, that Cobalt-alloy particle induced NLRP3 inflammasome danger signaling inflammatory responses were highly dominant relative to TLR4 activation, as measured respectively by IL-1β or TNF-α, IL-6, IL-10, tissue histology and quantitative bone loss measurement. Despite the lack of metal binding histidines H456 and H458 in murine TLR4, murine calvaria challenge with Cobalt alloy particles induced significant macrophage driven in vivo inflammation and bone loss inflammatory osteolysis, whereas LPS calvaria challenge alone did not. Additionally, no significant increase (p500pg/mL). Therefore, not only do the results of this investigation support Cobalt alloy danger signaling induced inflammation, but under normal homeostasis low levels of hematogenous PAMPs (alloy metal implant debris. This suggests the unique nature of Cobalt alloy particle bioreactivity is strong enough to illicit danger signaling that secondarily activate concomitant TLR activation, and may in part explain Cobalt particulate associated inflammatory and toxicity-like reactions of specific orthopedic implants.

  16. Auditory Brainstem Responses to Continuous Natural Speech in Human Listeners.

    Science.gov (United States)

    Maddox, Ross K; Lee, Adrian K C

    2018-01-01

    Speech is an ecologically essential signal, whose processing crucially involves the subcortical nuclei of the auditory brainstem, but there are few experimental options for studying these early responses in human listeners under natural conditions. While encoding of continuous natural speech has been successfully probed in the cortex with neurophysiological tools such as electroencephalography (EEG) and magnetoencephalography, the rapidity of subcortical response components combined with unfavorable signal-to-noise ratios signal-to-noise ratio has prevented application of those methods to the brainstem. Instead, experiments have used thousands of repetitions of simple stimuli such as clicks, tone-bursts, or brief spoken syllables, with deviations from those paradigms leading to ambiguity in the neural origins of measured responses. In this study we developed and tested a new way to measure the auditory brainstem response (ABR) to ongoing, naturally uttered speech, using EEG to record from human listeners. We found a high degree of morphological similarity between the speech-derived ABRs and the standard click-evoked ABR, in particular, a preserved Wave V, the most prominent voltage peak in the standard click-evoked ABR. Because this method yields distinct peaks that recapitulate the canonical ABR, at latencies too short to originate from the cortex, the responses measured can be unambiguously determined to be subcortical in origin. The use of naturally uttered speech to measure the ABR allows the design of engaging behavioral tasks, facilitating new investigations of the potential effects of cognitive processes like language and attention on brainstem processing.

  17. PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway

    Directory of Open Access Journals (Sweden)

    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.

  18. Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut

    NARCIS (Netherlands)

    Ganguli, K.; Collado, M.C.; Rautava, J.; Lu, L.; Satokari, R.M.; Ossowski, von I.; Reunanen, J.; Vos, de W.M.; Palva, A.; Isolauri, E.; Salminen, S.; Walker, W.A.; Rautava, S.

    2015-01-01

    BACKGROUND: Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human

  19. Toll-like receptor responses to Peste des petits ruminants virus in goats and water buffalo.

    Directory of Open Access Journals (Sweden)

    Sakthivel Dhanasekaran

    Full Text Available Ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the Peste des petits ruminants virus (PPRV. Differences in susceptibility to goat plague among different breeds and water buffalo exist. The host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as Toll like receptors (TLR. We investigated the role of TLR and cytokines in differential susceptibility of goat breeds and water buffalo to PPRV. We examined the replication of PPRV in peripheral blood mononuclear cells (PBMC of Indian domestic goats and water buffalo and demonstrated that the levels of TLR3 and TLR7 and downstream signalling molecules correlation with susceptibility vs resistance. Naturally susceptible goat breeds, Barbari and Tellichery, had dampened innate immune responses to PPRV and increased viral loads with lower basal expression levels of TLR 3/7. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (IL 10 levels were lower in PPRV resistant Kanni and Salem Black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected PBMC. Water buffalo produced higher levels of interferon (IFN α in comparison with goats at transcriptional and translational levels. Pre-treatment of Vero cells with human IFNα resulted in reduction of PPRV replication, confirming the role of IFNα in limiting PPRV replication. Treatment with IRS66, a TLR7 antagonist, resulted in the reduction of IFNα levels, with increased PPRV replication confirming the role of TLR7. Single nucleotide polymorphism analysis of TLR7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to PPRV. Analyzing other host genetic factors

  20. Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus

    Science.gov (United States)

    Wong-Baeza, Carlos; Tescucano, Alonso; Astudillo, Horacio; Reséndiz, Albany; Landa, Carla; España, Luis; Serafín-López, Jeanet; Estrada-García, Iris; Estrada-Parra, Sergio; Flores-Romo, Leopoldo; Wong, Carlos; Baeza, Isabel

    2015-01-01

    Systemic lupus erythematosus is characterized by dysregulated activation of T and B cells and autoantibodies to nuclear antigens and, in some cases, lipid antigens. Liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice, including IgM and IgG antibodies against nonbilayer phospholipid arrangements. As the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation, we evaluated the effect of these liposomes on Toll-like receptor (TLR) signaling, cytokine production, proinflammatory gene expression, and T, NKT, dendritic, and B cells. Liposomes induce TLR-4- and, to a lesser extent, TLR-2/TLR-6-dependent signaling in TLR-expressing human embryonic kidney (HEK) cells and bone marrow-derived macrophages. Mice with the lupus-like disease had increased serum concentrations of proinflammatory cytokines, C3a and C5a; they also had more TLR-4-expressing splenocytes, a higher expression of genes associated with TRIF-dependent TLR-4-signaling and complement activation, and a lower expression of apoptosis-related genes, compared to healthy mice. The percentage of NKT and the percentage and activation of dendritic and B2 cells were also increased. Thus, TLR-4 and TLR-2/TLR-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus-like disease in mice. PMID:26568960

  1. Nonbilayer Phospholipid Arrangements Are Toll-Like Receptor-2/6 and TLR-4 Agonists and Trigger Inflammation in a Mouse Model Resembling Human Lupus

    Directory of Open Access Journals (Sweden)

    Carlos Wong-Baeza

    2015-01-01

    Full Text Available Systemic lupus erythematosus is characterized by dysregulated activation of T and B cells and autoantibodies to nuclear antigens and, in some cases, lipid antigens. Liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice, including IgM and IgG antibodies against nonbilayer phospholipid arrangements. As the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation, we evaluated the effect of these liposomes on Toll-like receptor (TLR signaling, cytokine production, proinflammatory gene expression, and T, NKT, dendritic, and B cells. Liposomes induce TLR-4- and, to a lesser extent, TLR-2/TLR-6-dependent signaling in TLR-expressing human embryonic kidney (HEK cells and bone marrow-derived macrophages. Mice with the lupus-like disease had increased serum concentrations of proinflammatory cytokines, C3a and C5a; they also had more TLR-4-expressing splenocytes, a higher expression of genes associated with TRIF-dependent TLR-4-signaling and complement activation, and a lower expression of apoptosis-related genes, compared to healthy mice. The percentage of NKT and the percentage and activation of dendritic and B2 cells were also increased. Thus, TLR-4 and TLR-2/TLR-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus-like disease in mice.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. House dust mite allergen induces asthma via TLR4 triggering of airway structural cells

    Science.gov (United States)

    HAMMAD, Hamida; CHIEPPA, Marcello; PERROS, Frederic; WILLART, Monique A.; GERMAIN, Ronald N.; LAMBRECHT, Bart N.

    2009-01-01

    Barrier epithelial cells and airway dendritic cells (DC) make up the first line of defence against inhaled substances like house dust mite (HDM) allergen and endotoxin. We hypothesized that these cells need to communicate to cause allergic disease. Using irradiated chimeric mice, we demonstrate that TLR4 expression on radioresistant lung structural cells is required and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony stimulating factor, interleukin-25 and IL-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs. PMID:19330007

  4. A Lipopolysaccharide from Pantoea Agglomerans Is a Promising Adjuvant for Sublingual Vaccines to Induce Systemic and Mucosal Immune Responses in Mice via TLR4 Pathway

    Science.gov (United States)

    Kiyotoh, Eiji; Okazaki, Arimichi; Gomi, Yasuyuki; Tanimoto, Takeshi; Takeuchi, Osamu; Akira, Shizuo; Hori, Mitsuhiko

    2015-01-01

    A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens. PMID:25978818

  5. A Lipopolysaccharide from Pantoea Agglomerans Is a Promising Adjuvant for Sublingual Vaccines to Induce Systemic and Mucosal Immune Responses in Mice via TLR4 Pathway.

    Science.gov (United States)

    Fukasaka, Masahiro; Asari, Daisuke; Kiyotoh, Eiji; Okazaki, Arimichi; Gomi, Yasuyuki; Tanimoto, Takeshi; Takeuchi, Osamu; Akira, Shizuo; Hori, Mitsuhiko

    2015-01-01

    A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens.

  6. A Lipopolysaccharide from Pantoea Agglomerans Is a Promising Adjuvant for Sublingual Vaccines to Induce Systemic and Mucosal Immune Responses in Mice via TLR4 Pathway.

    Directory of Open Access Journals (Sweden)

    Masahiro Fukasaka

    Full Text Available A lipopolysaccharide from Pantoea agglomerans (LPSpa has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine with LPSpa induced both HA-specific IgG (systemic and IgA (mucosal antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens.

  7. Variability salt stress response analysis of Tunisian natural ...

    African Journals Online (AJOL)

    We evaluated the responses to salt stress of 106 Medicago truncatula lines from 11 Tunisian natural populations collected from areas that varied in soil composition, salinity and water availability. Five references lines were also included in this study. Plants were cultivated in two treatments (0 and 50 mM of NaCl) during a ...

  8. Treatment of autoimmune inflammation by a TLR7 ligand regulating the innate immune system.

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Toll-like receptor 3 (TLR3 plays a major role in the formation of rabies virus Negri Bodies.

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    Pauline Ménager

    2009-02-01

    Full Text Available Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3. TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G. The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs. NBs are not formed in the absence of TLR3, and TLR3(-/- mice -- in which brain tissue was less severely infected -- had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV-induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.

  12. Genetic predisposition of variants in TLR2 and its co-receptors to severe malaria in Odisha, India.

    Science.gov (United States)

    Panigrahi, Subhendu; Kar, Avishek; Tripathy, Sagnika; Mohapatra, Manoj K; Dhangadamajhi, Gunanidhi

    2016-02-01

    Although the role of TLRs signalling in malaria pathogenesis is well established, contribution of individual TLR to clinical outcome of malaria still remains inconclusive. Given the importance of TLR2 and its co-receptors in recognising distinct structural forms of key malaria toxins and mediating innate immune response, it is essential to delineate their genetic contribution. Variants in TLR1 (I602S) and TLR6 (P249S) were genotyped by PCR-RFLP methods, and TLR2 (I/D) was genotyped by PCR in 200 samples each from uncomplicated malaria (UM) and severe malaria (SM). Further, SM was categorised into its sub-clinical groups (CM and NCSM or SOD and MODS) and analysed. The results showed the PP genotype of TLR6 (P249S) to be significantly more common in UM (P genetic predisposition to SM and that its association with either TLR2 'D' or TLR1 '602S' modulates for CM development. The present study opens up several new avenues for their exploration and validation in future studies in different global settings for malaria.

  13. Physiologic TLR9-CpG-DNA interaction is essential for the homeostasis of the intestinal immune system.

    Science.gov (United States)

    Hofmann, Claudia; Dunger, Nadja; Doser, Kristina; Lippert, Elisabeth; Siller, Sebastian; Edinger, Matthias; Falk, Werner; Obermeier, Florian

    2014-01-01

    Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system. Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice. TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype. Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.

  14. Immunological basis of M13 phage vaccine: Regulation under MyD88 and TLR9 signaling.

    Science.gov (United States)

    Hashiguchi, Shuhei; Yamaguchi, Yuya; Takeuchi, Osamu; Akira, Shizuo; Sugimura, Kazuhisa

    2010-11-05

    Peptide-displaying bacteriophages induce mimotope-specific antibody responses, suggesting a novel application of phage-display library as bacteriophage vaccine. We examined the antibody response against M13 phage in mice induced by an i.p. administration of M13 phage in phosphate-buffered saline. We showed here that firstly, mice showed strong IgG antibody responses, particularly, in IgG2b, IgG2c, and IgG3 subclasses even in primary responses. Secondly, IgG production in primary response is totally dependent on MyD88 signaling. These responses were almost comparable, but slightly weaker, in TLR2-, TLR4- and TLR7-deficient mice relative to wild-type mice, suggesting that this enhancing effect is not due to plausible LPS contamination. Thirdly, although primary IgG1 response was not detected in wild-type mice, remarkable IgG1 response was induced in TLR9-deficient mice, suggesting that TLR9 pathway functions as regulatory, but not a simple augmenting signaling cascade, and furthermore, the enhanced IgG1 response was not due to adjuvant effect of single-stranded DNA derived from M13 phage. Thus, innate immunity including TLR regulation is crucial for M13 phage vaccine design. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Dynamic lipopolysaccharide transfer cascade to TLR4/MD2 complex via LBP and CD14.

    Science.gov (United States)

    Kim, Soo Jin; Kim, Ho Min

    2017-02-01

    Toll-like receptor 4 (TLR4) together with MD2, one of the key pattern recognition receptors for a pathogen-associated molecular pattern, activates innate immunity by recognizing lipopolysaccharide (LPS) of Gram-negative bacteria. Although LBP and CD14 catalyze LPS transfer to the TLR4/MD2 complex, the detail mechanisms underlying this dynamic LPS transfer remain elusive. Using negative-stain electron microscopy, we visualized the dynamic intermediate complexes during LPS transfer-LBP/LPS micelles and ternary CD14/LBP/LPS micelle complexes. We also reconstituted the entire cascade of LPS transfer to TLR4/MD2 in a total internal reflection fluorescence (TIRF) microscope for a single molecule fluorescence analysis. These analyses reveal longitudinal LBP binding to the surface of LPS micelles and multi-round binding/unbinding of CD14 to single LBP/LPS micelles via key charged residues on LBP and CD14. Finally, we reveal that a single LPS molecule bound to CD14 is transferred to TLR4/MD2 in a TLR4-dependent manner. These discoveries, which clarify the molecular mechanism of dynamic LPS transfer to TLR4/MD2 via LBP and CD14, provide novel insights into the initiation of innate immune responses. [BMB Reports 2017; 50(2): 55-57].

  16. The ubiquitin-like protein PLIC-1 or ubiquilin 1 inhibits TLR3-Trif signaling.

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    Nabanita Biswas

    Full Text Available The innate immune responses to virus infection are initiated by either Toll-like receptors (TLR3/7/8/9 or cytoplasmic double-stranded RNA (dsRNA-recognizing RNA helicases RIG-I and MDA5. To avoid causing injury to the host, these signaling pathways must be switched off in time by negative regulators.Through yeast-two hybrid screening, we found that an ubiquitin-like protein named protein linking integrin-associated protein to cytoskeleton 1(PLIC-1 or Ubiquilin 1 interacted with the Toll/interleukin-1 receptor (TIR domain of TLR4. Interestingly, PLIC-1 had modest effect on TLR4-mediated signaling, but strongly suppressed the transcriptional activation of IFN-β promoter through the TLR3-Trif-dependent pathway. Concomitantly, reduction of endogenous PLIC-1 by short-hairpin interfering RNA (shRNA enhanced TLR3 activation both in luciferase reporter assays as well as in new castle disease virus (NDV infected cells. An interaction between PLIC-1 and Trif was confirmed in co-immunoprecipitation (Co-IP and GST-pull-down assays. Subsequent confocal microscopic analysis revealed that PLIC-1 and Trif colocalized with the autophagosome marker LC3 in punctate subcellular structures. Finally, overexpression of PLIC-1 decreased Trif protein abundance in a Nocodazole-sensitive manner.Our results suggest that PLIC-1 is a novel inhibitor of the TLR3-Trif antiviral pathway by reducing the abundance of Trif.

  17. 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

  18. 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

  19. Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis.

    Science.gov (United States)

    Rogier, Rebecca; Ederveen, Thomas H A; Boekhorst, Jos; Wopereis, Harm; Scher, Jose U; Manasson, Julia; Frambach, Sanne J C M; Knol, Jan; Garssen, Johan; van der Kraan, Peter M; Koenders, Marije I; van den Berg, Wim B; van Hijum, Sacha A F T; Abdollahi-Roodsaz, Shahla

    2017-06-23

    Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further

  20. SNP marker discovery in koala TLR genes.

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    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.

  1. 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.

  2. Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices

    International Nuclear Information System (INIS)

    Switalla, S.; Lauenstein, L.; Prenzler, F.; Knothe, S.; Foerster, C.; Fieguth, H.-G.; Pfennig, O.; Schaumann, F.; Martin, C.; Guzman, C.A.; Ebensen, T.; Mueller, M.; Hohlfeld, J.M.; Krug, N.; Braun, A.; Sewald, K.

    2010-01-01

    Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1β, MIP-1β, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-γ, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation > 0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1β, and IFN-γ. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans.

  3. Tetra- and penta-acylated lipid A structures of Porphyromonas gingivalis LPS differentially activate TLR4-mediated NF-κB signal transduction cascade and immuno-inflammatory response in human gingival fibroblasts.

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    Thanuja D K Herath

    Full Text Available Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS, as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449 and penta-acylated (LPS1690 structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.This study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs. We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs.The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in

  4. 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.

  5. 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.

  6. Evaluation of in situ expression of effector and regulatory cytokines, TLR, galectins and matrix metalloproteinases in oral manifestations of paracoccidioidomycosis.

    Science.gov (United States)

    de Araújo, Marcelo Sivieri; Alves, Polyanna Miranda; de Lima, Lilian Margareth Biagioni; da Silva, Marcelo Fernandes; de Lima Pereira, Sanívia Aparecida; Rodrigues, Virmondes; Rodrigues, Denise Bertulucci Rocha

    2015-01-01

    Although the pathophysiology of paracoccidioidomycosis (PCM) is not completely understood, the study of immune response against fungus has provided insight into understanding the natural course of the disease and its clinical manifestations, hence contributing to the development of preventive measures and treatment proposals. The aim of this study was to evaluate the histopathological and immunological aspects involved in the role of different effector and regulatory responses, as well as the correlation between the TLRs, Galectins, Matrix Metalloproteinases and cytoplasmic proteases of mast cells in this infection. Sixteen biopsy specimens with oral lesions of chronic PCM, as well as 13 sections of normal oral mucosa were analyzed. Histopathological and immunological aspects involved in the role of different effector and regulatory responses were evaluated. Indirect immunohistochemistry was performed for IL-17, IL-10, IL-4, TGF-β, FoxP3, Gal-1, Gal-3, Gal-9, TLR-2, TLR-4, MMP-3 and MMP-9, as well as for chymase and tryptase for mast cells identification. Fibrosis was quantified using Picrosirius. There was a significant increase in the area of fibrosis and in the number of cells expressing IL-10, IL-4, IL-17, FoxP3, Gal-3, TLR-2, MMP3 and MMP9 in patients with PCM in comparison with patients in the group control. There was no difference in the expression of TGF-β, TLR-4, Gal-1 or Gal-9. Mast cells number was found to be significantly lower in oral chronic PCM when compared to control samples after quantification of mast cells and expression of chymase and tryptase. PCM granulomas were classified to the morphological aspects in organized ou non-organized. Expression of IL-4 in non-organized granulomas was significantly higher. The proteins studied herein appear to play an important role in the development and maintenance of oral lesions of PCM, as well as in the processes of development and progression of lesions caused by the fungus and by the immune response

  7. 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...

  8. 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...

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

    Directory of Open Access Journals (Sweden)

    Longbiao Zhu

    Full Text Available BACKGROUNDS: 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. METHODOLOGY/PRINCIPLE FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: This meta-analysis presented additional evidence for the association between TLR2 and TLR4 polymorphisms and cancer risk. Further well

  10. TLR2 and TLR4 co-activation utilizes distinct signaling pathways for the production of Th1/Th2/Th17 cytokines in neonatal immune cells.

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Møller-Larsen, S; Nyegaard, M; Haagerup, A

    2008-01-01

    of both TLR7 and 8 in all four phenotypes investigated: asthma, rhinitis, atopic dermatitis and increased specific IgE. The most significant association was seen for rs2407992 (TLR8) in asthma (p = 0.00023, sample A and B combined, recessive model). In TLR7, rs179008 showed the strongest association. Both...... rs179008 and rs2407992 are of putative functional significance, potentially affecting TLR7 processing and TLR8 splicing, respectively. Haplotypes comprising the major alleles of these two SNPs were overtransmitted to the affected offspring (eg, p = 0.00012 in asthma, combined sample, additive model...... 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...

  12. 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.

  13. The bile acid sensor FXR is required for immune-regulatory activities of TLR-9 in intestinal inflammation.

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    Barbara Renga

    Full Text Available BACKGROUND: Toll like receptors (TLRs sense the intestinal microbiota and regulate the innate immune response. A dysregulation of TLRs function participates into intestinal inflammation. Farnesoid X Receptor (FXR is a nuclear receptor and bile acid sensor highly expressed in entero-hepatic tissues. FXR regulates lipid metabolism and innate immunity. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have investigated whether FXR gene expression/function in the intestine is modulated by TLRs. We found that in human monocytes activation of membrane TLRs (i.e. TLR2, 4, 5 and 6 downregulates, while activation of intracellular TLRs (i.e. TLR3, 7, 8 and 9 upregulates the expression of FXR and its target gene SHP, small heterodimer partner. This effect was TLR9-dependent and TNFα independent. Intestinal inflammation induced in mice by TNBS downregulates the intestinal expression of FXR in a TLR9-dependent manner. Protection against TNBS colitis by CpG, a TLR-9 ligand, was lost in FXR(-/- mice. In contrast, activation of FXR rescued TLR9(-/- and MyD88(-/- mice from colitis. A putative IRF7 response element was detected in the FXR promoter and its functional characterization revealed that IRF7 is recruited on the FXR promoter under TLR9 stimulation. CONCLUSIONS/SIGNIFICANCE: Intestinal expression of FXR is selectively modulated by TLR9. In addition to its role in regulating type-I interferons and innate antiviral immunity, IRF-7 a TLR9-dependent factor, regulates the expression of FXR, linking microbiota-sensing receptors to host's immune and metabolic signaling.

  14. UP-REGULATION OF TLR3 IN RESPIRATORY EPITHELIAL CELLS MAY OCCUR THROUGH A POSITIVE FEEDBACK LOOP INVOLVING IFN-B

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    Toll-like receptor 3 (TLR3) plays an integral role in innate immunity through the recognition of and response to viral infections. Our previous findings have shown that exposure to diesel exhaust prior to viral infection causes an enhancement of TLR3 expression and signaling in ...

  15. Caenorhabditis elegans responses to bacteria from its natural habitats

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    Rowedder, Holli; Braendle, Christian; Félix, Marie-Anne; Ruvkun, Gary

    2016-01-01

    Most Caenorhabditis elegans studies have used laboratory Escherichia coli as diet and microbial environment. Here we characterize bacteria of C. elegans' natural habitats of rotting fruits and vegetation to provide greater context for its physiological responses. By the use of 16S ribosomal DNA (rDNA)-based sequencing, we identified a large variety of bacteria in C. elegans habitats, with phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria being most abundant. From laboratory assays using isolated natural bacteria, C. elegans is able to forage on most bacteria (robust growth on ∼80% of >550 isolates), although ∼20% also impaired growth and arrested and/or stressed animals. Bacterial community composition can predict wild C. elegans population states in both rotting apples and reconstructed microbiomes: alpha-Proteobacteria-rich communities promote proliferation, whereas Bacteroidetes or pathogens correlate with nonproliferating dauers. Combinatorial mixtures of detrimental and beneficial bacteria indicate that bacterial influence is not simply nutritional. Together, these studies provide a foundation for interrogating how bacteria naturally influence C. elegans physiology. PMID:27317746

  16. HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.

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    Zhen Luo

    2017-08-01

    Full Text Available Enterovirus 71 (EV71 is an RNA virus that causes hand-foot-mouth disease (HFMD, and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.

  17. HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.

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    Luo, Zhen; Ge, Maolin; Chen, Junbo; Geng, Qibin; Tian, Mingfu; Qiao, Zhi; Bai, Lan; Zhang, Qi; Zhu, Chengliang; Xiong, Ying; Wu, Kailang; Liu, Fang; Liu, Yingle; Wu, Jianguo

    2017-08-01

    Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs) induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER) to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.

  18. 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

  19. TLR2 Plays a Key Role in Platelet Hyperreactivity and Accelerated Thrombosis Associated With Hyperlipidemia.

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    Biswas, Sudipta; Zimman, Alejandro; Gao, Detao; Byzova, Tatiana V; Podrez, Eugene A

    2017-09-29

    Platelet hyperreactivity, which is common in many pathological conditions, is associated with increased atherothrombotic risk. The mechanisms leading to platelet hyperreactivity are complex and not yet fully understood. Platelet hyperreactivity and accelerated thrombosis, specifically in dyslipidemia, have been mechanistically linked to the accumulation in the circulation of a specific group of oxidized phospholipids (oxPC CD36 ) that are ligands for the platelet pattern recognition receptor CD36. In the current article, we tested whether the platelet innate immune system contributes to responses to oxPC CD36 and accelerated thrombosis observed in hyperlipidemia. Using in vitro approaches, as well as platelets from mice with genetic deletion of MyD88 (myeloid differentiation factor 88) or TLRs (Toll-like receptors), we demonstrate that TLR2 and TLR6 are required for the activation of human and murine platelets by oxPC CD36 . oxPC CD36 induce formation of CD36/TLR2/TLR6 complex in platelets and activate downstream signaling via TIRAP (Toll-interleukin 1 receptor domain containing adaptor protein)-MyD88-IRAK (interleukin-1 receptor-associated kinase)1/4-TRAF6 (TNF receptor-associated factor 6), leading to integrin activation via the SFK (Src family kinase)-Syk (spleen tyrosine kinase)-PLCγ2 (phospholipase Cγ2) pathway. Intravital thrombosis studies using ApoE -/- mice with genetic deficiency of TLR2 or TLR6 have demonstrated that oxPC CD36 contribute to accelerated thrombosis specifically in the setting of hyperlipidemia. Our studies reveal that TLR2 plays a key role in platelet hyperreactivity and the prothrombotic state in the setting of hyperlipidemia by sensing a wide range of endogenous lipid peroxidation ligands and activating innate immune signaling cascade in platelets. © 2017 American Heart Association, Inc.

  20. Association between TLR2 + 2477G/A polymorphism and bacterial meningitis: a meta-analysis.

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    Jin, Xiaochun; Yin, Shuzhou; Zhang, Youtao; Chen, Xu

    2018-02-19

    Toll-like receptor 2 (TLR2) is a key member of TLRs, which is crucial in the initial inflammatory response against bacteria. TLR2, is also the initial barrier against bacterial infection and plays an important role in recognising a variety of bacterial lipoproteins. Several studies have been performed to investigate the TLR2 + 2477G/A polymorphism and bacterial meningitis susceptibility. Unfortunately, the results of previous studies were controversial. Therefore, we performed a meta-analysis to derive a more precise estimation of the association. The association between the TLR2 + 2477G/A polymorphism and bacterial meningitis susceptibility was assessed by odds ratios together with their 95% confidence intervals (CI). Six studies were enrolled in the present meta-analysis. Overall, no significant association between TLR2 + 2477G/A polymorphism and bacterial meningitis risk were found under allele contrast (A vs. G: OR = 1.15, 95% CI = 0.93-1.43, P = 0.202), recessive genetic model (AA vs. OR = 1.12, 95% CI = 0.90-1.41, P = 0.313). The significant association was found between TLR2 + 2477G/A polymorphism and pneumococcal meningitis risk under allele contrast (A vs. G: OR = 1.54, 95% CI = 1.01-2.36, P = 0.046), recessive genetic model (AA vs. OR = 1.63, 95% CI = 1.03-2.57, P = 0.035). We conclude that TLR2 + 2477G/A polymorphism is not associated with meningococcal meningitis risk but contributes an increased risk of pneumococcal meningitis.

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

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    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.

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

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    Jia, Yin-Ping; Wang, Kun; Zhang, Zhu-Jun; Tong, Ya-Nan; Han, Dan; Hu, Chun-Yu; Li, Qian; Xiang, Yang; Mao, Xu-Hu; Tang, Bin

    2017-01-01

    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.

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

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    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.

  4. Cisplatin induces tolerogenic dendritic cells in response to TLR agonists via the abundant production of IL-10, thereby promoting Th2- and Tr1-biased T-cell immunity

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    Kim, Hongmin; Kwon, Kee Woong; Im, Sin-Hyeog; Lee, Bo Ryeong; Ha, Sang-Jun; Shin, Sung Jae

    2016-01-01

    Although many advantageous roles of cisplatin (cis-diamminedichloroplatinum (II), CDDP) have been reported in cancer therapy, the immunomodulatory roles of cisplatin in the phenotypic and functional alterations of dendritic cells (DCs) are poorly understood. Here, we investigated the effect of cisplatin on the functionality of DCs and the changes in signaling pathways activated upon toll-like receptor (TLR) stimulation. Cisplatin-treated DCs down-regulated the expression of cell surface molecules (CD80, CD86, MHC class I and II) and up-regulated endocytic capacity in a dose-dependent manner. Upon stimulation with various TLR agonists, cisplatin-treated DCs showed markedly increased IL-10 production through activation of the p38 MAPK and NF-κB signaling pathways without altering the levels of TNF-α and IL-12p70, indicating the cisplatin-mediated induction of tolerogenic DCs. This effect was dependent on the production of IL-10 from DCs, as neither DCs isolated from IL-10−/− mice nor IL-10-neutralized DCs generated tolerogenic DCs. Interestingly, DCs that were co-treated with cisplatin and lipopolysaccharide (LPS) exhibited a decreased immunostimulatory capacity for inducing the proliferation of Th1- and Th17-type T cells; instead, these DCs contributed to Th2-type T cell immunity. Furthermore, in vitro and in vivo investigations revealed a unique T cell population, IL-10-producing CD3+CD4+LAG-3+CD49b+CD25−Foxp3− Tr1 cells, that was significantly increased without altering the Foxp3+ regulatory T cell population. Taken together, our results suggest that cisplatin induces immune-suppressive tolerogenic DCs in TLR agonist-induced inflammatory conditions via abundant IL-10 production, thereby skewing Th cell differentiation towards Th2 and Tr1 cells. This relationship may provide cancer cells with an opportunity to evade the immune system. PMID:27172902

  5. Common TLR1 genetic variation is not associated with death from melioidosis, a common cause of sepsis in rural Thailand.

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    Narisara Chantratita

    Full Text Available Melioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G, rs4833095 (742A/G, and rs5743618 (1804G/T. We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory

  6. Combined effect of TLR2 gene polymorphism and early life stress on the age at onset of bipolar disorders.

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    José Oliveira

    Full Text Available Gene-environment interactions may play an important role in modulating the impact of early-life stressful events on the clinical course of bipolar disorder (BD, particularly associated to early age at onset. Immune dysfunction is thought to be an important mechanism linking childhood trauma with early-onset BD, thus the genetic diversity of immune-related loci may account for an important part of the interindividual susceptibility to this severe subform. Here we investigated the potential interaction between genetic variants of Toll-like receptors 2 (TLR2 and 4 (TLR4, major innate immune response molecules to pathogens, and the childhood trauma questionnaire (CTQ in age at onset of BD. We recruited 531 BD patients (type I and II or not otherwise specified, genotyped for the TLR2 rs4696480 and rs3804099 and TLR4 rs1927914 and rs11536891 single-nucleotide polymorphisms and recorded for history of childhood trauma using the CTQ. TLR2 and TLR4 risk genotype carrier state and history of childhood emotional, physical and sexual abuses were evaluated in relation to age at onset as defined by the age at first manic or depressive episode. We observed a combined effect of TLR2 rs3804099 TT genotype and reported sexual abuse on determining an earlier age at onset of BD by means of a Kaplan-Meier survival curve (p = 0.002; corrected p = 0.02. Regression analysis, however, was non-significant for the TLR2-CTQ sexual abuse interaction term. The negative effects of childhood sexual abuse on age at onset of BD may be amplified in TLR2 rs3804099 risk genotype carriers through immune-mediated pathways. Clinical characteristics of illness severity, immune phenotypes and history of early life infectious insults should be included in future studies involving large patient cohorts.

  7. The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.

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    Georgina L Hold

    Full Text Available Dysregulated Toll-Like Receptor (TLR signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.

  8. TLR3 Ligand Poly(I:C Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles

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    Mojca Frank-Bertoncelj

    2018-01-01

    Full Text Available Extracellular vesicles (EV can modulate the responses of cells to toll-like receptor (TLR ligation; conversely, TLR ligands such as double-stranded RNA (dsRNA can enhance the release of EV and influence of the composition and functions of EV cargos. Inflamed synovial joints in rheumatoid arthritis (RA are rich in EV and extracellular RNA; besides, RNA released from necrotic synovial fluid cells can activate the TLR3 signaling in synovial fibroblasts (SFs from patients with RA. Since EV occur prominently in synovial joints in RA and may contribute to the pathogenesis, we questioned whether EV can interact with dsRNA, a TLR3 ligand, and modify its actions in arthritis. We have used as model the effects on RA SFs, of EV released from monocyte U937 cells and peripheral blood mononuclear cells upon stimulation with Poly(I:C, a synthetic analog of dsRNA. We show that EV released from unstimulated cells and Poly(I:C-stimulated U937 cells [Poly(I:C EV] differ in size but bind similar amounts of Annexin V and express comparable levels of MAC-1, the receptor for dsRNA, on the vesicular membranes. Specifically, Poly(I:C EV contain or associate with Poly(I:C and at least partially protect Poly(I:C from RNAse III degradation. Poly(I:C EV shuttle Poly(I:C to SFs and reproduce the proinflammatory and antiviral gene responses of SFs to direct stimulation with Poly(I:C. Poly(I:C EV, however, halt the death receptor-induced apoptosis in SFs, thereby inverting the proapoptotic nature of Poly(I:C. These prosurvival effects sharply contrast with the high toxicity of cationic liposome-delivered Poly(I:C and may reflect the route of Poly(I:C delivery via EV or the fine-tuning of Poly(I:C actions by molecular cargo in EV. The demonstration that EV may safeguard extracellular dsRNA and allow dsRNA to exert antiapoptotic effects on SFs highlights the potential of EV to amplify the pathogenicity of dsRNA in arthritis beyond inflammation (by concurrently enhancing the

  9. TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles.

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    Frank-Bertoncelj, Mojca; Pisetsky, David S; Kolling, Christoph; Michel, Beat A; Gay, Renate E; Jüngel, Astrid; Gay, Steffen

    2018-01-01

    Extracellular vesicles (EV) can modulate the responses of cells to toll-like receptor (TLR) ligation; conversely, TLR ligands such as double-stranded RNA (dsRNA) can enhance the release of EV and influence of the composition and functions of EV cargos. Inflamed synovial joints in rheumatoid arthritis (RA) are rich in EV and extracellular RNA; besides, RNA released from necrotic synovial fluid cells can activate the TLR3 signaling in synovial fibroblasts (SFs) from patients with RA. Since EV occur prominently in synovial joints in RA and may contribute to the pathogenesis, we questioned whether EV can interact with dsRNA, a TLR3 ligand, and modify its actions in arthritis. We have used as model the effects on RA SFs, of EV released from monocyte U937 cells and peripheral blood mononuclear cells upon stimulation with Poly(I:C), a synthetic analog of dsRNA. We show that EV released from unstimulated cells and Poly(I:C)-stimulated U937 cells [Poly(I:C) EV] differ in size but bind similar amounts of Annexin V and express comparable levels of MAC-1, the receptor for dsRNA, on the vesicular membranes. Specifically, Poly(I:C) EV contain or associate with Poly(I:C) and at least partially protect Poly(I:C) from RNAse III degradation. Poly(I:C) EV shuttle Poly(I:C) to SFs and reproduce the proinflammatory and antiviral gene responses of SFs to direct stimulation with Poly(I:C). Poly(I:C) EV, however, halt the death receptor-induced apoptosis in SFs, thereby inverting the proapoptotic nature of Poly(I:C). These prosurvival effects sharply contrast with the high toxicity of cationic liposome-delivered Poly(I:C) and may reflect the route of Poly(I:C) delivery via EV or the fine-tuning of Poly(I:C) actions by molecular cargo in EV. The demonstration that EV may safeguard extracellular dsRNA and allow dsRNA to exert antiapoptotic effects on SFs highlights the potential of EV to amplify the pathogenicity of dsRNA in arthritis beyond inflammation (by concurrently enhancing the

  10. Natural IgM and TLR Agonists Switch Murine Splenic Pan-B to “Regulatory” Cells That Suppress Ischemia-Induced Innate Inflammation via Regulating NKT-1 Cells

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    Peter I. Lobo

    2017-08-01

    Full Text Available Natural IgM anti-leukocyte autoantibodies (IgM-ALAs inhibit inflammation by several mechanisms. Here, we show that pan-B cells and bone marrow-derived dendritic cells (BMDCs are switched to regulatory cells when pretreated ex vivo with IgM. B cells are also switched to regulatory cells when pretreated ex vivo with CpG but not with LPS. Pre-emptive infusion of such ex vivo induced regulatory cells protects C57BL/6 mice from ischemia-induced acute kidney injury (AKI via regulation of in vivo NKT-1 cells, which normally amplify the innate inflammatory response to DAMPS released after reperfusion of the ischemic kidney. Such ex vivo induced regulatory pan-B cells and BMDC express low CD1d and inhibit inflammation by regulating in vivo NKT-1 in the context of low-lipid antigen presentation and by a mechanism that requires costimulatory molecules, CD1d, PDL1/PD1, and IL10. Second, LPS and CpG have opposite effects on induction of regulatory activity in BMDC and B cells. LPS enhances regulatory activity of IgM-pretreated BMDC but negates the IgM-induced regulatory activity in B cells, while CpG, with or without IgM pretreatment, induces regulatory activity in B cells but not in BMDC. Differences in the response of pan-B and dendritic cells to LPS and CpG, especially in the presence of IgM-ALA, may have relevance during infections and inflammatory disorders where there is an increased IgM-ALA and release of TLRs 4 and 9 ligands. Ex vivo induced regulatory pan-B cells could have therapeutic relevance as these easily available cells can be pre-emptively infused to prevent AKI that can occur during open heart surgery or in transplant recipients receiving deceased donor organs.

  11. Animal responses to natural disturbance and climate extremes: a review

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    Sergio, Fabrizio; Blas, Julio; Hiraldo, Fernando

    2018-02-01

    Natural disturbances, such as droughts, fires or hurricanes, are key drivers of ecological heterogeneity and ecosystem function. The frequency and severity of these episodes is unequivocally expected to increase in the coming decades, through the concerted action of climate change and anthropogenic pressures. This will impose severe challenges for many biota through exposure to rapidly changing conditions never experienced in the preceding millennia. Thus, it is urgently needed to gain a thorough understanding of animal responses and adaptations to disturbances in order to better estimate potential future impacts. Here, we review such adjustments and find that animals may respond to disturbances through changes in: (1) behaviour, such as altered mobility, emigration, resource-switching, refuge use, suspended animation, or biotic interactions; (2) life history traits, such as survival, aging, longevity, recruitment, reproductive restraint, breeding output, phenology and bet-hedging tactics; (3) morphology, such as rapid evolution through size-dependent mortality or facultative metamorphosis; (4) physiology, such as altered body condition, pathogen prevalence and transmission, or adrenocortical modulation of stress responses to emergency conditions; (5) genetic structure, such as changes in frequency of polymorphic variants or diversity-modulation through mortality bottlenecks. Individual-level responses scale up to population and community responses, such as altered density, population dynamics, distribution, local extinction and colonization, or assemblage structure and diversity. Overall, disturbances have pervasive effects on individuals, populations and communities of vertebrates and invertebrates of all realms, biomes, continents and ecosystems. Their rapidly increasing incidence and severity will bring unique study opportunities for researchers and novel, unpredictable challenges for managers, while demanding tougher choices and more proactive crisis

  12. TLR3 signaling is either protective or pathogenic for the development of Theiler's virus-induced demyelinating disease depending on the time of viral infection

    Directory of Open Access Journals (Sweden)

    Jin Young-Hee

    2011-12-01

    Full Text Available Abstract Background We have previously shown that toll-like receptor 3 (TLR3-mediated signaling plays an important role in the induction of innate cytokine responses to Theiler's murine encephalomyelitis virus (TMEV infection. In addition, cytokine levels produced after TMEV infection are significantly higher in the glial cells of susceptible SJL mice compared to those of resistant C57BL/6 mice. However, it is not known whether TLR3-mediated signaling plays a protective or pathogenic role in the development of demyelinating disease. Methods SJL/J and B6;129S-Tlr3tm1Flv/J (TLR3KO-B6 mice, and TLR3KO-SJL mice that TLR3KO-B6 mice were backcrossed to SJL/J mice for 6 generations were infected with Theiler's murine encephalomyelitis virus (2 × 105 PFU with or without treatment with 50 μg of poly IC. Cytokine production and immune responses in the CNS and periphery of infected mice were analyzed. Results We investigated the role of TLR3-mediated signaling in the protection and pathogenesis of TMEV-induced demyelinating disease. TLR3KO-B6 mice did not develop demyelinating disease although they displayed elevated viral loads in the CNS. However, TLR3KO-SJL mice displayed increased viral loads and cellular infiltration in the CNS, accompanied by exacerbated development of demyelinating disease, compared to the normal littermate mice. Late, but not early, anti-viral CD4+ and CD8+ T cell responses in the CNS were compromised in TLR3KO-SJL mice. However, activation of TLR3 with poly IC prior to viral infection also exacerbated disease development, whereas such activation after viral infection restrained disease development. Activation of TLR3 signaling prior to viral infection hindered the induction of protective IFN-γ-producing CD4+ and CD8+ T cell populations. In contrast, activation of these signals after viral infection improved the induction of IFN-γ-producing CD4+ and CD8+ T cells. In addition, poly IC-pretreated mice displayed elevated PDL-1 and

  13. 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.

  14. Th2 Regulation of Viral Myocarditis in Mice: Different Roles for TLR3 versus TRIF in Progression to Chronic Disease

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    Eric D. Abston

    2012-01-01

    Full Text Available Viral infections are able to induce autoimmune inflammation in the heart. Here, we investigated the role of virus-activated Toll-like receptor (TLR3 and its adaptor TRIF on the development of autoimmune coxsackievirus B3 (CVB3 myocarditis in mice. Although TLR3- or TRIF-deficient mice developed similarly worse acute CVB3 myocarditis and viral replication compared to control mice, disease was significantly worse in TRIF compared to TLR3-deficient mice. Interestingly, TLR3-deficient mice developed an interleukin (IL-4-dominant T helper (Th2 response during acute CVB3 myocarditis with elevated markers of alternative activation, while TRIF-deficient mice elevated the Th2-associated cytokine IL-33. Treatment of TLR3-deficient mice with recombinant IL-33 improved heart function indicating that elevated IL-33 in the context of a classic Th2-driven response protects against autoimmune heart disease. We show for the first time that TLR3 versus TRIF deficiency results in different Th2 responses that uniquely influence the progression to chronic myocarditis.

  15. CD200R1 supports HSV-1 viral replication and licenses pro-inflammatory signaling functions of TLR2.

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    Roy J Soberman

    Full Text Available The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/- mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1 infection. CD200R1(-/- peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes in response to the TLR2 agonist Pam(2CSK(4 and to HSV-1. CD200R1(-/- macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/- mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/- mice and CD200R1(-/- fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.

  16. HSV-2 increases TLR4-dependent phosphorylated IRFs and IFN-β induction in cervical epithelial cells.

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    Hongya Liu

    Full Text Available Our previous studies demonstrated that HSV-2 infection up-regulates TLR4 expression and induces NF-kB activity, thereby facilitating innate immune response in human cervical epithelial cells. This process requires involvement of TLR4 adaptors, Mal and MyD88. In the current study, we found that HSV-2 infection increases levels of phosphoryalted IRF3 and IRF7, then regulating expression of type I IFN. As expected, these changes induced by HSV-2 infection depended upon TLR4. Knockdown of TRIF and/or TRAM by siRNAs indicated that TRIF/TRAM might be involved in expression of IFN-β. Our results demonstrate for the first time that IRF3 and IRF7 are both involved in inducing TLR4-dependent IFN-β expression in response to HSV-2 in its primary infected genital epithelial cells. Thus, TLR4-Mal/MyD88 and TLR4-TRIF/TRAM signaling may synergize and/or cooperate in innate immune response of cervical epithelial cells to HSV-2 infection.

  17. The Influence of Familiarity on Affective Responses to Natural Scenes

    Science.gov (United States)

    Sanabria Z., Jorge C.; Cho, Youngil; Yamanaka, Toshimasa

    This kansei study explored how familiarity with image-word combinations influences affective states. Stimuli were obtained from Japanese print advertisements (ads), and consisted of images (e.g., natural-scene backgrounds) and their corresponding headlines (advertising copy). Initially, a group of subjects evaluated their level of familiarity with images and headlines independently, and stimuli were filtered based on the results. In the main experiment, a different group of subjects rated their pleasure and arousal to, and familiarity with, image-headline combinations. The Self-Assessment Manikin (SAM) scale was used to evaluate pleasure and arousal, and a bipolar scale was used to evaluate familiarity. The results showed a high correlation between familiarity and pleasure, but low correlation between familiarity and arousal. The characteristics of the stimuli, and their effect on the variables of pleasure, arousal and familiarity, were explored through ANOVA. It is suggested that, in the case of natural-scene ads, familiarity with image-headline combinations may increase the pleasure response to the ads, and that certain components in the images (e.g., water) may increase arousal levels.

  18. Multifaceted effects of synthetic TLR2 ligand and Legionella pneumophilia on Treg-mediated suppression of T cell activation

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    Sutmuller Roger PM

    2011-03-01

    Full Text Available Abstract Background Regulatory T cells (Treg play a crucial role in maintaining immune homeostasis and self-tolerance. The immune suppressive effects of Tregs should however be limited in case effective immunity is required against pathogens or cancer cells. We previously found that the Toll-like receptor 2 (TLR2 agonist, Pam3CysSK4, directly stimulated Tregs to expand and temporarily abrogate their suppressive capabilities. In this study, we evaluate the effect of Pam3CysSK4 and Legionella pneumophila, a natural TLR2 containing infectious agent, on effector T (Teff cells and dendritic cells (DCs individually and in co-cultures with Tregs. Results TLR2 agonists can directly provide a co-stimulatory signal inducing enhanced proliferation and cytokine production of naive CD4+ Teff cells. With respect to cytokine production, DCs appear to be most sensitive to low amounts of TLR agonists. Using wild type and TLR2-deficient cells in Treg suppression assays, we accordingly show that all cells (e.g. Treg, Teff cells and DCs contributed to overcome Treg-mediated suppression of Teff cell proliferation. Furthermore, while TLR2-stimulated Tregs readily lost their ability to suppress Teff cell proliferation, cytokine production by Teff cells was still suppressed. Similar results were obtained upon stimulation with TLR2 ligand containing bacteria, Legionella pneumophila. Conclusions These findings indicate that both synthetic and natural TLR2 agonists affect DCs, Teff cells and Treg directly, resulting in multi-modal modulation of Treg-mediated suppression of Teff cells. Moreover, Treg-mediated suppression of Teff cell proliferation is functionally distinct from suppression of cytokine secretion.

  19. Molecular insights of the first gastropod TLR counterpart from disk abalone (Haliotis discus discus), revealing its transcriptional modulation under pathogenic stress.

    Science.gov (United States)

    Elvitigala, Don Anushka Sandaruwan; Premachandra, H K A; Whang, Ilson; Nam, Bo-Hye; Lee, Jehee

    2013-08-01

    Toll-like receptors (TLRs) are well-characterized pattern recognition receptors of innate immunity, known to induce immune responses against the pathogens by interacting with evolutionarily conserved pathogen-associated molecular patterns (PAMPs). In this study, a novel TLR homolog from disk abalone (Haliotis discus discus) was identified and characterized at molecular level. The open reading frame (ORF) of AbTLR is 3804 bp in length and encodes a 1268 amino acid peptide with a calculated molecular mass of 143.5 kDa. The deduced protein shows typical TLR domain architecture, with leucine rich repeats (LRR) and the toll-interleukin receptor (TIR) domain. Phylogenetic analysis revealed a close evolutionary relationship for AbTLR to its invertebrate counterparts, with close clustering to the molluscan homologs. Quantitative real-time PCR detected ubiquitous transcription of AbTLR in healthy tissues, but with highest levels in hemocytes. Differential transcriptional modulation of AbTLR was observed in abalone hemocytes and gills upon immune challenge, whereby Vibrio parahaemolyticus and purified lipopolysaccharide (LPS) enhanced the transcript level prominently. In addition, the viral hemorrhagic septicemia virus induced AbTLR transcription in hemocytes and gills, representing the first evidence of viral-induced immune response in mollusks to date. Collectively, our findings support a putative role for AbTLR in abalone antiviral and antibacterial defense. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR(2) ) is involved in vascular function.

    Science.gov (United States)

    Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

    2013-01-01

    Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR(2) and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR(2) activating peptide (AP) was used as a PAR(2) agonist. Aortas harvested from TLR4(-/-) mice were also used to characterize the PAR(2) response. PAR(2) , but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR(2) AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR(2) AP-induced vasorelaxation and PAR(2) AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4(-/-) mice, the expression of PAR(2) was reduced and the PAR(2) AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Cross-talk between PAR(2) and TLR4 contributes to vascular homeostasis. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  1. Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR2) is involved in vascular function

    Science.gov (United States)

    Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

    2013-01-01

    Background and Purpose Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR2 and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Experimental Approach Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR2 activating peptide (AP) was used as a PAR2 agonist. Aortas harvested from TLR4–/– mice were also used to characterize the PAR2 response. Key Results PAR2, but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR2AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR2AP-induced vasorelaxation and PAR2AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4–/– mice, the expression of PAR2 was reduced and the PAR2AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Conclusions and Implications Cross-talk between PAR2 and TLR4 contributes to vascular homeostasis. PMID:22957757

  2. Large granular lymphocyte leukemia: natural history and response to treatment.

    LENUS (Irish Health Repository)

    Fortune, Anne F

    2012-02-01

    Large granular lymphocyte leukemia (T-LGL) is an indolent T lymphoproliferative disorder that was difficult to diagnose with certainty until clonality testing of the T cell receptor gene became routinely available. We studied the natural history and response to treatment in 25 consecutive patients with T-LGL diagnosed between 2004 and 2008 in which the diagnosis was confirmed by molecular analysis, to define an effective treatment algorithm. The median age at diagnosis was 61 years (range 27-78), with a male to female ratio of 1:1.8 and presenting features of fatigue (n = 13), recurrent infections (n = 9), and\\/or abnormal blood counts (n = 5). Thirteen patients with symptomatic disease were treated as follows: pentostatin (nine patients), cyclosporine (six patients), methotrexate (three patients), and alemtuzumab in two patients in whom pentostatin was ineffective. Pentostatin was the single most effective therapy, with a response rate of 75% and minimal toxicity. The overall survival (OS) and progression-free survival (PFS) 37 months from diagnosis were 80% and 52%, respectively. Treatment of T-LGL should be reserved for patients with symptomatic disease, but in this series, pentostatin treatment was less toxic and more effective than cyclosporine or methotrexate.

  3. Antitumor Responses of Invariant Natural Killer T Cells

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    Jennie B. Altman

    2015-01-01

    Full Text Available Natural killer T (NKT cells are innate-like lymphocytes that were first described in the late 1980s. Since their initial description, numerous studies have collectively shed light on their development and effector function. These studies have highlighted the unique requirements for the activation of these lymphocytes and the functional responses that distinguish these cells from other effector lymphocyte populations such as conventional T cells and NK cells. This body of literature suggests that NKT cells play diverse nonredundant roles in a number of disease processes, including the initiation and propagation of airway hyperreactivity, protection against a variety of pathogens, development of autoimmunity, and mediation of allograft responses. In this review, however, we focus on the role of a specific lineage of NKT cells in antitumor immunity. Specifically, we describe the development of invariant NKT (iNKT cells and the factors that are critical for their acquisition of effector function. Next, we delineate the mechanisms by which iNKT cells influence and modulate the activity of other immune cells to directly or indirectly affect tumor growth. Finally, we review the successes and failures of clinical trials employing iNKT cell-based immunotherapies and explore the future prospects for the use of such strategies.

  4. Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

    Science.gov (United States)

    Huang, Hai-bo; Xiao, Ke; Lu, Shun; Yang, Ke-li; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Zhong, Juming; Liu, Hua-zhen; Peng, Ke-mei

    2015-01-01

    Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory

  5. Porphyromonas gingivalis Stimulates TLR2-PI3K Signaling to Escape Immune Clearance and Induce Bone Resorption Independently of MyD88

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    Hasnaa Makkawi

    2017-08-01

    Full Text Available Porphyromonas gingivalis is a gram-negative anaerobic periodontal pathogen that persists in dysbiotic mixed-species biofilms alongside a dense inflammatory infiltrate of neutrophils and other leukocytes in the subgingival areas of the periodontium. Toll-like receptor 2 (TLR2 mediates the inflammatory response to P. gingivalis and TLR2-deficient mice resist alveolar bone resorption following oral challenge with this organism. Although, MyD88 is an adaptor protein considered necessary for TLR2-induced inflammation, we now report for the first time that oral challenge with P. gingivalis leads to alveolar bone resorption in the absence of MyD88. Indeed, in contrast to prototypical TLR2 agonists, such as the lipopeptide Pam3CSK4 that activates TLR2 in a strictly MyD88-dependent manner, P. gingivalis strikingly induced TLR2 signaling in neutrophils and macrophages regardless of the presence or absence of MyD88. Moreover, genetic or antibody-mediated inactivation of TLR2 completely reduced cytokine production in P. gingivalis-stimulated neutrophils or macrophages, suggesting that TLR2 plays a non-redundant role in the host response to P. gingivalis. In the absence of MyD88, inflammatory TLR2 signaling in P. gingivalis-stimulated neutrophils or macrophages depended upon PI3K. Intriguingly, TLR2-PI3K signaling was also critical to P. gingivalis evasion of killing by macrophages, since their ability to phagocytose this pathogen was reduced in a TLR2 and PI3K-dependent manner. Moreover, within those cells that did phagocytose bacteria, TLR2-PI3K signaling blocked phago-lysosomal maturation, thereby revealing a novel mechanism whereby P. gingivalis can enhance its intracellular survival. Therefore, P. gingivalis uncouples inflammation from bactericidal activity by substituting TLR2-PI3K in place of TLR2-MyD88 signaling. These findings further support the role of P. gingivalis as a keystone pathogen, which manipulates the host inflammatory response in a way

  6. TLR3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of CNS infection in mice

    DEFF Research Database (Denmark)

    Reinert, Line; Harder, Louis Andreas; Holm, Christian

    2012-01-01

    , it is not known what cell type mediates the role of TLR3 in the immunological control of HSV, and it is not known whether TLR3 sensing occurs prior to or after CNS entry. Here, we show that in mice TLR3 provides early control of HSV-2 infection immediately after entry into the CNS by mediating type I IFN...... responses to HSV, but astrocytes were defective in HSV-induced type I IFN production. Thus, TLR3 acts in astrocytes to sense HSV-2 infection immediately after entry into the CNS, possibly preventing HSV from spreading beyond the neurons mediating entry into the CNS.......Herpes simplex viruses (HSVs) are highly prevalent neurotropic viruses. While they can replicate lytically in cells of the epithelial lineage, causing lesions on mucocutaneous surfaces, HSVs also establish latent infections in neurons, which act as reservoirs of virus for subsequent reactivation...

  7. Molecular characterization and expression profile of partial TLR4 gene in association to mastitis in crossbred cattle.

    Science.gov (United States)

    Panigrahi, Manjit; Sharma, Arjava; Bhushan, Bharat

    2014-01-01

    Crossbred cattle are more prone to mastitis in comparison to indigenous cattle. Toll-like receptor 4 (TLR4) recognizes pathogen ligands, for example, lipopolysaccharide (LPS) endotoxin from Escherichia coli and mediates signaling to initiate innate and adaptive immune responses. Mutations in TLR4 can compromise the host immune response to certain pathogens, so it may be a potential candidate for marker assisted selection to enhance mastitis resistance in dairy cattle. Hence, in this study role of bovine TLR4 gene in mastitis resistance was investigated by association as well as expression profiling analysis in crossbred cattle. The animals were divided into mastitis affected and unaffected groups on the basis of history of animals and California Mastitis Test (CMT). PCR-SSCP and Sequence analysis revealed three genotypes of coreceptor binding region 1 (CRBR1) fragment of TLR4 gene namely AA, AB, and BB in both groups of cattle. The logistic regression model did not show any significant effect of these genotypes on the occurrence of clinical mastitis. Moreover, in vitro challenge of peripheral blood mononuclear cells (PBMCs) with LPS failed to show any association of the genotypes with TLR4 gene expression. In a nutshell, in the present study enough evidence was not found for association of the SNP variants of CRBR1 fragment of TLR4 gene with mastitis susceptibility in crossbred cattle.

  8. 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

    BACKGROUND: Evidence exists that exposure to high levels of microbial agents such as endotoxin in the farm environment decreases the risk of atopic sensitization. Genetic variation in innate immunity genes may modulate the response to microbial agents and thus influence susceptibility to asthma a....../-651 promoter polymorphisms are associated with atopy prevalence among young adults exposed to farm environments. Udgivelsesdato: 2007-Nov...... 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...... 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...

  9. Urban nature as a response to stress of urban population

    Directory of Open Access Journals (Sweden)

    Vujčić Maja

    2017-01-01

    Full Text Available In everyday life, urban residents, especially the younger population, have given up some healthy habits of spending their free time outdoors in urban forests or parks. This study was conducted in order to understand how urban nature might help in reducing psychological stress and improving mental wellbeing. The participants were volunteer students of the Faculty of Forestry in Belgrade (n=47. These students were randomly recruited in the study and control group and self-tested using DASS 21 scale. The Arboretum of the Faculty of Forestry represented a research location and a special healing environment with a high variety of species. The study group stayed at the Arboretum during the study break period, while a control group was inside the Faculty. After the intervention, a slightly greater reduction in stress scale on the total score was recorded in the study group (F1.45 = 3.781; r < .058. This study has shown that urban green areas can have a positive impact on the mental well-being and reveal their role as a great response to the stress from urban population.

  10. Venezuelan policies and responses on climate change and natural hazards

    Science.gov (United States)

    Caponi, Claudio; Rosales, Anibal

    1992-06-01

    Venezuela is an intertropical country which has the fortune not to suffer the severities of natural hazards which are usual in other countries of this region. It is a developing country, whose economy is heavily dependent on oil production and exports. Its greenhouse gas emissions are relatively low, but it is expected that the planned industrialization development will bring an associated increase in emissions. As a nation, Venezuela has a highly developed environmental consciousness. The Ministry of environment, the first in Latin America, was created in 1977, and has been the main contributor to the national policy of Disaster Prevention and Reduction. As in many developing countries actions and responses in this regard have been rather limited in scope, and even though legislation has been developed, many problems arise for its enforcement. Several local warning systems, civil defense procedures, and infrastructural protection measures are operational, however they have not been designed, revised, or planned taking into consideration the potential impacts of climate change. Presently Venezuela is an active participant state in the negotiation for a framework convention on climate change. That is a very difficult negotiation for our country. Here we have to conciliate enviromental principles with national economic interests. The elements of our position in this contex are presented in this statement.

  11. Immunotherapeutic strategies targeting Natural killer T cell responses in cancer

    Science.gov (United States)

    Shissler, Susannah C.; Bollino, Dominique R.; Tiper, Irina V.; Bates, Joshua; Derakhshandeh, Roshanak; Webb, Tonya J.

    2017-01-01

    Natural killer T (NKT) cells are a unique subset of lymphocytes that bridge the innate and adaptive immune system. NKT cells possess a classic αβ T-cell receptor (TCR) that is able to recognize self and foreign glycolipid antigens presented by the nonclassical class I major histocompatibility complex (MHC) molecule, CD1d. Type I NKT cells (referred to as invariant NKT cells) express a semi-invariant Vα14Jα18 TCR in mice and Vα24Jα18 TCR in humans. Type II NKT cells are CD1d-restricted T cells that express a more diverse set of TCR α chains. The two types of NKT cells often exert opposing effects especially in tumor immunity, where Type II cells generally suppress tumor immunity while Type I NKT cells can enhance antitumor immune responses. In this review, we focus on the role of NKT cells in cancer. We discuss their effector and suppressive functions, as well as describe preclinical and clinical studies utilizing therapeutic strategies focused on harnessing their potent anti-tumor effector functions, and conclude with a discussion on potential next steps for the utilization of NKT cell targeted therapies for the treatment of cancer. PMID:27393665

  12. 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.

  13. 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

  14. TLR4 Signaling in MPP+-Induced Activation of BV-2 Cells

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    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.

  15. Trauma hemorrhagic shock-induced lung injury involves a gut-lymph-induced TLR4 pathway in mice.

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    Diego C Reino

    Full Text Available Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI and multiple organ dysfunction syndrome (MODS. Since Toll-like receptors (TLR act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS mediate gut-induced lung injury via TLR4 activation.The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD lung permeability and myeloperoxidase (MPO levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.

  16. MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers.

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    Franzenburg, Sören; Fraune, Sebastian; Künzel, Sven; Baines, John F; Domazet-Loso, Tomislav; Bosch, Thomas C G

    2012-11-20

    Toll-like receptor (TLR) signaling is one of the most important signaling cascades of the innate immune system of vertebrates. Studies in invertebrates have focused on the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, and there is little information regarding the evolutionary origin and ancestral function of TLR signaling. In Drosophila, members of the Toll-like receptor family are involved in both embryonic development and innate immunity. In C. elegans, a clear immune function of the TLR homolog TOL-1 is controversial and central components of vertebrate TLR signaling including the key adapter protein myeloid differentiation primary response gene 88 (MyD88) and the transcription factor NF-κB are not present. In basal metazoans such as the cnidarians Hydra magnipapillata and Nematostella vectensis, all components of the vertebrate TLR signaling cascade are present, but their role in immunity is unknown. Here, we use a MyD88 loss-of-function approach in Hydra to demonstrate that recognition of bacteria is an ancestral function of TLR signaling and that this process contributes to both host-mediated recolonization by commensal bacteria as well as to defense against bacterial pathogens.

  17. DNase Sda1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition.

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    Satoshi Uchiyama

    Full Text Available Group A Streptococcus (GAS has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

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

    Science.gov (United States)

    2013-01-01

    downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1β and iNOS on microglia post-hypoxia. Conclusion Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries. PMID:23388509

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

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    Yao Linli

    2013-02-01

    NO in BV-2 cells. TLR4 downregulation-mediated inhibition of inflammatory cytokines in primary microglia and BV-2 cells was accompanied by the suppression of NF-κB activation. Furthermore, HIF-1α antibody neutralization attenuated the increase of TLR4 expression in hypoxic BV-2 cells. TLR4 inhibition in vivo attenuated the immunoexpression of TNF-α, IL-1β and iNOS on microglia post-hypoxia. Conclusion Activated microglia TLR4 expression mediated neuroinflammation via a NF-κB signaling pathway in response to hypoxia. Hence, microglia TLR4 presents as a potential therapeutic target for neonatal hypoxia brain injuries.

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

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    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

  1. Environmental psychology: Human responses and relationships to natural landscapes

    Science.gov (United States)

    Daniel R. Williams

    2004-01-01

    The purpose of this chapter is to present a thorough assessment of environmental psychology as a way to understand relationships between people and natural landscapes, and to describe how this knowledge can be applied to natural resource management. Environmental psychology seeks to clarify how individuals perceive, experience and create meaning in the environment. In...

  2. 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.

  3. Activation of autoreactive B cells by endogenous TLR7 and TLR3 RNA ligands.

    Science.gov (United States)

    Green, Nathaniel M; Moody, Krishna-Sulayman; Debatis, Michelle; Marshak-Rothstein, Ann

    2012-11-16

    The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.

  4. Surfactant protein-A modulates LPS-induced TLR4 localization and signaling via β-arrestin 2.

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    Vicky Sender

    Full Text Available The soluble C-type lectin surfactant protein (SP-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM, the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT mice, but not in β-arrestin 2(-/- AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2(-/- mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A(-/- mice is significantly reduced compared to SP-A(+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A(-/- mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.

  5. Gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by TLR2.

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    Landry Blanc

    Full Text Available Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs, of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses.

  6. Long-term activation of TLR3 by Poly(I:C induces inflammation and impairs lung function in mice

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    Alexopoulou Lena

    2009-06-01

    Full Text Available Abstract Background The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. Methods TLR3 knock-out (KO mice and C57B6 (WT mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C. Results There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFα were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C, the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. Conclusion These findings demonstrate that TLR3 activation by poly(I:C modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

  7. Desert dust induces TLR signaling to trigger Th2-dominant lung allergic inflammation via a MyD88-dependent signaling pathway

    International Nuclear Information System (INIS)

    He, Miao; Ichinose, Takamichi; Song, Yuan; Yoshida, Yasuhiro; Bekki, Kanae; Arashidani, Keiichi; Yoshida, Seiichi; Nishikawa, Masataka; Takano, Hirohisa; Shibamoto, Takayuki; Sun, Guifan

    2016-01-01

    Asian sand dust (ASD) is known to exacerbate asthma, although its mechanism is not yet well understood. In this study, when the effects on inflammatory response by LPS present in ASD was investigated by measuring the gene expression of cytokines and chemokines in RAW264.7 cells treated with ASD and/or polymyxin B (PMB), the ASD effects were attenuated by PMB, but not completely. When an in vitro study was performed using bone marrow-derived macrophages (BMDMs) from WT, TLR2 −/− , TLR4 −/− , and MyD88 −/− BALB/c mice and BMDMs from WT, TLR2 −/− , TLR4 −/− , TLR2/4 −/− , TLR7/9 −/− , and MyD88 −/− C57BL/6J mice, cytokine (IL-6, IL-12) production in BMDMs was higher in ASD-stimulated TLR2 −/− cells than in TLR4 −/− cells, whereas it was lower or undetectable in TLR2/4 −/− and MyD88 −/− cells. These results suggest that ASD causes cytokine production predominantly in a TLR4/MyD88-dependent pathway. When WT and TLRs 2 −/− , 4 −/− , and MyD88 −/− BALB/c mice were intratracheally challenged with OVA and/or ASD, ASD caused exacerbation of lung eosinophilia along with Th2 cytokine and eosinophil-relevant chemokine production. Serum OVA-specific IgE and IgG1 similar to WT was observed in TLRs 2 −/− , 4 −/− mice, but not in MyD88 −/− mice. The Th2 responses in TLR2 −/− mice were attenuated remarkably by PMB. These results indicate that ASD exacerbates lung eosinophilia in a MyD88-dependent pathway. TLRs 2 and 4 signaling may be important in the increase in lung eosinophilia. Also, the TLR4 ligand LPS and TLR2 ligand like β-glucan may be strong candidates for exacerbation of lung eosinophilia. - Highlights: • ASD enhanced Th2 response in TLR2 −/− , TLR4 −/− and WT mice, but not in MyD88 −/− . • Th2 responses in TLR2 −/− mice were attenuated by LPS inhibitor polymyxin B. • TLR2 and TLR4 signaling is important in allergic lung disease aggravation by ASD. • MyD88 is the key

  8. TLR 9 involvement in early protection induced by immunization with rPb27 against Paracoccidioidomycosis.

    Science.gov (United States)

    Morais, Elis Araujo; Chame, Daniela Ferreira; Melo, Eliza Mathias; de Carvalho Oliveira, Junnia Alvarenga; de Paula, Ana Cláudia Chagas; Peixoto, Andiara Cardoso; da Silva Santos, Lílian; Gomes, Dawidson Assis; Russo, Remo Castro; de Goes, Alfredo Miranda

    2016-02-01

    Paracoccidioidomycosis is caused by fungi of the Paracoccidioides genus and constitutes the most prevalent deep mycosis in Latin America. Toll-like receptors promote immune response against infectious agents. Recently, it was reported that TLR9 is crucial for mice survival during the first 48 h of P. brasiliensis infection. In this study, we used CPG oligodeoxynucleotide motif as an adjuvant with and without rPb27 to immunize mice against Paracoccidioidomycosis. CPG adjuvant induced differential recruitment of lymphocytes in the inflammatory process and a lower recruitment of neutrophils. In addition, CPG induced the production of pro-inflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-12; increased phagocytic ability and microbicidal activity by macrophages; and induced differential production of lgG2a and lgG2b, subtypes of Ig. Knockout mice for TLR9 and IL-12 showed higher fungal loads and rates of mortality compared to control mice after 30 days of infection. The association between CPG and rPb27 induced a high level of protection against Paracoccidioidomycosis after the first 30 days of infection but not at 60 days. Our findings demonstrate that TLR 9 plays a role in the protection induced by immunization with rPb27 and confirms the importance of TLR9 in the initial protection against Paracoccidioidomycosis. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  9. 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.

  10. Gold-quercetin nanoparticles prevent metabolic endotoxemia-induced kidney injury by regulating TLR4/NF-κB signaling and Nrf2 pathway in high fat diet fed mice.

    Science.gov (United States)

    Xu, Min-Xuan; Wang, Ming; Yang, Wei-Wei

    2017-01-01

    High-fat diet-induced metabolic syndrome followed by chronic kidney disease caused by intestinal endotoxemia have received extensive attention. Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) and oxidative stress-related Nrf2/Keap1 were regarded as the key target points involved in metabolic inflammation and kidney injury. However, the molecular mechanism of interaction between TLR4/NF-κB and Nrf2 activation in high-fat diet-induced renal injury is not absolutely understood. Quercetin, a natural product, has been reported to possess antitumor and anti-inflammatory effects. In this regard, this study attempted to prepare poly(d,l-lactide- co -glycolide)-loaded gold nanoparticles precipitated with quercetin (GQ) to investigate the anti-inflammatory and anti-oxidative stress effects in high-fat diet-induced kidney failure. For this study, C57BL/6 mice fed fat-rich fodder were used as the metabolic syndrome model to evaluate the protective effects of GQ on kidney injury and to determine whether TLR4/NF-κB and Nrf2 pathways were associated with the process. Moreover, histological examinations, enzyme-linked immunosorbent assay, Western blot, and basic blood tests and systemic inflammation-related indicators were used to investigate the inhibitory effects of GQ and underlying molecular mechanism by which it may reduce renal injury. Of note, podocyte injury was found to participate in endotoxin-stimulated inflammatory response. TLR4/NF-κB and Nrf2 pathways were upregulated with high-fat diet intake in mice, resulting in reduction of superoxide dismutase activity and increase in superoxide radical, H 2 O 2 , malondialdehyde, XO, XDH, and XO/XDH ratio. In addition, upregulation of TLR4/NF-κB and oxidative stress by endotoxin were observed in vitro, which were suppressed by GQ administration, ultimately alleviating podocyte injury. These findings indicated that GQ could restore the metabolic disorders caused by high-fat diet, which suppresses insulin

  11. NASP and ISPA Response to the Japanese Natural Disaster

    Science.gov (United States)

    Pfohl, Bill; Cowan, Katherine

    2011-01-01

    The authors have worked together with the NASP (National Association of School Psychologists) National Emergency Assistance Team (NEAT) for a decade to help coordinate communications around large-scale crisis response efforts. The massive earthquake and tsunami that devastated the northeastern part of Japan and the subsequent response represented…

  12. mRNA expression of TLR4, TLR9 and NF-κB in a neonatal murine model of necrotizing enterocolitis.

    Science.gov (United States)

    Yin, Yiyu; Liu, Fengli; Li, Yiping; Tang, Ruze; Wang, Jian

    2016-09-01

    A neonatal model of necrotizing enterocolitis (NEC) in mice was established to examine the role of Toll-like receptors (TLRs) 4 and 9, and of nuclear factor (NF)‑κB by quantitative detection of their mRNAs in intestinal tissue during the occurrence of NEC, and thus aid in the understanding of the basic pathogenesis of NEC. A total of 50 newborn BALB/c mice (specific pathogen-free level) ranging in age from 7 to 10 days, of either gender, and weighing 4.8‑5.4 g were selected and randomly divided into a control and test group, n=25 mice per group. Mice in the control group were kept in the same cage with the mother who fed them, free from any interventions. Mice in the test group were separated from their mother 48 h following birth and placed in an incubator, artificially fed with milk substitutes, and regularly treated with hypoxia and cold stimulation (100% nitrogen anoxia for 90 sec, cold stimulation at 4˚C for 10 min, 3 times a day for 3 days) to induce the neonatal NEC. The general state and body weight variations of the mice were recorded, the mice were sacrificed and the intestinal tissue necrosis was evaluated visually, the degree of intestinal injury was determined by histopathological staining, and the mRNA expression levels of intestinal tissue TLR4, TLR9 and NF‑κB were quantified. Of the 25 mice in the test group, 3 died a natural death and 22 were sacrificed; their general state was worse than that of the mice in the control group, and the body weight variations among them were considerably larger. NEC was confirmed in 12 cases by visual inspection, and the average histological scores of the mice in the test group were 3.5±0.6, significantly higher than that in the control group (P<0.05). The mRNA expression of TLR4 and NF‑κB in the test group were significantly higher than in the control group. By contrast, the mRNA expression of TLR9 was significantly lower in the test group, and differences were statistically

  13. Natural hazards research and response; international decade for reducing loss from natural disasters

    Science.gov (United States)

    Hays, W.W.

    1992-01-01

    Worldwide losses from natural disasters are increasing rapidly due to population growth, urban sprawl, and increasing concentration of new construction in high-risk areas. to deal with these problems, the United Nations has designated the 1990's as the International Decade for Natural Disaster Reduction (IDNDR). The United States and more than 150 other nations signed the IDNDR resolution at the 44th General Assembly of the United Nations. The resolution calls on all nations to develop programs to reduce loss of life, economic impact, and human suffering caused by natural disasters. 

  14. Meat and fiber intake and interaction with pattern recognition receptors (TLR1, TLR2, TLR4, and TLR10) in relation to colorectal cancer in a Danish prospective, case-cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Vogel, Ulla; Tjonneland, Anne

    2018-01-01

    Background: Meat and dietary fiber are associated with increased and decreased risk of colorectal cancer (CRC), respectively. Toll-like receptors (TLRs) regulate the intestinal immune response in a complex interplay between the mucosal epithelium and the microbiota and may therefore be important...... modulators of diet-induced CRC together with other inflammatory mediators. Objective: Our aim was to investigate the association between functional TLR polymorphisms and risk of CRC and the interaction with dietary factors. Additionally, interactions with previously studied polymorphisms in IL10, IL1B, PTGS2......, and NFKB1 were assessed in order to examine possible biological pathways in meat-induced CRC. Design: A nested case-cohort study of 897 CRC cases and 1689 randomly selected participants from the Danish prospective "Diet, Cancer and Health" study encompassing 57,053 persons was performed using Cox...

  15. Milk matters: soluble Toll-like receptor 2 (sTLR2 in breast milk significantly inhibits HIV-1 infection and inflammation.

    Directory of Open Access Journals (Sweden)

    Bethany M Henrick

    Full Text Available The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM. Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2 for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam(3CSK(4 lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001 increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1 immunomodulating pro-inflammatory responses to bacterial ligands, and (2 directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

  16. Otitis media induced by peptidoglycan-polysaccharide (PGPS) in TLR2-deficient (Tlr2(-/-)) mice for developing drug therapy.

    Science.gov (United States)

    Zhang, Xiaolin; Zheng, Tihua; Sang, Lu; Apisa, Luke; Zhao, Hongchun; Fu, Fenghua; Wang, Qingzhu; Wang, Yanfei; Zheng, Qingyin

    2015-10-01

    Toll like receptor 2 (TLR2) signaling can regulate the pathogenesis of otitis media (OM). However, the precise role of TLR2 signaling in OM has not been clarified due to the lack of an optimal animal model. Peptidoglycan-polysaccharide (PGPS) of the bacterial cell wall can induce inflammation by activating the TLR2 signaling. This study aimed at examining the pathogenic characteristics of OM induced by PGPS in Tlr2(-/-) mice, and the potential therapeutic effect of sodium aescinate (SA) in this model. Wild-type (WT) and Tlr2(-/-) mice were inoculated with streptococcal PGPS into their middle ears (MEs) and treated intravenously with vehicle or SA daily beginning at 3days prior to PGPS for 6 consecutive days. The pathologic changes of individual mice were evaluated longitudinally. In comparison with WT mice, Tlr2(-/-) mice were susceptible to PGPS-induced OM. Tlr2(-/-) mice displayed greater hearing loss, tympanic membrane damage, ME mucosal thickening, longer inflammation state, cilia and goblet cell loss. SA-treatment decreased neutrophil infiltration, modulated TLR2-related gene expression and improved ciliary organization. PGPS induced a relatively stable OM in Tlr2(-/-) mice, providing a new model for OM research. Treatment with SA mitigated the pathogenic damage in the ME and may be valuable for intervention of OM. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer 1

    Science.gov (United States)

    Liang, Shuang; Hosur, Kavita B.; Lu, Shanyun; Nawar, Hesham F.; Weber, Benjamin R.; Tapping, Richard I.; Connell, Terry D.; Hajishengallis, George

    2009-01-01

    LT-IIb-B5, a doughnut-shaped oligomeric protein from enterotoxigenic Escherichia coli, is known to activate the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B5 interaction with TLR2/1 in order to define the structure-function relationship of LT-IIb-B5 and, moreover, to gain an insight into how TLR2/1 recognizes large, non-acylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam3CSK4 lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B5 pore: Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate antigen-presenting cells, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam3CSK4 complex, resulted in diminished activation by both Pam3CSK4 and LT-IIb-B5. Docking analysis of the LT-IIb-B5 interaction with this apparently “predominant” activation conformation of TLR2/1 revealed that LT-IIb-B5 may primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B5 interface is relatively smaller, the leucine-rich repeat motifs 9–12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B5. Moreover, the putative LT-IIb-B5 binding site overlaps partially with that of Pam3CSK4; consistent with this, Pam3CSK4 suppressed TLR2 binding of LT-IIb-B5, albeit not as potently as self-competitive inhibition. In conclusion, we identified the upper pore region of LT-IIb-B5 as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, though overlaps with, that of Pam3CSK4. PMID:19234193

  18. 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.

  19. Diversity and Response of Benthic Macroinvertebrates to Natural ...

    African Journals Online (AJOL)

    User

    and aesthetically less attractive species. Invertebrate conservation, however, does not rely only on public ... 22(1), 2014 stressors and, thus, provide a broad measure of their aggregate impact (Reynoldson et al., ..... nature of a freshwater environment is a major determinant of its invertebrate composition. Preference for lotic ...

  20. 3.5 square meters: Constructive responses to natural disasters

    Science.gov (United States)

    Vinitsky, Maya

    2017-01-01

    Natural disasters and their consequences dominate the news almost on a daily basis. Quick-impact preventive and aid measures are essential for the victims to survive. This volume presents a selection of projects which demonstrate impressively how both cutting-edge technology and locally available materials and resources can be used for this purpose.

  1. Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Sakuma, Chisato [Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 (Japan); Sato, Mitsuru, E-mail: mitsuru.sato@affrc.go.jp [Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 (Japan); Oshima, Takuma [Department of Biological Science and Technology, Graduate School of Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 (Japan); Takenouchi, Takato [Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 (Japan); Chiba, Joe [Department of Biological Science and Technology, Graduate School of Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 (Japan); Kitani, Hiroshi [Animal Immune and Cell Biology Research Unit, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634 (Japan)

    2015-02-27

    Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow–derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1β in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages. - Highlights: • The interaction between WASP and Btk is critical for TLR3, TLR7, and TLR9 signaling. • Anti-WASP intrabodies inhibited several TLR pathways that led to cytokine expression. • Phosphorylation of NF-κB via TLR signaling was inhibited by anti-WASP intrabodies. • WASP phosphorylation via several TLR ligands was inhibited by anti-WASP intrabodies.

  2. Activated NKT Cells Can Condition Different Splenic Dendritic Cell Subsets To Respond More Effectively to TLR Engagement and Enhance Cross-Priming.

    Science.gov (United States)

    Osmond, Taryn L; Farrand, Kathryn J; Painter, Gavin F; Ruedl, Christiane; Petersen, Troels R; Hermans, Ian F

    2015-08-01

    The function of dendritic cells (DCs) can be modulated through multiple signals, including recognition of pathogen-associated molecular patterns, as well as signals provided by rapidly activated leukocytes in the local environment, such as innate-like T cells. In this article, we addressed the possibility that the roles of different murine DC subsets in cross-priming CD8(+) T cells can change with the nature and timing of activatory stimuli. We show that CD8α(+) DCs play a critical role in cross-priming CD8(+) T cell responses to circulating proteins that enter the spleen in close temporal association with ligands for TLRs and/or compounds that activate NKT cells. However, if NKT cells are activated first, then CD8α(-) DCs become conditioned to respond more vigorously to TLR ligation, and if triggered directly, these cells can also contribute to priming of CD8(+) T cell responses. In fact, the initial activation of NKT cells can condition multiple DC subsets to respond more effectively to TLR ligation, with plasmacytoid DCs making more IFN-α and both CD8α(+) and CD8α(-) DCs manufacturing more IL-12. These results suggest that different DC subsets can contribute to T cell priming if provided appropriately phased activatory stimuli, an observation that could be factored into the design of more effective vaccines. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Reconstruction of LPS Transfer Cascade Reveals Structural Determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfer.

    Science.gov (United States)

    Ryu, Je-Kyung; Kim, Soo Jin; Rah, Sang-Hyun; Kang, Ji In; Jung, Hi Eun; Lee, Dongsun; Lee, Heung Kyu; Lee, Jie-Oh; Park, Beom Seok; Yoon, Tae-Young; Kim, Ho Min

    2017-01-17

    Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, binds Toll-like receptor 4 (TLR4)-MD2 complex and activates innate immune responses. LPS transfer to TLR4-MD2 is catalyzed by both LPS binding protein (LBP) and CD14. To define the sequential molecular interactions underlying this transfer, we reconstituted in vitro the entire LPS transfer process from LPS micelles to TLR4-MD2. Using electron microscopy and single-molecule approaches, we characterized the dynamic intermediate complexes for LPS transfer: LBP-LPS micelles, CD14-LBP-LPS micelle, and CD14-LPS-TLR4-MD2 complex. A single LBP molecule bound longitudinally to LPS micelles catalyzed multi-rounds of LPS transfer to CD14s that rapidly dissociated from LPB-LPS complex upon LPS transfer via electrostatic interactions. Subsequently, the single LPS molecule bound to CD14 was transferred to TLR4-MD2 in a TLR4-dependent manner. The definition of the structural determinants of the LPS transfer cascade to TLR4 may enable the development of targeted therapeutics for intervention in LPS-induced sepsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Dynamic Nature of Noncoding RNA Regulation of Adaptive Immune Response

    Directory of Open Access Journals (Sweden)

    Franca Citarella

    2013-08-01

    Full Text Available Immune response plays a fundamental role in protecting the organism from infections; however, dysregulation often occurs and can be detrimental for the organism, leading to a variety of immune-mediated diseases. Recently our understanding of the molecular and cellular networks regulating the immune response, and, in particular, adaptive immunity, has improved dramatically. For many years, much of the focus has been on the study of protein regulators; nevertheless, recent evidence points to a fundamental role for specific classes of noncoding RNAs (ncRNAs in regulating development, activation and homeostasis of the immune system. Although microRNAs (miRNAs are the most comprehensive and well-studied, a number of reports suggest the exciting possibility that long ncRNAs (lncRNAs could mediate host response and immune function. Finally, evidence is also accumulating that suggests a role for miRNAs and other small ncRNAs in autocrine, paracrine and exocrine signaling events, thus highlighting an elaborate network of regulatory interactions mediated by different classes of ncRNAs during immune response. This review will explore the multifaceted roles of ncRNAs in the adaptive immune response. In particular, we will focus on the well-established role of miRNAs and on the emerging role of lncRNAs and circulating ncRNAs, which all make indispensable contributions to the understanding of the multilayered modulation of the adaptive immune response.

  5. Synergy between TLR-2 and TLR-3 signaling in primary human nasal epithelial cells

    NARCIS (Netherlands)

    van Tongeren, Joost; Golebski, Korneliusz; van Egmond, Danielle; de Groot, Esther J.; Fokkens, Wytske J.; van Drunen, Cornelis M.

    2015-01-01

    Although we have a detailed understanding of how single microbial derived triggers activate specialized Toll-like receptors (TLR) on airway epithelial cells, we know little of how these receptors react in a more complex environment. In everyday life, nasal epithelial cells are exposed to multiple

  6. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling.

    Science.gov (United States)

    Yazji, Ibrahim; Sodhi, Chhinder P; Lee, Elizabeth K; Good, Misty; Egan, Charlotte E; Afrazi, Amin; Neal, Matthew D; Jia, Hongpeng; Lin, Joyce; Ma, Congrong; Branca, Maria F; Prindle, Thomas; Richardson, Ward M; Ozolek, John; Billiar, Timothy R; Binion, David G; Gladwin, Mark T; Hackam, David J

    2013-06-04

    Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS(-/-) mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate--a precursor for enteral generation of nitrite and nitric oxide--and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.

  7. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling

    Science.gov (United States)

    Yazji, Ibrahim; Sodhi, Chhinder P.; Lee, Elizabeth K.; Good, Misty; Egan, Charlotte E.; Afrazi, Amin; Neal, Matthew D.; Jia, Hongpeng; Lin, Joyce; Branca, Maria F.; Prindle, Thomas; Richardson, Ward M.; Ozolek, John; Billiar, Timothy R.; Binion, David G.; Gladwin, Mark T.; Hackam, David J.

    2013-01-01

    Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS−/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate—a precursor for enteral generation of nitrite and nitric oxide—and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate–nitrite–NO signaling. PMID:23650378

  8. MF59 oil-in-water emulsion in combination with a synthetic TLR4 agonist (E6020) is a potent adjuvant for a combination Meningococcus vaccine.

    Science.gov (United States)

    Singh, Manmohan; Kazzaz, Jina; Ugozzoli, Mildred; Baudner, Barbara; Pizza, Mariagrazia; Giuliani, Marzia; Hawkins, Lynn D; Otten, Gillis; O'Hagan, Derek T

    2012-04-01

    The inclusion of a potent TLR4 immune potentiator to a recombinant antigen vaccine formulation enhances both the magnitude and the breadth of the engendered immune response. One such immune potentiator (TLR4 agonist E6020) was evaluated with recombinant Men B antigens delivered in MF59 sub-micron adjuvant emulsion. The ability of this formulation to enhance serum antibody and bactercidal titers was investigated. The co-delivery of E6020 within MF59 enhanced both the serum and bactericidal titers for Men B antigens and for Men B antigens combined with Men ACWY-CRM conjugate vaccine. The delivery of TLR4 agonist within MF59 emulsion oil droplets leads to a more potent response in comparison to the TLR4 when admixed with MF59 emulsion.

  9. Hyperspectral Cubesat Constellation for Rapid Natural Hazard Response

    Science.gov (United States)

    Mandl, Daniel; Huemmrich, Karl; Crum, Gary; Ly, Vuong; Handy, Matthew; Ong, Lawrence

    2015-01-01

    Earth Observing 1 (E0-1) satellite has an imaging spectrometer (hyperspectral) instrument called Hyperion. The satellite is able to image any spot on Earth in the nadir looking direction every 16 days. With slewing of the satellite and allowing for up to a 23 degree view angle, any spot on the Earth can be imaged approximately every 2 to 3 days. EO-1 has been used to track many natural hazards such as wildfires, volcanoes and floods. An enhanced capability that is sought is the ability to image natural hazards in a daily time series for space based imaging spectrometers. The Hyperion can not provide this capability on EO-1 with the present polar orbit. However, a constellation of cubesats, each with the same imaging spectrometer, positioned strategically in the same orbit, can be used to provide daily coverage, cost-effectively.

  10. Responses of primate frontal cortex neurons during natural vocal communication

    OpenAIRE

    Miller, Cory T.; Thomas, A. Wren; Nummela, Samuel U.; de la Mothe, Lisa A.

    2015-01-01

    The role of primate frontal cortex in vocal communication and its significance in language evolution have a controversial history. While evidence indicates that vocalization processing occurs in ventrolateral prefrontal cortex neurons, vocal-motor activity has been conjectured to be primarily subcortical and suggestive of a distinctly different neural architecture from humans. Direct evidence of neural activity during natural vocal communication is limited, as previous studies were performed ...

  11. 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.

  12. TLR and NKG2D signaling pathways mediate CS-induced pulmonary pathologies.

    Directory of Open Access Journals (Sweden)

    Brian W Wortham

    Full Text Available Long-term exposure to cigarette smoke (CS can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.

  13. Sublingual flagellin protects against acute pneumococcal pneumonia in a TLR5-dependent and NLRC4-independent fashion.

    Science.gov (United States)

    Muñoz-Wolf, Natalia; Rial, Analía; Fougeron, Delphine; Tabareau, Julien; Sirard, Jean-Claude; Chabalgoity, José A

    2016-09-01

    To evaluate efficacy of sublingual flagellin to treat acute pneumonia. Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection. Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo. Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia.

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

    Indian Academy of Sciences (India)

    similar to Oncorhynchus mykiss TLRs. 74180 similar to Takifugu rubripes TLR9. 102021 similar to Strongylocentrotus purpuratus TLR3 precursor. Table 2. Database accession numbers of V-TLRs, V-TIRs, V-LRRs and P-Tolls used for phylogenetic analysis. The accession numbers of. NCBI are listed. Branchiostoma floridae ...

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

    African Journals Online (AJOL)

    Ilakkia Sivaji

    2016-01-20

    Jan 20, 2016 ... implicated the genetic variations (polymorphisms) in TLR genes to influence the host susceptibility to infectious diseases. However, the available literature on TLR polymorphism and susceptibility to typhoid fever is unclear. Aim: This study aimed to investigate the polymorphism of TLRs 1, 2, 4 and 5 in ...

  16. The Nature of Ritalin: A Response to Cooter.

    Science.gov (United States)

    Mick, Lori Bell

    1991-01-01

    In response to Robert Cooter, Jr. (EC 202 670), who questioned the widespread use of Ritalin with children having attention deficit hyperactivity disorders, this commentary considers four specific points: how Ritalin works, Ritalin's side effects and relationship to Tourette syndrome, the assumption that Ritalin leads to drug abuse, and the…

  17. Diversity and response of Benthic Macroinvertebrates to Natural and ...

    African Journals Online (AJOL)

    The diversity and response of benthic macroinvertebartes were used in assessing the biological water quality and health status of the stream. Samples were collected from four different stations using the Kick Sampling Technique. All the specimens collected were preserved in 70% alcohol solution and later identified in the ...

  18. TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

    Directory of Open Access Journals (Sweden)

    Pinder Margaret

    2009-03-01

    Full Text Available Abstract Background During malaria infection the Toll-like receptor 9 (TLR9 is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. Methods This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual. Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE assay on a panel of HapMap cell lines was carried out. Results No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. Conclusion By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals.

  19. Natural and Induced Humoral Responses to MUC1

    International Nuclear Information System (INIS)

    Mensdorff-Pouilly, Silvia von; Moreno, Maria; Verheijen, René H. M.

    2011-01-01

    MUC1 is a membrane-tethered mucin expressed on the ductal cell surface of glandular epithelial cells. Loss of polarization, overexpression and aberrant glycosylation of MUC1 in mucosal inflammation and in adenocarcinomas induces humoral immune responses to the mucin. MUC1 IgG responses have been associated with a benefit in survival in patients with breast, lung, pancreatic, ovarian and gastric carcinomas. Antibodies bound to the mucin may curb tumor progression by restoring cell-cell interactions altered by tumor-associated MUC1, thus preventing metastatic dissemination, as well as counteracting the immune suppression exerted by the molecule. Furthermore, anti-MUC1 antibodies are capable of effecting tumor cell killing by antibody-dependent cell-mediated cytotoxicity. Although cytotoxic T cells are indispensable to achieve anti-tumor responses in advanced disease, abs to tumor-associated antigens are ideally suited to address minimal residual disease and may be sufficient to exert adequate immune surveillance in an adjuvant setting, destroying tumor cells as they arise or maintaining occult disease in an equilibrium state. Initial evaluation of MUC1 peptide/glycopeptide mono and polyvalent vaccines has shown them to be immunogenic and safe; anti-tumor responses are scarce. Progress in carbohydrate synthesis has yielded a number of sophisticated substrates that include MUC1 glycopeptide epitopes that are at present in preclinical testing. Adjuvant vaccination with MUC1 glycopeptide polyvalent vaccines that induce strong humoral responses may prevent recurrence of disease in patients with early stage carcinomas. Furthermore, prophylactic immunotherapy targeting MUC1 may be a strategy to strengthen immune surveillance and prevent disease in subjects at hereditary high risk of breast, ovarian and colon cancer

  20. Natural and Induced Humoral Responses to MUC1

    Energy Technology Data Exchange (ETDEWEB)

    Mensdorff-Pouilly, Silvia von, E-mail: s.vonmensdorff@vumc.nl; Moreno, Maria [Department of Obstetrics and Gynecology, VU University Medical Center, De Boelelaan 1117, Amsterdam 1081 HV (Netherlands); Verheijen, René H. M. [Department of Woman & Baby, Division of Surgical & Oncological Gynaecology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands)

    2011-07-29

    MUC1 is a membrane-tethered mucin expressed on the ductal cell surface of glandular epithelial cells. Loss of polarization, overexpression and aberrant glycosylation of MUC1 in mucosal inflammation and in adenocarcinomas induces humoral immune responses to the mucin. MUC1 IgG responses have been associated with a benefit in survival in patients with breast, lung, pancreatic, ovarian and gastric carcinomas. Antibodies bound to the mucin may curb tumor progression by restoring cell-cell interactions altered by tumor-associated MUC1, thus preventing metastatic dissemination, as well as counteracting the immune suppression exerted by the molecule. Furthermore, anti-MUC1 antibodies are capable of effecting tumor cell killing by antibody-dependent cell-mediated cytotoxicity. Although cytotoxic T cells are indispensable to achieve anti-tumor responses in advanced disease, abs to tumor-associated antigens are ideally suited to address minimal residual disease and may be sufficient to exert adequate immune surveillance in an adjuvant setting, destroying tumor cells as they arise or maintaining occult disease in an equilibrium state. Initial evaluation of MUC1 peptide/glycopeptide mono and polyvalent vaccines has shown them to be immunogenic and safe; anti-tumor responses are scarce. Progress in carbohydrate synthesis has yielded a number of sophisticated substrates that include MUC1 glycopeptide epitopes that are at present in preclinical testing. Adjuvant vaccination with MUC1 glycopeptide polyvalent vaccines that induce strong humoral responses may prevent recurrence of disease in patients with early stage carcinomas. Furthermore, prophylactic immunotherapy targeting MUC1 may be a strategy to strengthen immune surveillance and prevent disease in subjects at hereditary high risk of breast, ovarian and colon cancer.

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

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    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.

  2. Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex.

    Science.gov (United States)

    Silva, E; Leitão, S; Henriques, S; Kowalewski, M P; Hoffmann, B; Ferreira-Dias, G; da Costa, L Lopes; Mateus, L

    2010-01-01

    Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia-pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE(2) and PGF(2alpha) endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE(2) and PGF(2alpha) content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE(2) and PGF(2alpha), which may further regulate the local inflammatory response. 2009 Elsevier Ireland Ltd. All rights reserved.

  3. 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

  4. The nature of immune responses to urinary tract infections

    Science.gov (United States)

    Abraham, Soman N.; Miao, Yuxuan

    2016-01-01

    The urinary tract is constantly exposed to microorganisms that inhabit the gastrointestinal tract, but generally the urinary tract resists infection by gut microorganisms. This resistance to infection is mainly ascribed to the versatility of the innate immune defences in the urinary tract as the adaptive immune responses are limited, particularly when only the lower urinary tract is infected. In recent years, as the strengths and weaknesses of the immune system of the urinary tract have emerged and as the virulence attributes of uropathogens are recognized, several potentially effective and unconventional strategies to contain or prevent urinary tract infections have emerged. PMID:26388331

  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. Analysis of TLR2, TLR4, and TLR9 single nucleotide polymorphisms in children with bacterial meningitis and their healthy family members.

    Science.gov (United States)

    Gowin, Ewelina; Świątek-Kościelna, Bogna; Kałużna, Ewelina; Nowak, Jerzy; Michalak, Michał; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

    2017-07-01

    The aim was to analyse TLR2 rs5743708, TLR2 rs4696480, TLR4 rs4986790, TLR9 rs5743836, and TLR9 rs352140 single nucleotide polymorphisms (SNPs) in children with pneumococcal and meningococcal meningitis and their family members. The study group consisted of 39 children with bacterial meningitis (25 with meningococcal meningitis and 14 with pneumococcal meningitis) and 49 family members. Laboratory test results and the course of the diseases were analyzed. Genomic DNA was extracted from 1.2ml of peripheral blood in order to analyze the five SNPs. Patients with pneumococcal and meningococcal meningitis showed a similar male/female ratio, mean age, and duration of symptoms. There were no statistically significant differences in biochemical markers between the two groups. All patients possessed at least one polymorphic variant of the analyzed SNPs. The most common SNP was TLR9 rs352140, detected in 89.7% of patients. No significant differences in SNP frequency were found between patients, family members, and the general population. The allele frequencies in the population studied are in accordance with the literature data. The study did not find an association between the analyzed SNPs and susceptibility to bacterial meningitis. The role of SNPs in genes coding toll-like receptors and the interactions between them in controlling inflammation in the central nervous system needs further evaluation. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  7. Ethic of responsibility and the future of nature

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    Miso Kulic

    2012-07-01

    Since the question of science is at the same time the one of the production of reality, it is obvious that the question "what is science?" does not amount to a self-evident question asked by a scientist regarding his scientific field. It is not only a question concerning the nature of scientific knowledge, or of scientific methods of scientific results achieved. What is at stake here is the insight concerning social and political usage of science, that the reality, which is produced by the sciences, reveals to us even in the forms of its deification, manipulation, ideologization and virtualization. Is persevering in its science-Enlightenment paradigm of human emancipation or does it, on the wave of critical self-reflection spanning all the way through the 20th century, more and more question, as Paul Feyerabend (Against Method does, the extent of constraints imposed on free thought which it produces itself? Of course, the other side of the questioning itself belongs here too: scientific progress can be evaluated regardless of its consequences, of the dangerous threats it poses to our future: nuclear annihilation, ecological pollution or climate changes which endanger the survival of the living world ?

  8. Coastal bacterioplankton community dynamics in response to a natural disturbance.

    Directory of Open Access Journals (Sweden)

    Sara K Yeo

    Full Text Available In order to characterize how disturbances to microbial communities are propagated over temporal and spatial scales in aquatic environments, the dynamics of bacterial assemblages throughout a subtropical coastal embayment were investigated via SSU rRNA gene analyses over an 8-month period, which encompassed a large storm event. During non-perturbed conditions, sampling sites clustered into three groups based on their microbial community composition: an offshore oceanic group, a freshwater group, and a distinct and persistent coastal group. Significant differences in measured environmental parameters or in the bacterial community due to the storm event were found only within the coastal cluster of sampling sites, and only at 5 of 12 locations; three of these sites showed a significant response in both environmental and bacterial community characteristics. These responses were most pronounced at sites close to the shoreline. During the storm event, otherwise common bacterioplankton community members such as marine Synechococcus sp. and members of the SAR11 clade of Alphaproteobacteria decreased in relative abundance in the affected coastal zone, whereas several lineages of Gammaproteobacteria, Betaproteobacteria, and members of the Roseobacter clade of Alphaproteobacteria increased. The complex spatial patterns in both environmental conditions and microbial community structure related to freshwater runoff and wind convection during the perturbation event leads us to conclude that spatial heterogeneity was an important factor influencing both the dynamics and the resistance of the bacterioplankton communities to disturbances throughout this complex subtropical coastal system. This heterogeneity may play a role in facilitating a rapid rebound of regions harboring distinctly coastal bacterioplankton communities to their pre-disturbed taxonomic composition.

  9. Kaempferol alleviates LPS-induced neuroinflammation and BBB dysfunction in mice via inhibiting HMGB1 release and down-regulating TLR4/MyD88 pathway.

    Science.gov (United States)

    Cheng, Xiao; Yang, Ying-Lin; Yang, Huan; Wang, Yue-Hua; Du, Guan-Hua

    2018-03-01

    Kaempferol is a natural flavonoid with many biological activities including anti-oxidation and anti-inflammation. Nevertheless, its anti-neuroinflammation role and the relevant mechanism remain unclear. The present study was to investigate effects of kaempferol against LPS-induced neuroinflammation and blood-brain barrier dysfunction as well as the mechanism in mice. BALB/c mice were treated with LPS 5mg/kg to induce inflammation after pre-treatment with kaempferol 25, 50, or 100mg/kg for 7days. The results showed that kaempferol reduced the production of various pro-inflammatory factors and inflammatory proteins including IL-1β, IL-6, TNF-α, MCP-1, COX-2 and iNOS in brain tissues. In addition, kaempferol also protected BBB integrity and increased BBB related proteins including occludin-1, claudin-1 and CX43 in brain of LPS-induced mice. Furthermore, kaempferol significantly reduced HMGB1 level and suppressed TLR4/MyD88 inflammatory pathway in both transcription level and translation level. These results collectively suggested that kaempferol might be a promising neuroprotective agent for alleviating inflammatory responses and BBB dysfunction by inhibiting HMGB1 release and down-regulating TLR4/MyD88 inflammatory pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Experiencing Nature through Immersive Virtual Environments: Environmental Perceptions, Physical Engagement, and Affective Responses during a Simulated Nature Walk

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    Giovanna Calogiuri

    2018-01-01

    Full Text Available By combining physical activity and exposure to nature, green exercise can provide additional health benefits compared to physical activity alone. Immersive Virtual Environments (IVE have emerged as a potentially valuable supplement to environmental and behavioral research, and might also provide new approaches to green exercise promotion. However, it is unknown to what extent green exercise in IVE can provide psychophysiological responses similar to those experienced in real natural environments. In this study, 26 healthy adults underwent three experimental conditions: nature walk, sitting-IVE, and treadmill-IVE. The nature walk took place on a paved trail along a large river. In the IVE conditions, the participants wore a head-mounted display with headphones reproducing a 360° video and audio of the nature walk, either sitting on a chair or walking on a manually driven treadmill. Measurements included environmental perceptions (presence and perceived environmental restorativeness – PER, physical engagement (walking speed, heart rate, and perceived exertion, and affective responses (enjoyment and affect. Additionally, qualitative information was collected through open-ended questions. The participants rated the IVEs with satisfactory levels of ‘being there’ and ‘sense of reality,’ but also reported discomforts such as ‘flatness,’ ‘movement lag’ and ‘cyber sickness.’ With equivalent heart rate and walking speed, participants reported higher perceived exertion in the IVEs than in the nature walk. The nature walk was associated with high enjoyment and enhanced affect. However, despite equivalent ratings of PER in the nature walk and in the IVEs, the latter were perceived as less enjoyable and gave rise to a poorer affect. Presence and PER did not differ between the two IVEs, although in the treadmill-IVE the negative affective responses had slightly smaller magnitude than in the sitting-IVE. In both the IVEs, the negative

  11. Key Role of Toll-Like Receptor 2 in the Inflammatory Response and Major Histocompatibility Complex Class II Downregulation in Brucella abortus-Infected Alveolar Macrophages

    Science.gov (United States)

    Ferrero, Mariana C.; Hielpos, M. Soledad; Carvalho, Natalia B.; Barrionuevo, Paula; Corsetti, Patricia P.; Giambartolomei, Guillermo H.; Oliveira, Sergio C.

    2014-01-01

    Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1β (IL-1β), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival. PMID:24478078

  12. TLR2-dependent MyD88 signaling contributes to early host defense in murine Enterococcus faecium peritonitis

    NARCIS (Netherlands)

    Leendertse, Masja; Willems, Rob J. L.; Giebelen, Ida A. J.; van den Pangaart, Petra S.; Wiersinga, W. Joost; de Vos, Alex F.; Florquin, Sandrine; Bonten, Marc J. M.; van der Poll, Tom

    2008-01-01

    The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling,

  13. Relationship between TLR4 signalling alterations and effective human cytomegalovirus infection

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    Germini D.

    2014-09-01

    Full Text Available Toll-like receptors (TLR, the main class of immune-sensor molecules triggering the innate immunity pathways, are known to be involved in the infection of different RNA and DNA viruses, including herpesviruses. Human cytomegalovirus (HCMV is a widespread human beta-herpesvirus that infects 80–90 % of the world’s population and it can cause severe and even fatal diseases in immunocompromised patients and it is also responsible for birth defects as a consequence of congenital infection. Aim of this review is to discuss the existing data regarding the role of TLRs in HCMV concentrating mainly on TLR4. A better understanding in this relationship could be exploited for the development of efficient early diagnosis methodologies and anti viral therapies.

  14. CpG oligodeoxynucleotides as TLR9 agonists: therapeutic applications in cancer.

    Science.gov (United States)

    Murad, Yanal M; Clay, Timothy M

    2009-01-01

    Toll-like receptors (TLRs) are part of the innate immune system, and they belong to the pattern recognition receptors (PRR) family. The PRR family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are not generated by the host and are restricted and essential to micro-organisms. TLR9, which recognizes unmethylated CpG (cytosine guanosine dinucleotide), is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells and B cells, and results in potent T helper-1 (T(h)1)-type immune responses and antitumor responses in mouse tumor models and in patients. Several pharmaceutical companies, such as Pfizer, Idera, and Dynavax, are developing CpG oligodeoxynucleotides (ODNs) for the treatment of cancer, along with other conditions, such as infections and allergy. CpG ODNs have shown promising results as vaccine adjuvants and in combination with cancer immunotherapy. Several TLR9 agonists are being developed and have entered clinical trials to evaluate their safety and efficacy for the treatment of several hematopoietic and solid tumors. In this review, we discuss the use of CpG ODNs in several phase I and II clinical trials for the treatment of NHL, renal cell carcinoma, melanoma, and non-small cell lung cancer, either alone or in combination with other agents.

  15. TLR4-dependent recognition of lipopolysaccharide by epithelial cells requires sCD14.

    Science.gov (United States)

    Bäckhed, Fredrik; Meijer, Lisa; Normark, Staffan; Richter-Dahlfors, Agneta

    2002-08-01

    Epithelial cells lining the urinary bladder mucosa are engaged in numerous functions that act in concert to prevent exposure of the sensitive upper urinary tract to bacteria. This protective effect was recently suggested to be achieved mainly by compartmentalized, organ-specific expression of the lipopolysaccharide (LPS) receptor Toll-like receptor (TLR) 4 within epithelial cells of the urogenital tract. Here, we show that bladder epithelial cells recognize similarly low amounts of LPS as macrophages. LPS responsiveness measured as secretion of the chemoattractant interleukin 8 demonstrates a dependency on TLR4 in epithelial cells, which is similar to the situation in macrophages. The TLR4-mediated LPS response in bladder epithelial cells also uses the co-receptor CD14 for efficient LPS signalling. However, bladder epithelial cells do not express endogenous CD14 and are therefore dependent on the soluble form of CD14 that is present in body fluids. Furthermore, we demonstrate that epithelial chemokine production is augmented by type 1-mediated attachment of uropathogenic Escherichia coli in the absence, but not in the presence, of CD14. Collectively, our findings strengthen the role for bladder epithelial cells as important players in the innate immune system within the urinary tract.

  16. Inflammation, longevity, and cardiovascular diseases: role of polymorphisms of TLR4.

    Science.gov (United States)

    Candore, Giuseppina; Aquino, Alessandra; Balistreri, Carmela Rita; Bulati, Matteo; Di Carlo, Daniele; Grimaldi, Maria Paola; Listì, Florinda; Orlando, Valentina; Vasto, Sonya; Caruso, Marco; Colonna-Romano, Giuseppina; Lio, Domenico; Caruso, Calogero

    2006-05-01

    The total burden of infection at various sites may affect the progression of atherosclerosis, the risk being modulated by host genotype. The role of lipopolysaccaride receptor TLR4 is paradigmatic. It initiates the innate immune response against gram-negative bacteria; and TLR4 polymorphisms, as ASP299GLY, suggested to attenuate receptor signaling, have been described. We demonstrated that TLR4 ASP299GLY polymorphism shows a significantly lower frequency in patients affected by myocardial infarction compared to controls, whereas centenarians show a higher frequency. Thus, people genetically predisposed to developing weak inflammatory activity, seem to have fewer chances of developing cardiovascular diseases (CVD) and, subsequently, live longer if they do not become affected by serious infectious diseases. These results are in agreement with our other data demonstrating how genetic background may exert the opposite effect with respect to inflammatory components in CVD and longevity. In the present report, to validate this hypothesis, the levels of interleukin (IL)-6, a pro-inflammatory cytokine involved in atherosclerosis and longevity, were determined by an enzyme-linked immuno-sorbent assay (ELISA) in supernatants from a whole blood assay after stimulation with subliminal doses of lipopolysaccaride (LPS) from Escherichia coli (E. coli). The samples, genotyped for the ASP299GLY polymorphism, were challenged with LPS for 4, 24, and 48 h. What we found was that Il-6 values were significantly lower in carriers bearing TLR4 mutation. Therefore, the pathogen burden, by interacting with host genotype, determines the type and intensity of the immune-inflammatory responses accountable for pro-inflammatory status, CVD, and unsuccessful aging. On the other hand, our present data seem to explain the inconclusive results obtained in case-control studies taking into account the role of functional IL-6 polymorphisms in successful and unsuccessful aging. In fact, IL6 levels seem

  17. Association of TLR variants with susceptibility to Plasmodium vivax malaria and parasitemia in the Amazon region of Brazil.

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    Allyson Guimarães Costa

    Full Text Available Plasmodium vivax malaria (Pv-malaria is still considered a neglected disease despite an alarming number of individuals being infected annually. Malaria pathogenesis occurs with the onset of the vector-parasite-host interaction through the binding of pathogen-associated molecular patterns (PAMPs and receptors of innate immunity, such as toll-like receptors (TLRs. The triggering of the signaling cascade produces an elevated inflammatory response. Genetic polymorphisms in TLRs are involved in susceptibility or resistance to infection, and the identification of genes involved with Pv-malaria response is important to elucidate the pathogenesis of the disease and may contribute to the formulation of control and elimination tools.A retrospective case-control study was conducted in an intense transmission area of Pv-malaria in the state of Amazonas, Brazil. Genetic polymorphisms (SNPs in different TLRs, TIRAP, and CD14 were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP analysis in 325 patients infected with P. vivax and 274 healthy individuals without malaria history in the prior 12 months from the same endemic area. Parasite load was determined by qPCR. Simple and multiple logistic/linear regressions were performed to investigate association between the polymorphisms and the occurrence of Pv-malaria and parasitemia. The C/T (TLR5 R392StopCodon and T/T (TLR9 -1486C/T genotypes appear to be risk factors for infection by P. vivax (TLR5: C/C vs. C/T [OR: 2.116, 95% CI: 1.054-4.452, p = 0.031]; TLR9: C/C vs. T/T [OR: 1.919, 95% CI: 1.159-3.177, p = 0.010]; respectively. Fever (COEF = 7599.46, 95% CI = 3063.80-12135.12, p = 0.001 and the C/C genotype of TLR9 -1237C/T (COEF = 17006.63, 95% CI = 3472.83-30540.44, p = 0.014 were independently associated with increased parasitemia in patients with Pv-malaria.Variants of TLRs may predispose individuals to infection by P. vivax. The TLR5 R392StopCodon and TLR9 -1486C

  18. BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation.

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    Ying-Yu Wu

    Full Text Available The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.

  19. A presumed antagonistic LPS identifies distinct functional organization of TLR4 in mouse microglia.

    Science.gov (United States)

    Döring, Christin; Regen, Tommy; Gertig, Ulla; van Rossum, Denise; Winkler, Anne; Saiepour, Nasrin; Brück, Wolfgang; Hanisch, Uwe-Karsten; Janova, Hana

    2017-07-01

    Microglia as principle innate immune cells of the central nervous system (CNS) are the first line of defense against invading pathogens. They are capable of sensing infections through diverse receptors, such as Toll-like receptor 4 (TLR4). This receptor is best known for its ability to recognize bacterial lipopolysaccharide (LPS), a causative agent of gram-negative sepsis and septic shock. A putative, naturally occurring antagonist of TLR4 derives from the photosynthetic bacterium Rhodobacter sphaeroides. However, the antagonistic potential of R. sphaeroides LPS (Rs-LPS) is no universal feature, since several studies suggested agonistic rather than antagonistic actions of this molecule depending on the investigated mammalian species. Here we show the agonistic versus antagonistic potential of Rs-LPS in primary mouse microglia. We demonstrate that Rs-LPS efficiently induces the release of cytokines and chemokines, which depends on TLR4, MyD88, and TRIF, but not CD14. Furthermore, Rs-LPS is able to regulate the phagocytic capacity of microglia as agonist, while it antagonizes Re-LPS-induced MHC I expression. Finally, to our knowledge, we are the first to provide in vivo evidence for an agonistic potential of Rs-LPS, as it efficiently triggers the recruitment of peripheral immune cells to the endotoxin-challenged CNS. Together, our results argue for a versatile and complex organization of the microglial TLR4 system, which specifically translates exogenous signals into cellular functions. Importantly, as demonstrated here for microglia, the antagonistic potential of Rs-LPS needs to be considered with caution, as reactions to Rs-LPS not only differ by cell type, but even by function within one cell type. © 2017 Wiley Periodicals, Inc.

  20. Regulation of Toll-like receptors-dependent inflammatory response 

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    Ewa Kowalczyk

    2013-03-01

    Full Text Available Toll-like receptors (TLRs are a pivotal part of our innate immune response. They recognize a wide variety of pathogens and instigate an immune response, thus facilitating the removal of the disease-causing agent. Due to the intense nature of this response its strict control is of keyimportance, as a prolonged inflammatory signal leads to carcinogenesis and autoimmune disorders. The signaling cascade initiated by the activated TLR is complex and consists of multiple stages. It involves a variety of adaptor proteins, protein kinases and effector transcription factors. The number of stages in this process enables many possible checkpoints and ways of regulation. Signal modulation involves differentiated expression of TLRs, splicing variants of their adaptorproteins, enzymes modifying proteins engaged in the cascade and many more. This review focuses on endogenous factors responsible for controlling the TLR-dependent inflammatory response as well as on pharmacological therapies designed for regulating the innate immune response.  

  1. Role of TLR5 and flagella in bacillus intraocular infection.

    Directory of Open Access Journals (Sweden)

    Salai Madhumathi Parkunan

    Full Text Available B. cereus possesses flagella which allow the organism to migrate within the eye during a blinding form of intraocular infection called endophthalmitis. Because flagella is a ligand for Toll-like receptor 5 (TLR5, we hypothesized that TLR5 contributed to endophthalmitis pathogenesis. Endophthalmitis was induced in C57BL/6J and TLR5-/- mice by injecting 100 CFU of B. cereus into the mid-vitreous. Eyes were analyzed for intraocular bacterial growth, retinal function, and inflammation by published methods. Purified B. cereus flagellin was also injected into the mid-vitreous of wild type C57BL/6J mice and inflammation was analyzed. TLR5 activation by B. cereus flagellin was also analyzed in vitro. B. cereus grew rapidly and at similar rates in infected eyes of C57BL/6J and TLR5-/- mice. A significant loss in retinal function in both groups of mice was observed at 8 and 12 hours postinfection. Retinal architecture disruption and acute inflammation (neutrophil infiltration and proinflammatory cytokine concentrations increased and were significant at 8 and 12 hours postinfection. Acute inflammation was comparable in TLR5-/- and C57BL/6J mice. Physiological concentrations of purified B. cereus flagellin caused significant inflammation in C57BL/6J mouse eyes, but not to the extent of that observed during active infection. Purified B. cereus flagellin was a weak agonist for TLR5 in vitro. These results demonstrated that the absence of TLR5 did not have a significant effect on the evolution of B. cereus endophthalmitis. This disparity may be due to sequence differences in important TLR5 binding domains in B. cereus flagellin or the lack of flagellin monomers in the eye to activate TLR5 during infection. Taken together, these results suggest a limited role for flagellin/TLR5 interactions in B. cereus endophthalmitis. Based on this and previous data, the importance of flagella in this disease lies in its contribution to the motility of the organism within the

  2. Characterizing Response-Reinforcer Relations in the Natural Environment: Exploratory Matching Analyses

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    Sy, Jolene R.; Borrero, John C.; Borrero, Carrie S. W.

    2010-01-01

    We assessed problem and appropriate behavior in the natural environment from a matching perspective. Problem and appropriate behavior were conceptualized as concurrently available responses, the occurrence of which was thought to be determined by the relative rates or durations of reinforcement. We also assessed whether response allocation could…

  3. More on the Nature of Scientific Thought: Responses to Professors Lederman and Ohlsson.

    Science.gov (United States)

    Suchting, W. A.

    1996-01-01

    Provides author comments in response to the papers of Professors Lederman and Ohlsson in "Science and Education," Vol. 4, No. 4, 1995. These papers were in response to the paper "On the Nature of Scientific Thought" ("Science and Education," Vol. 4, No. 1). Includes 19 references. (DDR)

  4. Tourists’ Environmentally Responsible Behavior in Response to Climate Change and Tourist Experiences in Nature-Based Tourism

    OpenAIRE

    Ju Hyoung Han; Min Jae Lee; Yun-Seop Hwang

    2016-01-01

    Nature-based tourism destinations—locations in which economic viability and environmental responsibility are sought—are sensitive to climate change and its effects on important environmental components of the tourism areas. To meet the dual roles, it is important for destination marketers and resources managers to provide quality experiences for tourists and to induce tourists’ environmentally responsible behavior in such destinations. This study documents the importance of perceptions toward...

  5. A meta-analysis of crop pest and natural enemy response to landscape complexity.

    Science.gov (United States)

    Chaplin-Kramer, Rebecca; O'Rourke, Megan E; Blitzer, Eleanor J; Kremen, Claire

    2011-09-01

    Many studies in recent years have investigated the relationship between landscape complexity and pests, natural enemies and/or pest control. However, no quantitative synthesis of this literature beyond simple vote-count methods yet exists. We conducted a meta-analysis of 46 landscape-level studies, and found that natural enemies have a strong positive response to landscape complexity. Generalist enemies show consistent positive responses to landscape complexity across all scales measured, while specialist enemies respond more strongly to landscape complexity at smaller scales. Generalist enemy response to natural habitat also tends to occur at larger spatial scales than for specialist enemies, suggesting that land management strategies to enhance natural pest control should differ depending on whether the dominant enemies are generalists or specialists. The positive response of natural enemies does not necessarily translate into pest control, since pest abundances show no significant response to landscape complexity. Very few landscape-scale studies have estimated enemy impact on pest populations, however, limiting our understanding of the effects of landscape on pest control. We suggest focusing future research efforts on measuring population dynamics rather than static counts to better characterise the relationship between landscape complexity and pest control services from natural enemies. © 2011 Blackwell Publishing Ltd/CNRS.

  6. TLR9-dependent systemic interferon-beta production by intravenous injection of plasmid DNA/cationic liposome complex in mice.

    Science.gov (United States)

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Mizuno, Yumiko; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Rei; Takakura, Yoshinobu

    2009-08-01

    The type I interferon (IFN) response to DNA/cationic liposome complex, or lipoplex, has been reported in cultured cells, but little is known about the response in vivo. Studies of the pro-inflammatory cytokine response to lipoplex have shown the importance of the unmethylated CpG dinucleotide (CpG motif) and its receptor, Toll-like receptor (TLR)-9. CpG- and non-CpG lipoplex consisting of CpG- or non-CpG plasmid DNA, respectively, and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol liposomes were intravenously injected into mice. IFN-beta and interleukin (IL)-6 in the serum and organs were measured by the enzyme-linked immunosorbent assay. The involvement of TLR9, phagocytic cells and the spleen in the responses was evaluated using TLR9(-/-), clodronate liposome-treated-, and splenectomized mice, respectively. Accumulation of blood cells in the lung was evaluated histologically. CpG lipoplex induced a large increase in the levels of IFN-beta and IL-6 in the serum, liver, spleen, lung and kidney, whereas non-CpG lipoplex scarcely had any effect. Neither formulation led to significant cytokine production in TLR9(-/-) mice. Clodronate liposome-treated mice showed a large reduction in both IFN-beta and IL-6 levels. Splenectomized mice receiving CpG lipoplex also showed a significantly low production of IL-6 but a similar level of IFN-beta production to that of unsplenectomized mice. A large number of monocytes were found in the capillary vessels around the pulmonary alveoli of mice receiving lipoplex. These findings indicate that, in contrast to the production of IL-6 from splenic macrophages, IFN-beta is produced from phagocytic cells other than splenic macrophages after the injection of CpG lipoplex through the TLR9-dependent pathway.

  7. Differences in innate cytokine responses between European and African children.

    Science.gov (United States)

    Labuda, Lucja A; de Jong, Sanne E; Meurs, Lynn; Amoah, Abena S; Mbow, Moustapha; Ateba-Ngoa, Ulysse; van der Ham, Alwin J; Knulst, André C; Yazdanbakhsh, Maria; Adegnika, Ayola A

    2014-01-01

    Although differences in immunological responses between populations have been found in terms of vaccine efficacy, immune responses to infections and prevalence of chronic inflammatory diseases, the mechanisms responsible for these differences are not well understood. Therefore, innate cytokine responses mediated by various classes of pattern-recognition receptors including Toll-like receptors (TLR), C-type lectin receptors (CLRs) and nucleotide-binding oligomerisation domain-like receptors (NLRs) were compared between Dutch (European), semi-urban and rural Gabonese (African) children. Whole blood was stimulated for 24 hours and the pro-inflammatory tumor necrosis factor (TNF) and the anti-inflammatory/regulatory interleukin-10 (IL-10) cytokines in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Gabonese children had a lower pro-inflammatory response to poly(I:C) (TLR3 ligand), but a higher pro-inflammatory response to FSL-1 (TLR2/6 ligand), Pam3 (TLR2/1 ligand) and LPS (TLR4 ligand) compared to Dutch children. Anti-inflammatory responses to Pam3 were also higher in Gabonese children. Non-TLR ligands did not induce substantial cytokine production on their own. Interaction between various TLR and non-TLR receptors was further assessed, but no differences were found between the three populations. In conclusion, using a field applicable assay, significant differences were observed in cytokine responses between European and African children to TLR ligands, but not to non-TLR ligands.

  8. Differences in innate cytokine responses between European and African children.

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    Lucja A Labuda

    Full Text Available Although differences in immunological responses between populations have been found in terms of vaccine efficacy, immune responses to infections and prevalence of chronic inflammatory diseases, the mechanisms responsible for these differences are not well understood. Therefore, innate cytokine responses mediated by various classes of pattern-recognition receptors including Toll-like receptors (TLR, C-type lectin receptors (CLRs and nucleotide-binding oligomerisation domain-like receptors (NLRs were compared between Dutch (European, semi-urban and rural Gabonese (African children. Whole blood was stimulated for 24 hours and the pro-inflammatory tumor necrosis factor (TNF and the anti-inflammatory/regulatory interleukin-10 (IL-10 cytokines in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA. Gabonese children had a lower pro-inflammatory response to poly(I:C (TLR3 ligand, but a higher pro-inflammatory response to FSL-1 (TLR2/6 ligand, Pam3 (TLR2/1 ligand and LPS (TLR4 ligand compared to Dutch children. Anti-inflammatory responses to Pam3 were also higher in Gabonese children. Non-TLR ligands did not induce substantial cytokine production on their own. Interaction between various TLR and non-TLR receptors was further assessed, but no differences were found between the three populations. In conclusion, using a field applicable assay, significant differences were observed in cytokine responses between European and African children to TLR ligands, but not to non-TLR ligands.

  9. Natural Killer Dendritic Cells Enhance Immune Responses Elicited by α-Galactosylceramide-Stimulated Natural Killer T Cells

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    Sung Won Lee

    2013-01-01

    Full Text Available Natural killer dendritic cells (NKDCs possess potent anti-tumor activity, but the cellular effect of NKDC interactions with other innate immune cells is unclear. In this study, we demonstrate that the interaction of NKDCs and natural killer T (NKT cells is required for the anti-tumor immune responses that are elicited by α-galactosylceramide (α-GC in mice. The rapid and strong expression of interferon-γ by NKDCs after α-GC stimulation was dependent on NKT cells. Various NK and DC molecular markers and cytotoxic molecules were up-regulated following α-GC administration. This up-regulation could improve NKDC presentation of tumor antigens and increase cytotoxicity against tumor cells. NKDCs were required for the stimulation of DCs, NK cells, and NKT cells. The strong anti-tumor immune responses elicited by α-GC may be due to the down-regulation of regulatory T cells. Furthermore, the depletion of NKDCs dampened the tumor clearance mediated by α-GC-stimulated NKT cells in vivo. Taken together, these results indicate that complex interactions of innate immune cells might be required to achieve optimal anti-tumor immune responses during the early stages of tumorigenesis.

  10. Mycobacterial Phenolic Glycolipids Selectively Disable TRIF-Dependent TLR4 Signaling in Macrophages

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    Reid Oldenburg

    2018-01-01

    Full Text Available Phenolic glycolipids (PGLs are cell wall components of a subset of pathogenic mycobacteria, with immunomodulatory properties. Here, we show that in addition, PGLs exert antibactericidal activity by limiting the production of nitric oxide synthase (iNOS in mycobacteria-infected macrophages. PGL-mediated downregulation of iNOS was complement receptor 3-dependent and comparably induced by bacterial and purified PGLs. Using Mycobacterium leprae PGL-1 as a model, we found that PGLs dampen the toll-like receptor (TLR4 signaling pathway, with macrophage exposure to PGLs leading to significant reduction in TIR-domain-containing adapter-inducing interferon-β (TRIF protein level. PGL-driven decrease in TRIF operated posttranscriptionally and independently of Src-family tyrosine kinases, lysosomal and proteasomal degradation. It resulted in the defective production of TRIF-dependent IFN-β and CXCL10 in TLR4-stimulated macrophages, in addition to iNOS. Our results unravel a mechanism by which PGLs hijack both the bactericidal and inflammatory responses of host macrophages. Moreover, they identify TRIF as a critical node in the crosstalk between CR3 and TLR4.

  11. 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

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

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    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.

  13. Tourists’ Environmentally Responsible Behavior in Response to Climate Change and Tourist Experiences in Nature-Based Tourism

    Directory of Open Access Journals (Sweden)

    Ju Hyoung Han

    2016-07-01

    Full Text Available Nature-based tourism destinations—locations in which economic viability and environmental responsibility are sought—are sensitive to climate change and its effects on important environmental components of the tourism areas. To meet the dual roles, it is important for destination marketers and resources managers to provide quality experiences for tourists and to induce tourists’ environmentally responsible behavior in such destinations. This study documents the importance of perceptions toward climate change and tourist experiences in determining tourists’ environmentally responsible behavior while enjoying holidays at nature-based tourism destinations in Jeju Island, South Korea. Two hundred and eleven Korean and 204 Chinese tourists marked dominant tourist arrivals to the island, and responded to the survey questionnaire. Results showed that perceptions toward climate change and tourist experiences affect Korean tourists’ environmentally responsible behavior intentions, whereas tourist experiences—not perceptions toward climate change—only significantly affect Chinese tourists’ behavior intention. In a nature-based tourism context under the pressure of climate change and adverse environmental effects as consequences of tourism activities, resources managers and destination marketers need to develop environmental campaigns or informative tourist programs to formulate environmentally responsible behavior as well as to increase tourist quality experiences among domestic and international tourists.

  14. Expression of BMP2, TLR3, TLR4 and COX2 in colorectal polyps, adenoma and adenocarcinoma.

    Science.gov (United States)

    Xiang, Li; Wang, Shiqi; Jin, Xianqing; Duan, Wenjuan; Ding, Xionghui; Zheng, Chang

    2012-11-01

    The initiation and development of colorectal cancer is closely associated with the malignant transformation of colorectal polyps. The aim of this study was to analyze the expression of the bone morphogenetic protein-2 (BMP2), toll-like receptor 3 (TLR3), TLR4 and cyclooxygenase-2 (COX2) proteins in colorectal polyps, adenoma and adenocarcinoma. An immunohistochemical streptavidin-peroxidase (SP) method was used to examine the expression of MBP2, TLR4, TLR3 and COX2 in 20 colorectal juvenile polyps and 15 colorectal polyps of hamartomatous polyposis obtained from children, and 20 colorectal adenomas and 20 colorectal adenocarcinomas obtained from adults. A comparison of the expression levels of TLR3 among the groups revealed a gradual downward trend from the colorectal juvenile polyp group to the colorectal hamartomatous polyposis, adenoma and adenocarcinoma groups, respectively. The expression level of TLR3 was significantly lower in the colorectal adenocarcinoma group (ppolyp, hamartomatous polyposis, adenoma and adenocarcinoma groups. These three protein molecules may be significant in the development and malignant transformation of colorectal polyps.

  15. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation

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    Belén Borrego

    2015-07-01

    Full Text Available The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV genome (ncRNAs, to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs.

  16. Endometrial response to IVF hormonal manipulation: Comparative analysis of menopausal, down regulated and natural cycles

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    Gayer Nalini

    2004-04-01

    Full Text Available Abstract Background Uterine luminal epithelial cell response to different hormonal strategies was examined to determine commonality when an endometrium attains a receptive, stimulated, morphological profile that may lead to successful implantation. Methods Endometrial biopsies from 3 cohorts of patients were compared. The tissue samples taken from these patients were categorized into 8 different groups according to their baseline and the hormone regime used. Results Pre-treatment natural cycle tissue was variable in appearance. Downregulation with a GnRH analogue tissue appeared menopausal in character. HRT after downregulation resulted in tissue uniformity. HRT in menopause resulted in a 'lush' epithelial surface. HST in the natural cycle improved the morphology with significant difference in secretion between the two regimes examined. Conclusions Down regulation plus HRT standardized surface appearance but tissue response is significantly different from the natural cycle, natural cycle plus HRT or menopause plus HRT. HRT in menopause reinstates tissue to a state similar to a natural cycle but significantly different from a natural cycle plus HST. HST with a natural cycle is similar to tissue from the natural cycle but significant differences reflect the influence of the particular hormones present (at any point within the cycle.

  17. Endometrial response to IVF hormonal manipulation: Comparative analysis of menopausal, down regulated and natural cycles

    Science.gov (United States)

    Adams, Susan M; Terry, Vera; Hosie, Margot J; Gayer, Nalini; Murphy, Christopher R

    2004-01-01

    Background Uterine luminal epithelial cell response to different hormonal strategies was examined to determine commonality when an endometrium attains a receptive, stimulated, morphological profile that may lead to successful implantation. Methods Endometrial biopsies from 3 cohorts of patients were compared. The tissue samples taken from these patients were categorized into 8 different groups according to their baseline and the hormone regime used. Results Pre-treatment natural cycle tissue was variable in appearance. Downregulation with a GnRH analogue tissue appeared menopausal in character. HRT after downregulation resulted in tissue uniformity. HRT in menopause resulted in a 'lush' epithelial surface. HST in the natural cycle improved the morphology with significant difference in secretion between the two regimes examined. Conclusions Down regulation plus HRT standardized surface appearance but tissue response is significantly different from the natural cycle, natural cycle plus HRT or menopause plus HRT. HRT in menopause reinstates tissue to a state similar to a natural cycle but significantly different from a natural cycle plus HST. HST with a natural cycle is similar to tissue from the natural cycle but significant differences reflect the influence of the particular hormones present (at any point) within the cycle. PMID:15117407

  18. TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation.

    Science.gov (United States)

    Ni, Weihua; Wang, Fang; Liu, Guomu; Zhang, Nannan; Yuan, Hongyan; Jie, Jing; Tai, Guixiang

    2016-11-01

    Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4 + T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Inactivated influenza vaccine adjuvanted with Bacterium-like particles induce systemic and mucosal influenza A virus specific T-cell and B-cell responses after nasal administration in a TLR2 dependent fashion

    NARCIS (Netherlands)

    Keijzer, C.; Haijema, B. J.; Meijerhof, T.; Voorn, P.; de Haan, A.; Leenhouts, K.; van Roosmalen, M. L.; van Eden, W.; Broere, F.

    2014-01-01

    Background: Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies

  20. Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix

    NARCIS (Netherlands)

    Berbée, Jimmy F. P.; Coomans, Claudia P.; Westerterp, Marit; Romijn, Johannes A.; Havekes, Louis M.; Rensen, Patrick C. N.

    2010-01-01

    Timely sensing of lipopolysaccharide (LPS) is critical for the host to fight invading Gram-negative bacteria. We recently showed that apolipoprotein CI (apoCI) (apoCI1-57) avidly binds to LPS, involving an LPS-binding motif (apoCI48-54), and thereby enhances the LPS-induced inflammatory response.

  1. TLR accessory molecule RP105 (CD180 is involved in post-interventional vascular remodeling and soluble RP105 modulates neointima formation.

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    Jacco C Karper

    Full Text Available BACKGROUND: RP105 (CD180 is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/- mice resulting in a higher proliferation rate. In RP105(-/- mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2 vs.1947 ± 278 µm(2,p = 0.0014. Local LPS application augmented neointima formation in both groups, but in RP105(-/- mice this effect was more pronounced (10316±1243 µm(2 vs.4208 ± 555 µm(2,p = 0.0002, suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2,p<0.05, whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.

  2. Enhanced inflammatory responses to toll-like receptor 2/4 stimulation in type 1 diabetic coronary artery endothelial cells: the effect of insulin

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    Ao Lihua

    2010-12-01

    Full Text Available Abstract Background Endothelial inflammatory responses mediated by Toll-like receptors (TLRs, particularly TLR2 and TLR4, play an important role in atherogenesis. While Type 1 diabetes (T1D promotes the development and progression of atherosclerosis, the effect of T1D on TLR2/4-mediated inflammatory responses in coronary artery endothelial cells (CAECs remains unclear. Methods We tested the hypothesis that diabetic CAECs have enhanced inflammatory responses to TLR2/4 stimulation. Non-diabetic and diabetic CAECs were treated with TLR2 agonist peptidoglycan and TLR4 agonist lipopolysaccharide. The expression of ICAM-1, IL-6 and IL-8 were analyzed by real-time PCR, immunoblotting and ELISA, and NF-κB activation by immunoblotting and immunostaining. In additional experiments, insulin was added before TLR stimulation to determine whether insulin deficiency alone is responsible for the alteration of TLR2/4-mediated inflammatory responses. Results Stimulation of TLR2 or TLR4 induced NF-κB activation, and the expression of ICAM-1, IL-6 and IL-8. Interestingly, the expression of inflammatory mediators was significantly enhanced in diabetic cells. The enhanced inflammatory responses correlated with augmented NF-κB activation in the absence of a change in TLR2 or TLR4 protein levels. Further, pretreatment of diabetic cells with insulin failed to suppress the enhanced inflammatory responses. Conclusions Diabetic CAECs have enhanced inflammatory responses to stimulation of TLR2 or TLR4, and insulin alone is insufficient to correct the hyper-inflammatory responses. The mechanism underlying the enhanced inflammatory responses appears to be augmentation of pro-inflammatory signaling, rather than up-regulation of levels of TLR2 and TLR4. These findings suggest that diabetic CAECs adopt a hyper-inflammatory phenotype and that this endothelial phenotypic change may predispose coronary artery to atherogenesis.

  3. Induction of TLR-2 and TLR-5 expression by Helicobacter pylori switches cagPAI-dependent signalling leading to the secretion of IL-8 and TNF-α.

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    Suneesh Kumar Pachathundikandi

    2011-05-01

    Full Text Available Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI encoding a type-IV secretion system (T4SS for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5 during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression

  4. TNF-α Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism

    Science.gov (United States)

    Lee, Jiyoung; Tian, Yongjun; Chan, Stephanie Tze; Kim, Ja Yeon; Cho, Cecilia; Ou, Jing-hsiung James

    2015-01-01

    Invasion by infectious pathogens can elicit a range of cytokine responses from host cells. These cytokines provide the initial host defense mechanism. In this report, we demonstrate that TNF-α, a pro-inflammatory cytokine, can be induced by hepatitis C virus (HCV) in its host cells in a biphasic manner. The initial induction of TNF-α by HCV was prompt and could be blocked by the antibody directed against the HCV E2 envelope protein and by chemicals that inhibit endocytosis, indicating the specificity of endocytic uptake of HCV in this induction. Further studies indicated that the induction of TNF-α was dependent on toll-like receptors 7 and 8 (TLR7/8) but not on other intracellular pattern recognition receptors. Consistently, siRNA-mediated gene silencing of the downstream effectors in the TLR7/8 signaling pathway including MyD88, IRAK1, TRAF6, TAK1 and p65 NF-κB suppressed the expression of TNF-α. The role of p65 NF-κB in the induction of TNF-α via transcriptional up-regulation was further confirmed by the chromatin immunoprecipitation assay. TNF-α induced by HCV could activate its own receptor TNFR1 on hepatocytes to suppress HCV replication. This suppressive effect of TNF-α on HCV was due to its role in supporting interferon signaling, as the suppression of its expression led to the loss of IFNAR2 and impaired interferon signaling and the induction of interferon-stimulated genes. In conclusion, our results indicate that hepatocytes can sense HCV infection via TLR7/8 to induce the expression of TNF-α, which inhibits HCV replication via an autocrine mechanism to support interferon signaling. PMID:26023919

  5. Human physiological benefits of viewing nature: EEG responses to exact and statistical fractal patterns.

    Science.gov (United States)

    Hagerhall, C M; Laike, T; Küller, M; Marcheschi, E; Boydston, C; Taylor, R P

    2015-01-01

    Psychological and physiological benefits of viewing nature have been extensively studied for some time. More recently it has been suggested that some of these positive effects can be explained by nature's fractal properties. Virtually all studies on human responses to fractals have used stimuli that represent the specific form of fractal geometry found in nature, i.e. statistical fractals, as opposed to fractal patterns which repeat exactly at different scales. This raises the question of whether human responses like preference and relaxation are being driven by fractal geometry in general or by the specific form of fractal geometry found in nature. In this study we consider both types of fractals (statistical and exact) and morph one type into the other. Based on the Koch curve, nine visual stimuli were produced in which curves of three different fractal dimensions evolve gradually from an exact to a statistical fractal. The patterns were shown for one minute each to thirty-five subjects while qEEG was continuously recorded. The results showed that the responses to statistical and exact fractals differ, and that the natural form of the fractal is important for inducing alpha responses, an indicator of a wakefully relaxed state and internalized attention.

  6. Natural responses to Quaternary climatic change in the Nevada Test Site region

    International Nuclear Information System (INIS)

    Gibson, J.D.

    1993-01-01

    Migration of hazardous contaminants within geologic settings depends on natural processes. Climatic fluctuations can affect the magnitudes and rates of many of these processes. In any long-term environmental evaluation of natural processes, responses to climatic change must be considered. Four generalized categories of natural responses to Quaternary climatic change are recognized for the Nevada Test Site (NTS) region of southwestern Nevada and adjacent California: (1) biologic, (2) geomorphic, (3) hydrologic (including surface and subsurface) and (4) pedologic/diagenetic. Specific examples that correspond to the four categories illustrate the broad range of complex natural processes the are affected by climatic change. These responses dictate the potential effects of climatic change on contaminant transport, effects that are being examined by existing and planned environmental-restoration and waste-management programs within the region. Regulatory requirements for many of these programs include long-term (>10,000-year) waste isolation because of radiologic components. The purpose here is not to be exhaustive in documenting all known natural responses to climatic change in the NTS region, but rather to give a flavor of the scope of interdisciplinary and interrelated fields of Quaternary science that must be considered in evaluating the possible effects of climatic change on long-term environmental programs

  7. TLR4 accessory molecule RP105 (CD180 regulates monocyte-driven arteriogenesis in a murine hind limb ischemia model.

    Directory of Open Access Journals (Sweden)

    Antonius J N M Bastiaansen

    Full Text Available AIMS: We investigated the role of the TLR4-accessory molecule RP105 (CD180 in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia. METHODS AND RESULTS: RP105-/- and wild type (WT mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice. CONCLUSIONS: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

  8. The synthesis and the electric-responsiveness of hydrogels entrapping natural polyelectrolyte

    International Nuclear Information System (INIS)

    Sutani, Kouichi; Kaetsu, Isao; Uchida, Kumao

    2001-01-01

    A mixture of vinyl monomer, a natural polyelectrolyte--hyaluronic acid--and crosslinker was polymerized and crosslinked to entrap the natural polymer into the synthetic gel. The controlled release of the model drug from the obtained gel was studied under the on-off switching of electric field. It was proved that electric-responsive drug releases were possible using hyaluronic acid entrapping gel and the electro-responsiveness was greatly affected by various factors such as degree of swelling, crosslinking density, kind and composition of vinyl monomer and crosslinkers

  9. The synthesis and the electric-responsiveness of hydrogels entrapping natural polyelectrolyte

    Science.gov (United States)

    Sutani, Kouichi; Kaetsu, Isao; Uchida, Kumao

    2001-04-01

    A mixture of vinyl monomer, a natural polyelectrolyte—hyaluronic acid—and crosslinker was polymerized and crosslinked to entrap the natural polymer into the synthetic gel. The controlled release of the model drug from the obtained gel was studied under the on-off switching of electric field. It was proved that electric-responsive drug releases were possible using hyaluronic acid entrapping gel and the electro-responsiveness was greatly affected by various factors such as degree of swelling, crosslinking density, kind and composition of vinyl monomer and crosslinkers.

  10. Burn-induced alterations in toll-like receptor-mediated responses by bronchoalveolar lavage cells.

    Science.gov (United States)

    Oppeltz, Richard F; Rani, Meenakshi; Zhang, Qiong; Schwacha, Martin G

    2011-09-01

    Burn is associated with profound inflammation and activation of the innate immune system in multiple organ beds, including the lung. Similarly, toll-like receptors (TLR) are associated with innate immune activation. Nonetheless, it is unclear what impact burn has on TLR-induced inflammatory responses in the lung. Male C57BL/6 mice were subjected to burn (3rd degree, 25% TBSA) or sham procedure and 1, 3 or 7 days thereafter, bronchoalveolar lavage (BAL) fluid was collected and cells were isolated and cultured in vitro with specific TLR agonists as follows: Zymosan (TLR-2), LPS (TLR-4) and CpG-ODN (TLR-9). Supernatants were collected 48 h later and assayed for inflammatory cytokine levels (IL-1β, IL-6, IL-10, IL-17, TNF-α, KC, MCP-1, MIP-1α, MIP-1β and RANTES) by Bioplex. BAL fluid from sham and burn mice did not contain detectable cytokine levels. BAL cells, irrespective of injury, were responsive to TLR-2 and TLR-4 activation. Seven days after burn, TLR-2 and TLR-4 mediated responses by BAL cells were enhanced as evidenced by increased production of IL-6, IL-17, TNF-α, MCP-1, MIP-1β and RANTES. Burn-induced changes in TLR-2 and TLR-4 reactivity may contribute to the development of post-burn complications, such as acute lung injury (ALI) and adult respiratory distress syndrome (ARDS). Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Gold-quercetin nanoparticles prevent metabolic endotoxemia-induced kidney injury by regulating TLR4/NF-kB signaling and Nrf2 pathway in high fat diet fed mice

    Directory of Open Access Journals (Sweden)

    Xu MX

    2017-01-01

    Full Text Available Min-Xuan Xu,1,2,* Ming Wang,3,* Wei-Wei Yang4 1Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 2College of Engineering and Applied Sciences, Nanjing University, Nanjing, 3Department of Urology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 4Department of Nephrology, Huai’an First People’s Hospital, Nanjing Medical University, Jiangsu, People’s Republic of China *These authors contributed equally to this work Abstract: High-fat diet-induced metabolic syndrome followed by chronic kidney disease caused by intestinal endotoxemia have received extensive attention. Toll-like receptor 4 (TLR4/nuclear factor-kappa B (NF-κB and oxidative stress-related Nrf2/Keap1 were regarded as the key target points involved in metabolic inflammation and kidney injury. However, the molecular mechanism of interaction between TLR4/NF-κB and Nrf2 activation in high-fat diet-induced renal injury is not absolutely understood. Quercetin, a natural product, has been reported to possess antitumor and anti-inflammatory effects. In this regard, this study attempted to prepare poly(d,l-lactide-co-glycolide-loaded gold nanoparticles precipitated with quercetin (GQ to investigate the anti-inflammatory and anti-oxidative stress effects in high-fat diet-induced kidney failure. For this study, C57BL/6 mice fed fat-rich fodder were used as the metabolic syndrome model to evaluate the protective effects of GQ on kidney injury and to determine whether TLR4/NF-κB and Nrf2 pathways were associated with the process. Moreover, histological examinations, enzyme-linked immunosorbent assay, Western blot, and basic blood tests and systemic inflammation-related indicators were used to investigate the inhibitory effects of GQ and underlying molecular mechanism by which it may reduce renal injury. Of note, podocyte