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Sample records for single tlr ligand

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

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    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.

  2. 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.

  3. 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.

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

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    Tomoko Hayashi

    Full Text Available The Toll-like receptors (TLR have been advocated as attractive therapeutic targets because TLR signaling plays dual roles in initiating adaptive immune responses and perpetuating inflammation. Paradoxically, repeated stimulation of bone marrow mononuclear cells with a synthetic TLR7 ligand 9-benzyl-8-hydroxy-2-(2-methoxyethoxy adenine (called 1V136 leads to subsequent TLR hyporesponsiveness. Further studies on the mechanism of action of this pharmacologic agent demonstrated that the TLR7 ligand treatment depressed dendritic cell activation, but did not directly affect T cell function. To verify this mechanism, we utilized experimental allergic encephalitis (EAE as an in vivo T cell dependent autoimmune model. Drug treated SJL/J mice immunized with proteolipid protein (PLP(139-151 peptide had attenuated disease severity, reduced accumulation of mononuclear cells in the central nervous system (CNS, and limited demyelination, without any apparent systemic toxicity. Splenic T cells from treated mice produced less cytokines upon antigenic rechallenge. In the spinal cords of 1V136-treated EAE mice, the expression of chemoattractants was also reduced, suggesting innate immune cell hyposensitization in the CNS. Indeed, systemic 1V136 did penetrate the CNS. These experiments indicated that repeated doses of a TLR7 ligand may desensitize dendritic cells in lymphoid organs, leading to diminished T cell responses. This treatment strategy might be a new modality to treat T cell mediated autoimmune diseases.

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

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    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.

  6. 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.

  7. 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.

  8. Molecular modeling-based evaluation of hTLR10 and identification of potential ligands in Toll-like receptor signaling.

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    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.

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

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    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

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

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    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.

  11. Synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands as influenza virus vaccine adjuvants induce rapid, sustained, and broadly protective responses.

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    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

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

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    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.

  13. 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.

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

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    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.

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

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    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.

  16. Expression, purification, and functional characterisation of Flagellin, a TLR5-ligand

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    Irshad Ahmed Hajam

    2013-06-01

    Full Text Available Flagellin, a Toll-like receptor 5 (TLR5-ligand, is known for its activities like adjuvant, induction of pro-inflammatory cytokines and innate immunity. In this context, fliC gene of Salmonella Typhimurium was cloned into pET32a expression plasmid using in-house designed gene specific primers. The frame and orientation of the inserted fliC gene was confirmed upon colony PCR, restriction enzyme analysis and sequencing. Sequence analysis of fliC revealed proper orientation of the gene and had 1,485 nucleotides. Following transformation of pET-fliC plasmid into Escherichia coli BL21 (DE3 cells, the gene was expressed after inducing with IPTG (Isopropylβ-D-1-thiogalactopyranoside. The polyHis-tag-fliC was ~70kDa as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. The identity/authenticity of the recombinant-fliC was confirmed by its specific reactivity with commercial anti-fliC MAb of S. Typhimurium. Further, the antigenic and functional properties of recombinant-fliC were determined espousing its ability to induce antigen specific antibodies in G pigs and increased m-RNA expression of certain pro-inflammatory mediators like TNF-α and GM-CSF in vitro.

  17. TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding

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    Sakaguchi, Masakiyo; Murata, Hitoshi; Yamamoto, Ken-ichi; Ono, Tomoyuki; Sakaguchi, Yoshihiko; Motoyama, Akira; Hibino, Toshihiko; Kataoka, Ken; Huh, Nam-ho

    2011-01-01

    The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions. PMID:21829704

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

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    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.

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

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    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. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

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    Bikash Ranjan Sahoo

    2013-01-01

    Full Text Available Toll-like receptors (TLRs play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN and lipoteichoic acid (LTA of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs of the extracellular domain (ECD and important amino acids in TLR2-TIR (toll/interleukin-1 receptor domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond molecular dynamics simulation (MDS for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD–ligands (PGN, LTA, and zymosan docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR—protein (MyD88-TIR interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling.

  6. 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

  7. Chirality of TLR-2 ligand Pam3CysSK4 in fully synthetic peptide conjugates critically influences the induction of specific CD8+ T-cells.

    Science.gov (United States)

    Khan, Selina; Weterings, Jimmy J; Britten, Cedrik M; de Jong, Ana R; Graafland, Dirk; Melief, Cornelis J M; van der Burg, Sjoerd H; van der Marel, Gijs; Overkleeft, Hermen S; Filippov, Dmitri V; Ossendorp, Ferry

    2009-03-01

    Covalent conjugation of synthetic Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides provides well-defined constructs that have significantly improved capacity to induce efficient priming of CD8(+) T lymphocytes in vivo. We have recently explored the cellular mechanisms underlying the efficient induction of a CD8(+) cytotoxic T lymphocyte response by such synthetic model vaccines [Khan, S., Bijker, M.S., Weterings, J.J., Tanke, H.J., Adema, G.J., van, H.T., Drijfhout, J.W., Melief, C.J., Overkleeft, H.S., van der Marel, G.A., Filippov, D.V., van der Burg, S.H., Ossendorp, F., 2007. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells. J. Biol. Chem. 282, 21145-21159.]. In the current study we have investigated the behaviour of two diastereomers of the TLR-2 ligand Pam(3)CSK(4) (Pam) derivatives, namely the R- and S-epimers at C-2 of the glycerol moiety. Other studies have shown that the Pam(3)Cys based lipopeptides of R-configuration (Pam(R)) in the glycerol moiety enhanced macrophage and B-cell activation compared to those with S-configuration (Pam(S)). Here we report that Pam(R)-conjugates lead to better activation of dendritic cells than the Pam(S)-conjugates as judged by higher IL-12 secretion, upregulation of relevant markers for dendritic cell maturation. In contrast both epimers were internalized equally efficient in a clathrin-dependent manner indicating no qualitative difference in the uptake of the two stereoisomeric Pam-conjugates. We conclude that the enhanced DC activation is due to enhanced TLR-2 triggering by the Pam(R)-conjugate in contrast to the Pam(S)-conjugate. Importantly, induction of specific CD8(+) T-cells was significantly higher in mice injected with the Pam(R)-conjugates compared to mice injected with the Pam(S)-conjugate. In summary we show that the favourable effects of the Pam(R)-configuration of TLR-2 ligand can be attributed to

  8. Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

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    Estefanía R Zacca

    Full Text Available The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

  9. Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

    Science.gov (United States)

    Zacca, Estefanía R; Crespo, María I; Acland, Rachel P; Roselli, Emiliano; Núñez, Nicolás G; Maccioni, Mariana; Maletto, Belkys A; Pistoresi-Palencia, María C; Morón, Gabriel

    2015-01-01

    The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

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

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

  12. 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.

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

    Science.gov (United States)

    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

  14. Ligand Exchange and 1H NMR Quantification of Single- and Mixed-Moiety Thiolated Ligand Shells on Gold Nanoparticles.

    Science.gov (United States)

    Smith, Ashley M; Millstone, Jill E

    2017-01-01

    The use of nanoparticles in biomedicine critically depends on their surface chemistry. For metal nanoparticles, a common way to tune this surface chemistry is through mass action ligand exchange, where ligand exchange can be used to expand the functionality of the resulting nanoparticle conjugates. Specifically, the quantity, identity, and arrangement of the molecules in the resulting ligand shell each can be tuned significantly. Here, we describe methods to exchange and quantify thiolated and non-thiolated ligands on gold nanoparticle surfaces. Importantly, these strategies allow the quantification of multiple ligand types within a single ligand shell, simultaneously providing ligand composition and ligand density information. These results are crucial for both designing and assigning structure-function relationships in bio-functionalized nanoparticles, and these methods can be applied to a broad range of nanoparticle cores and ligand types including peptides, small molecule drugs, and oligonucleotides.

  15. Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

    International Nuclear Information System (INIS)

    Riener, Christian K.; Kienberger, Ferry; Hahn, Christoph D.; Buchinger, Gerhard M.; Egwim, Innocent O.C.; Haselgruebler, Thomas; Ebner, Andreas; Romanin, Christoph; Klampfl, Christian; Lackner, Bernd; Prinz, Heino; Blaas, Dieter; Hinterdorfer, Peter; Gruber, Hermann J.

    2003-01-01

    Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2 -PEG-COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2 -PEG-COOH; (iii) in particular, a new PEG crosslinker with one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether His 6 -tagged protein molecules to AFM tips via noncovalent NTA-Ni 2+ -His 6 bridges. The new crosslinker was applied to link a recombinant His 6 -tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin β1 was studied in detail by SMRFM, using the new crosslinker to link His 6 -tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553-557

  16. MDP-Induced selective tolerance to TLR4 ligands: impairment in NOD2 mutant Crohn's disease patients.

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    Cantó, Elisabet; Moga, Esther; Ricart, Elena; Garcia-Bosch, Orlando; Garcia-Planella, Esther; Juarez, Candido; Vidal, Silvia

    2009-11-01

    Pathogen infection is a complex process in which several pathogen-recognition receptor (PRR) pathways are activated to induce proinflammatory mediators. The activation of multiple PRRs suggests an interaction between Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptor (NOD) signaling pathways. To understand the modulation induced by NOD2 signals on successive responses to pathogen-associated molecular patterns (PAMPs), we examined how muramyl dipeptide (MDP) pretreatment reprograms the MDP+LPS (lipopolysaccharide) response of monocytes from human peripheral blood. Preexposure to bacterial MDP components induced selective tolerance to a subsequent NOD2+TLR4 stimulation. MDP pretreatment inhibited the production of tumor necrosis factor alpha (TNFalpha) and interleuken 10 (IL10), whereas IL6 and IL8 remained unaffected. MDP-induced tolerance was independent of receptor downregulation but was associated with reduced levels of phosphorylated TAK1 and abrogated phosphorylation of the downstream MAPK.Since Nod2 mutations have been associated with susceptibility to develop Crohn's disease (CD), we compared the MDP-induced tolerance in healthy donors and CD patients with compound heterozygous Nod2 mutations (Mut-Nod2) expressing variant NOD2 proteins. MDP-induced tolerance in Mut-Nod2 patients reduced IL10 but not TNFalpha production. In contrast with healthy donors, a p38-independent TNFalpha production was observed during the kinetics of the MDP+LPS response in Mut-Nod2 patients. Our findings suggest that the selective tolerance induced by MDP in healthy donors was related to the modulation of a convergent nub of NOD2 and TLR4 signaling pathways. This MDP-induced tolerance was impaired in Mut-Nod2 CD patients, resulting in a p38-independent TNFalpha production and an imbalance between pro- and antiinflammatory cytokines that could be partly responsible for the pathogenesis of CD.

  17. Delivery of the TLR ligand poly(I:C) to liver cells in vitro and in vivo by calcium phosphate nanoparticles leads to a pronounced immunostimulation.

    Science.gov (United States)

    Sokolova, Viktoriya; Shi, Zou; Huang, Shunmei; Du, Yanqin; Kopp, Mathis; Frede, Annika; Knuschke, Torben; Buer, Jan; Yang, Dongliang; Wu, Jun; Westendorf, Astrid Maria; Epple, Matthias

    2017-12-01

    The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action was discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. A modular system where the immunoactive toll-like-receptor ligand 3 (TLR-3) poly(I:C) was incorporated into calcium phosphate nanoparticles was developed. The nanoparticles had a hydrodynamic diameter of 275nm and a zeta potential of +20mV, measured by dynamic light scattering. The diameter of the solid core was 120nm by scanning electron microscopy. In vitro, the nanoparticle uptake was investigated after 1 and 24h of incubation of THP-1 cells (macrophages) with nanoparticles by fluorescence microscopy. After intravenous injection into BALB/c and C57BL/6J mice, respectively, the in vivo uptake was especially prominent in lung and liver, 1 and 3h after the injection. Pronounced immunostimulatory effects of the nanoparticles were found in vitro with primary liver cells, i.e. Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild-type C57BL/6J mice. Thus, they represent a suitable alternative to hydrodynamic injection treatments for future vaccination concepts. The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action has been discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. We have developed a modular system where poly(I:C) was incorporated into calcium phosphate nanoparticles. The uptake into relevant liver cells was studied both in vitro and in vivo. After intravenous injection into mice, the in vivo uptake was especially prominent in lung

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Immunopotentiation of trivalent influenza vaccine when given with VAX102, a recombinant influenza M2e vaccine fused to the TLR5 ligand flagellin.

    Directory of Open Access Journals (Sweden)

    H Keipp Talbot

    Full Text Available BACKGROUND: Currently controversy exists about the immunogenicity of seasonal trivalent influenza vaccine in certain populations, especially the elderly. STF2.4×M2e (VAX102 is a recombinant fusion protein that links four copies of the ectodomain of influenza virus matrix protein 2 (M2e antigen to Salmonella typhimurium flagellin, a TLR5 ligand. The objectives of this study were to assess the feasibility of giving VAX102 and TIV in combination in an effort to achieve greater immunogenicity and to provide cross-protection. METHODOLOGY/PRINCIPAL FINDINGS: Eighty healthy subjects, 18-49 years old, were enrolled in May and June 2009 in a double-blind, randomized, controlled trial at two clinical sites. Subjects were randomized to receive either TIV + VAX102 or TIV + placebo. Both arms tolerated the vaccines. Pain at the injection site was more severe with TIV + VAX102. Two weeks after immunization the HAI responses to the H1 and H3 antigens of TIV were higher in those that received TIV + VAX102 than in TIV + placebo (309 vs 200 and 269 vs 185, respectively, although statistically non-significant. There was no difference in the HAI of the B antigen. In the TIV + VAX102 arm, the geometric mean M2e antibody concentration was 0.5 µg/ml and 73% seroconverted. CONCLUSIONS/SIGNIFICANCE: The combination of TIV + VAX102 has the potential to increase the immune response to the influenza A components of TIV and to provide M2e immunity which may protect against influenza A strains not contained in seasonal TIV. TRIAL REGISTRATION: ClinicalTrials.gov NCT00921973.

  20. Distinct uptake mechanisms but similar intracellular processing of two different toll-like receptor ligand-peptide conjugates in dendritic cells.

    Science.gov (United States)

    Khan, Selina; Bijker, Martijn S; Weterings, Jimmy J; Tanke, Hans J; Adema, Gosse J; van Hall, Thorbald; Drijfhout, Jan W; Melief, Cornelis J M; Overkleeft, Hermen S; van der Marel, Gijsbert A; Filippov, Dmitri V; van der Burg, Sjoerd H; Ossendorp, Ferry

    2007-07-20

    Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam(3)CysSK(4) (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG approximately conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam approximately conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG approximately conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8(+) T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen

  1. A non-synonymous single-nucleotide polymorphism in the gene encoding Toll-like Receptor 3 (TLR3) is associated with sero-negative Rheumatoid Arthritis (RA) in a Danish population

    DEFF Research Database (Denmark)

    Laska, Magdalena Janina; Hansen, Bettina; Troldborg, Anne

    2014-01-01

    in a predominantly Caucasian population from Denmark using a case-control approach. FINDINGS: DNA samples (3 university hospital outpatient clinics) were obtained from patients with RA (n = 704) and healthy controls (n = 639) in a Danish population. TLR single nucleotide polymorphisms (SNPs) were selected based...... in the TLR3 locus may be implicated in the pathogenesis of sero-negative RA. Since this TLR3 SNP has previously been associated with systemic lupus erythematous (SLE), the present findings support the notion that TLR3 genetic variants may represent a common risk factor in different chronic inflammatory......BACKGROUND: It has been suggested that polymorphisms in Toll-like Receptors (TLRs) are associated with Rheumatoid Arthritis (RA), but the implicated alleles have differed between studies. The aim of this investigation was to explore whether polymorphisms of TLR genes are associated with RA...

  2. Effect of ligand substitution on the exchange interactions in {Mn12}-type single-molecule magnets

    OpenAIRE

    Boukhvalov, D. W.; Dobrovitski, V. V.; Kögerler, P.; Al-Saqer, M.; Katsnelson, M. I.; Lichtenstein, A. I.; Harmon, B. N.

    2010-01-01

    We investigate how the ligand substitution affects the intra-molecular spin exchange interactions, studying a prototypal family of single-molecule magnets comprising dodecanuclear cluster molecules [Mn12O12(COOR)16]. We identify a simple scheme based on accumulated Pauling electronegativity numbers (a.e.n.) of the carboxylate ligand groups (R). The redistribution of the electron density, controlled by a.e.n. of a ligand, changes the degree of hybridization between 3d electrons of manganese an...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. 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

  6. 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

  7. Development of radioiodinated receptor ligands for cerebral single photon emission tomography

    International Nuclear Information System (INIS)

    Knapp, F.F. Jr.; McPherson, D.W.

    1992-01-01

    In the last decade the use of radiolabeled ligands for the imaging of cerebral receptors by emission computed tomography (ECT) has seen rapid growth. The opportunity to routinely perform cerebral single photon emission tomography (SPET) with iodine-123-labeled ligands depends on the availability of receptor ligands into which iodine can be introduced without decreasing the required high target receptor specificity. The use of iodine-123-labeled receptor-specific ligands also depends on the availability of high purity iodine-123 at reasonable costs and the necessary imaging instrumentation. In this paper, the development and current stage of evaluation of various iodine-123-labeled ligands for SPET imaging of dopaminergic, serotonergic and muscarinic acetylcholinergic receptor classes are discussed

  8. Ligand-tailored single-site silica supported titanium catalysts: Synthesis, characterization and towards cyanosilylation reaction

    International Nuclear Information System (INIS)

    Xu, Wei; Li, Yani; Yu, Bo; Yang, Jindou; Zhang, Ying; Chen, Xi; Zhang, Guofang; Gao, Ziwei

    2015-01-01

    A successive anchoring of Ti(NMe 2 ) 4 , cyclopentadiene and a O-donor ligand, 1-hydroxyethylbenzene (PEA), 1,1′-bi-2-naphthol (Binol) or 2,3-dihydroxybutanedioic acid diethyl ester (Tartrate), on silica was conducted by SOMC strategy in moderate conditions. The silica, monitored by in-situ Fourier transform infrared spectroscopy (in-situ FT-IR), was pretreated at different temperatures (200, 500 and 800 °C). The ligand tailored silica-supported titanium complexes were characterized by in-situ FT-IR, 13 C CP MAS-NMR, X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES) and elemental analysis in detail, verifying that the surface titanium species are single sited. The catalytic activity of the ligand tailored single-site silica supported titanium complexes was evaluated by a cyanosilylation of benzaldehyde. The results showed that the catalytic activity is dependent strongly on the dehydroxylation temperatures of silica and the configuration of the ligands. - Graphical abstract: The ligand-tailored silica supported “single site” titanium complexes were synthesized by SOMC strategy and fully characterized. Their catalytic activity were evaluated by benzaldehyde silylcyanation. - Highlights: • Single-site silica supported Ti active species was prepared by SOMC technique. • O-donor ligand tailored Ti surface species was synthesized. • The surface species was characterized by XPS, 13 C CP-MAS NMR, XANES etc. • Catalytic activity of the Ti active species in silylcyanation reaction was evaluated

  9. Modifying the properties of 4f single-ion magnets by peripheral ligand functionalisation

    DEFF Research Database (Denmark)

    Pedersen, Kasper Steen; Ungur, Liviu; Sigrist, Marc

    2014-01-01

    We study the ligand-field splittings and magnetic properties of three ErIII single-ion magnets which differ in the peripheral ligand sphere but exhibit similar first coordination spheres by inelastic neutron scattering (INS) and SQUID magnetometry. The INS spectra of the three compounds are profo......We study the ligand-field splittings and magnetic properties of three ErIII single-ion magnets which differ in the peripheral ligand sphere but exhibit similar first coordination spheres by inelastic neutron scattering (INS) and SQUID magnetometry. The INS spectra of the three compounds...... allows for the extraction of the sign and magnitude of all symmetry-allowed Stevens parameters. The parameter values and the energy spectrum derived from INS are compared to the results of state-of-the-art ab initio CASSCF calculations. Temperature-dependent alternating current (ac) susceptibility...... measurements suggest that the magnetisation relaxation in the investigated temperature range of 1.9 K

  10. Identification of small heat shock protein B8 (HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis.

    NARCIS (Netherlands)

    Roelofs, M.F.; Boelens, W.C.; Joosten, L.A.B.; Abdollahi-Roodsaz, S.; Geurts, J.; Wunderink, L.U.; Schreurs, B.W.; Berg, W.B. van den; Radstake, T.R.D.J.

    2006-01-01

    Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in

  11. 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...

  12. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  13. Human beta-defensin-2 and -3 enhance pro-inflammatory cytokine expression induced by TLR ligands via ATP-release in a P2X7R dependent manner.

    Science.gov (United States)

    Wanke, Daniela; Mauch-Mücke, Katrin; Holler, Ernst; Hehlgans, Thomas

    2016-11-01

    Our previous results indicate that HBD2 and HBD3 are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner. In this study we report that pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3 results in a synergistic, enhanced expression of pro-inflammatory cytokines and chemokines induced by TLR ligand re-stimulation. Experiments using specific inhibitors of the ATP-gated channel receptor P2X7 or its functional ligand ATP, suggest that the enhanced expression of pro-inflammatory cytokines and chemokines seems to be mediated by P2X7R. Furthermore, our data provide evidence that beta-defensins do not directly interact with P2X7R but rather induce the release of intracellular ATP. Interference with ATP release abrogated the synergistic effect mediated by HBD2 and HBD3 pre-stimulation in THP-1 cells. However, extracellular ATP alone seems not to be sufficient to elicit the enhanced synergistic effect on cytokine and chemokine expression observed by pre-stimulation of primary human macrophages or THP-1 cells with HBD2 or HBD3. Collectively, our findings provide new insights into the molecular mechanisms how HBD2 and HBD3 interact with cells of myeloid origin and demonstrate their immuno-modulating functions during innate immune responses. Copyright © 2016 Elsevier GmbH. All rights reserved.

  14. Ligand-tailored single-site silica supported titanium catalysts: Synthesis, characterization and towards cyanosilylation reaction

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Wei; Li, Yani; Yu, Bo; Yang, Jindou; Zhang, Ying; Chen, Xi; Zhang, Guofang, E-mail: gfzhang@snnu.edu.cn; Gao, Ziwei, E-mail: zwgao@snnu.edu.cn

    2015-01-15

    A successive anchoring of Ti(NMe{sub 2}){sub 4}, cyclopentadiene and a O-donor ligand, 1-hydroxyethylbenzene (PEA), 1,1′-bi-2-naphthol (Binol) or 2,3-dihydroxybutanedioic acid diethyl ester (Tartrate), on silica was conducted by SOMC strategy in moderate conditions. The silica, monitored by in-situ Fourier transform infrared spectroscopy (in-situ FT-IR), was pretreated at different temperatures (200, 500 and 800 °C). The ligand tailored silica-supported titanium complexes were characterized by in-situ FT-IR, {sup 13}C CP MAS-NMR, X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES) and elemental analysis in detail, verifying that the surface titanium species are single sited. The catalytic activity of the ligand tailored single-site silica supported titanium complexes was evaluated by a cyanosilylation of benzaldehyde. The results showed that the catalytic activity is dependent strongly on the dehydroxylation temperatures of silica and the configuration of the ligands. - Graphical abstract: The ligand-tailored silica supported “single site” titanium complexes were synthesized by SOMC strategy and fully characterized. Their catalytic activity were evaluated by benzaldehyde silylcyanation. - Highlights: • Single-site silica supported Ti active species was prepared by SOMC technique. • O-donor ligand tailored Ti surface species was synthesized. • The surface species was characterized by XPS, {sup 13}C CP-MAS NMR, XANES etc. • Catalytic activity of the Ti active species in silylcyanation reaction was evaluated.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Ligand-tailored single-site silica supported titanium catalysts: Synthesis, characterization and towards cyanosilylation reaction

    Science.gov (United States)

    Xu, Wei; Li, Yani; Yu, Bo; Yang, Jindou; Zhang, Ying; Chen, Xi; Zhang, Guofang; Gao, Ziwei

    2015-01-01

    A successive anchoring of Ti(NMe2)4, cyclopentadiene and a O-donor ligand, 1-hydroxyethylbenzene (PEA), 1,1‧-bi-2-naphthol (Binol) or 2,3-dihydroxybutanedioic acid diethyl ester (Tartrate), on silica was conducted by SOMC strategy in moderate conditions. The silica, monitored by in-situ Fourier transform infrared spectroscopy (in-situ FT-IR), was pretreated at different temperatures (200, 500 and 800 °C). The ligand tailored silica-supported titanium complexes were characterized by in-situ FT-IR, 13C CP MAS-NMR, X-ray photoelectron spectroscopy (XPS), X-ray absorption near edge structure (XANES) and elemental analysis in detail, verifying that the surface titanium species are single sited. The catalytic activity of the ligand tailored single-site silica supported titanium complexes was evaluated by a cyanosilylation of benzaldehyde. The results showed that the catalytic activity is dependent strongly on the dehydroxylation temperatures of silica and the configuration of the ligands.

  20. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. TLR9 ligand (CpG oligodeoxynucleotide induces CLL B-cells to differentiate into CD20+ antibody-secreting cells

    Directory of Open Access Journals (Sweden)

    Hussein eGhamlouch

    2014-06-01

    Full Text Available B-cell chronic lymphocytic leukemia (CLL is the most frequent adult leukemia in the Western world. It is a heterogeneous disease characterized by clonal proliferation and the accumulation of CD5+ mature B lymphocytes. However, the normal counterpart from which the latter cells arise has not yet been identified. CD27 expression and gene expression profiling data suggest that CLL cells are related to memory B-cells. In vitro, memory B-cells differentiate into plasma cells when stimulated with CpG oligodeoxynucleotide (CpG. The objective of the present study was therefore to investigate the ability of CpG, in the context of CD40 ligation, to induce the differentiation of CLL B-cells into antibody-secreting cells (ASCs. CD20+CD38− CLL B-cells were stimulated with a combination of CpG, CD40 ligand and cytokines (CpG/CD40L/c in a two-step, seven-day culture system. We found that the CpG/CD40L/c culture system prompted CLL B-cells to differentiate into CD19+CD20+CD27+CD38- ASCs. These cells secreted large amounts of IgM and had the same shape as plasma cells. However, only IgMs secreted by ASCs that had differentiated from unmutated CLL B-cells were poly/autoreactive. Class-switch recombination to IgG and IgA was detected in cells expressing the activation-induced cytidine deaminase gene (AICDA. Although these ASCs expressed high levels of the transcription factors PRDM1 (BLIMP1, IRF4 and XBP1s, they did not downregulate expression of PAX5. Our results suggest that CLL B-cells can differentiate into ASCs, undergo class-switch recombination and produce poly/autoreactive antibodies. Furthermore, our findings may be relevant for (i identifying the normal counterpart of CLL B-cells and (ii developing novel treatment strategies in CLL.

  5. A single glycine-alanine exchange directs ligand specificity of the elephant progestin receptor.

    Directory of Open Access Journals (Sweden)

    Michael Wierer

    Full Text Available The primary gestagen of elephants is 5α-dihydroprogesterone (DHP, which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR. Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD, we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.

  6. 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.

  7. Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution.

    Science.gov (United States)

    Haller, Andrea; Altman, Roger B; Soulière, Marie F; Blanchard, Scott C; Micura, Ronald

    2013-03-12

    Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5' and 3' ends of the aptamer domain, adopts a predominantly folded structure in the presence of Mg(2+) alone. However, even at saturating concentrations of Mg(2+) and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/P5 forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation.

  8. Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments.

    Science.gov (United States)

    Liu, Shih-Wei; Chu, Jen-Fei; Tsai, Cheng-Ting; Fang, Hung-Chih; Chang, Ta-Chau; Li, Hung-Wen

    2013-05-15

    G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  9. Cell type-specific regulatory effects of glucocorticoids on cutaneous TLR2 expression and signalling.

    Science.gov (United States)

    Su, Qi; Pfalzgraff, Anja; Weindl, Günther

    2017-07-01

    Glucocorticoids (GCs) induce Toll-like receptor (TLR) 2 expression and synergistically upregulate TLR2 with pro-inflammatory cytokines or bacteria. These paradoxical effects have drawn attention to the inflammatory initiating or promoting effects of GCs, as GC treatment can provoke inflammatory skin diseases. Here, we aimed to investigate the regulatory effects of GCs in human skin cells of different epidermal and dermal layers. We found that Dex induced TLR2 expression mainly in undifferentiated and less in calcium-induced differentiated keratinocytes but not in HaCaT cells or fibroblasts, however, Dex reduced TLR1/6 expression. Stimulation with Dex under inflammatory conditions further increased TLR2 but not TLR1 or TLR6 levels in keratinocytes. Increased ligand-induced interaction of TLR2 with MyD88 and expression of the adaptor protein TRAF6 indicated enhanced TLR2 signalling, whereas TLR2/1 or TLR2/6 signalling was not increased in Dex-pretreated keratinocytes. GC-increased TLR2 expression was negatively regulated by JNK MAPK signalling when stimulated with Propionibacterium acnes. Our results provide novel insights into the molecular mechanisms of glucocorticoid-mediated expression and function of TLR2 in human skin cells and the understanding of the mechanisms of corticosteroid side effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. 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

  11. A single-ligand ultra-microporous MOF for precombustion CO2 capture and hydrogen purification.

    Science.gov (United States)

    Nandi, Shyamapada; De Luna, Phil; Daff, Thomas D; Rother, Jens; Liu, Ming; Buchanan, William; Hawari, Ayman I; Woo, Tom K; Vaidhyanathan, Ramanathan

    2015-12-01

    Metal organic frameworks (MOFs) built from a single small ligand typically have high stability, are rigid, and have syntheses that are often simple and easily scalable. However, they are normally ultra-microporous and do not have large surface areas amenable to gas separation applications. We report an ultra-microporous (3.5 and 4.8 Å pores) Ni-(4-pyridylcarboxylate)2 with a cubic framework that exhibits exceptionally high CO2/H2 selectivities (285 for 20:80 and 230 for 40:60 mixtures at 10 bar, 40°C) and working capacities (3.95 mmol/g), making it suitable for hydrogen purification under typical precombustion CO2 capture conditions (1- to 10-bar pressure swing). It exhibits facile CO2 adsorption-desorption cycling and has CO2 self-diffusivities of ~3 × 10(-9) m(2)/s, which is two orders higher than that of zeolite 13X and comparable to other top-performing MOFs for this application. Simulations reveal a high density of binding sites that allow for favorable CO2-CO2 interactions and large cooperative binding energies. Ultra-micropores generated by a small ligand ensures hydrolytic, hydrostatic stabilities, shelf life, and stability toward humid gas streams.

  12. A single-ligand ultra-microporous MOF for precombustion CO2 capture and hydrogen purification

    Science.gov (United States)

    Nandi, Shyamapada; De Luna, Phil; Daff, Thomas D.; Rother, Jens; Liu, Ming; Buchanan, William; Hawari, Ayman I.; Woo, Tom K.; Vaidhyanathan, Ramanathan

    2015-01-01

    Metal organic frameworks (MOFs) built from a single small ligand typically have high stability, are rigid, and have syntheses that are often simple and easily scalable. However, they are normally ultra-microporous and do not have large surface areas amenable to gas separation applications. We report an ultra-microporous (3.5 and 4.8 Å pores) Ni-(4-pyridylcarboxylate)2 with a cubic framework that exhibits exceptionally high CO2/H2 selectivities (285 for 20:80 and 230 for 40:60 mixtures at 10 bar, 40°C) and working capacities (3.95 mmol/g), making it suitable for hydrogen purification under typical precombustion CO2 capture conditions (1- to 10-bar pressure swing). It exhibits facile CO2 adsorption-desorption cycling and has CO2 self-diffusivities of ~3 × 10−9 m2/s, which is two orders higher than that of zeolite 13X and comparable to other top-performing MOFs for this application. Simulations reveal a high density of binding sites that allow for favorable CO2-CO2 interactions and large cooperative binding energies. Ultra-micropores generated by a small ligand ensures hydrolytic, hydrostatic stabilities, shelf life, and stability toward humid gas streams. PMID:26824055

  13. Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery.

    Science.gov (United States)

    Honegr, Jan; Dolezal, Rafael; Malinak, David; Benkova, Marketa; Soukup, Ondrej; Almeida, Joyce S F D de; Franca, Tanos C C; Kuca, Kamil; Prymula, Roman

    2018-01-04

    In order to identify novel lead structures for human toll-like receptor 4 ( h TLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the h TLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate h TLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

  14. A mononuclear uranium(IV) single-molecule magnet with an azobenzene radical ligand

    Energy Technology Data Exchange (ETDEWEB)

    Antunes, Maria A.; Coutinho, Joana T.; Santos, Isabel C.; Marcalo, Joaquim; Almeida, Manuel; Pereira, Laura C.J. [C" 2TN, Instituto Superior Tecnico, Universidade de Lisboa, Bobadela (Portugal); Baldovi, Jose J.; Gaita-Arino, Alejandro; Coronado, Eugenio [Instituto de Ciencia Molecular, Universitat de Valencia, Paterna (Spain)

    2015-12-01

    A tetravalent uranium compound with a radical azobenzene ligand, namely, [{(SiMe_2NPh)_3-tacn}U{sup IV}(η{sup 2}-N{sub 2}Ph{sub 2{sup .}})] (2), was obtained by one-electron reduction of azobenzene by the trivalent uranium compound [U{sup III}{(SiMe_2NPh)_3-tacn}] (1). Compound 2 was characterized by single-crystal X-ray diffraction and {sup 1}H NMR, IR, and UV/Vis/NIR spectroscopy. The magnetic properties of 2 and precursor 1 were studied by static magnetization and ac susceptibility measurements, which for the former revealed single-molecule magnet behaviour for the first time in a mononuclear U{sup IV} compound, whereas trivalent uranium compound 1 does not exhibit slow relaxation of the magnetization at low temperatures. A first approximation to the magnetic behaviour of these compounds was attempted by combining an effective electrostatic model with a phenomenological approach using the full single-ion Hamiltonian. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  15. Small-molecule inhibition of TLR8 through stabilization of its resting state.

    Science.gov (United States)

    Zhang, Shuting; Hu, Zhenyi; Tanji, Hiromi; Jiang, Shuangshuang; Das, Nabanita; Li, Jing; Sakaniwa, Kentaro; Jin, Jin; Bian, Yanyan; Ohto, Umeharu; Shimizu, Toshiyuki; Yin, Hang

    2018-01-01

    Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. In this manuscript, we identified the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. As a proof of concept of their therapeutic potential, we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    Lindenmaier Werner

    2010-04-01

    as TNFα translation and release by macrophages were largely abrogated upon transduction of αT2ib. αT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdVαT2ib. Systemic transduction applying AdVαT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. Conclusion The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of αT2ib in microbial or viral infections.

  18. 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

    well as TNFalpha translation and release by macrophages were largely abrogated upon transduction of alphaT2ib. alphaT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdValphaT2ib. Systemic transduction applying AdValphaT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of alphaT2ib in microbial or viral infections.

  19. Compact halo-ligand-conjugated quantum dots for multicolored single-molecule imaging of overcrowding GPCR proteins on cell membranes.

    Science.gov (United States)

    Komatsuzaki, Akihito; Ohyanagi, Tatsuya; Tsukasaki, Yoshikazu; Miyanaga, Yukihiro; Ueda, Masahiro; Jin, Takashi

    2015-03-25

    To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    profile of chemokines. In conclusion, it was demonstrated that TLR4 is constitutively expressed by podocytes and is upregulated in MPGN, where it may mediate glomerular injury by modulating expression of chemokines; therefore, TLR4 may link podocytes with the innate immune system to mediate MPGN triggered......Toll-like receptors (TLR) classically recognize pathogen-associated danger signals but are also activated via endogenous ligands. For evaluation of their role in inflammatory kidney disease, the function of TLR was analyzed in two mouse models of cryoglobulinemic membranoproliferative...... by the deposition of immune complexes....

  1. TLR accessory molecule RP105 (CD180 is involved in post-interventional vascular remodeling and soluble RP105 modulates neointima formation.

    Directory of Open Access Journals (Sweden)

    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. Luminescent single-ion magnets from Lanthanoid(III) complexes with monodentate ketone ligands

    Energy Technology Data Exchange (ETDEWEB)

    Kanetomo, Takuya; Ishida, Takayuki, E-mail: takayuki.ishida@uec.ac.jp [Department of Engineering Science, The University of Electro-Communications, Tokyo (Japan)

    2016-02-01

    We synthesized [Ln{sup III}(hfac){sub 3}(H{sub 2}O)(L)] (abbreviated as Ln-L; Ln = Gd, Tb, Eu; L = DTBK (di-t-butyl ketone), BP (benzophenone)), in which the carbonyl oxygen atom was coordinated to the Ln ion center, despite of such bulky substituents. Their crystal structures were determined by means of X-ray diffraction study. Gd-DTBK is completely isomorphous to the di-t-butyl nitroxide derivative and accordingly can be regarded as a model with the ligand spin masked. The ac magnetic susceptibility measurements on Tb-DTBK and -BP showed frequency dependence, characteristic of single-ion magnets. They also displayed photoluminescence in the solid state at room temperature. The quantum yields of the luminescence of Tb-DTBK and -BP (λ{sub ex} = 360 nm) were improved to 57 and 35%, respectively, from that of the starting material [TbI{sup III}(hfac){sub 3}(H{sub 2}O){sub 2}] (28% at λ{sub ex} = 370 nm). Similarly, the quantum yields for Eu-DTBK and -BP were 8 and 15%, respectively, with λ{sub ex} = 400 nm, while that of the starting material [EuI{sup III}(hfac){sub 3}(H{sub 2}O){sub 2}] was 4% at λ{sub ex}=400 nm.

  3. Luminescent single-ion magnets from Lanthanoid(III) complexes with monodentate ketone ligands

    Science.gov (United States)

    Kanetomo, Takuya; Ishida, Takayuki

    2016-02-01

    We synthesized [LnIII(hfac)3(H2O)(L)] (abbreviated as Ln-L; Ln = Gd, Tb, Eu; L = DTBK (di-t-butyl ketone), BP (benzophenone)), in which the carbonyl oxygen atom was coordinated to the Ln ion center, despite of such bulky substituents. Their crystal structures were determined by means of X-ray diffraction study. Gd-DTBK is completely isomorphous to the di-t-butyl nitroxide derivative and accordingly can be regarded as a model with the ligand spin masked. The ac magnetic susceptibility measurements on Tb-DTBK and -BP showed frequency dependence, characteristic of single-ion magnets. They also displayed photoluminescence in the solid state at room temperature. The quantum yields of the luminescence of Tb-DTBK and -BP (λex = 360 nm) were improved to 57 and 35%, respectively, from that of the starting material [TbIIII(hfac)3(H2O)2] (28% at λex = 370 nm). Similarly, the quantum yields for Eu-DTBK and -BP were 8 and 15%, respectively, with λex = 400 nm, while that of the starting material [EuIIII(hfac)3(H2O)2] was 4% at λex=400 nm.

  4. TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

    Science.gov (United States)

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

    2011-03-01

    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.

  5. Application of X-ray single crystal diffractometry to investigation of Np(5) complexes with n-donor ligands

    International Nuclear Information System (INIS)

    Andreev, G.

    2007-01-01

    Full text of publication follows. We present here some results of application of conventional X-ray single crystal diffractometry to the research on the interaction of Np(V) with N-donor ligands. Compounds that can coordinate to actinides through one or several nitrogen atoms are of a great variety and occur widely in the biosphere. For example, imidazole, pyridine and their derivatives are the building blocks of many biologically important molecules; triazines are known to occur in some aquatic plants. The presence of anthropogenic organic agents like amine-N-carboxylic acids in surface waters has the potential to re-mobilize metals from sediments and aquifers and to influence their bioavailability. The interaction of radionuclides with such ligands needs to be studied in detail to give fundamental understanding the conditions of the incorporation of long lived a-emitters (Np and Pu primarily) into the food chain. Another aspect of the same problem is the design of new chelating ligands for selective co-ordination of actinide ions as an alternative to the traditional sequestering agents. The problem of the separation of long-lived minor actinides and their transmutation also calls for design of new highly selective ligands for solvent extraction. Polydentate N-donor ligands are now considered to be very promising. A detailed study of structural chemistry is crucial for understanding the relationship between the architecture of the ligands and their binding affinity for actinides. The X-ray single crystal diffractometry became conventional technique as applied to the investigation of actinides in spite of difficulties regarding safe handling of radionuclides. This technique provides unambiguous information about modes of the ligand co-ordination to the metal ion and geometrical parameters of complexes. Moreover, the employment of a synchrotron radiation shows considerable promise for determination of solid state structures as well as obtaining structural

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Science.gov (United States)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  8. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Metal-ligand cooperation by aromatization-dearomatization as a tool in single bond activation.

    Science.gov (United States)

    Milstein, David

    2015-03-13

    Metal-ligand cooperation (MLC) plays an important role in bond activation processes, enabling many chemical and biological catalytic reactions. A recent new mode of activation of chemical bonds involves ligand aromatization-dearomatization processes in pyridine-based pincer complexes in which chemical bonds are broken reversibly across the metal centre and the pincer-ligand arm, leading to new bond-making and -breaking processes, and new catalysis. In this short review, such processes are briefly exemplified in the activation of C-H, H-H, O-H, N-H and B-H bonds, and mechanistic insight is provided. This new bond activation mode has led to the development of various catalytic reactions, mainly based on alcohols and amines, and to a stepwise approach to thermal H2 and light-induced O2 liberation from water. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  11. 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

  12. 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

  13. 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.

  14. Colloidal Quantum Dot Inks for Single-Step-Fabricated Field-Effect Transistors: The Importance of Postdeposition Ligand Removal.

    Science.gov (United States)

    Balazs, Daniel M; Rizkia, Nisrina; Fang, Hong-Hua; Dirin, Dmitry N; Momand, Jamo; Kooi, Bart J; Kovalenko, Maksym V; Loi, Maria Antonietta

    2018-02-14

    Colloidal quantum dots are a class of solution-processed semiconductors with good prospects for photovoltaic and optoelectronic applications. Removal of the surfactant, so-called ligand exchange, is a crucial step in making the solid films conductive, but performing it in solid state introduces surface defects and cracks in the films. Hence, the formation of thick, device-grade films have only been possible through layer-by-layer processing, limiting the technological interest for quantum dot solids. Solution-phase ligand exchange before the deposition allows for the direct deposition of thick, homogeneous films suitable for device applications. In this work, fabrication of field-effect transistors in a single step is reported using blade-coating, an upscalable, industrially relevant technique. Most importantly, a postdeposition washing step results in device properties comparable to the best layer-by-layer processed devices, opening the way for large-scale fabrication and further interest from the research community.

  15. Single-molecule analysis of ligand efficacy in beta(2)AR-G-protein activation

    DEFF Research Database (Denmark)

    Gregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel

    2017-01-01

    G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail ...

  16. Reversible Single-Crystal-to-Single-Crystal Structural Transformation in a Mixed-Ligand 2D Layered Metal-Organic Framework: Structural Characterization and Sorption Study

    Directory of Open Access Journals (Sweden)

    Chih-Chieh Wang

    2017-12-01

    Full Text Available A 3D supramolecular network, [Cd(bipy(C4O4(H2O2]·3H2O (1 (bipy = 4,4′-bipyridine and C4O42− = dianion of H2C4O4, constructed by mixed-ligand two-dimensional (2D metal-organic frameworks (MOFs has been reported and structurally determined by the single-crystal X-ray diffraction method and characterized by other physicochemical methods. In 1, the C4O42− and bipy both act as bridging ligands connecting the Cd(II ions to form a 2D layered MOF, which are then extended to a 3D supramolecular network via the mutually parallel and interpenetrating arrangements among the 2D-layered MOFs. Compound 1 shows a two-step dehydration process with weight losses of 11.0% and 7.3%, corresponding to the weight-loss of three guest and two coordinated water molecules, respectively, and exhibits an interesting reversible single-crystal-to-single-crystal (SCSC structural transformation upon de-hydration and re-hydration for guest water molecules. The SCSC structural transformation have been demonstrated and monitored by single-crystal and X-ray powder diffraction, and thermogravimetic analysis studies.

  17. Probing force-induced unfolding intermediates of a single staphylococcal nuclease molecule and the effect of ligand binding

    International Nuclear Information System (INIS)

    Ishii, Takaaki; Murayama, Yoshihiro; Katano, Atsuto; Maki, Kosuke; Kuwajima, Kunihiro; Sano, Masaki

    2008-01-01

    Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics

  18. M tuberculosis in the adjuvant modulates time of appearance of CNS-specific effector T cells in the spleen through a polymorphic site of TLR2.

    Directory of Open Access Journals (Sweden)

    Chiara Nicolò

    Full Text Available DC deliver information regulating trafficking of effector T cells along T-cell priming. However, the role of pathogen-derived motives in the regulation of movement of T cells has not been studied. We hereinafter report that amount of M tuberculosis in the adjuvant modulates relocation of PLP139-151 specific T cells. In the presence of a low dose of M tuberculosis in the adjuvant, T cells (detected by CDR3 BV-BJ spectratyping, the so-called "immunoscope" mostly reach the spleen by day 28 after immunization ("late relocation" in the SJL strain, whereas T cells reach the spleen by d 14 with a high dose of M tuberculosis ("early relocation". The C57Bl/6 background confers a dominant "early relocation" phenotype to F1 (SJL×C57Bl/6 mice, allowing early relocation of T cells in the presence of low dose M tuberculosis. A single non-synonymous polymorphism of TLR2 is responsible for "early/late" relocation phenotype. Egress of T lymphocytes is regulated by TLR2 expressed on T cells. Thus, pathogens engaging TLR2 on T cells regulate directly T-cell trafficking, and polymorphisms of TLR2 condition T-cell trafficking upon a limiting concentration of ligand.

  19. Low TLR7 gene expression in atherosclerotic plaques is associated with major adverse cardio- and cerebrovascular events

    DEFF Research Database (Denmark)

    Karadimou, Glykeria; Folkersen, Lasse; Berg, Martin

    2016-01-01

    cells, macrophages and endothelial cells in capillaries, as shown by immunohistochemistry. In short-term tissue cultures, ex vivo treatment of plaques with the TLR7 ligand imiquimod elicited dose-dependent secretion of IL-10, TNF-α, GM-CSF, and IL-12/IL-23p40. This secretion was blocked with a TLR7...... inhibitor. Immunofluorescent tissue analysis after TLR7 stimulation showed IL-10 expression in T cells, macrophages and vascular smooth muscle cells. TLR7 mRNA levels in the plaques were correlated with IL-10 receptor (r=0.4031, p

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

    Directory of Open Access Journals (Sweden)

    Katherine L Irvine

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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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

  3. 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.

  4. Ligand-based transport resonances of single-molecule magnet spin filters: Suppression of the Coulomb blockade and determination of the orientation of the magnetic easy axis

    OpenAIRE

    Renani, Fatemeh Rostamzadeh; Kirczenow, George

    2011-01-01

    We investigate single molecule magnet transistors (SMMTs) with ligands that support transport resonances. We find the lowest unoccupied molecular orbitals of Mn12-benzoate SMMs (with and without thiol or methyl-sulfide termination) to be on ligands, the highest occupied molecular orbitals being on the Mn12 magnetic core. We predict gate controlled switching between Coulomb blockade and coherent resonant tunneling in SMMTs based on such SMMs, strong spin filtering by the SMM in both transport ...

  5. Identification of single nucleotide polymorphisms in the bovine Toll-like receptor 1 gene and association with health traits in cattle

    Directory of Open Access Journals (Sweden)

    Russell Christopher D

    2012-03-01

    Full Text Available Abstract Bovine mastitis remains the most common and costly disease of dairy cattle worldwide. A complementary control measure to herd hygiene and vaccine development would be to selectively breed cattle with greater resistance to mammary infection. Toll-like receptor 1 (TLR1 has an integral role for the initiation and regulation of the immune response to microbial pathogens, and has been linked to numerous inflammatory diseases. The objective of this study was to investigate whether single nucleotide polymorphisms (SNPs within the bovine TLR1 gene (boTLR1 are associated with clinical mastitis (CM. Selected boTLR1 SNPs were analysed within a Holstein Friesian herd. Significant associations were found for the tagging SNP -79 T > G and the 3'UTR SNP +2463 C > T. We observed favourable linkage of reduced CM with increased milk fat and protein, indicating selection for these markers would not be detrimental to milk quality. Furthermore, we present evidence that some of these boTLR1 SNPs underpin functional variation in bovine TLR1. Animals with the GG genotype (from the tag SNP -79 T > G had significantly lower boTLR1 expression in milk somatic cells when compared with TT or TG animals. In addition, stimulation of leucocytes from GG animals with the TLR1-ligand Pam3csk4 resulted in significantly lower levels of CXCL8 mRNA and protein. SNPs in boTLR1 were significantly associated with CM. In addition we have identified a bovine population with impaired boTLR1 expression and function. This may have additional implications for animal health and warrants further investigation to determine the suitability of identified SNPs as markers for disease susceptibility.

  6. 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.

  7. GABA(A)-benzodiazepine receptor complex ligands and stress-induced hyperthermia in singly housed mice.

    NARCIS (Netherlands)

    Olivier, B.; Bouwknecht, J.A.; Pattij, T.; Leahy, C.; Oorschot, R. van; Zethof, T.J.

    2002-01-01

    Stress-induced hyperthermia (SIH) in singly housed mice, in which the rectal temperature of a mouse is measured twice with a 10-min interval, enables to study the effects of a drug on the basal (T(1)) and on the stress-enhanced temperature (T(2)), 10 min later, using the rectal procedure as

  8. Engagement of Platelet Toll-Like Receptor 9 by Novel Endogenous Ligands Promotes Platelet Hyper-Reactivity and Thrombosis

    Science.gov (United States)

    Panigrahi, Soumya; Ma, Yi; Hong, Li; Gao, Detao; West, Xiaoxia Z.; Salomon, Robert G.; Byzova, Tatiana V.; Podrez, Eugene A.

    2012-01-01

    Rationale A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance of altered-self ligands in circulation that can be recognized by Toll-like receptors (TLR). Platelets express a number of TLR, including TLR9, however, the role of TLR in platelet function and thrombosis is poorly understood. Objective To investigate the biological activities of carboxy(alkylpyrrole) protein adducts (CAPs), an altered self-ligand generated in oxidative stress, on platelet function and thrombosis. Methods and Results In this study we show that CAPs represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that CAPs promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and is Src kinase dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. Conclusions Our study demonstrates that platelet TLR9 is a functional platelet receptor that links oxidative stress, innate immunity, and thrombosis. PMID:23071157

  9. Single-molecule imaging of platinum ligand exchange reaction reveals reactivity distribution.

    Science.gov (United States)

    Esfandiari, N Melody; Wang, Yong; Bass, Jonathan Y; Cornell, Trevor P; Otte, Douglas A L; Cheng, Ming H; Hemminger, John C; McIntire, Theresa M; Mandelshtam, Vladimir A; Blum, Suzanne A

    2010-11-03

    Single-molecule fluorescence microscopy provided information about the real-time distribution of chemical reactivity on silicon oxide supports at the solution-surface interface, at a level of detail which would be unavailable from a traditional ensemble technique or from a technique that imaged the static physical properties of the surface. Chemical reactions on the surface were found to be uncorrelated; that is, the chemical reaction of one metal complex did not influence the location of a future chemical reaction of another metal complex.

  10. 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...

  11. 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.

  12. Transforming single domain magnetic CoFe2O4 nanoparticles from hydrophobic to hydrophilic by novel mechanochemical ligand exchange

    International Nuclear Information System (INIS)

    Munjal, Sandeep; Khare, Neeraj

    2017-01-01

    Single-phase uniform-sized (~9 nm) cobalt ferrite (CFO) nanoparticles have been synthesized by hydrothermal synthesis using oleic acid as a surfactant. The as-synthesized oleic acid-coated CFO (OA-CFO) nanoparticles were well dispersible in nonpolar solvents but not dispersible in water. The OA-CFO nanoparticles have been successfully transformed to highly water-dispersible citric acid-coated CFO (CA-CFO) nanoparticles using a novel single-step ligand exchange process by mechanochemical milling, in which small chain citric acid molecules replace the original large chain oleic acid molecules available on CFO nanoparticles. The OA-CFO nanoparticle’s hexane solution and CA-CFO nanoparticle’s water solution remain stable even after 6 months and show no agglomeration and their dispersion stability was confirmed by zeta-potential measurements. The contact angle measurement shows that OA-CFO nanoparticles are hydrophobic whereas CA-CFO nanoparticles are superhydrophilic in nature. The potentiality of as-synthesized OA-CFO and mechanochemically transformed CA-CFO nanoparticles for the demulsification of highly stabilized water-in-oil and oil-in-water emulsions has been demonstrated.

  13. Synthesis, characterization, single crystal X-ray determination, fluorescence and electrochemical studies of new dinuclear nickel(II) and oxovanadium(IV) complexes containing double Schiff base ligands.

    Science.gov (United States)

    Shafaatian, Bita; Ozbakzaei, Zahra; Notash, Behrouz; Rezvani, S Ahmad

    2015-04-05

    A series of new bimetallic complexes of nickel(II) and vanadium(IV) have been synthesized by the reaction of the new double bidentate Schiff base ligands with nickel acetate and vanadyl acetylacetonate in 1:1 M ratio. In nickel and also vanadyl complexes the ligands were coordinated to the metals via the imine N and enolic O atoms. The complexes have been found to possess 1:1 metals to ligands stoichiometry and the molar conductance data revealed that the metal complexes were non-electrolytes. The nickel and vanadyl complexes exhibited distorted square planar and square pyramidal coordination geometries, respectively. The emission spectra of the ligands and their complexes were studied in methanol. Electrochemical properties of the ligands and their metal complexes were also investigated in DMSO solvent at 150 mV s(-1) scan rate. The ligands and metal complexes showed both quasi-reversible and irreversible processes at this scan rate. The Schiff bases and their complexes have been characterized by FT-IR, 1H NMR, UV/Vis spectroscopies, elemental analysis and conductometry. The crystal structure of the nickel complex has been determined by single crystal X-ray diffraction. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Songül Tosun

    2016-05-01

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

  15. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  16. Ligand-based transport resonances of single-molecule-magnet spin filters: Suppression of Coulomb blockade and determination of easy-axis orientation

    Science.gov (United States)

    Rostamzadeh Renani, Fatemeh; Kirczenow, George

    2011-11-01

    We investigate single-molecule-magnet transistors (SMMTs) with ligands that support transport resonances. We find the lowest unoccupied molecular orbitals of Mn12-benzoate SMMs (with and without thiol or methyl-sulfide termination) to be on ligands, the highest occupied molecular orbitals being on the Mn12 magnetic core. We predict gate-controlled switching between Coulomb blockade and coherent resonant tunneling in SMMTs based on such SMMs, strong spin filtering by the SMM in both transport regimes, and that if such switching is observed, then the magnetic easy axis of the SMM is parallel to the direction of the current through the SMM.

  17. TLR2 Controls Intestinal Carcinogen Detoxication by CYP1A1

    DEFF Research Database (Denmark)

    Do, Khoa; Fink, Lisbeth Nielsen; Jensen, Thomas Elbenhardt

    2012-01-01

    of ligands for TLR2 of bacterial origin seems to be crucial for detoxication of luminal carcinogens by CYP1A1 in the intestine. This unprecedented finding indicates a complex interplay between the immune system of the host and intestinal bacteria with detoxication mechanisms. This highlights the relevance...

  18. Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.

    Directory of Open Access Journals (Sweden)

    Sahil Talwar

    Full Text Available Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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

  1. 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.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. 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

  6. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

    Science.gov (United States)

    Tillib, S V; Privezentseva, M E; Ivanova, T I; Vasilev, L F; Efimov, G A; Gursky, Y G; Georgiev, G P; Goldman, I L; Sadchikova, E R

    2014-02-15

    Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. TLR-dependent human mucosal epithelial cell responses to microbial pathogens.

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  9. 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.

  10. Scanning near-field optical microscopy-based study of local dynamics of receptor-ligand interactions at the single molecule level

    Science.gov (United States)

    Mensi, M.; Dukenbayev, K.; Sekatskii, S. K.; Dietler, G.

    2010-01-01

    A scanning near-field optical microscope (SNOM)—based modification of the method to study the dynamics of single molecule receptor—ligand interactions exploiting the fluorescence imaging by total internal reflection fluorescence microscopy is introduced. The main advantage of this approach consists in the possibility to study the single molecule interaction dynamics with a subwavelength spatial resolution and a submillisecond time resolution. Additionally, due to the much smaller irradiation area and some other technical features, such a modification enables to enlarge the scope of the receptor—ligand pairs to be investigated and to improve the temporal resolution. We briefly discuss corresponding experimental set up with a special accent on the SNOM operation in liquid and present some preliminary results of related investigations.

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

    Directory of Open Access Journals (Sweden)

    Ahmed R El-Awady

    2015-02-01

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

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

    Science.gov (United States)

    El-Awady, Ahmed R; Miles, Brodie; Scisci, Elizabeth; Kurago, Zoya B; Palani, Chithra D; Arce, Roger M; Waller, Jennifer L; Genco, Caroline A; Slocum, Connie; Manning, Matthew; Schoenlein, Patricia V; Cutler, Christopher W

    2015-02-01

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

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

    Science.gov (United States)

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

    2014-10-01

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

  14. Field-Induced Co(II) Single-Ion Magnets with mer-Directing Ligands but Ambiguous Coordination Geometry.

    Science.gov (United States)

    Peng, Yan; Mereacre, Valeriu; Anson, Christopher E; Zhang, Yiquan; Bodenstein, Tilmann; Fink, Karin; Powell, Annie K

    2017-06-05

    Three air-stable Co(II) mononuclear complexes with different aromatic substituents have been prepared and structurally characterized by single-crystal X-ray diffraction. The mononuclear complexes [Co(H 2 L1) 2 ]·2THF (1), [Co(HL2) 2 ] (2), and [Co(H 2 L3) 2 ]·CH 2 Cl 2 (3) (where H 3 L1, H 2 L2, and H 3 L3 represent 3-hydroxy-naphthalene-2-carboxylic acid (6-hydroxymethyl-pyridin-2-ylmethylene) hydrazide, nicotinic acid (6-hydroxymethyl-pyridin-2-ylmethylene) hydrazide, and 2-hydroxy-benzoic acid (6-hydroxymethyl-pyridin-2-ylmethylene) hydrazide, respectively) feature a distorted mer octahedral coordination geometry. Detailed magnetic studies of 1-3 have been conducted using direct and alternating current magnetic susceptibility data. Field-induced slow magnetic relaxation was observed for these three complexes. There are few examples of such behavior in (distorted) octahedral coordination geometry (OC) Co(II) mononuclear complexes with uniaxial anisotropy. Analysis of the six-coordinate Co(II) mononuclear single-ion magnets (SIMs) in the literature using the SHAPE program revealed that they all show what is best described as distorted trigonal prismatic (TRP) coordination geometry, and in general, these show negative D zero-field splitting (ZFS) values. On the other hand, all the Co(II) mononuclear complexes displaying what is best approximated as distorted octahedral (OC) coordination geometry show positive D values. In the new Co(II) mononuclear complexes we describe here, there is an ambiguity, since the rigid tridentate ligands confer what is best described for an octahedral complex as a mer coordination geometry, but the actual shape of the first coordination sphere is between octahedral and trigonal prismatic. The negative D values observed experimentally and supported by high-level electronic structure calculations are thus in line with a trigonal prismatic geometry. However, a consideration of the rhombicity as indicated by the E value of the ZFS in

  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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    LENUS (Irish Health Repository)

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  19. 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].

  20. Hyaluronan Inhibits Tlr-4-Dependent RANKL Expression in Human Rheumatoid Arthritis Synovial Fibroblasts.

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    Tatsuo Watanabe

    Full Text Available The Toll-like receptor (TLR signaling pathway is activated in synovial fibroblast cells in patients with rheumatoid arthritis (RA. The receptor activator of nuclear factor-κB (RANK and its ligand, RANKL, are key molecules involved in the differentiation of osteoclasts and joint destruction in RA. Hyaluronan (HA is a major extracellular component and an important immune regulator. In this study, we show that lipopolysaccharide (LPS stimulation significantly increases RANKL expression via a TLR-4 signaling pathway. We also demonstrate that HA suppresses LPS-induced RANKL expression, which is dependent on CD44, but not intercellular adhesion molecule-1 (ICAM-1. Our study provides evidence for HA-mediated suppression of TLR-4-dependent RANKL expression. This could present an alternative target for the treatment of destructed joint bones and cartilages in RA.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Stimulation of duodenal biopsies and whole blood from dogs with food-responsive chronic enteropathy and healthy dogs with Toll-like receptor ligands and probiotic Enterococcus faecium.

    Science.gov (United States)

    Schmitz, S; Henrich, M; Neiger, R; Werling, D; Allenspach, K

    2014-08-01

    The composition of the microbiome plays a significant role in the pathogenesis of inflammatory bowel disease (IBD) in humans and chronic enteropathies (CE) in dogs. The administration of probiotic micro-organisms is one way of modulating the microbiome, but experiments elucidating mechanisms of action of probiotics in the intestine of healthy and CE dogs are lacking. The aim of our study was to investigate the effects of different Toll-like receptor (TLR) ligands and Enterococcus faecium (EF) on ex vivo cultured duodenal samples and whole blood (WB) from dogs with food-responsive chronic enteropathy (FRE) when compared to healthy dogs. Biopsy stimulation was performed in 17 FRE and 11 healthy dogs; WB stimulation was performed in 16 FRE and 16 healthy dogs. Expression of TLR2, 4, 5 and 9, IL-17A, IL-22, IFNy, TNFα, IL-4, IL-10, TGFβ and PPARy was determined in biopsies by quantitative polymerase chain reaction (PCR). In addition, production of TNFα, IL-10, IFNy and IL-17A protein in WB and biopsy supernatants was assessed by ELISA. Treatment with individual TLR ligands or EF induced a variety of changes in the expression of different TLRs and cytokines, but not necessarily a consistent change with a single stimulating agent. Even though cytokine protein could not be detected in supernatants from ex vivo stimulated biopsies, we found TNFα protein responses in blood to be opposite of the transcriptional responses seen in the biopsies. Stimulation of canine duodenal biopsies with TLR ligands can potentially induce anti-inflammatory gene expression, especially in healthy tissue, whereas the effects of EF were limited. © 2014 John Wiley & Sons Ltd.

  6. 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.

  7. Characterization of chicken thrombocyte responses to Toll-like receptor ligands.

    Directory of Open Access Journals (Sweden)

    Michael St Paul

    Full Text Available Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR, to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS, which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL-1β, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. A single midcycle dose of levonorgestrel similar to emergency contraceptive does not alter the expression of the L-selectin ligand or molecular markers of endometrial receptivity.

    Science.gov (United States)

    Palomino, Wilder Alberto; Kohen, Paulina; Devoto, Luigi

    2010-10-01

    To examine the effects of a single-dose of 1.5 mg of levonorgestrel (commonly used as emergency contraceptive) on endometrial receptivity biomarkers through the oral or vaginal route. Prospective randomized single-blinded trial. Affiliated Hospital and University Research Center. Fertile normal women previously sterilized by tubal ligation. Levonorgestrel (1.5 mg) was administered on the day of LH surge either orally (n = 14) or vaginally (n = 13). Molecular assessment of endometrial progesterone receptors, L-selectin ligand, glicodelin-A and αvβ3 integrin by Immunohistochemistry and reverse transcriptase-polymerase chain reaction. Plasma progesterone concentration and endometrial dating were not different. The pattern of progesterone receptors and glycodelin-A expression was not affected during the early and midsecretory phase. Some endometrial biopsies from the group in which levonorgetrel was orally administered showed areas of glandular atrophy and stromal decidualization. However, the expression of the progesterone receptor, L-selectin ligand, αvβ3 integrin, and glycodelin-A were not different between the groups. Levonorgestrel, given as emergency contraceptive on the day of LH surge, does not disrupt either ovulation or progesterone production by the corpus luteum. The contraceptive mechanism of levonorgestrel at the time of LH surge does not include changes in the progesterone receptors or the endometrial receptivity biomarkers. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  11. End-to-end single cyanato and thiocyanato bridged Cu(II) polymers with a new tridentate Schiff base ligand: crystal structure and magnetic properties.

    Science.gov (United States)

    Talukder, Pritha; Datta, Amitabha; Mitra, Samiran; Rosair, Georgina; El Fallah, M Salah; Ribas, Joan

    2004-12-21

    A new tridentate Schiff base ligand HL (L = C14H19N2O), derived from the condensation of benzoylacetone and 2-dimethylaminoethylamine in a 1:1 ratio, reacts with copper(ii) acetate and cyanate, thiocyanate or azide, to give rise to several end-to-end polymeric complexes of formulae [CuL(mu(1,3)-NCO)]n 1, [CuL(mu(1,3)-NCS)]n 2 and the complex 3 has two crystallographically independent units of formula [CuL(N3)] in the asymmetric unit cell. Complex 3 exists in dimeric form rather than as a polymeric chain. Compound 1 is the first report of a singly end-to-end cyanate bridged polymeric chain of Cu(II) with a Schiff base as a co-ligand. There are many examples of double NCS bridged polymeric chains, but fewer singly bridged ones such as compound 2. We have characterized these complexes by analytical, spectroscopic, structural and variable temperature magnetic susceptibility measurements. The coordination geometry around the Cu(II) centers is distorted square pyramidal for 1 and 2 and square planar for complex 3. The magnetic susceptibility data show slight antiferromagnetic coupling for the polymers having J values -0.19 and -0.57 cm(-1) for complexes 1 and 2 respectively. The low values of J are consistent with the equatorial-axial disposition of the bridges in the polymers.

  12. 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.

  13. 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.

  14. 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

  15. Polymorphisms at Locus 4p14 of Toll-Like Receptors TLR-1 and TLR-10 Confer Susceptibility to Gastric Carcinoma in Helicobacter pylori Infection.

    Directory of Open Access Journals (Sweden)

    M Ravishankar Ram

    Full Text Available Helicobacter pylori (H. pylori -induced gastric inflammation impacts the functions of leptin- and ghrelin-producing cells in the gastroduodenum. Inflammation resulting from H. pylori sensing via Toll-like receptors (TLRs and the associated downstream signaling largely remain ambiguous. Here, we investigated the role of gut hormones, pro-inflammatory cytokines and single nucleotide polymorphisms (SNPs associated with TLR 4p14 in H. pylori disease in 30 subjects with non-ulcer dyspepsia (NUD, 40 with peptic ulcer disease (PUD and 15 with gastric cancer (GC subjects positive and negative for H. pylori infection. The level of pro-inflammatory cytokines was directly proportional to the severity of gastritis, and disease status influenced the levels of gut hormones and pro-inflammatory cytokines. TLR-1 SNPs rs4833095 and TLR-10 SNPs rs10004195 and were directly associated with H. pylori disease, and were up-regulated in the presence of H. pylori in a genotype-independent manner. We concluded that TLR-1 rs4833095 and TLR10 rs10004195 confer susceptibility to development of gastroduodenal disease, especially GC in H.pylori disease.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  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. Targeting TLR2 for Vaccine Development

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. Atopic dermatitis: Interaction between genetic variants of GSTP1, TNF, TLR2 & TLR4 and air pollution in early life.

    Science.gov (United States)

    Hüls, Anke; Klümper, Claudia; MacIntyre, Elaina A; Brauer, Michael; Melén, Erik; Bauer, Mario; Berdel, Dietrich; Bergström, Anna; Brunekreef, Bert; Chan-Yeung, Moira; Fuertes, Elaine; Gehring, Ulrike; Gref, Anna; Heinrich, Joachim; Standl, Marie; Lehmann, Irina; Kerkhof, Marjan; Koppelman, Gerard H; Kozyrskyj, Anita L; Pershagen, Göran; Carlsten, Christopher; Krämer, Ursula; Schikowski, Tamara

    2018-04-06

    Associations between traffic-related air pollution (TRAP) and childhood atopic dermatitis (AD) remain inconsistent, possibly due to unexplored gene-environment interactions. The aim of this study was to examine whether a potential effect of TRAP on AD prevalence in children is modified by selected single nucleotide polymorphisms (SNPs) related to oxidative stress and inflammation. Doctor-diagnosed AD up to age 2 years and at 7-8 years, as well as AD symptoms up to age 2 years, were assessed using parental-reported questionnaires in six birth cohorts (N=5,685). Associations of nitrogen dioxide (NO2) estimated at the home address of each child at birth, and nine SNPs within the GSTP1, TNF, TLR2, or TLR4 genes with AD were examined. Weighted genetic risk scores (GRS) were calculated from the above SNPs and used to estimate combined marginal genetic effects of oxidative stress and inflammation on AD and its interaction with TRAP. GRS was associated with childhood AD and modified the association between NO2 and doctor-diagnosed AD up to the age of 2 years (p(interaction)=0.029). This interaction was mainly driven by a higher susceptibility to air pollution in TNF rs1800629 minor allele (A) carriers. TRAP was not associated with the prevalence of AD in the general population. The marginal genetic association of a weighted GRS from GSTP1, TNF, TLR2, and TLR4 SNPs and its interaction with air pollution supports the role of oxidative stress & inflammation in AD. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  5. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    International Nuclear Information System (INIS)

    Highlights: → Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. → These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. → The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  6. Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

    Energy Technology Data Exchange (ETDEWEB)

    Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

    2011-05-20

    Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

  7. Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation

    Directory of Open Access Journals (Sweden)

    Miguel Angel Burguillos

    2015-03-01

    Full Text Available Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4 in microglia’s inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3 released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain.

  8. Evolutionary analysis of TLR9 genes reveals the positive selection of extant teleosts in Perciformes.

    Science.gov (United States)

    Zhu, Zhihuang; Sun, Yuena; Wang, Rixin; Xu, Tianjun

    2013-08-01

    The innate immune system can recognize non-self through pattern recognition receptors. Toll-like receptors were the best-known members of these receptors, and they could sense, recognize, and bind pathogen-associated molecular patterns. TLRs played an important role in innate immune system and were conserved in both invertebrate and vertebrate lineages. Thereinto, TLR9 could detect unmethylated CpG motifs in dsDNA and was expected to undergo coevolution with its microbial ligands. It was known that aquatic and terrestrial organisms dwelled in different environments which contained different pathogens, and they had to adapt to their local environmental conditions. Therefore, we collected TLR9 genes from invertebrate to vertebrate to further explore whether the huge differences between aquatic and terrestrial environments affected the TLR9s evolution between aquatic and terrestrial organisms. Molecular evolution analysis detected positively selected sites in the ancestral lineages of vertebrates, teleosts, and Perciformes but not in the ancestral lineage of mammals. In PAML, site model revealed that extant mammalian TLR9 genes underwent positive selection. However, the positive selection of extant teleosts appeared primarily in Perciformes in which there were 14 positively selected sites. Among these sites, two of them were located on the amino acid insertions of the leucine-rich repeats which could create DNA binding sites, three were found on the convex surface which might possibly affect the flexibility of the TLR solenoids, and six were located on the β-face of concave surface which contained the ligand-binding sites of the TLR solenoids. In other ML methods, we also found three sites under selection that coincided with the codons identified by M8 and these sites were all located in LRRs. The diverse aquatic and terrestrial environments might possess different pathogens to make the living organisms adapt to their local environmental conditions. The positive

  9. 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.

  10. 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...

  11. 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...

  12. Expression of TLR4 in Non-Small Cell Lung Cancer Is Associated with PD-L1 and Poor Prognosis in Patients Receiving Pulmonectomy

    Directory of Open Access Journals (Sweden)

    Xiubao Ren

    2017-04-01

    Full Text Available Currently, the effect of inflammation on tumorigenesis and progression has been widely noted. As a member of pattern recognition receptors, toll-like receptor 4 (TLR4 plays a pivotal role in tumor immune microenvironment and has been increasingly investigated. In the present study, we evaluated TLR4 expression and its association with programmed cell death ligand 1 (PD-L1 in non-small cell lung cancer (NSCLC tissues and assessed the predicting value of TLR4 on postoperative outcome. A total of 126 NSCLC patients receiving complete pulmonary resection and systematic lymph node dissection between April 2008 and August 2014 were enrolled. All the patients had integrated clinicopathological records and follow-up data. TLR4 and PD-L1 expression on NSCLC samples were determined by immunohistochemistry, and serum soluble TLR4 (sTLR4 levels were measured by enzyme-linked immunosorbent assay. Results showed that TLR4 expression level in cancer tissue was significantly higher than that in para-cancer tissue. Elevated TLR4 expression was significantly associated with histological type (adenocarcinoma higher than squamous cell carcinoma, P = 0.041, increased clinical TNM stage (P < 0.001, and presence of lymphatic invasion (P < 0.001. Besides, TLR4 expression level in cancer samples was inversely correlated with serum sTLR4 level in patients with early-stage NSCLC (r = −0.485, P = 0.003. TLR4 expression level was also positively correlated with the PD-L1 expression level (r = 0.545, P < 0.0001. Multivariate analysis showed that expression level of TLR4 was an independent prognostic factor and TLR4 overexpression indicated a poor overall survival and disease-free survival. Taken together, we conclude that expression of TLR4 in lung cancer is associated with PD-L1 and could predict the outcome of patients with NSCLC receiving pulmonary resection for cancer.

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

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    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

  14. 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...

  15. 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.

  16. 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.

  17. Structural and EPR studies on single-crystal and polycrystalline samples of copper(II) and cobalt(II) complexes with N2S2-based macrocyclic ligands.

    Science.gov (United States)

    Tamayo, Abel; Casabó, Jaume; Escriche, Lluís; González, Pablo; Lodeiro, Carlos; Rizzi, Alberto C; Brondino, Carlos D; Passeggi, M C G; Kivekäs, Raikko; Sillanpää, Reijo

    2007-07-09

    The properties of Cu(II) and Co(II) complexes with oxygen- or nitrogen-containing macrocycles have been extensively studied; however, less attention has been paid to the study of complexes containing sulfur atoms in the first coordination sphere. Herein we present the interaction between these two metal ions and two macrocyclic ligands with N2S2 donor sets. Cu(II) and Co(II) complexes with the pyridine-containing 14-membered macrocycles 3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L) and 7-(9-anthracenylmethyl)-3,11-dithia-7,17-diazabicyclo[11.3.1]heptadeca-1(17),13,15-triene (L1) have been synthesized. The X-ray structural analysis of {[Co(ClO4)(H2O)(L)][Co(H2O)2(L)]}(ClO4)3 shows two different metal sites in octahedral coordination. The EPR spectra of powdered samples of this compound are typical of distorted six-coordinated Co(II) ions in a high-spin (S=3/2) configuration, with the ground state being S=1/2 (g1=5.20, g2=3.20, g3=1.95). The EPR spectrum of [Cu(ClO4)(L)](ClO4) was simulated assuming an axial g tensor (g1=g2=2.043, g3=2.145), while that of [Cu(ClO4)(L1)](ClO4) slightly differs from an axial symmetry (g1=2.025, g2=2.060, g3=2.155). These results are compatible with a Cu(II) ion in square-pyramidal coordination with N2S2 as basal ligands. Single-crystal EPR experiment performed on [Cu(ClO4)(L1)](ClO4) allowed determining the eigenvalues of the molecular g tensor associated with the copper site, as well as the two possible orientations for the tensor. On the basis of symmetry arguments, an assignment in which the eigenvectors are nearly along the Cu(II)-ligand bonds is chosen.

  18. NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis.

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    T Eoin West

    2014-09-01

    Full Text Available Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice and type III secretion system components (in mice and humans. In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4(-/- mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.

  19. Transforming single domain magnetic CoFe{sub 2}O{sub 4} nanoparticles from hydrophobic to hydrophilic by novel mechanochemical ligand exchange

    Energy Technology Data Exchange (ETDEWEB)

    Munjal, Sandeep; Khare, Neeraj, E-mail: nkhare@physics.iitd.ernet.in [Indian Institute of Technology Delhi, Department of Physics (India)

    2017-01-15

    Single-phase uniform-sized (~9 nm) cobalt ferrite (CFO) nanoparticles have been synthesized by hydrothermal synthesis using oleic acid as a surfactant. The as-synthesized oleic acid-coated CFO (OA-CFO) nanoparticles were well dispersible in nonpolar solvents but not dispersible in water. The OA-CFO nanoparticles have been successfully transformed to highly water-dispersible citric acid-coated CFO (CA-CFO) nanoparticles using a novel single-step ligand exchange process by mechanochemical milling, in which small chain citric acid molecules replace the original large chain oleic acid molecules available on CFO nanoparticles. The OA-CFO nanoparticle’s hexane solution and CA-CFO nanoparticle’s water solution remain stable even after 6 months and show no agglomeration and their dispersion stability was confirmed by zeta-potential measurements. The contact angle measurement shows that OA-CFO nanoparticles are hydrophobic whereas CA-CFO nanoparticles are superhydrophilic in nature. The potentiality of as-synthesized OA-CFO and mechanochemically transformed CA-CFO nanoparticles for the demulsification of highly stabilized water-in-oil and oil-in-water emulsions has been demonstrated.

  20. 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

  1. 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

  2. Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10.

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    Carmen A Ambarus

    Full Text Available BACKGROUND: Costimulation of murine macrophages with immune complexes (ICs and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages. MATERIALS AND METHODS: Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs. Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR. RESULTS: HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ(IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2. The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6. CONCLUSION: HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.

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

    Directory of Open Access Journals (Sweden)

    Yin-Ping Jia

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

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

    Science.gov (United States)

    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.

  5. 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.

  6. TLR4 and RAGE: similar routes leading to inflammation in type 2 diabetic patients.

    Science.gov (United States)

    Veloso, C A; Fernandes, J S; Volpe, C M O; Fagundes-Netto, F S; Reis, J S; Chaves, M M; Nogueira-Machado, J A

    2011-09-01

    The present study investigates the interaction of TLR4 and RAGE with their respective ligands as inducers of the inflammatory markers IL-6 and TNF-α. Also, the reactivity of peripheral blood mononuclear cells (PBMNC) from type 2 diabetic (T2D) patients and non-diabetic healthy controls (ND) were comparatively studied. Concentrations of IL-6 and TNF-α were measured by sandwich Elisa, using kits supplied by Assay Designs (Ann Arbor, MI, USA). PBMNC from T2D and ND were incubated in the presence or absence of LPS, anti-TLR4 or anti-RAGE for 72 hours at 37°C under 5% CO(2). The final volume was adjusted to 300 μL in DMEM supplemented with 10% fetal bovine serum. After incubation, the cells were centrifuged, the supernatant collected and the cytokines measured. PBMNC from T2D were more sensitive to innate immune stimulation with LPS and monoclonal agonist anti-TLR4 than were cells from ND. The actions of LPS, anti-TLR4 and anti-RAGE potentiated the production of IL-6 and TNF-α in both groups. The simultaneous activation of monoclonal anti-RAGE and anti-TLR4 suggests that both antibodies used different receptors on the cell surface, but converged on the same PBMNC signaling metabolic pathways. This simultaneous activation induced a higher production of IL-6 and TNF-α in PBMNC from the T2D patients than from the ND subjects. Our results clearly show an exacerbation of innate immunity in PBMNC with T2D that was possibly hyperglycaemia-induced. These data, when analyzed together, suggest the importance of innate immunity in the pathogenesis of T2D. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  7. Heterometallic Zn3 Ln3 Ensembles Containing (μ6 -CO3 ) Ligand and Triangular Disposition of Ln3+ ions: Analysis of Single-Molecule Toroic (SMT) and Single-Molecule Magnet (SMM) Behavior.

    Science.gov (United States)

    Goura, Joydeb; Colacio, Enrique; Herrera, Juan Manuel; Suturina, Elizaveta A; Kuprov, Ilya; Lan, Yanhua; Wernsdorfer, Wolfgang; Chandrasekhar, Vadapalli

    2017-11-21

    Two new heterometallic Zn 3 Ln 3 (Ln 3+ =Dy, Tb) complexes, with a double triangular topology of the metal ions, have been assembled from the polytopic Mannich base ligand 6,6'-{[2-(dimethylamino)ethylazanediyl]bis(methylene)}bis(2-methoxy-4-methylphenol) (H 2 L) with the aid of an in situ generated carbonate ligand from atmospheric CO 2 fixation. Theoretical calculations indicate axial ground states for the Ln 3+ ions in these complexes, with their local magnetic moments being almost coplanar and tangential to the Ln 3+ atoms that define the equilateral triangle. Therefore, they can be considered as single-molecule toroics (SMTs) with almost zero total magnetic moment. Micro-SQUID measurements on the Dy 3+ counterpart show hysteresis loops below 3 K that have an S-shape, with large coercive fields opening upon cooling. This behavior is typical of a single molecule magnet (SMM) with very slow zero-field relaxation. At around ±0.35 T, the loops have a broad step, which is due to a direct relaxation process and corresponds to an acceleration of the relaxation of the magnetization, also observed at this magnetic field from ac susceptibility measurements. Simulations suggest that the broad step corresponds to two level avoidance of crossing points where the spin chiral Kramers doublet meets excited states of the coupled manifold, whose position is defined by exchange and dipole interactions. The Tb 3+ counterpart does not exhibit SMM behavior, which is due to the fact that the degeneracy of the ground state of the exchange coupled system is lifted at zero field, thus favoring quantum tunneling of magnetization (QTM). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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. 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.

  10. HLA-A*0206 with TLR3 polymorphisms exerts more than additive effects in Stevens-Johnson syndrome with severe ocular surface complications.

    Directory of Open Access Journals (Sweden)

    Mayumi Ueta

    Full Text Available BACKGROUND: Stevens-Johnson syndrome (SJS is an acute inflammatory vesiculobullous reaction of the skin and mucosa, often including the ocular surface, and toxic epidermal necrolysis (TEN occurs with its progression. Although SJS/TEN is thought to be initiated by certain types of medication coupled with possible infection. In the present study we examined the multiplicative interaction(s between HLA-A*0206 and 7 Toll-like receptor 3 (TLR3 Single-nucleotide polymorphisms (SNPs in patients with SJS/TEN. PRINCIPAL FINDINGS: We analyzed the genotypes for HLA-A and 7 TLR3 SNPs in 110 Japanese SJS/TEN patients with severe ocular complications and 206 healthy volunteers to examine the interactions between the two loci. We found that HLA-A*0206 exhibited a high odds ratio for SJS/TEN (carrier frequency: OR = 5.1; gene frequency: OR = 4.0 and that there was a strong association with TLR3 rs.5743312T/T SNP (OR = 7.4, TLR3 rs.3775296T/T SNP (OR = 5.8, TLR3 rs.6822014G/G SNP (OR = 4.8, TLR3 rs.3775290A/A SNP (OR = 2.9, TLR3 rs.7668666A/A SNP (OR = 2.7, TLR3 rs.4861699G/G SNP (OR = 2.3, and TLR3 rs.11732384G/G SNP (OR = 1.9. There was strong linkage disequilibrium (LD between rs.3775296 and rs.5743312 and between rs.7668666 and rs.3775290. The results of interaction analysis showed that the pair, HLA-A*0206 and TLR3 SNP rs3775296T/T, which exhibited strong LD with TLR3 rs.5743312, exerted more than additive effects (OR = 47.7. The other pairs, HLA-A*0206 and TLR3 rs.3775290A/A SNP (OR = 11.4 which was in strong LD with TLR3 rs7668666A/A SNP, and TLR3 rs4861699G/G SNP (OR = 7.6 revealed additive effects. Moreover, the combination HLA-A*0206 and TLR3 rs3775296T/T was stronger than the TLR3 rs6822014G/G and TLR3 rs3775290A/A pair, which reflected the interactions within the TLR3 gene alone. SIGNIFICANCE: By interaction analysis, HLA-A*0206 and TLR3 SNP rs3775296T/T, which were in strong LD with TLR3 SNP rs5743312T/T, manifested more than additive effects that

  11. I. Enabling Single-Chain Surfactants to Form Vesicles by Nonamphiphilic Liquid Crystals in Water II. Controlling Attachment and Ligand-Mediated Adherence of Candida albicans on Monolayers

    Science.gov (United States)

    Varghese, Nisha

    This dissertation describes a fundamental study of weak noncovalent interactions and surface forces that exist at the interfaces of various interacting moieties (small molecules or microbes), and its relevance to colloidal and material chemistry. Chapter 1 presents an emulsion system that enables single-chain anionic or nonionic surfactants to sequester and encapsulate certain water-soluble organic salts, leading to the formation of vesicles in water. The water-soluble organic salt in the system comprises of disodium cromoglycate crystals that are emulsified by surfactants in water to form stable liquid crystal droplets. The work provides an exception to the rule of geometric packing factor that dictates formation of micelles by the surfactants in water. Chapter 2 shows that the odd or even number of carbon atoms present in the aliphatic chain of surfactants affect the ability of surfactants to emulsify aqueous-based liquid crystals of disodium cromoglycate. Such an odd-even effect is frequently observed for solid state properties like melting point, heat of fusion and refractive index but is rarely observed for molecules present in solution. When mixed in water, anionic single-chain surfactants with odd number of carbon atoms emulsifies disodium cromoglycate to form liquid crystal droplets, while surfactants with even number of carbon atoms fail to emulsify disodium cromoglycate. Chapter 3 Bolaamphiphiles usually form vesicles only in extreme conditions or in the presence of surfactants. Here, we explore the co-assembly system of synthesized bolaamphiphiles and disodium cromoglycate in water. The combination of the self-assembly forces of the bolaamphiphile and self-associating property of disodium cromoglycate liquid crystals act together at the interface form a unique microemulsion of liquid crystal droplets of disodium cromoglycate embedded in liquid crystal phase. Chapter 4 describes a key event (adhesion) that precedes infections caused by Candida albicans

  12. Reactivation of latent HIV-1 in central memory CD4+ T cells through TLR-1/2 stimulation

    OpenAIRE

    Novis, Camille L; Archin, Nancie M; Buzon, Maria J; Verdin, Eric; Round, June L; Lichterfeld, Mathias; Margolis, David M; Planelles, Vicente; Bosque, Alberto

    2013-01-01

    Abstract Background Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied. ...

  13. 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.

  14. Optical and dielectric studies of KH2PO4 crystal influenced by organic ligand of citric acid and l-valine: A single crystal growth and comparative study

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    Mohd Anis

    Full Text Available In the present study pure, citric acid (CA and l-valine (LV doped potassium dihydrogen phosphate (KDP crystals have been grown with the aim to investigate the nonlinear optical applications facilitated by UV–visible, third order nonlinear optical (TONLO and dielectric properties. The structural parameters of grown crystals have been confirmed by single crystal X-ray diffraction analysis. The enhancement in optical transparency of KDP crystal due to addition of CA and LV has been examined within 200–900 nm by means of UV–visible spectral analysis. In addition, the transmittance data have been used to evaluate the effect of dopants on reflectance, refractive index and extinction coefficient of grown crystals in the visible region. The Z-scan analysis has been performed at 632.8 nm to identify the nature of photoinduced nonlinear refraction and nonlinear absorption in doped KDP crystals. The influence of π-bonded ligand of dopant CA and LV on TONLO susceptibility (χ3, refractive index (n2 and absorption coefficient (β of KDP crystals has been evaluated to discuss laser assisted device applications. The decrease in dielectric constant and dielectric loss of KDP crystal due to addition of CA and LV has been explored using the temperature dependent dielectric studies. Keywords: Crystal growth, Nonlinear optical materials, UV–visible studies, Z-scan analysis, Dielectric studies

  15. 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

  16. Design of a Family of Ln3 Triangles with the HAT Ligand (1,4,5,8,9,12-Hexaazatriphenylene): Single-Molecule Magnetism.

    Science.gov (United States)

    Díaz-Ortega, Ismael F; Herrera, Juan Manuel; Gupta, Tulika; Rajaraman, Gopalan; Nojiri, Hiroyuki; Colacio, Enrique

    2017-05-15

    A series of trinuclear Ln 3 complexes (Ln III = Yb (1), Er (2), Dy (3) and Gd (4)) were prepared from the tris-chelate bidentate ligand 1,4,5,8,9,12-hexaazatriphenylene (HAT). 1 and 2 exhibited field-induced single-molecule-magnet (SMM) behavior with estimated U eff values of 21.30 and 13.86 K, respectively. Complex 3 behaved as a SMM even at zero field, and two different thermally assisted relaxation processes were detected with U eff values of 29.6 K (fast relaxation process, FR) and 69 K (slow relaxation process, SR) due to the existence of two magnetically different Dy III centers in the molecule. Ab initio studies reveal that all the Dy 3+ centers have almost an Ising ground state. The local anisotropy axes are not coplanar but form angles with the Dy3 plane in the range 58-78°. The magnetic interaction between the anisotropic Dy 3+ ions is antiferromagnetic in nature and very weak in magnitude. However, due to the extreme feebleness of the magnetic interaction with regard to the local excitation energies, the magnetization blockade is most probably of single-ion origin. Calculations support the existence of two relaxation processes, which take place through the first excited state following an Orbach/Raman mechanism. Finally, for complex 4, the magnetocaloric effect was simulated using the magnetic parameters extracted from the fit of the magnetization and susceptibility data and demonstrated that the simulated -ΔS m values were almost coincident with those extracted from the integration of the field dependence of the magnetization. The simulated MCE value at 2 K and 5 T (20.46 J kg -1 K -1 ) makes complex 4 an attractive candidate for cryogenic magnetization.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Reactivation of latent HIV-1 in central memory CD4+ T cells through TLR-1/2 stimulation

    Science.gov (United States)

    2013-01-01

    Background Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied. Results We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4+ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation. Conclusions Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs. PMID:24156240

  3. 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.

  4. 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.

  5. Effective innate and adaptive antimelanoma immunity through localized TLR7/8 activation.

    Science.gov (United States)

    Singh, Manisha; Khong, Hiep; Dai, Zhimin; Huang, Xue-Fei; Wargo, Jennifer A; Cooper, Zachary A; Vasilakos, John P; Hwu, Patrick; Overwijk, Willem W

    2014-11-01

    Intratumoral immune activation can induce local and systemic antitumor immunity. Imiquimod is a cream-formulated, TLR7 agonist that is Food and Drug Administration approved for the treatment of nonmelanoma skin cancers, but it has limited activity against melanoma. We studied the antitumor activity and mechanism of action of a novel, injectable, tissue-retained TLR7/8 agonist, 3M-052, which avoids systemic distribution. Intratumoral administration of 3M-052 generated systemic antitumor immunity and suppressed both injected and distant, uninjected wild-type B16.F10 melanomas. Treated tumors showed that an increased level of CCL2 chemokines and infiltration of M1 phenotype-shifted macrophages, which could kill tumor cells directly through production of NO and CCL2, were essential for the antitumor activity of 3M-052. CD8(+) T cells, B cells, type I IFN, IFN-γ, and plasmacytoid dendritic cells were contributed to efficient tumor suppression, whereas perforin, NK cells, and CD4 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-programmed death ligand 1 Abs, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

  6. 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.

  7. Genetic polymorphisms in the bovine toll-like receptor 4 (TLR4) and monocyte chemo attractant protein-1(CCL2) genes: SNPs distribution analysis in Bos indicus Sahiwal cattle breed.

    Science.gov (United States)

    Behl, Jyotsna Dhingra; Sharma, Anurodh; Kataria, R S; Verma, N K; Kimothi, Shiv Prasad; Bhatia, Avnish Kumar; Sodhi, Monika; Behl, Rahul; Joshi, B K

    2014-01-01

    Toll-like receptor 4 gene (TLR4) that recognizes the Gram negative bacterial ligand LPS was sequenced in the Bos indicus Sahiwal cattle breed. Ninety four single nucleotide polymorphisms (SNPs) were detected within 10.8 kb gene region. Seventeen of the SNPs were in the coding regions and the one at position 9589(A > G) in exon3 resulted in an amino acid change from Valine to Isoleucine. These SNPs led to generation of 27 TLR4 gene haplotypes. All the Sahiwal animals studied presently showed the occurrence of the genotype CC at gene position 9662, which codes for the amino acid threonine at position 674 of the TLR4 protein, and which had been reported to be associated with lower somatic cell score and, therefore, a lower susceptibility to mastitis, in Taurus cattle. This nucleotide configuration of the Toll-like receptor 4 gene of the Bos indicus Sahiwal cattle breed could possibly indicate toward a lower susceptibility to mastitis in the Sahiwal animals. Monocyte chemo-attractant protein-1 (CCL2) gene encoding for small inducible cytokine A2 that belongs to the CC chemokine family was also sequence characterized in these Sahiwal animals. The CCL2 gene was observed to have 12 polymorphic sites in 3.3 kb region of which one SNP at position 2500 (A > G) in exon 3 resulted in amino acid change from Valine to Isoleucine at position 46 of the mature CCL2 peptide. Seventeen haplotypes of the CCL2 gene were predicted corresponding to 12 genotypes detected.

  8. TLR4 Deficiency Protects against Hepatic Fibrosis and Diethylnitrosamine-Induced Pre-Carcinogenic Liver Injury in Fibrotic Liver.

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    Susanne Nicole Weber

    Full Text Available The development of hepatocellular carcinoma (HCC is a common consequence of advanced liver fibrosis but the interactions between fibrogenesis and carcinogenesis are still poorly understood. Recently it has been shown that HCC promotion depends on Toll-like receptor (TLR 4. Pre-cancerogenous events can be modelled in mice by the administration of a single dose of diethylnitrosamine (DEN, with HCC formation depending amongst others on interleukin (IL 6 production. Mice lacking the hepatocanalicular phosphatidylcholine transporter ABCB4 develop liver fibrosis spontaneously, resemble patients with sclerosing cholangitis due to mutations of the orthologous human gene, and represent a valid model to study tumour formation in pre-injured cholestatic liver. The aim of this study was to investigate DEN-induced liver injury in TLR4-deficient mice with biliary fibrosis.ABCB4-deficient mice on the FVB/NJ genetic background were crossed to two distinct genetic backgrounds (TLR4-sufficient C3H/HeN and TLR4-deficient C3H/HeJ for more than 10 generations. The two congenic knockout and the two corresponding wild-type mouse lines were treated with a single dose of DEN for 48 hours. Phenotypic differences were assessed by measuring hepatic collagen contents, inflammatory markers (ALT, CRP, IL6 as well as hepatic apoptosis (TUNEL and proliferation (Ki67 rates.Hepatic collagen accumulation is significantly reduced in ABCB4-/-:TLR4-/-double-deficient mice. After DEN challenge, apoptosis, proliferation and inflammatory markers are decreased in TLR4-deficient in comparison to TLR4-sufficient mice. When combining ABCB4 and TLR4 deficiency with DEN treatment, hepatic IL6 expression and proliferation rates are lowest in fibrotic livers from the double-deficient line. Consistent with these effects, selective digestive tract decontamination in ABCB4-/- mice also led to reduced tumor size and number after DEN.This study demonstrates that liver injury upon DEN challenge

  9. 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.

  10. 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

  11. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

    Science.gov (United States)

    Raschi, Elena; Chighizola, Cecilia B; Grossi, Claudia; Ronda, Nicoletta; Gatti, Rita; Meroni, Pier Luigi; Borghi, M Orietta

    2014-12-01

    The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts

  12. 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.

  13. 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.

  14. Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

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    Perdijk, Olaf; van Neerven, R J Joost; Meijer, Ben; Savelkoul, Huub F J; Brugman, Sylvia

    2018-03-01

    The human milk oligosaccharide 3'-sialyllactose (3'SL) has previously been shown to activate murine dendritic cells (DC) in a Toll-like receptor (TLR) 4-mediated manner ex vivo. In this study we aimed to investigate whether 3'SL has similar immunomodulatory properties on human DC. 3'SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3'SL from different suppliers. After the removal of LPS from 3'SL, we studied its ability to modify DC differentiation in vitro. In contrast to LPS and 3'SL, LPS-free 3'SL did not induce functional and phenotypical changes on immature DC (iDC). iDC that were differentiated in the presence of LPS or 3'SL showed a semi-mature phenotype (i.e., fewer CD83+CD86+ DC), produced IL-10 and abrogated IL-12p70 and tumor necrosis factor-alpha levels upon stimulation with several TLR ligands. Differentiation into these tolerogenic DC was completely abrogated by LPS removal from 3'SL. In contrast to previous reports in mice, we found that LPS-free 3'SL does not activate NF-κB via human TLR4. In conclusion, removing LPS from (oligo)saccharide preparations is necessary to study their potential immunomodulatory function.

  15. 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

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    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.

  16. Methylation of 23S rRNA caused by tlrA (ermSF), a tylosin resistance determinant from Streptomyces fradiae.

    Science.gov (United States)

    Zalacain, M; Cundliffe, E

    1989-01-01

    Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus). Images PMID:2753855

  17. Synthesis and Characterization of a Ru(II Complex with Functionalized Phenanthroline Ligands Having Single-Double Linked Anthracenyl and 1-Methoxy-1-buten-3-yne Moieties

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    Adewale O. Adeloye

    2010-10-01

    Full Text Available Two series of bidentate polypyridine ligands, made of phenanthroline chelating subunits having substituted mono-and di-anthracenyl groups, and 1-methoxy-1-buten-3-yne at the 4 and 7-positions with the corresponding heteroleptic Ru(II complex have been synthesized and characterized. The complex is formulated as [(Ru(L1(L2(NCS2], (where L1 = 4-(9-dianthracenyl-10-(2,3-dimethylacrylic acid-7-(9-anthracenyl-10-(2,3-dimethylacrylic acid-1,10-phenanthroline and L2 = 4,7-bis(1-methoxy-1-buten-3-yne-1,10-phenanthroline. The Ru(II complex shows characteristic broad and intense metal-to-ligand charge transfer (MLCT bands absorption and appreciable photoluminescence spanning the visible region. The ligands and complex were characterized by FT-IR, 1H, 13C NMR spectroscopy, UV-Vis, photoluminescence and elemental analysis (see in supplementary materials. The anchoring groups in both ligands have allowed an extended delocalization of acceptor orbital of the metal-to-ligand charge-transfer (MLCT excited state.

  18. TNF is required for TLR ligand–mediated but not protease-mediated allergic airway inflammation

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    Whitehead, Gregory S.; Thomas, Seddon Y.; Shalaby, Karim H.; Nakano, Keiko; Moran, Timothy P.; Ward, James M.; Flake, Gordon P.; Cook, Donald N.

    2017-01-01

    Asthma is associated with exposure to a wide variety of allergens and adjuvants. The extent to which overlap exists between the cellular and molecular mechanisms triggered by these various agents is poorly understood, but it might explain the differential responsiveness of patients to specific therapies. In particular, it is unclear why some, but not all, patients benefit from blockade of TNF. Here, we characterized signaling pathways triggered by distinct types of adjuvants during allergic sensitization. Mice sensitized to an innocuous protein using TLR ligands or house dust extracts as adjuvants developed mixed eosinophilic and neutrophilic airway inflammation and airway hyperresponsiveness (AHR) following allergen challenge, whereas mice sensitized using proteases as adjuvants developed predominantly eosinophilic inflammation and AHR. TLR ligands, but not proteases, induced TNF during allergic sensitization. TNF signaled through airway epithelial cells to reprogram them and promote Th2, but not Th17, development in lymph nodes. TNF was also required during the allergen challenge phase for neutrophilic and eosinophilic inflammation. In contrast, TNF was dispensable for allergic airway disease in a protease-mediated model of asthma. These findings might help to explain why TNF blockade improves lung function in only some patients with asthma. PMID:28758900

  19. Association of TLR7 variants with AIDS-like disease and AIDS vaccine efficacy in rhesus macaques.

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    Roman A Siddiqui

    Full Text Available In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV-infected male rhesus macaques, including an 'MHC adjusted' subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T, located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype.

  20. Bruton's tyrosine kinase and protein kinase C µ are required for TLR7/9-induced IKKα and IRF-1 activation and interferon-β production in conventional dendritic cells.

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    Yan-Feng Li

    Full Text Available Stimulation of TLR7/9 by their respective ligands leads to the activation of IκB kinase α (IKKα and Interferon Regulatory Factor 1 (IRF-1 and results in interferon (IFN-β production in conventional dendritic cells (cDC. However, which other signaling molecules are involved in IKKα and IRF-1 activation during TLR7/9 signaling pathway are not known. We and others have shown that Bruton's Tyrosine Kinase (BTK played a part in TLR9-mediated cytokine production in B cells and macrophages. However, it is unclear if BTK participates in TLR7/9-induced IFN-β production in cDC. In this study, we show that BTK is required for IFN-β synthesis in cDC upon TLR7/9 stimulation and that stimulated BTK-deficient cDC are defective in the induction of IKKα/β phosphorylation and IRF-1 activation. In addition, we demonstrate that Protein Kinase C µ (PKCµ is also required for TLR7/9-induced IRF-1 activation and IFN-β upregulation in cDC and acts downstream of BTK. Taken together, we have uncovered two new molecules, BTK and PKCµ, that are involved in TLR7/9-triggered IFN-β production in cDC.

  1. TLR2 Elicits IL-17-Mediated RANKL Expression, IL-17, and OPG Production in Neutrophils from Arthritic Mice

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    Viktoriya Milanova

    2014-01-01

    Full Text Available We investigated the ability of neutrophils to express receptor activator of nuclear factor kappa-B ligand (RANKL, to secrete osteoprotegerin (OPG, and to produce IL-17. Arthritis was induced by intra-articular injection of zymosan, a ligand for Toll-like receptor 2 (TLR2. Frequencies of neutrophils in bone marrow (BM, blood and synovial fluid (SF, receptor expression, and cytokine production were evaluated by flow cytometry. 1A8 antibody (1A8 Ab was used to deplete neutrophils in zymosan-injected SCID mice. IL-17, RANKL, and OPG amounts in SF, serum, or cell cultures were determined by ELISA. The development of arthritis was associated with increased secretion of IL-17, RANKL, and OPG in serum and SF, elevated frequencies of Ly6G+CD11b+ cells in BM, blood, and SF and upregulated RANKL expression. Both IL-17 and OPG were absent in serum and SF after neutrophil depletion; therefore we assume that they were released by neutrophils. In vitro blood Ly6G+CD11b+ cells from arthritic mice produced spontaneously IL-17, IFN-γ, and OPG and expressed RANKL. This phenotype was sustained by IL-17. TLR2 engagement increased IL-17 and IFN-γ production, potentiated IL-17-mediated RANKL expression, and inhibited OPG secretion. We conclude that TLR2 regulates the destructive potential of neutrophils and its targeting might limit joint alterations in arthritis.

  2. IL-6 amplifies TLR mediated cytokine and chemokine production: implications for the pathogenesis of rheumatic inflammatory diseases.

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    Ivan Caiello

    Full Text Available The role of Interleukin(IL-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA and systemic juvenile idiopathic arthritis (s-JIA has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs, synovial fluid mononuclear cells from JIA patients (SFMCs and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R. SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ. Cells were stimulated with LPS, S100A8-9, poly(I-C, CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C, CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic

  3. Incidence and dynamics of active cytomegalovirus infection in allogeneic stem cell transplant patients according to single nucleotide polymorphisms in donor and recipient CCR5, MCP-1, IL-10, and TLR9 genes.

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    Corrales, Isabel; Giménez, Estela; Solano, Carlos; Amat, Paula; de la Cámara, Rafael; Nieto, José; Garcia-Noblejas, Ana; Navarro, David

    2015-02-01

    Single nucleotide polymorphisms (SNPs) in genes involved in the activation or regulation of innate and adaptive immune responses may modulate the susceptibility to and the natural history of certain chronic viral infections. The current study aimed to investigate whether donor and recipient SNPs in the chemokine receptor 5 (rs1800023), monocyte chemoattractant protein 1 (rs13900), interleukin-10 (rs1878672), and Toll-like receptor 9 (rs352140) genes would exert any influence on the rate of incidence and features of CMV DNAemia in the allogeneic stem cell transplantation setting. This was a retrospective observational multicenter study. The cohort consisted of 102 non-consecutive allogeneic stem cell transplant recipients. SNP genotyping was performed by allele-specific real-time PCR. CMV surveillance was performed by the pp65 antigenemia assay/and or by real-time PCR. Seventy-three patients developed CMV DNAemia within the first 100 days after transplantation (71.5%). Neither donor nor recipient SNPs were associated significantly with the rate of incidence of active CMV infection, nor with the need for pre-emptive antiviral therapy. Both the duration of CMV DNAemia and the plasma CMV DNA peak load during episodes were significantly higher in patients harboring the donor (but not the recipient) chemokine receptor 5 A/A genotype, than in their A/G and G/G counterparts (P = 0.022 and P = 0.045, respectively). The data reported suggest that SNPs in chemokine receptor 5 may influence the dynamics of CMV infection in the Allo-SCT setting. © 2014 Wiley Periodicals, Inc.

  4. Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands

    NARCIS (Netherlands)

    Weterings, Josephus Johannes

    2008-01-01

    Toll-like receptors (TLRs) are receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. TLRs can be found at cells that play a role in the immune system. Binding of the TLR with its corresponding ligand

  5. 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.

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

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    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.

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

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    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.

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

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    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.

  9. Identification of BCAP-{sub L} as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Takayuki [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Oyama, Masaaki; Kozuka-Hata, Hiroko [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Ishikawa, Kosuke; Inoue, Takafumi [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Muta, Tatsushi [Laboratory of Cell Recognition and Response, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578 (Japan); Semba, Kentaro, E-mail: ksemba@waseda.jp [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan); Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639 (Japan)

    2010-09-17

    Research highlights: {yields} Twenty five tyrosine-phosphorylated proteins in LPS-stimulated macrophages were determined. {yields} BCAP is a novel tyrosine-phosphorylated protein in LPS-stimulated macrophages. {yields} BCAP-{sub L} inhibits IL-6 and IL-10 production in LPS-stimulated macrophages. -- Abstract: Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here, we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-{sub L}) and an alternatively initiated or spliced (Bcap-{sub S}) mRNA, and little is known about the differential functions of the BCAP-{sub L} and BCAP-{sub S} proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-{sub L} enhanced IL-6 and IL-10 production but not TNF-{alpha} production in TLR ligand-stimulated macrophages. We propose that BCAP-{sub L} (but not BCAP-{sub S}) is a negative regulator of the TLR-mediated host defense system in macrophages.

  10. 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...

  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-mediated albuminuria needs TNFα-mediated cooperativity between TLRs present in hematopoietic tissues and CD80 present on non-hematopoietic tissues in mice.

    Science.gov (United States)

    Jain, Nidhi; Khullar, Bhavya; Oswal, Neelam; Banoth, Balaji; Joshi, Prashant; Ravindran, Balachandran; Panda, Subrat; Basak, Soumen; George, Anna; Rath, Satyajit; Bal, Vineeta; Sopory, Shailaja

    2016-06-01

    Transient albuminuria induced by pathogen-associated molecular patterns (PAMPs) in mice through engagement of Toll-like receptors (TLRs) is widely studied as a partial model for some forms of human nephrotic syndrome (NS). In addition to TLRs, CD80 has been shown to be essential for PAMP-mediated albuminuria. However, the mechanistic relationships between TLRs, CD80 and albuminuria remain unclear. Here, we show that albuminuria and CD80-uria induced in mice by many TLR ligands are dependent on the expression of TLRs and their downstream signalling intermediate MyD88 exclusively in hematopoietic cells and, conversely, on CD80 expression exclusively in non-hematopoietic cells. TNFα is crucial for TLR-mediated albuminuria and CD80-uria, and induces CD80 expression in cultured renal podocytes. IL-10 from hematopoietic cells ameliorates TNFα production, albuminuria and CD80-uria but does not prevent TNFα-mediated induction of podocyte CD80 expression. Chitohexaose, a small molecule originally of parasite origin, mediates TLR4-dependent anti-inflammatory responses, and blocks TLR-mediated albuminuria and CD80-uria through IL-10. Thus, TNFα is a prominent mediator of renal CD80 induction and resultant albuminuria in this model, and small molecules modulating TLR-mediated inflammatory activation might have contributory or adjunct therapeutic potential in some contexts of NS development. © 2016. Published by The Company of Biologists Ltd.

  13. NOD-2 and TLR-4 Signaling Reinforces the Efficacy of Dendritic Cells and Reduces the Dose of TB Drugs against Mycobacterium tuberculosis.

    Science.gov (United States)

    Khan, Nargis; Pahari, Susanta; Vidyarthi, Aurobind; Aqdas, Mohammad; Agrewala, Javed N

    2016-01-01

    Tuberculosis (TB) is one of the leading killer infectious diseases. TB patients are inflicted with devastating side effects and the toxicity of a lengthy drug regime, accentuating an urgent need to explore newer and safer treatment methods. Recently, an improved understanding of host-pathogen interaction has opened new avenues for TB treatment, including immunotherapy. This has emboldened us to devise a novel strategy to restrict Mycobacterium tuberculosis(Mtb) growth by activating dendritic cells (DCs) through the NOD-2 and TLR-4 molecules of innate immunity. Triggered DCs show a robust release of cytokines and nitric oxide, autophagy and improved migration towards the lymph nodes, and consequently impede the intracellular survival of Mtb. Of note, this approach enhanced the efficacy of TB drugs by reducing their dose to a 5-fold lesser concentration than recommended. In vivo administration of ligands of NOD-2 (NOD-2L) and TLR-4 (TLR-4L) substantially increased the pool of effector memory CD4 and CD8 T cells. Additionally, NOD-2L and TLR-4L, in conjunction with the reduced dose of isoniazid, substantially declined the Mtb burden in the lungs. In the future, adjunct therapy involving NOD-2L, TLR-4L and TB drugs may have enough potential to reduce the dose and duration of treatment of TB patients. © 2015 S. Karger AG, Basel.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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 ...

  16. 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.

  17. 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 ...

  18. 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

  19. Molecular pharmacology and ligand docking studies reveal a single amino acid difference between mouse and human serotonin 5-HT2A receptors that impacts behavioral translation of novel 4-phenyl-2-dimethylaminotetralin ligands.

    Science.gov (United States)

    Canal, Clinton E; Cordova-Sintjago, Tania; Liu, Yue; Kim, Myong S; Morgan, Drake; Booth, Raymond G

    2013-12-01

    During translational studies to develop 4-phenyl-2-dimethylaminotetralin (PAT) compounds for neuropsychiatric disorders, the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of the analog 6-hydroxy-7-chloro-PAT (6-OH-7-Cl-PAT) demonstrated unusual pharmacology at serotonin (5-HT) 5-HT2 G protein-coupled receptors (GPCRs). The enantiomers had similar affinities (Ki) at human (h) 5-HT2A receptors (≈ 70 nM). In an in vivo mouse model of 5-HT2A receptor activation [(±)-(2,5)-dimethoxy-4-iodoamphetamine (DOI)-elicited head twitch], however, (-)-6-OH-7-Cl-PAT was about 5-fold more potent than the (+)-enantiomer at attenuating the DOI-elicited response. It was discovered that (+)-6-OH-7-Cl-PAT (only) had ≈ 40-fold-lower affinity at mouse (m) compared with h5-HT2A receptors. Molecular modeling and computational ligand docking studies indicated that the 6-OH moiety of (+)- but not (-)-6-OH-7-Cl-PAT could form a hydrogen bond with serine residue 5.46 of the h5-HT2A receptor. The m5-HT2A as well as m5-HT2B, h5-HT2B, m5-HT2C, and h5-HT2C receptors have alanine at position 5.46, obviating this interaction; (+)-6-OH-7-Cl-PAT also showed ≈ 50-fold lower affinity than (-)-6-OH-7-Cl-PAT at m5-HT2C and h5-HT2C receptors. Mutagenesis studies confirmed that 5-HT2A S5.46 is critical for (+)- but not (-)-6-OH-7-Cl-PAT binding, as well as function. The (+)-6-OH-7-Cl-PAT enantiomer showed partial agonist effects at h5-HT2A wild-type (WT) and m5-HT2A A5.46S point-mutated receptors but did not activate m5-HT2A WT and h5-HT2A S5.46A point-mutated receptors, or h5-HT2B, h5-HT2C, and m5-HT2C receptors; (-)-6-OH-7-Cl-PAT did not activate any of the 5-HT2 receptors. Experiments also included the (2R,4S)-trans-(+)- and (2S,4R)-trans-(-)-enantiomers of 6-methoxy-7-chloro-PAT to validate hydrogen bonding interactions proposed for the corresponding 6-OH analogs. Results indicate that PAT ligand three-dimensional structure impacts target receptor binding and translational outcomes

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

    Directory of Open Access Journals (Sweden)

    Nadigel Jessica

    2011-11-01

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

  1. 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

  2. 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.

  3. 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...

  4. 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....

  5. Role of Lanthanide-Ligand bonding in the magnetization relaxation ...

    Indian Academy of Sciences (India)

    Ligand bonding. Our calculations transpire comparatively improved Single-Ion Magnet (SIM) behaviour for carbene analogues due to the more axially compressed trigonal prismatic ligand environment. Furthermore, our detailed Mulliken charge, ...

  6. 123I-5-I-R91150, a new single-photon emission tomography ligand for 5-HT2A receptors: influence of age and gender in healthy subjects

    International Nuclear Information System (INIS)

    Baeken, C.; D'haenen, H.; Flamen, P.; Bossuyt, A.; Mertens, J.; Terriere, D.; Chavatte, K.; Boumon, R.

    1998-01-01

    5-HT 2A receptors have been implicated in the pathophysiology of mood disorders and in the therapeutic effect of the so-called atypical antipsychotics. Recently, a new radioiodinated ligand with high affinity and selectivity for serotonin 5-HT 2A receptors, 123 iodinated 4-amino-N-1-[3-(4-fluorophenoxy)propyl-4-methyl-4-piperidinyl] 5-iodo-2-methoxybenzamide ( 123 I-5-I-R91150), has been developed and has been shown to be suitable for single-photon emission tomography (SPET) imaging. In this study the influence of age and gender on the ligand binding was investigated in normal volunteers. One hundred and fifty MBq of 123 I-5-I-R91150 was administered to 26 normal volunteers (13 females and 13 males) with an age range of 23-60 years. SPET imaging was performed with a triple-headed gamma camera. For semi-quantitative analysis, ratios of ligand binding in different regions of interest to the binding in the cerebellum were calculated. Mean ratios of 1.7 were obtained. No gender difference was demonstrated. 5-HT 2A binding was shown to decline with age. Over an age range of 40 years a reduction in ligand binding of 42%±7% was found. These results are in agreement with in vitro and positron emission tomography findings of a decline in 5-HT 2A receptor binding with age. The findings confirm the suitability of 123 I-5-I-R91150 for SPET imaging of 5-HT 2A receptors, and highlight the necessity for age-matched controls in clinical studies. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Jincheng Chen

    2015-07-01

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

  8. 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

  9. The tylosin resistance gene tlrB of Streptomyces fradiae encodes a methyltransferase that targets G748 in 23S rRNA

    DEFF Research Database (Denmark)

    Liu, M; Kirpekar, F; Van Wezel, G P

    2000-01-01

    tlrB is one of four resistance genes encoded in the operon for biosynthesis of the macrolide tylosin in antibiotic-producing strains of Streptomyces fradiae. Introduction of tlrB into Streptomyces lividans similarly confers tylosin resistance. Biochemical analysis of the rRNA from the two...... Streptomyces species indicates that in vivo TlrB modifies nucleotide G748 within helix 35 of 23S rRNA. Purified recombinant TlrB retains its activity and specificity in vitro and modifies G748 in 23S rRNA as well as in a 74 nucleotide RNA containing helix 35 and surrounding structures. Modification...... is dependent on the presence of the methyl group donor, S-adenosyl methionine. Analysis of the 74-mer RNA substrate by biochemical and mass spectrometric methods shows that TlrB adds a single methyl group to the base of G748. Homologues of TlrB in other bacteria have been revealed through database searches...

  10. Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation.

    Science.gov (United States)

    Huang, Zijing; Zhou, Tian; Sun, Xiaowei; Zheng, Yingfeng; Cheng, Bing; Li, Mei; Liu, Xialin; He, Chang

    2018-01-01

    Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases.

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

    Directory of Open Access Journals (Sweden)

    James F Curtin

    2009-01-01

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

  12. Ruthenium(II) bipyridine complexes bearing quinoline-azoimine (NN'N″) tridentate ligands: synthesis, spectral characterization, electrochemical properties and single-crystal X-ray structure analysis.

    Science.gov (United States)

    Al-Noaimi, Mousa; Abdel-Rahman, Obadah S; Fasfous, Ismail I; El-khateeb, Mohammad; Awwadi, Firas F; Warad, Ismail

    2014-05-05

    Four octahedral ruthenium(II) azoimine-quinoline complexes having the general molecular formula [Ru(II)(L-Y)(bpy)Cl](PF6) {L-Y=YC6H4N=NC(COCH3)=NC9H6N, Y=H (1), CH3 (2), Br (3), NO2 (4) and bpy=2,2'-bipyrdine} were synthesized. The azoimine-quinoline based ligands behave as NN'N″ tridentate donors and coordinated to ruthenium via azo-N', imine-N' and quinolone-N″ nitrogen atoms. The composition of the complexes has been established by elemental analysis, spectral methods (FT-IR, electronic, (1)H NMR, UV/Vis and electrochemical (cyclic voltammetry) techniques. The crystal structure of complex 1 is reported. The Ru(II) oxidation state is greatly stabilized by the novel tridentate ligands, showing Ru(III/II) couples ranging from 0.93-1.27 V vs. Cp2Fe/Cp2Fe(+). The absorption spectrum of 1 in dichloromethane was modeled by time-dependent density functional theory (TD-DFT). Copyright © 2014 Elsevier B.V. All rights reserved.

  13. KIR alloreactivity based on the receptor-ligand model is associated with improved clinical outcomes of allogeneic hematopoietic stem cell transplantation: Result of single center prospective study.

    Science.gov (United States)

    Park, Silvia; Kim, Kihyun; Jang, Jun Ho; Kim, Seok Jin; Kim, Won Seog; Kang, Eun-Suk; Jung, Chul Won

    2015-09-01

    Receptors on natural killer (NK) cells, named killer immunoglobulin-like receptors (KIRs), recognize HLA class I alleles. Patients (n=59) who received allogeneic hematopoietic stem cell transplantation (HSCT) from either a related (n=17) or unrelated donor (n=42) in Samsung Medical Center (Seoul, South Korea) were included. KIR mismatch was defined as incompatibility between the donor KIR and recipient KIR ligand (receptor-ligand model), and all cases were classified into the two broad haplotypes of KIR A and B. Patients with acute leukemia (n=51, 86.4%) or myelodysplastic syndrome (n=8, 13.6%) were included. Peripheral blood was used as the source of stem cells in all patients. Kaplan-Meier survival curves for overall survival (OS), disease-free survival (DFS), and cumulative incidence of relapse (CIR) favored recipients with a KIR-mismatched donor, although the differences were not statistically significant. In multivariate analysis, KIR mismatch was an independent prognostic indicator of a better OS (P=0.010, HR=0.148, 95% CI 0.034-0.639), DFS (P=0.022, HR=0.237, 95% CI 0.069-0.815), and CIR (P=0.031, HR=0.117, 95% CI 0.017-0.823). OS, DFS, and CIR did not differ significantly between the KIR A and B haplotypes. Copyright © 2015. Published by Elsevier Inc.

  14. 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).

  15. 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.

  16. TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Guan-Lin [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Wu, Jing-Yiing [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Yeh, Chang-Ching [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Kuo, Cheng-Chin, E-mail: kuocc@nhri.org.tw [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2016-05-13

    Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

  17. Reactivation of latent HIV-1 in central memory CD4⁺ T cells through TLR-1/2 stimulation.

    Science.gov (United States)

    Novis, Camille L; Archin, Nancie M; Buzon, Maria J; Verdin, Eric; Round, June L; Lichterfeld, Mathias; Margolis, David M; Planelles, Vicente; Bosque, Alberto

    2013-10-24

    Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4⁺ T cells. Memory CD4⁺ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4⁺ T cells has not been studied. We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4⁺ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation. Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.

  18. Scavenger Receptor SREC-I Mediated Entry of TLR4 into Lipid Microdomains and Triggered Inflammatory Cytokine Release in RAW 264.7 Cells upon LPS Activation

    Science.gov (United States)

    Murshid, Ayesha; Gong, Jianlin; Prince, Thomas; Borges, Thiago J.; Calderwood, Stuart K.

    2015-01-01

    Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages. PMID:25836976

  19. NOD1 cooperates with TLR2 to enhance T cell receptor-mediated activation in CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Blandine C Mercier

    Full Text Available Pattern recognition receptors (PRR, like Toll-like receptors (TLR and NOD-like receptors (NLR, are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR. This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions.

  20. Four new single nucleotide polymorphisms (SNPs) of toll-like ...

    African Journals Online (AJOL)

    In order to reveal the single nucleotide polymorphisms (SNPs), genotypes and allelic frequencies of each mutation site of TLR7 gene in Chinese native duck breeds, SNPs of duck TLR7 gene were detected by DNA sequencing. The genotypes of 465 native ducks from eight key protected duck breeds were determined by ...

  1. 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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Laleh Sharifi

    2017-07-01

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

  4. 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.

  5. 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.

  6. Antagonism of antiviral and allogeneic activity of a human public CTL clonotype by a single altered peptide ligand: implications for allograft rejection

    Energy Technology Data Exchange (ETDEWEB)

    Ely, Lauren K.; Green, Katherine J.; Beddoe, Travis; Clements, Craig S.; Miles, John J.; Bottomley, Stephen P.; Zernich, Danielle; Kjer-Nielsen, Lars; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R. (Monash)

    2010-06-30

    Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801{sup +} individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801{sup FLRGRAYGL}. Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 {micro}M, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Hassan Alizadeh

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

  9. 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

  10. 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.

    Directory of Open Access Journals (Sweden)

    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

  11. Neonatal BCG vaccination influences cytokine responses to Toll-like receptor ligands and heterologous antigens.

    Science.gov (United States)

    Freyne, B; Donath, S; Germano, S; Gardiner, K; Casalaz, D; Robins-Browne, R M; Amenyogbe, N; Messina, N L; Netea, M G; Flanagan, K L; Kollmann, T; Curtis, N

    2018-02-03

    Bacille Calmette-Guérin (BCG) vaccination is associated with a reduction in all-cause infant mortality in high-mortality settings. The underlying mechanisms remain uncertain but long-term modulation of the innate immune response (trained immunity) may be involved. Whole blood, collected 7 days post randomisation from 212 neonates enrolled in a randomised trial of neonatal BCG vaccination, was stimulated with killed pathogens and Toll-like receptor (TLR) ligands to interrogate cytokine responses. BCG-vaccinated infants had increased production of IL-6 in unstimulated samples and decreased production of IL-1ra, IL-6, and IL-10 and the chemokines MIP-1α, MIP-1β, MCP-1 following stimulation with peptidoglycan (TLR2) and R848 (TLR7/8). BCG-vaccinated infants also had decreased MCP-1 responses following stimulation with heterologous pathogens. Sex and maternal BCG vaccination status interacted with neonatal BCG vaccination. Neonatal BCG vaccination influences cytokine responses to TLR ligands and heterologous pathogens. This effect is characterised by decreased anti-inflammatory cytokine and chemokine responses in the context of higher levels of IL-6 in unstimulated samples. This supports the hypothesis that BCG vaccination modulates the innate immune system. Further research is warranted to determine if there is an association between these findings and the beneficial non-specific (heterologous) effects of BCG vaccine on all-cause mortality.

  12. Direct versus ligand-exchange synthesis of [PtAg28(BDT)12(TPP)4]4-nanoclusters: effect of a single-atom dopant on the optoelectronic and chemical properties.

    Science.gov (United States)

    Bootharaju, Megalamane S; Kozlov, Sergey M; Cao, Zhen; Harb, Moussab; Parida, Manas R; Hedhili, Mohamed N; Mohammed, Omar F; Bakr, Osman M; Cavallo, Luigi; Basset, Jean-Marie

    2017-07-13

    Heteroatom doping of atomically precise nanoclusters (NCs) often yields a mixture of doped and undoped products of single-atom difference, whose separation is extremely difficult. To overcome this challenge, novel synthesis methods are required to offer monodisperse doped NCs. For instance, the direct synthesis of PtAg 28 NCs produces a mixture of [Ag 29 (BDT) 12 (TPP) 4 ] 3- and [PtAg 28 (BDT) 12 (TPP) 4 ] 4- NCs (TPP: triphenylphosphine; BDT: 1,3-benzenedithiolate). Here, we designed a ligand-exchange (LE) strategy to synthesize single-sized, Pt-doped, superatomic Ag NCs [PtAg 28 (BDT) 12 (TPP) 4 ] 4- by LE of [Pt 2 Ag 23 Cl 7 (TPP) 10 ] NCs with BDTH 2 (1,3-benzenedithiol). The doped NCs were thoroughly characterized by optical and photoelectron spectroscopy, mass spectrometry, total electron count, and time-dependent density functional theory (TDDFT). We show that the Pt dopant occupies the center of the PtAg 28 cluster, modulates its electronic structure and enhances its photoluminescence intensity and excited-state lifetime, and also enables solvent interactions with the NC surface. Furthermore, doped NCs showed unique reactivity with metal ions - the central Pt atom of PtAg 28 could not be replaced by Au, unlike the central Ag of Ag 29 NCs. The achieved synthesis of single-sized PtAg 28 clusters will facilitate further applications of the LE strategy for the exploration of novel multimetallic NCs.

  13. Direct versus ligand-exchange synthesis of [PtAg28(BDT)12(TPP)4]4− nanoclusters: effect of a single-atom dopant on the optoelectronic and chemical properties

    KAUST Repository

    Bootharaju, Megalamane Siddaramappa

    2017-06-07

    Heteroatom doping of atomically precise nanoclusters (NCs) often yields a mixture of doped and undoped products of single-atom difference, whose separation is extremely difficult. To overcome this challenge, novel synthesis methods are required to offer monodisperse doped NCs. For instance, the direct synthesis of PtAg28 NCs produces a mixture of [Ag29(BDT)12(TPP)4]3- and [PtAg28(BDT)12(TPP)4]4- NCs (TPP: triphenylphosphine; BDT: 1,3-benzenedithiolate). Here, we designed a ligand-exchange (LE) strategy to synthesize single-sized, Pt-doped, superatomic Ag NCs [PtAg28(BDT)12(TPP)4]4- by LE of [Pt2Ag23Cl7(TPP)10] NCs with BDTH2 (1,3-benzenedithiol). The doped NCs were thoroughly characterized by optical and photoelectron spectroscopy, mass spectrometry, total electron count, and time-dependent density functional theory (TDDFT). We show that the Pt dopant occupies the center of the PtAg28 cluster, modulates its electronic structure and enhances its photoluminescence intensity and excited-state lifetime, and also enables solvent interactions with the NC surface. Furthermore, doped NCs showed unique reactivity with metal ions - the central Pt atom of PtAg28 could not be replaced by Au, unlike the central Ag of Ag29 NCs. The achieved synthesis of single-sized PtAg28 clusters will facilitate further applications of the LE strategy for the exploration of novel multimetallic NCs.

  14. Lability and Basicity of Bipyridine-Carboxylate-Phosphonate Ligand Accelerate Single-Site Water Oxidation by Ruthenium-Based Molecular Catalysts.

    Science.gov (United States)

    Shaffer, David W; Xie, Yan; Szalda, David J; Concepcion, Javier J

    2017-11-01

    A critical step in creating an artificial photosynthesis system for energy storage is designing catalysts that can thrive in an assembled device. Single-site catalysts have an advantage over bimolecular catalysts because they remain effective when immobilized. Hybrid water oxidation catalysts described here, combining the features of single-site bis-phosphonate catalysts and fast bimolecular bis-carboxylate catalysts, have reached turnover frequencies over 100 s -1 , faster than both related catalysts under identical conditions. The new [(bpHc)Ru(L) 2 ] (bpH 2 cH = 2,2'-bipyridine-6-phosphonic acid-6'-carboxylic acid, L = 4-picoline or isoquinoline) catalysts proceed through a single-site water nucleophilic attack pathway. The pendant phosphonate base mediates O-O bond formation via intramolecular atom-proton transfer with a calculated barrier of only 9.1 kcal/mol. Additionally, the labile carboxylate group allows water to bind early in the catalytic cycle, allowing intramolecular proton-coupled electron transfer to lower the potentials for oxidation steps and catalysis. That a single-site catalyst can be this fast lends credence to the possibility that the oxygen evolving complex adopts a similar mechanism.

  15. Lability and Basicity of Bipyridine-Carboxylate-Phosphonate Ligand Accelerate Single-Site Water Oxidation by Ruthenium-Based Molecular Catalysts

    International Nuclear Information System (INIS)

    Shaffer, David W.; Xie, Yan; Szalda, David J.; Concepcion, Javier J.

    2017-01-01

    Here, a critical step in creating an artificial photosynthesis system for energy storage is designing catalysts that can thrive in an assembled device. Single-site catalysts have an advantage over bimolecular catalysts because they remain effective when immobilized. Hybrid water oxidation catalysts described here, combining the features of single-site bis-phosphonate catalysts and fast bimolecular bis-carboxylate catalysts, have reached turnover frequencies over 100 s –1 , faster than both related catalysts under identical conditions. The new [(bpHc)Ru(L) 2 ] (bpH 2 cH = 2,2'-bipyridine-6-phosphonic acid-6'-carboxylic acid, L = 4-picoline or isoquinoline) catalysts proceed through a single-site water nucleophilic attack pathway. The pendant phosphonate base mediates O–O bond formation via intramolecular atom-proton transfer with a calculated barrier of only 9.1 kcal/mol. Additionally, the labile carboxylate group allows water to bind early in the catalytic cycle, allowing intramolecular proton-coupled electron transfer to lower the potentials for oxidation steps and catalysis. That a single-site catalyst can be this fast lends credence to the possibility that the oxygen evolving complex adopts a similar mechanism.

  16. MD-2 Determinants of Nickel and Cobalt-Mediated Activation of Human TLR4

    Science.gov (United States)

    Oblak, Alja; Pohar, Jelka; Jerala, Roman

    2015-01-01

    Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the –independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation. PMID:25803856

  17. MD-2 determinants of nickel and cobalt-mediated activation of human TLR4.

    Directory of Open Access Journals (Sweden)

    Alja Oblak

    Full Text Available Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the -independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Steffen Bank

    Full Text Available The inflammatory bowel diseases (IBD, Crohn's disease (CD and ulcerative colitis (UC, result from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the genetic heritage.Using a candidate gene approach, 39 mainly functional single nucleotide polymorphisms (SNPs in 26 genes regulating inflammation were assessed in a clinical homogeneous group of severely diseased patients consisting of 624 patients with CD, 411 patients with UC and 795 controls. The results were analysed using logistic regression.Sixteen polymorphisms in 13 genes involved in regulation of inflammation were associated with risk of CD and/or UC (p ≤ 0.05. The polymorphisms TLR2 (rs1816702, NFKB1 (rs28362491, TNFRSF1A (rs4149570, IL6R (rs4537545, IL23R (rs11209026 and PTPN22 (rs2476601 were associated with risk of CD and the polymorphisms TLR2 (rs1816702, TLR4 (rs1554973 and rs12377632, TLR9 (rs352139, LY96 (rs11465996, NFKBIA (rs696, TNFA (rs1800629, TNFRSF1A (rs4149570, IL10 (rs3024505, IL23R (rs11209026, PTPN22 (rs2476601 and PPARG (rs1801282 were associated with risk of UC. When including all patients (IBD the polymorphisms TLR2 (rs4696480 and rs1816702, TLR4 (rs1554973 and rs12377632, TLR9 (rs187084, TNFRSF1A (rs4149570, IL6R (rs4537545, IL10 (rs3024505, IL23R (rs11209026 and PTPN22 (rs2476601 were associated with risk. After Bonferroni correction for multiple testing, both the homozygous and the heterozygous variant genotypes of IL23R G>A(rs11209026 (OR(CD,adj: 0.38, 95% CI: 0.21-0.67, p = 0.03; OR(IBD,adj 0.43, 95% CI: 0.28-0.67, p = 0.007 and PTPN22 1858 G>A(rs2476601 (OR(CD,unadj 0.54, 95% CI: 0.41-0.72, p = 7*10-4; OR(IBD,unadj: 0.61, 95% CI: 0.48-0.77, p = 0.001 were associated with reduced risk of CD.The biological effects of the studied polymorphisms suggest that genetically determined high inflammatory response was associated with increased risk of CD. The many SNPs found in TLRs

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

    OpenAIRE

    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 obstruction 1 . 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 bacteria 2 . The ability of bacterial products to act as adjuvants 3 suggests they might promote asthma by priming allergic sensitization to inhaled allergens. In support of this idea, house dust e...

  1. DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands

    DEFF Research Database (Denmark)

    End, Caroline; Bikker, Floris; Renner, Marcus

    2009-01-01

    Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-kappaB activation and to the pathogenesis of Crohn's disease. Here, we aimed ...... that DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-kappaB activation....

  2. Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation.

    Science.gov (United States)

    Ullah, Md Ashik; Loh, Zhixuan; Gan, Wan Jun; Zhang, Vivian; Yang, Huan; Li, Jian Hua; Yamamoto, Yasuhiko; Schmidt, Ann Marie; Armour, Carol L; Hughes, J Margaret; Phipps, Simon; Sukkar, Maria B

    2014-08-01

    The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization. To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization. TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively. The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation. The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  3. TLR2, TLR4 and CD14 recognize venom-associated molecular patterns from Tityus serrulatus to induce macrophage-derived inflammatory mediators.

    Directory of Open Access Journals (Sweden)

    Karina Furlani Zoccal

    Full Text Available Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense Tityus serrulatus venom (TsV and its major component, toxin 1 (Ts1, to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.

  4. Cytosolic phospholipase A2 modulates TLR2 signaling in synoviocytes.

    Directory of Open Access Journals (Sweden)

    Randi M Sommerfelt

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

  5. Modulation of TLR3/TLR4 inflammatory signaling by the GABAB receptor agonist baclofen in glia and immune cells: relevance to therapeutic effects in multiple sclerosis

    Directory of Open Access Journals (Sweden)

    Tadhg eCrowley

    2015-07-01

    Full Text Available The GABAB receptor agonist, baclofen, is used to treat muscle tightness and cramping caused by spasticity in a number of disorders including Multiple Sclerosis (MS, but its precise mechanism of action is unknown. Neuroinflammation drives the central pathology in MS and is mediated by both immunoreactive glial cells and invading lymphocytes. Furthermore, a body of data indicates that the Toll-like receptor (TLR family of innate immune receptors is implicated in MS progression. In the present study we investigated whether modulation of GABAB receptors using baclofen can exert anti-inflammatory effects by targeting TLR3 and(or TLR4-induced inflammatory signaling in murine glial cells and human peripheral blood mononuclear cells (PBMCs isolated from healthy control individuals and patients with the relapse-remitting (RR form of MS. TLR3 and TLR4 stimulation promoted the nuclear sequestration of NF-κB and pro-inflammatory cytokine expression in murine glia, while TLR4, but not TLR3, promoted pro-inflammatory cytokine expression in PBMCs isolated from both healthy donors and RR-MS patients. Importantly, this effect was exacerbated in RR-MS patient immune cells. We present further evidence that baclofen dose-dependently attenuated TLR3- and TLR4-induced inflammatory signaling in primary glial cells. Pre-exposure of PBMCs isolated from healthy donors to baclofen attenuated TLR4-induced TNF-α expression, but did not affect TLR4-induced TNF-α expression in RR-MS patient PBMCs. Interestingly, mRNA expression of the GABAB receptor was reduced in PBMCs from RR-MS donors when compared to healthy controls, an effect that might contribute to the differential sensitivity to baclofen seen in healthy and RR-MS patient cells. Overall these findings indicate that baclofen differentially regulates TLR3 and TLR4 signaling in glia and immune cells, and offers insight on the role of baclofen in the treatment of neuroinflammatory disease states including MS.

  6. Ligands in PSI structures

    International Nuclear Information System (INIS)

    Kumar, Abhinav; Chiu, Hsiu-Ju; Axelrod, Herbert L.; Morse, Andrew; Elsliger, Marc-André; Wilson, Ian A.; Deacon, Ashley

    2010-01-01

    A survey of the types and frequency of ligands that are bound to PSI structures is analyzed as well as their utility in functional annotation of previously uncharacterized proteins. Approximately 65% of PSI structures report some type of ligand(s) that is bound in the crystal structure. Here, a description is given of how such ligands are handled and analyzed at the JCSG and a survey of the types, variety and frequency of ligands that are observed in the PSI structures is also compiled and analyzed, including illustrations of how these bound ligands have provided functional clues for annotation of proteins with little or no previous experimental characterization. Furthermore, a web server was developed as a tool to mine and analyze the PSI structures for bound ligands and other identifying features

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

    African Journals Online (AJOL)

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

  8. DMPD: New insights into the regulation of TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16698941 New insights into the regulation of TLR signaling. Miggin SM, O'Neill LA. ...J Leukoc Biol. 2006 Aug;80(2):220-6. Epub 2006 May 12. (.png) (.svg) (.html) (.csml) Show New insights into the regulation of TLR sig...naling. PubmedID 16698941 Title New insights into the regulation of TLR signaling.

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

    Lifescience Database Archive (English)

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

  10. DMPD: Signal integration between IFNgamma and TLR signalling pathways in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16920490 Signal integration between IFNgamma and TLR signalling pathways in macroph...tml) (.csml) Show Signal integration between IFNgamma and TLR signalling pathways in macrophages. PubmedID 1...6920490 Title Signal integration between IFNgamma and TLR signalling pathways in

  11. DMPD: TLR3 in antiviral immunity: key player or bystander? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16027039 TLR3 in antiviral immunity: key player or bystander? Schroder M, Bowie AG.... Trends Immunol. 2005 Sep;26(9):462-8. (.png) (.svg) (.html) (.csml) Show TLR3 in antiviral immunity: key pl...ayer or bystander? PubmedID 16027039 Title TLR3 in antiviral immunity: key player or bystander? Authors Schr

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

    African Journals Online (AJOL)

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

  13. Substituted biurets as uranophilic ligands: A facile DMSO-induced conversion of a 1:1 into a 2:1 uranyl-ligand complex

    Energy Technology Data Exchange (ETDEWEB)

    Potts, K.T.; O' Brien, J.J.; Tham, F.S. (Rensselaer Polytechnic Inst., Troy, NY (United States))

    1990-01-01

    1,5-Bis(6-(1-ethoxycarbonyl-3-thioureido)-2-pyridindiyl)biuret and uranyl acetate gave a crystalline 1:1 uranyl-ligand complex which, on crystallization from DMSO, underwent rearrangement to a crystalline 2:1 uranyl-ligand complex and a stoichiometric amount of the uncomplexed ligand. Spectral characteristics of these ligands and their uranyl complexes together with single crystal x-ray data for the uranyl-ligand complexes are described.

  14. Flavonoids Affect Host-Microbiota Crosstalk through TLR Modulation

    Directory of Open Access Journals (Sweden)

    Francisco J. Pérez-Cano

    2014-10-01

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

  15. Toll-like receptor-4 (TLR-4) expression on polymorphonuclear ...

    African Journals Online (AJOL)

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

  16. Toll-like receptor-4 (TLR-4) expression on polymorphonuclear ...

    African Journals Online (AJOL)

    reading 5

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

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

    Indian Academy of Sciences (India)

    Domain combination of the vertebrate-like TLR gene family: implications for their origin and evolution. Baojun Wu, Tianxiao Huan, Jing Gong, Pin Zhou and Zengliang Bai. J. Genet. 90, 401–408. Figure 1. Unrooted NJ phylogenetic tree of P-Toll proteins from selected phyla based on TIR domains. The putative V-TIR protein.

  18. Synthesis and characterization of radioiodinated MD-230254. A new ligand for potential imaging of monoamine oxidase B activity by single photon emission computed tomography

    International Nuclear Information System (INIS)

    Hirata, Masahiko; Kagawa, Shinya; Yoshimoto, Mitsuyoshi; Ohmomo, Yoshiro

    2002-01-01

    A series of iodinated analogues of MD-230254 was synthesized and evaluated for inhibitory potency and selectivity toward monoamine oxidase B (MAO-B). Among them, 5-[4-(2-iodobenzyloxy)phenyl]-3(cyanoethyl)-1,3,4-oxadiazole-2(3H)one (2-IBPO) was found to have high inhibitory potency and selectivity toward MAO-B (IC 50 =2.0 n M , MAO-A/MAO-B>50000). Analysis of the inhibition kinetics indicated that 2-IBPO acts in a two-step mechanism as a competitive, slow, and tight-binding inhibitor of MAO-B with a Ki value of 2.4 n M and an overall Ki value at an equilibrium of 3.8 n M . The new radioligand for MAO-B, [ 125 I]2-IBPO was conveniently synthesized from a tributylstannyl precursor by an iododestannylation reaction using sodium [ 125 I]iodide and hydrogen peroxide with high radiochemical yield. The in vivo tissue distribution studies of [ 125 I]2-IBPO demonstrated its high initial uptake and prolonged retention in the brain. A selective interaction of [ 125 I]2-IBPO with MAO-B was confirmed by the pretreatment experiment with well known MAO specific inhibitors, l-deprenyl, Ro-16-6491, clorgyline, and Ro-41-1049. These very desirable characteristics of [ 125 I]2-IBPO suggested that a 123 I-labeled counterpart, [ 123 I]2-IBPO, would have great potential in vivo studies of MAO-B in the human brain with single photon emission computed tomography (SPECT). (author)

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

    Science.gov (United States)

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

    2016-08-01

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

  20. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts.

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    Olga N Karpus

    Full Text Available CD55 (decay-accelerating factor is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS. CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS.FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (dsRNA sensors melanoma differentiation-associated gene 5 (MDA5 and retinoic acid-inducible gene-I (RIG-I. CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry.CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA, osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1β and especially by the TLR3 ligand poly(I:C. Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55.Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocytes.

  1. 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.

  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. Mycobacterium tuberculosis and TLR2 Agonists Inhibit Induction of Type I IFN and Class I MHC Antigen Cross Processing by TLR9

    OpenAIRE

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

    2010-01-01

    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-α/β), which enhances MHC-I Ag cross processing, but lipoproteins that signal through TLR2 do not induce IFN-α/β. In these studies we observed that M. tuberculosis, which expresses agonists of both TLR9 and TLR2, did not induce product...

  4. Association of TLR4 polymorphisms with subclinical mastitis in Brazilian holsteins

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    Adriano Queiroz de Mesquita

    2012-06-01

    Full Text Available The identification of dairy cows with greater or lower potential to develop mastits has been pursued for many years among different segments of the milk industry, including governmental organizations. Genomic studies have suggested that Single Nucleotide Polymorphisms (SNPs within the pattern recognition receptors (PRR could lead to different responses to pathogens, and consequently result in mastitis resistance or susceptibility. To investigate whether toll like receptor 4 (TLR4 gene is associated with subclinical mastitis in Holstein cows from a property in the state of Goiás, Brazil, TaqMan allelic discrimination and somatic cell count were performed. One hundred and fifty milk samples were analyzed for SCC and centesimal composition. Twenty percent of those samples with SCC above 200,000 (n=13 were screened for real-time PCR identification of microorganisms and blood samples were genotyped for TLR4 SNPs. There was a higher prevalence of Gram-positive bacteria in the analyzed samples (88.9% and animals that had the combined genotypes AACCCC, GGTCGG and GACCGC presented the lowest somatic cell scores, and consequently those genotypes have the potential to be applied as molecular markers for assisted animal selection to improve milk quality.

  5. Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

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    Semeraro, Fabrizio; Ammollo, Concetta T.; Morrissey, James H.; Dale, George L.; Friese, Paul; Esmon, Naomi L.

    2011-01-01

    The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. PMID:21673343

  6. 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.

  7. 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.

  8. A TLR3-Specific Adjuvant Relieves Innate Resistance to PD-L1 Blockade without Cytokine Toxicity in Tumor Vaccine Immunotherapy

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    Yohei Takeda

    2017-05-01

    Full Text Available Cancer patients having anti-programmed cell death-1 (PD-1/PD ligand 1 (L1-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs via Toll-like receptor 3 (TLR3. The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF3-IFN-β axis in DCs exclusively participated in CD8+ T cell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK cell recruitment and functionality. Human ex vivo studies disclosed that ARNAX+antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs, supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.

  9. Single nucleotide polymorphisms of Toll-like receptor 4 decrease the risk of development of hepatocellular carcinoma.

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    Shi Minmin

    Full Text Available BACKGROUND: Toll-like receptor 4 (TLR4 is a key innate immunity receptor that initiates an inflammatory response. Growing evidence suggests that mutation of TLR4 gene may play a role in the development of cancers. This study aimed to investigate the temporal relationship of single nucleotide polymorphisms of TLR4 and the risk of hepatocellular carcinoma, a single center-based case-control study was conducted. METHODS: A systematic genetic analysis of sequence variants of TLR4 by evaluating ten single-nucleotide polymorphisms was performed from 216 hepatocellular carcinoma cases and 228 controls. RESULTS: Six single nucleotide polymorphisms of the TLR4 in the 5'-untranslated region and intron were associated with risk of hepatocellular carcinoma. Individuals carrying the heterozygous genotypes for the rs10759930, rs2737190, rs10116253, rs1927914, rs12377632 and rs1927911 had significantly decreased risk of hepatocellular carcinoma (adjusted odds ratio [OR], from 0.527 to 0.578, P<0.01 comparing with those carrying wild-type homozygous genotypes. In haplotype analysis, one haplotype (GCCCTTAG of TLR4 was associated significantly with decrease of the occurrence of hepatocellular carcinoma (OR, 0.556, 95% confidence interval [CI], 0.407-0.758, P = 0.000. CONCLUSIONS: Collectively, these results suggested that the risk of hepatocellular carcinoma was associated with TLR4 sequence variation. TLR4 single nucleotide polymorphisms may play an important protective role in the development of hepatocellular carcinoma.

  10. 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.

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

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    Feixiang Wu

    2012-01-01

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

  12. TLR9 expression in glioma tissues correlated to glioma progression and the prognosis of GBM patients

    International Nuclear Information System (INIS)

    Wang, Chao; Cao, Shouqiang; Yan, Ying; Ying, Qiao; Jiang, Tao; Xu, Ke; Wu, Anhua

    2010-01-01

    Our study aims to evaluate the expression of TLR9 in glioma tissues, examine the association between TLR9 expression, clinicopathological variables, and glioma patient outcome, we further characterized the direct effects of TLR9 agonist CpG ODN upon the proliferation and invasion of glioma cells in vitro. RT-PCR and immunofluorescence were used to determine the expression of TLR9 in glioma cell lines and clinical glioma samples. Tissue microarry and immunohistochemistry were applied to evaluated TLR9 expression in 292 newly diagnosed glioma and 13 non-neoplastic brain tissues. We further investigated the effect of CpG ODN on the proliferation and invasion of glioma cells in vitro with MTT assays and matrigel transwell assay respectively. RT-PCR showed that TLR9 expressed in all the glioma samples and glioma cell lines we examined. The tissue array analysis indicated that TLR9 expression is correlated with malignancy of glioma (p < 0.01). Multivariate Cox regression analysis revealed that TLR9 expression is an independent prognostic factor for PFS of GBM patients(P = 0.026). TLR9 agonist CpG ODN has no significant effect on glioma proliferation, but matrigel transwell analysis showed that TLR9 agonist CpG ODN can significantly enhance glioma invasion in vitro. Our data indicated that TLR9 expression increases according to the histopathological grade of glioma, and the TLR9 expression level is related to the PFS of GBM patients. In addition, our findings warrant caution in the directly injection of TLR9 agonist CpG ODN into glioma tissues for the glioma immunotherapy

  13. Pleiotropic effects of Blastocystis spp. Subtypes 4 and 7 on ligand-specific toll-like receptor signaling and NF-κB activation in a human monocyte cell line.

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    Joshua D W Teo

    Full Text Available Blastocystis spp. is a common enteric stramenopile parasite that colonizes the colon of hosts of a diverse array of species, including humans. It has been shown to compromise intestinal epithelial cell barrier integrity and mediate the production of pro-inflammatory cytokines and chemokines. Mucosal epithelial surfaces, including the intestinal epithelium, are increasingly recognized to perform a vital surveillance role in the context of innate immunity, through the expression of pathogen recognition receptors, such as Toll-like receptors (TLRs. In this study, we use the human TLR reporter monocytic cell line, THP1-Blue, which expresses all human TLRs, to investigate effects of Blastocystis on TLR activation, more specifically the activation of TLR-2, -4 and -5. We have observed that live Blastocystis spp. parasites and whole cell lysate (WCL alone do not activate TLRs in THP1-Blue. Live ST4-WR1 parasites inhibited LPS-mediated NF-κB activation in THP1-Blue. In contrast, ST7-B WCL and ST4-WR1 WCL induced pleiotropic modulation of ligand-specific TLR-2 and TLR-4 activation, with no significant effects on flagellin-mediated TLR-5 activation. Real time-qPCR analysis on SEAP reporter gene confirmed the augmenting effect of ST7-B on LPS-mediated NF-κB activation in THP1-Blue. Taken together, this is the first study to characterize interactions between Blastocystis spp. and host TLR activation using an in vitro reporter model.

  14. Radioiodinated ligands for dopamine receptors

    International Nuclear Information System (INIS)

    Kung, H.F.

    1994-01-01

    The dopamine receptor system is important for normal brain function; it is also the apparent action site for various neuroleptic drugs for the treatment of schizophrenia and other metal disorders. In the past few years radioiodinated ligands for single photon emission tomography (SPECT) have been successfully developed and tested in humans: [ 123 I]TISCH for D1 dopamine receptors; [ 123 I]IBZM, epidepride, IBF and FIDA2, four iodobenzamide derivatives, for D2/D3 dopamine receptors. In addition, [ 123 I]β-CIT (RTI-55) and IPT, cocaine derivatives, for the dopamine reuptake site are potentially useful for diagnosis of loss of dopamine neurons. The first iodinated ligand, (R)trans-7-OH-PIPAT, for D3 dopamine receptors, was synthesized and characterized with cloned cell lines (Spodoptera frugiperda, Sf9) expressing the D2 and D3 dopamine receptors and with rat basal forebrain membrane preparations. Most of the known iodobenzamides displayed similar potency in binding to both D2 and D3 dopamine receptors expressed in the cell lines. Initial studies appear to suggest that by fine tuning the structures it may be possible to develop agents specific for D2 and D3 dopamine receptors. It is important to investigate D2/D3 selectivity for this series of potent ligands

  15. 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

  16. Heme oxygenase-1 protects rat liver against warm ischemia/reperfusion injury via TLR2/TLR4-triggered signaling pathways.

    Science.gov (United States)

    Huang, Han-Fei; Zeng, Zhong; Wang, Kun-Hua; Zhang, Hai-Yan; Wang, Shuai; Zhou, Wen-Xiang; Wang, Zhan-Bo; Xu, Wang-Gang; Duan, Jian

    2015-03-14

    To investigate the efficacy and molecular mechanisms of induced heme oxygenase (HO)-1 in protecting liver from warm ischemia/reperfusion (I/R) injury. Partial warm ischemia was produced in the left and middle hepatic lobes of SD rats for 75 min, followed by 6 h of reperfusion. Rats were treated with saline, cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP) at 24 h prior to the ischemia insult. Blood and samples of ischemic lobes subjected to ischemia were collected at 6 h after reperfusion. Serum transaminases level, plasma lactate dehydrogenase and myeloperoxidase activity in liver were measured. Liver histological injury and inflammatory cell infiltration were evaluated by tissue section and liver immunohistochemical analysis. We used quantitative reverse transcription polymerase chain reaction to analyze liver expression of inflammatory cytokines and chemokines. The cell lysates were subjected to immunoprecipitation with anti-Toll-IL-1R-containing adaptor inducing interferon-β (TRIF) and anti-myeloid differentiation factor 88 (MyD88), and then the immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. HO-1 protected livers from I/R injury, as evidenced by diminished liver enzymes and well-preserved tissue architecture. In comparison with ZnPP livers 6 h after surgery, CoPP treatment livers showed a significant increase inflammatory cell infiltration of lymphocytes, plasma cells, neutrophils and macrophages. The Toll-like receptor (TLR)-4 and TANK binding kinase 1 protein levels of rats treated with CoPP significantly reduced in TRIF-immunoprecipitated complex, as compared with ZnPP treatment. In addition, pretreatment with CoPP reduced the expression levels of TLR2, TLR4, IL-1R-associated kinase (IRAK)-1 and tumor necrosis factor receptor-associated factor 6 in MyD88-immunoprecipitated complex. The inflammatory cytokines and chemokines mRNA expression rapidly decreased in CoPP-pretreated liver, compared with the

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

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    Carrie E. Lasky

    2016-11-01

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

  18. THREE-DIMENSIONAL MAPPING OF DIFFERENTIAL AMINO ACIDS OF HUMAN, MURINE, CANINE AND EQUINE TLR4/MD-2 RECEPTOR COMPLEXES CONFERRING ENDOTOXIC ACTIVATION BY LIPID A, ANTAGONISM BY ERITORAN AND SPECIES-DEPENDENT ACTIVITIES OF LIPID IVA IN THE MAMMALIAN LPS SENSOR SYSTEM

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    Thomas Scior

    2013-05-01

    Full Text Available A literature review concerning the unexpected species differences of the vertebrate innate immune response to lipid IVA was published in CSBJ prior to the present computational study to address the unpaired activity-sequence correlation of prototypic E. coli -type lipid A and its precursor lipid IVA regarding human, murine, equine and canine species. To this end, their sequences and structures of hitherto known Toll-like receptor 4 (TLR4 and myeloid differentiation factor 2 (MD-2 complexes were aligned and their differential side chain patterns studied. If required due to the lack of the corresponding X-ray crystallographic data, three-dimensional models of TLR4/MD-2/ligand complexes were generated using mono and dimeric crystal structures as templates and in silico docking of the prototypic ligands lipid A, lipid IVA and Eritoran. All differential amino acids were mapped to pinpoint species dependency on an atomic scale, i.e. the possible concert of mechanistically relevant side chains. In its most abstract and general form the three-dimensional (3D- models devise a triangular interface or “wedge” where molecular interactions between TLR4, MD-2 and ligand itself take place. This study identifies two areas in the wedge related to either agonism or antagonism reflecting why ligands like lipid IVA can possess a species dependent dual activity. Lipid IVA represents an imperfect (underacylated and backbone-flipped, low affinity ligand of mammalian TLR4/MD-2 complexes. Its specific but weak antagonistic activity in the human system is in particular due to the loss of phosphate attraction in the wedge-shaped region conferred by nonhomologous residue changes when compared to crystal and modeled structures of the corresponding murine and equine TLR4/MD-2 complexes. The counter-TLR4/MD-2 unit was also taken into account since agonist-mediated dimerization in a defined m-shaped complex composed of two TLR4/MD-2/agonist subunits triggers intracellular

  19. TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth

    Science.gov (United States)

    Dowling, David J.; van Haren, Simon D.; Scheid, Annette; Bergelson, Ilana; Kim, Dhohyung; Mancuso, Christy J.; Foppen, Willemina; Fresh, Lynn; Theriot, Terese B.; Lackner, Andrew A.; Fichorova, Raina N.; Smirnov, Dmitri; Vasilakos, John P.; Beaurline, Joe M.; Tomai, Mark A.; Midkiff, Cecily C.; Alvarez, Xavier; Blanchard, James L.; Gilbert, Margaret H.; Aye, Pyone Pyone

    2017-01-01

    Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197–specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide–specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10–100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development. PMID:28352660

  20. 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.

  1. 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

  2. TLR4 and NFκB signaling is critical for taxol resistance in ovarian carcinoma cells.

    Science.gov (United States)

    Sun, Nian-Kang; Huang, Shang-Lang; Chang, Ting-Chang; Chao, Chuck C-K

    2018-03-01

    We report here that toll-like receptor 4 (TLR4) and ABCB1 are upregulated in SKOV3 ovarian carcinoma cells that acquired resistance to the anticancer drug taxol. Silencing of TLR4 using short-hairpin RNA sensitized taxol-resistant SKOV3 cells to taxol (4.6 fold), whereas ectopic expression of TLR4 in parental, taxol-sensitive SKOV3 cells or TLR4-null HEK293 cells induced taxol resistance (∼2 fold). A sub-lethal dose of taxol induced ABCB1 protein expression in taxol-resistant SKOV3 cells. Inactivation of TLR4 using chemical inhibitors (CLI-095 and AO-I) downregulated ABCB1 protein expression and enhanced the cytotoxic activity of taxol in taxol-resistant SKOV3 cells. While the sensitization effect of TLR4 inactivation was also detected in TOV21G ovarian cancer cells, which express moderate level of TLR4, ectopic expression of ABCB1 prevented the sensitization effect in these cells. Notably, the NFκB pathway was significantly activated by taxol, and inhibition of this pathway suppressed TLR4-regulated ABCB1 expression. Furthermore, taxol-induced NFκB signaling was reduced following TLR4 silencing in taxol-resistant SKOV3 cells. Consistent with these results, ectopic expression of TLR4 in taxol-sensitive SKOV3 cells enhanced ABCB1 expression and conferred resistance to taxol. The protective effect of exogenous TLR4 expression against taxol was reduced by treatment with NFκB inhibitor in these cells. These results demonstrate that taxol activates the TLR4-NFκB pathway which in turn induces ABCB1 gene expression. This cellular pathway thus represents a novel target to limit resistance to taxol in ovarian cancer cells. © 2017 Wiley Periodicals, Inc.

  3. Toll-like receptor ligands induce human T cell activation and death, a model for HIV pathogenesis.

    Directory of Open Access Journals (Sweden)

    Nicholas Funderburg

    2008-04-01

    Full Text Available Recently, heightened systemic translocation of microbial products was found in persons with chronic HIV infection and this was linked to immune activation and CD4(+ T cell homeostasis.We examined here the effects of microbial Toll-like receptor (TLR ligands on T cell activation in vitro.We show that exposure to TLR ligands results in activation of memory and effector CD4(+ and CD8(+ T cells. After exposure to each of 8 different ligands that activate TLRs 2, 3, 4, 5, 7, 8, and 9, CD8(+ T cells are activated and gain expression of the C type lectin CD69 that may promote their retention in lymphoid tissues. In contrast, CD4(+ T cells rarely increase CD69 expression but instead enter cell cycle. Despite activation and cell cycle entry, CD4(+ T cells divide poorly and instead, disproportionately undergo activation-induced cell death. Systemic exposure to TLR agonists may therefore increase immune activation, effector cell sequestration in lymphoid tissues and T cell turnover. These events may contribute to the pathogenesis of immune dysfunction and CD4+ T cell losses in chronic infection with the human immunodeficiency virus.

  4. Dockomatic - automated ligand creation and docking.

    Science.gov (United States)

    Bullock, Casey W; Jacob, Reed B; McDougal, Owen M; Hampikian, Greg; Andersen, Tim

    2010-11-08

    The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI) application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

  5. Dockomatic - automated ligand creation and docking

    Directory of Open Access Journals (Sweden)

    Hampikian Greg

    2010-11-01

    Full Text Available Abstract Background The application of computational modeling to rationally design drugs and characterize macro biomolecular receptors has proven increasingly useful due to the accessibility of computing clusters and clouds. AutoDock is a well-known and powerful software program used to model ligand to receptor binding interactions. In its current version, AutoDock requires significant amounts of user time to setup and run jobs, and collect results. This paper presents DockoMatic, a user friendly Graphical User Interface (GUI application that eases and automates the creation and management of AutoDock jobs for high throughput screening of ligand to receptor interactions. Results DockoMatic allows the user to invoke and manage AutoDock jobs on a single computer or cluster, including jobs for evaluating secondary ligand interactions. It also automates the process of collecting, summarizing, and viewing results. In addition, DockoMatic automates creation of peptide ligand .pdb files from strings of single-letter amino acid abbreviations. Conclusions DockoMatic significantly reduces the complexity of managing multiple AutoDock jobs by facilitating ligand and AutoDock job creation and management.

  6. 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.

  7. 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.

  8. 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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-04-09

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

  11. 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.

  12. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.

    Science.gov (United States)

    Pan, Xichun; Li, Bin; Kuang, Mei; Liu, Xin; Cen, Yanyan; Qin, Rongxin; Ding, Guofu; Zheng, Jiang; Zhou, Hong

    2016-02-22

    Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  13. Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

    Directory of Open Access Journals (Sweden)

    Xichun Pan

    2016-02-01

    Full Text Available Toll-like receptor (TLR 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN. Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS- and lipopeptide (PAM3CSK4-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

  14. Calculating the mean time to capture for tethered ligands and its effect on the chemical equilibrium of bound ligand pairs.

    Science.gov (United States)

    Shen, Lu; Decker, Caitlin G; Maynard, Heather D; Levine, Alex J

    2016-09-01

    We present here the calculation of the mean time to capture of a tethered ligand to the receptor. This calculation is then used to determine the shift in the partitioning between (1) free, (2) singly bound, and (3) doubly bound ligands in chemical equilibrium as a function of the length of the tether. These calculations are used in the research article Fibroblast Growth Factor 2 Dimer with Superagonist in vitro Activity Improves Granulation Tissue Formation During Wound Healing (Decker et al., in press [1]) to explain quantitatively how changes in polymeric linker length in the ligand dimers modifies the efficacy of these molecules relative to that of free ligands.

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

    Directory of Open Access Journals (Sweden)

    Rebeca Santaolalla

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

  16. 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.

  17. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-21

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

  19. Histamine Receptor 2 is Required to Suppress Innate Immune Responses to Bacterial Ligands in Patients with Inflammatory Bowel Disease.

    Science.gov (United States)

    Smolinska, Sylwia; Groeger, David; Perez, Noelia Rodriguez; Schiavi, Elisa; Ferstl, Ruth; Frei, Remo; Konieczna, Patrycja; Akdis, Cezmi A; Jutel, Marek; OʼMahony, Liam

    2016-07-01

    Histamine is a key immunoregulatory mediator in immediate-type hypersensitivity reactions and chronic inflammatory responses, in particular histamine suppresses proinflammatory responses to bacterial ligands, through histamine receptor 2 (H2R). The aim of this study was to investigate the effects of histamine and H2R on bacteria-induced inflammatory responses in patients with IBD. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Crohn's disease, patients with ulcerative colitis, and healthy controls. PBMC histamine receptor expression was evaluated by flow cytometry. Cytokine secretion following Toll-like receptor (TLR)-2, TLR-4, TLR-5, or TLR-9 stimulation in the presence or absence of histamine or famotidine (H2R antagonist) was quantified. Biopsy histamine receptor gene expression was evaluated using reverse transcription-polymerase chain reaction. The in vivo role of H2R was evaluated in the T-cell transfer murine colitis model. The percentage of circulating H2R monocytes was significantly reduced in patients with IBD. Histamine effectively suppressed TLR-induced cytokine secretion from healthy volunteer PBMCs but not for PBMCs from patients with IBD. Famotidine reversed this suppressive effect. H1R, H2R, and H4R gene expression was increased in inflamed gastrointestinal mucosa compared with noninflamed mucosa from the same patient and expression levels correlated with proinflammatory cytokine gene expression. Mice receiving lymphocytes from H2R donors, or treated with famotidine, displayed more severe weight loss, higher disease scores and increased numbers of mucosal IFN-γ and IL-17 T cells. Patients with IBD display dysregulated expression of histamine receptors, with diminished anti-inflammatory effects associated with H2R signaling. Deliberate manipulation of H2R signaling may suppress excessive TLR responses to bacteria within the gut.

  20. Systematic study of ligand structures of metal oxide EUV nanoparticle photoresists

    KAUST Repository

    Jiang, Jing

    2015-03-19

    Ligand stabilized metal oxide nanoparticle resists are promising candidates for EUV lithography due to their high sensitivity for high-resolution patterning and high etching resistance. As ligand exchange is responsible for the patterning mechanism, we systematically studied the influence of ligand structures of metal oxide EUV nanoparticles on their sensitivity and dissolution behavior. ZrO2 nanoparticles were protected with various aromatic ligands with electron withdrawing and electron donating groups. These nanoparticles have lower sensitivity compared to those with aliphatic ligands suggesting the structures of these ligands is more important than their pka on resist sensitivity. The influence of ligand structure was further studied by comparing the nanoparticles’ solubility for a single type ligand to mixtures of ligands. The mixture of nanoparticles showed improved pattern quality. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

  1. 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.

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

    Science.gov (United States)

    Omotuyi, Olaposi; Matsunaga, Hayato; Ueda, Hiroshi

    2015-01-01

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

  3. TLR signaling-induced CD103-expressing cells protect against intestinal inflammation

    NARCIS (Netherlands)

    Wittmann, Alexandra; Bron, Peter A.; Swam, Van Iris I.; Kleerebezem, Michiel; Adam, Patrick; Gronbach, Kerstin; Menz, Sarah; Flade, Isabell; Bender, Annika; Schäfer, Andrea; Korkmaz, Ali Giray; Parusel, Raphael; Autenrieth, Ingo B.; Frick, Julia Stefanie

    2015-01-01

    Background: Toll-like receptor (TLR) expression in patients with inflammatory bowel disease is increased when compared with healthy controls. However, the impact of TLR signaling during inflammatory bowel disease is not fully understood. Methods: In this study, we used a murine model of acute

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

    African Journals Online (AJOL)

    huis

    Human models, in particular, suggest TLR4 to be a candidate gene associated with resistance to sepsis, immunodeficiencies, atherosclerosis and asthma. In this study the bovine TLR4 mRNA and genomic DNA were sequenced and their gene structures were determined through alignment of the genomic DNA sequence to ...

  5. Role of TLR4 gene polymorphisms in the colorectal cancer risk ...

    African Journals Online (AJOL)

    Saniya Nissar

    2016-05-26

    May 26, 2016 ... Colorectal cancer;. Kashmir;. Polymorphism;. RFLP;. TLR4. Abstract Background: Colorectal carcinogenesis has been found to be associated with the poly- morphic status ..... Table 3 Association of TLR4 Thr299Ile polymorphism with various clinic-pathological characteristics in CRC cases. Characteristics.

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

    Directory of Open Access Journals (Sweden)

    Angela Castoldi

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

  9. HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2014-07-25

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

  11. Purinergic signaling to terminate TLR responses in macrophages

    Directory of Open Access Journals (Sweden)

    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.

  12. DMPD: TLR pathways and IFN-regulatory factors: to each its own. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17273997 TLR pathways and IFN-regulatory factors: to each its own. Colonna M. Eur J... Immunol. 2007 Feb;37(2):306-9. (.png) (.svg) (.html) (.csml) Show TLR pathways and IFN-regulatory factors: ...to each its own. PubmedID 17273997 Title TLR pathways and IFN-regulatory factors: to each its own. Authors C

  13. 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.

  14. Evaluation of TLR agonists as potential mucosal adjuvants for HIV gp140 and tetanus toxoid in mice.

    Directory of Open Access Journals (Sweden)

    Viviana Buffa

    Full Text Available In the present study we investigate the impact of a range of TLR ligands and chitosan as potential adjuvants for different routes of mucosal immunisation (sublingual (SL, intranasal (IN, intravaginal (IVag and a parenteral route (subcutaneous (SC in the murine model. We assess their ability to enhance antibody responses to HIV-1 CN54gp140 (gp140 and Tetanus toxoid (TT in systemic and vaginal compartments. A number of trends were observed by route of administration. For non-adjuvanted antigen, SC>SL>IN immunisation with respect to systemic IgG responses, where endpoint titres were greater for TT than for gp140. In general, co-administration with adjuvants increased specific IgG responses where IN = SC>SL, while in the vaginal compartment IN>SL>SC for specific IgA. In contrast, for systemic and mucosal IgA responses to antigen alone SL>IN = SC. A number of adjuvants increased specific systemic IgA responses where in general IN>SL>SC immunisation, while for mucosal responses IN = SL>SC. In contrast, direct intravaginal immunisation failed to induce any detectable systemic or mucosal responses to gp140 even in the presence of adjuvant. However, significant systemic IgG responses to TT were induced by intravaginal immunisation with or without adjuvant, and detectable mucosal responses IgG and IgA were observed when TT was administered with FSL-1 or Poly I∶C. Interestingly some TLRs displayed differential activity dependent upon the route of administration. MPLA (TLR4 suppressed systemic responses to SL immunisation while enhancing responses to IN or SC immunisation. CpG B enhanced SL and IN responses, while having little or no impact on SC immunisation. These data demonstrate important route, antigen and adjuvant effects that need to be considered in the design of mucosal vaccine strategies.

  15. Sterically demanding iminopyridine ligands

    NARCIS (Netherlands)

    Irrgang, Torsten; Keller, Sandra; Maisel, Heidi; Kretschmer, Winfried; Kempe, Rhett

    Two sterically demanding iminopyridine ligands, (2,6-diisopropylphenyl)[6-(2,4,6-triisopropylphenyl)pyridin-2-ylmeth- ylene]amine and (2,6-diisopropylphenyl)]6-(2,6-dimethylphenyl)pyridin-2-ylmethylene]amine, were prepared by a two-step process: first, condensation of 6-bromopyridine-2-carbaldehyde

  16. New monodentate amidine superbasic ligands with a single configuration in fac-[Re(CO)3(5,5'- or 6,6'-Me2bipyridine)(amidine)]BF4 complexes.

    Science.gov (United States)

    Abhayawardhana, Pramuditha; Marzilli, Patricia A; Perera, Theshini; Fronczek, Frank R; Marzilli, Luigi G

    2012-07-02

    Treatment of two precursors, fac-[Re(CO)(3)(L)(CH(3)CN)]BF(4) [L = 5,5'-dimethyl-2,2'-bipyridine (5,5'-Me(2)bipy) (1) and 6,6'-dimethyl-2,2'-bipyridine (6,6'-Me(2)bipy) (2)], with five C(2)-symmetrical saturated heterocyclic amines yielded 10 new amidine complexes, fac-[Re(CO)(3)(L)(HNC(CH(3))N(CH(2)CH(2))(2)Y)]BF(4) [Y = CH(2), (CH(2))(2), (CH(2))(3), NH, or O]. All 10 complexes possess the novel feature of having only one isomer (amidine E configuration), as established by crystallographic and (1)H NMR spectroscopic methods. We are confident that NMR signals of the other possible isomer (amidine Z configuration) would have been detected, if it were present. Isomers are readily detected in closely related amidine complexes because the double-bond character of the amidine C-N3 bond (N3 is bound to Re) leads to slow E to Z isomer interchange. The new fac-[Re(CO)(3)(L)(HNC(CH(3))N(CH(2)CH(2))(2)Y)]BF(4) complexes have C-N3 bonds with essentially identical double-bond character. However, the reason that the Z isomer is so unstable as to be undetectable in the new complexes is undoubtedly because of unfavorable clashes between the equatorial ligands and the bulky N(CH(2)CH(2))(2)Y ring moiety of the axial amidine ligand. The amidine formation reactions in acetonitrile (25 °C) proceeded more easily with 2 than with 1, indicating that the distortion in 6,6'-Me(2)bipy resulting from the proximity of the methyl substituents to the inner coordination sphere enhanced the reactivity of the coordinated CH(3)CN. Reaction times for 1 and 2 exhibited a similar dependence on the basicity and ring size of the heterocyclic amine reactants. Moreover, when the product of the reaction of 1 with piperidine, fac-[Re(CO)(3)(5,5'-Me(2)bipy)(HNC(CH(3))N(CH(2)CH(2))(2)CH(2))]BF(4), was challenged in acetonitrile-d(3) or CDCl(3) with a 5-fold excess of the strong 4-dimethylaminopyridine ligand, there was no evidence for replacement of the amidine ligand after two months, thus establishing

  17. Polymorphisms of Dectin-1 and TLR2 Predispose to Invasive Fungal Disease in Patients with Acute Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Mike Fischer

    Full Text Available Patients with acute myeloid leukemia (AML who undergo induction chemotherapy are at high risk for invasive fungal disease (IFD. Dectin-1, a C-type lectin family member represents one of the most important pattern recognition receptors of the innate immune system and single nucleotide polymorphisms (SNPs in the Dectin-1 gene have been associated with an increased risk of infectious complications. We sought to investigate the impact of three different Dectin-1 SNPs and one TLR2 SNP on developing IFD in 186 adult patients with newly diagnosed AML following anthracycline-based induction chemotherapy.Genotyping of Dectin-1 SNPs (rs16910526, rs3901533 and rs7309123 and TLR2 SNP (rs5743708 was performed by TaqMan method and pyrosequencing. IFD was defined according to the EORTC/MSG consensus guidelines. Multiple logistic regression analyses were applied to evaluate the association between the polymorphisms and the occurrence of pulmonary infections. Dectin-1 expression studies with SNP genotyped human monocytes were performed to elucidate susceptibility to IFD following chemotherapy.We could demonstrate that patients carrying the Dectin-1 SNP rs7309123 G/G (n = 47 or G/G and C/G (n = 133 genotype revealed a significant higher risk for developing both pneumonia in general (adjusted odds ratio (OR: 2.5; p = 0.014 and OR: 3.0, p = 0.004 and pulmonary IFD (OR: 2.6; p = 0.012 and OR: 2.4, p = 0.041, respectively. Patients carrying the TLR2 SNP rs5743708 (R753Q, GA/AA genotype, n = 12 also revealed a significantly higher susceptibility to pneumonia including IFD. Furthermore, Dectin-1 mRNA expression in human monocytes was lower following chemotherapy.To our best knowledge, this study represents the first analysis demonstrating that harbouring polymorphisms of Dectin-1 (rs7309123 or TLR2 (rs5743708 represents an independent risk factor of developing IFD in patients with AML undergoing induction chemotherapy.

  18. TLR4/CD14 Variants-Related Serologic and Immunologic Dys-Regulations Predict Severe Sepsis in Febrile De-Compensated Cirrhotic Patients.

    Directory of Open Access Journals (Sweden)

    Wen-Chien Fan

    Full Text Available Genetic variants and dysfunctional monocyte had been reported to be associated with infection susceptibility in advanced cirrhotic patients. This study aims to explore genetic predictive markers and relevant immune dysfunction that contributed to severe sepsis in febrile acute de-compensated cirrhotic patents. Polymorphism analysis of candidate genes was undergone in 108 febrile acute de-compensated cirrhotic patients and 121 healthy volunteers. Various plasma inflammatory/regulatory cytokines, proportion of classical (CD 16-, phagocytic and non-classical (CD16+, inflammatory monocytes, lipopolysaccharide (LPS-stimulated toll-like receptor 4 (TLR4 and intracellular/extracellular cytokines on cultured non-classical monocytes, mCD14/HLA-DR expression and phagocytosis of classical monocytes were measured. For TLR4+896A/G variant allele carriers with severe sepsis, high plasma endotoxin/IL-10 inhibits HLA-DR expression and impaired phagocytosis were noted in their classical monocyte. In the same group, increased non-classical monocyte subset, enhanced LPS-stimulated TLR4 expression and TNFα/nitrite production, and systemic inflammation [high plasma soluble CD14 (sCD14 and total nitric oxide (NOx levels] were noted. For CD14-159C/T variant allele carriers with severe sepsis, persist endotoxemia inhibited mCD14/HLA-DR expression and impaired phagocytosis of their classical monocyte. In the same group, increased non-classical monocyte subset up-regulated TLR4-NFκB-iNOS and p38MAPK pathway, stimulated TNFα/nitrite production and elicited systemic inflammation. In febrile acute de-compensated cirrhotic patients, TLR4+896A/G and CD14-159C/T polymorphisms-related non-classical and classical monocytes dysfunction resulted in increased severe sepsis risk. Malnutrition, high plasma endotoxin and sCD14 levels, single TLR4+896A/G or CD14-159C/T variant allele carriers and double variant allele carriers are significant predictive factors for the development

  19. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    Science.gov (United States)

    Von Dreele, Robert B [Los Alamos, NM

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  20. Studying the Effect of Downregulating Autophagy-Related Gene LC3 on TLR3 Apoptotic Pathway Mediated by dsRNA in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Wang, Guilan; Zhang, Maona; Li, Yunlong; Zhou, Jiaming; Chen, Li

    2017-01-01

    The purpose of this study is to examine the role of the double-stranded RNA (dsRNA) activated Toll-interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) signal pathway in triggering apoptosis in hepatocellular carcinoma (HCC) cells. First, siRNA targeted autophagy-related gene LC3 (pU6H1-LC3 siRNA and siLC3) and a dsRNA used as a Toll-like receptor 3 (TLR3) ligand was constructed and synthesized, respectively. Then, a human HCC cell line was transfected with dsRNA, siLC3, and cotransfected with siLC3 and dsRNA (siLC3+dsRNA), respectively. Finally, quantification real-time polymerase chain reaction, western blotting, and immunofluorescence staining were used in the HCC line (SMMC7721), and MTT assay, flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling, and transmission electron microscopy were used in an HCC xenograft model of nude mice. Human umbilical vein endothelial cell tube forming assay, color Doppler ultrasonographic flow image examination, and CD34-positive microvessel density were used in vitro and in vivo . Compared with untreated cells, the protein and mRNA expression of TLR3 and TRIF was up-regulated, in order, siLC3+dsRNA, dsRNA, and siLC3. Expression of LC3 was obviously down-regulated and the autophagosomes were significantly decreased in siLC3+dsRNA and siLC3, whereas in dsRNA (p protein, which can promote triggering of apoptosis by the TLR3-TRIF pathway. dsRNA and siLC3 could play anticancer roles in coordination.

  1. Pulmonary Epithelial TLR4 Activation Leads to Lung Injury in Neonatal Necrotizing Enterocolitis.

    Science.gov (United States)

    Jia, Hongpeng; Sodhi, Chhinder P; Yamaguchi, Yukihiro; Lu, Peng; Martin, Laura Y; Good, Misty; Zhou, Qinjie; Sung, Jungeun; Fulton, William B; Nino, Diego F; Prindle, Thomas; Ozolek, John A; Hackam, David J

    2016-08-01

    We seek to define the mechanisms leading to the development of lung disease in the setting of neonatal necrotizing enterocolitis (NEC), a life-threatening gastrointestinal disease of premature infants characterized by the sudden onset of intestinal necrosis. NEC development in mice requires activation of the LPS receptor TLR4 on the intestinal epithelium, through its effects on modulating epithelial injury and repair. Although NEC-associated lung injury is more severe than the lung injury that occurs in premature infants without NEC, the mechanisms leading to its development remain unknown. In this study, we now show that TLR4 expression in the lung gradually increases during postnatal development, and that mice and humans with NEC-associated lung inflammation express higher levels of pulmonary TLR4 than do age-matched controls. NEC in wild-type newborn mice resulted in significant pulmonary injury that was prevented by deletion of TLR4 from the pulmonary epithelium, indicating a role for pulmonary TLR4 in lung injury development. Mechanistically, intestinal epithelial TLR4 activation induced high-mobility group box 1 release from the intestine, which activated pulmonary epithelial TLR4, leading to the induction of the neutrophil recruiting CXCL5 and the influx of proinflammatory neutrophils to the lung. Strikingly, the aerosolized administration of a novel carbohydrate TLR4 inhibitor prevented CXCL5 upregulation and blocked NEC-induced lung injury in mice. These findings illustrate the critical role of pulmonary TLR4 in the development of NEC-associated lung injury, and they suggest that inhibition of this innate immune receptor in the neonatal lung may prevent this devastating complication of NEC. Copyright © 2016 by The American Association of Immunologists, Inc.

  2. 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...

  3. Total glucosides of paeony attenuated functional maturation of dendritic cells via blocking TLR4/5 signaling in vivo.

    Science.gov (United States)

    Zhou, Zhou; Lin, Jinpiao; Huo, Rongfen; Huang, Wenkang; Zhang, Jian; Wang, Li; Sun, Yue; Shen, Baihua; Li, Ningli

    2012-11-01

    It is well known that dendritic cells (DCs) play a critical role in the initiation and development of an immune response. Inhibitory effect on DC maturation alters immune-mediated inflammatory reaction in vivo. Total glucosides of paeony (TGP) are active compounds extracted from the roots of Paeonia lactiflora and have been widely used to ameliorate inflammation in therapy for autoimmune diseases. However, whether TGP act on DC maturation remains unknown. In this study, we investigated the effect of TGP on DC maturation in ovalbumin (OVA) immunized mice. Ear inflammation was inhibited by TGP (150 mgkg(-1), i.p.×11 days) obviously. The antigen presenting capacity of DC derived from TGP-treated mice was arrested. Meanwhile, OVA specific T cell proliferation was inhibited. In addition, we found that maturation of DCs was decreased by TGP treatment. Furthermore, OVA specific T cell proliferation was rescued by the adoptive transfer of mature DCs (mDCs) into TGP treated OVA-challenged mice. The research on the mechanism showed that TGP significantly inhibited activation of TLR4/5 singling. All these results demonstrated that TGP inhibited DC maturation and function by selectively blocking TLR4/5 activation in vivo, which in turn leads to reduce immune-mediated inflammation in vivo, adding a novel mechanism and therapeutic target of TGP for inflammatory and autoimmune disease treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

    Directory of Open Access Journals (Sweden)

    Nathalie Geurts

    Full Text Available The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO and knock-in mice, has become an established tool to study gene function in many animal models for human diseases. However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

  5. Radiobiology with DNA ligands

    International Nuclear Information System (INIS)

    Weinreich, R.; Argentini, M.; Guenther, I.; Koziorowski, J.; Larsson, B.; Nievergelt-Egido, M.C.; Salt, C.; Wyer, L.; Dos Santos, D.F.; Hansen, H.J.

    1997-01-01

    The paper deals with the following topics: labelling of DNA ligands and other tumour-affinic compounds with 4.15-d 124 I, radiotoxicity of Hoechst 33258 and 33342 and of iodinated Hoechst 33258 in cell cultures, preparation of 76 Br-, 123 I-, and 221 At-labelled 5-halo-2'-deoxyuridine, chemical syntheses of boron derivatives of Hoechst 33258.III., Gadolinium neutron capture therapy

  6. Therapeutic androgen receptor ligands

    Science.gov (United States)

    Allan, George F.; Sui, Zhihua

    2003-01-01

    In the past several years, the concept of tissue-selective nuclear receptor ligands has emerged. This concept has come to fruition with estrogens, with the successful marketing of drugs such as raloxifene. The discovery of raloxifene and other selective estrogen receptor modulators (SERMs) has raised the possibility of generating selective compounds for other pathways, including androgens (that is, selective androgen receptor modulators, or SARMs). PMID:16604181

  7. 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...

  8. 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.

  9. Ligand activation of the prokaryotic pentameric ligand-gated ion channel ELIC.

    Directory of Open Access Journals (Sweden)

    Iwan Zimmermann

    2011-06-01

    Full Text Available While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.

  10. 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.

  11. TLR2-dependent induction of IL-10 and Foxp3+ CD25+ CD4+ regulatory T cells prevents effective anti-tumor immunity induced by Pam2 lipopeptides in vivo.

    Directory of Open Access Journals (Sweden)

    Sayuri Yamazaki

    Full Text Available 16 S-[2,3-bis(palmitoylpropyl]cysteine (Pam2 lipopeptides act as toll-like receptor (TLR2/6 ligands and activate natural killer (NK cells and dendritic cells (DCs to produce inflammatory cytokines and cytotoxic NK activity in vitro. However, in this study, we found that systemic injection of Pam2 lipopeptides was not effective for the suppression of NK-sensitive B16 melanomas in vivo. When we investigated the immune suppressive mechanisms, systemic injection of Pam2 lipopeptides induced IL-10 in a TLR2-dependent manner. The Pam2 lipopeptides increased the frequencies of Foxp3(+CD4(+ regulatory T (T reg cells in a TLR2- and IL-10- dependent manner. The T reg cells from Pam2-lipopeptide injected mice maintained suppressor activity. Pam2 lipopeptides, plus the depletion of T reg with an anti-CD25 monoclonal antibody, improved tumor growth compared with Pam2 lipopeptides alone. In conclusion, our data suggested that systemic treatment of Pam2 lipopeptides promoted IL-10 production and T reg function, which suppressed the effective induction of anti-tumor immunity in vivo. It is necessary to develop an adjuvant that does not promote IL-10 and T reg function in vivo for the future establishment of an anti-cancer vaccine.

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

    Lifescience Database Archive (English)

    Full Text Available 15608698 LPS, TLR4 and infectious disease diversity. Miller SI, Ernst RK, Bader MW. Nat Rev Microbiol... Bader MW. Publication Nat Rev Microbiol. 2005 Jan;3(1):36-46. Pathway - PNG File

  13. Role of TLR4 gene polymorphisms in the colorectal cancer risk ...

    African Journals Online (AJOL)

    Role of TLR4 gene polymorphisms in the colorectal cancer risk modulation in ethnic Kashmiri population – A case–control study. Saniya Nissar, Aga Syed Sameer, Roohi Rasool, Qurteeba Qadri, Nissar A. Chowdri, Fouzia Rashid ...

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

    African Journals Online (AJOL)

    Jane

    2011-08-08

    . The genetic difference in these receptor genes can cause difference in the resistance to various pathogens (Werling and Jungi, 2003; Machida et al., 2006; Akira and Takeda, 2004). Recognition ability of. TLR4 to LPS among ...

  15. 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

  16. Bexarotene ligand pharmaceuticals.

    Science.gov (United States)

    Hurst, R E

    2000-12-01

    Bexarotene (LGD-1069), from Ligand, was the first retinoid X receptor (RXR)-selective, antitumor retinoid to enter clinical trials. The company launched the drug for the treatment of cutaneous T-cell lymphoma (CTCL), as Targretin capsules, in the US in January 2000 [359023]. The company filed an NDA for Targretin capsules in June 1999, and for topical gel in December 1999 [329011], [349982] specifically for once-daily oral administration for the treatment of patients with early-stage CTCL who have not tolerated other therapies, patients with refractory or persistent early stage CTCL and patients with refractory advanced stage CTCL. The FDA approved Targretin capsules at the end of December 1999 for once-daily oral treatment of all stages of CTCL in patients refractory to at least one prior systemic therapy, at an initial dose of 300 mg/m2/day. After an NDA was submitted in December 1999 for Targretin gel, the drug received Priority Review status for use as a treatment of cutaneous lesions in patients with stage IA, IB or IIA CTCL [354836]. The FDA issued an approvable letter in June 2000, and granted marketing clearance for CTCL in the same month [370687], [372768], [372769], [373279]. Ligand had received Orphan Drug designation for this indication [329011]. At the request of the FDA, Ligand agreed to carry out certain post-approval phase IV and pharmacokinetic studies [351604]. The company filed an MAA with the EMEA for Targretin Capsules to treat lymphoma in November 1999 [348944]. The NDA for Targretin gel is based on a multicenter phase III trial that was conducted in the US, Canada, Europe and Australia involving 50 patients and a multicenter phase I/II clinical program involving 67 patients. Targretin gel was evaluated for the treatment of patients with early stage CTCL (IA-IIA) who were refractory to, intolerant to, or reached a response plateau for at least 6 months on at least two prior therapies. Efficacy results exceeded the protocol-defined response

  17. Lipid raft localization of TLR2 and its co-receptors is independent of membrane lipid composition

    Directory of Open Access Journals (Sweden)

    Christine Hellwing

    2018-01-01

    Full Text Available Background Toll like receptors (TLRs are an important and evolutionary conserved class of pattern recognition receptors associated with innate immunity. The recognition of Gram-positive cell wall constituents strongly depends on TLR2. In order to be functional, TLR2 predominantly forms a heterodimer with TLR1 or TLR6 within specialized membrane microdomains, the lipid rafts. The membrane lipid composition and the physicochemical properties of lipid rafts are subject to modification by exogenous fatty acids. Previous investigations of our group provide evidence that macrophage enrichment with polyunsaturated fatty acids (PUFA induces a reordering of lipid rafts and non-rafts based on the incorporation of supplemented PUFA as well as their elongation and desaturation products. Methods In the present study we investigated potential constraining effects of membrane microdomain reorganization on the clustering of TLR2 with its co-receptors TLR1 and TLR6 within lipid rafts. To this end, RAW264.7 macrophages were supplemented with either docosahexaenoic acid (DHA or arachidonic acid (AA and analyzed for receptor expression and microdomain localization in context of TLR stimulation. Results and Conclusions Our analyses showed that receptor levels and microdomain localization were unchanged by PUFA supplementation. The TLR2 pathway, in contrast to the TLR4 signaling cascade, is not affected by exogenous PUFA at the membrane level.

  18. LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway

    Directory of Open Access Journals (Sweden)

    Qianran Yin

    2017-12-01

    Full Text Available Background/Aims: Lipopolysaccharide (LPS is a potent activator of vascular smooth muscle cells (VSMCs proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4 and Ras-related C3 botulinum toxin substrate 1 (Rac1 expression using small interfering RNA (siRNA in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation. Methods: VSMCs proliferation was monitored by 5-ethynyl-2’-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA, smooth muscle 22α (SM22α, myosin heavy chain (MYH and transient receptor potential channel 1 (TRPC1 were detected by qRT-PCR. The expression of total Akt, p-Akt (308, p-Akt (473, SM22α, MYH and TRPC1 protein was analysed by Western blot. Results: Treatment with TLR4 siRNA (siTLR4 or Rac1 siRNA (siRac1 significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1. Conclusion: This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect.

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

    OpenAIRE

    Kim, Soo Jin; Kim, Ho Min

    2017-01-01

    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 mice...

  20. LPS Promotes Vascular Smooth Muscle Cells Proliferation Through the TLR4/Rac1/Akt Signalling Pathway.

    Science.gov (United States)

    Yin, Qianran; Jiang, Dehua; Li, Lei; Yang, Yu; Wu, Pei; Luo, Yuanyuan; Yang, Rongli; Li, Dongye

    2017-01-01

    Lipopolysaccharide (LPS) is a potent activator of vascular smooth muscle cells (VSMCs) proliferation, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) and Ras-related C3 botulinum toxin substrate 1 (Rac1) expression using small interfering RNA (siRNA) in order to investigate the effects and possible mechanisms of LPS-induced VSMCs proliferation. VSMCs proliferation was monitored by 5-ethynyl-2'-deoxyuridine staining, and Rac1 activity was measured via Glutathione S-transferase pull-down assay. mRNAs encoding proliferating cell nuclear antigen (PCNA), smooth muscle 22α (SM22α), myosin heavy chain (MYH) and transient receptor potential channel 1 (TRPC1) were detected by qRT-PCR. The expression of total Akt, p-Akt (308), p-Akt (473), SM22α, MYH and TRPC1 protein was analysed by Western blot. Treatment with TLR4 siRNA (siTLR4) or Rac1 siRNA (siRac1) significantly decreased LPS-induced VSMCs proliferation. Moreover, LPS-induced activation of Rac1 through TLR4 was observed. Western blot analysis revealed that transfection with siTLR4 or siRac1 inhibited LPS-induced Akt phosphorylation. We discovered that LPS stimulated VSMCs proliferation via phenotypic modulation and that this effect was partially inhibited by pre-treatment with siTLR4 or siRac1. Further, TLR4 and Rac1 are involved in LPS-induced activation of TRPC1. This study suggests that LPS exerts an effect on VSMCs proliferation and that the TLR4/Rac1/Akt signalling pathway mediates this effect. © 2017 The Author(s). Published by S. Karger AG, Basel.

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

    Science.gov (United States)

    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.

  2. 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.

  3. Combating Drug Abuse by Targeting Toll-Like Receptor 4 (TLR)

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-12-1-0345 PROJECT TITLE: Combating drug abuse by targeting toll-like receptor 4 (TLR) PRINCIPAL INVESTIGATOR: Dr. Linda...5a. CONTRACT NUMBER not applicable Combating drug abuse by targeting toll-like receptor 4 (TLR) 5b. GRANT NUMBER W81XWH-12-1-0345 5c. PROGRAM...naltrexone; drug abuse ; glial activation; therapeutic approach to treating drug abuse ; opioids; cocaine 16. SECURITY CLASSIFICATION OF: 17. LIMITATION

  4. Expression of TLR-2, TLR-4, NOD2 and pNF-kappaB in a neonatal rat model of necrotizing enterocolitis.

    Directory of Open Access Journals (Sweden)

    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

  5. Significance of TLR4/MyD88 expression in breast cancer

    Science.gov (United States)

    Chen, Xiangjin; Zhao, Feng; Zhang, Huihao; Zhu, Youzhi; Wu, Kunlin; Tan, Guozheng

    2015-01-01

    Objective: To investigate the expression of TLR4/MyD88 in breast cancer, and explore the relationship between their expression and breast cancer tumor growth and invasion. Methods: We examined the protein expression of TLR4 and MyD88 in 60 cases of histologically confirmed breast cancer. The relationship of their protein expressions with clinical features including age at diagnosis, tumor size and stage, lymph node metastasis and distant metastasis were analyzed. Results: The IHC results showed that TLR4 and MyD88 were expressed in 63.3% (38/60) and 58.3% (35/60) of malignant breast tumors respectively. TLR4 expression in breast cancer were significantly higher than in fibroadenoma (n = 4, 20.0%) and adjacent normal tissues (n = 2, 10.0%) (P fibroadenoma (n = 4, 20.0%) and adjacent normal tissue (n = 3, 15.0%) (P fibroadenoma and adjacent normal tissues (P < 0.05). The protein expressions of TLR4 and MyD88 were also significantly associated with poor clinical features (P < 0.05). Conclusion: TLR4 and MyD88 expression might be associated with breast cancer growth and regional and distant metastases. PMID:26261595

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries.

    Science.gov (United States)

    Zhao, Alyssa; Blackburn, Corinne; Chin, Judith; Srinivasan, Mythily

    2014-08-31

    Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cariogenic bacteria and modulate the disease process. The objective of this study is to determine the potential of salivary sTLR-2 and sCD14 as biomarkers of caries activity and indirect measures of the cariogenic bacterial burden. Unstimulated whole saliva was collected from twenty caries free and twenty caries active children between the ages of 5 and 13 years. The concentration of sCD14 and sTLR-2 together with that of the cytokine IL-8 reported to be increased in dental caries was assessed by the enzyme linked immunosorbent assay. While the level of sCD14 and that of IL-8 was equivocal between the two groups, the sTLR-2 concentration in caries active saliva was significantly higher than that in caries free saliva. The sTLR-2 in saliva could serve as a potential biomarker for caries activity.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Effect of tick saliva on signalling pathways activated by TLR-2 ligand and Borrelia afzelii in dendritic cells

    Czech Academy of Sciences Publication Activity Database

    Lieskovská, Jaroslava; Kopecký, Jan

    2012-01-01

    Roč. 35, č. 8-9 (2012), s. 421-429 ISSN 0141-9838 R&D Projects: GA MŠk(CZ) LC06009; GA ČR GAP302/12/2208 Institutional support: RVO:60077344 Keywords : Borrelia * dendritic cells * tick saliva * Toll-like receptor signalling Subject RIV: EC - Immunology Impact factor: 2.208, year: 2012 http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3024.2012.01375.x/abstract

  11. 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.

  12. Effects of electrostatic interactions on ligand dissociation kinetics

    Science.gov (United States)

    Erbaş, Aykut; de la Cruz, Monica Olvera; Marko, John F.

    2018-02-01

    We study unbinding of multivalent cationic ligands from oppositely charged polymeric binding sites sparsely grafted on a flat neutral substrate. Our molecular dynamics simulations are suggested by single-molecule studies of protein-DNA interactions. We consider univalent salt concentrations spanning roughly a 1000-fold range, together with various concentrations of excess ligands in solution. To reveal the ionic effects on unbinding kinetics of spontaneous and facilitated dissociation mechanisms, we treat electrostatic interactions both at a Debye-Hückel (DH) (or implicit ions, i.e., use of an electrostatic potential with a prescribed decay length) level and by the more precise approach of considering all ionic species explicitly in the simulations. We find that the DH approach systematically overestimates unbinding rates, relative to the calculations where all ion pairs are present explicitly in solution, although many aspects of the two types of calculation are qualitatively similar. For facilitated dissociation (FD) (acceleration of unbinding by free ligands in solution) explicit-ion simulations lead to unbinding at lower free-ligand concentrations. Our simulations predict a variety of FD regimes as a function of free-ligand and ion concentrations; a particularly interesting regime is at intermediate concentrations of ligands where nonelectrostatic binding strength controls FD. We conclude that explicit-ion electrostatic modeling is an essential component to quantitatively tackle problems in molecular ligand dissociation, including nucleic-acid-binding proteins.

  13. 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.

  14. Cocaine-mediated induction of microglial activation involves the ER stress-TLR2 axis.

    Science.gov (United States)

    Liao, Ke; Guo, Minglei; Niu, Fang; Yang, Lu; Callen, Shannon E; Buch, Shilpa

    2016-02-09

    Neuroinflammation associated with advanced human immunodeficiency virus (HIV)-1 infection is often exacerbated by chronic cocaine abuse. Cocaine exposure has been demonstrated to mediate up-regulation of inflammatory mediators in in vitro cultures of microglia. The molecular mechanisms involved in this process, however, remain poorly understood. In this study, we sought to explore the underlying signaling pathways involved in cocaine-mediated activation of microglial cells. BV2 microglial cells were exposed to cocaine and assessed for toll-like receptor (TLR2) expression by quantitative polymerase chain reaction (qPCR), western blot, flow cytometry, and immunofluorescence staining. The mRNA and protein levels of cytokines (TNFα, IL-6, MCP-1) were detected by qPCR and ELISA, respectively; level of reactive oxygen species (ROS) production was examined by the Image-iT LIVE Green ROS detection kit; activation of endoplasmic reticulum (ER)-stress pathways were detected by western blot. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of activating transcription factor 4 (ATF4) with the TLR2 promoter. Immunoprecipitation followed by western blotting with tyrosine antibody was used to determine phosphorylation of TLR2. Cocaine-mediated up-regulation of TLR2 expression and microglial activation was validated in cocaine-injected mice. Exposure of microglial cells to cocaine resulted in increased expression of TLR2 with a concomitant induction of microglial activation. Furthermore, this effect was mediated by NADPH oxidase-mediated rapid accumulation of ROS with downstream activation of the ER-stress pathways as evidenced by the fact that cocaine exposure led to up-regulation of pPERK/peIF2α/ATF4 and TLR2. The novel role of ATF4 in the regulation of TLR2 expression was confirmed using genetic and pharmacological approaches. xThe current study demonstrates that cocaine-mediated activation of microglia involves up-regulation of TLR2 through the

  15. Lanosterol Modulates TLR4-Mediated Innate Immune Responses in Macrophages

    Directory of Open Access Journals (Sweden)

    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.

  16. Ligand Depot: a data warehouse for ligands bound to macromolecules.

    Science.gov (United States)

    Feng, Zukang; Chen, Li; Maddula, Himabindu; Akcan, Ozgur; Oughtred, Rose; Berman, Helen M; Westbrook, John

    2004-09-01

    Ligand Depot is an integrated data resource for finding information about small molecules bound to proteins and nucleic acids. The initial release (version 1.0, November, 2003) focuses on providing chemical and structural information for small molecules found as part of the structures deposited in the Protein Data Bank. Ligand Depot accepts keyword-based queries and also provides a graphical interface for performing chemical substructure searches. A wide variety of web resources that contain information on small molecules may also be accessed through Ligand Depot. Ligand Depot is available at http://ligand-depot.rutgers.edu/. Version 1.0 supports multiple operating systems including Windows, Unix, Linux and the Macintosh operating system. The current drawing tool works in Internet Explorer, Netscape and Mozilla on Windows, Unix and Linux.

  17. DMPD: Viral immunity: cross-priming with the help of TLR3. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15886091 Viral immunity: cross-priming with the help of TLR3. Salio M, Cerundolo V.... Curr Biol. 2005 May 10;15(9):R336-9. (.png) (.svg) (.html) (.csml) Show Viral immunity: cross-priming with ...the help of TLR3. PubmedID 15886091 Title Viral immunity: cross-priming with the help of TLR3. Authors Salio

  18. Melatonin: functions and ligands.

    Science.gov (United States)

    Singh, Mahaveer; Jadhav, Hemant R

    2014-09-01

    Melatonin is a chronobiotic substance that acts as synchronizer by stabilizing bodily rhythms. Its synthesis occurs in various locations throughout the body, including the pineal gland, skin, lymphocytes and gastrointestinal tract (GIT). Its synthesis and secretion is controlled by light and dark conditions, whereby light decreases and darkness increases its production. Thus, melatonin is also known as the 'hormone of darkness'. Melatonin and analogs that bind to the melatonin receptors are important because of their role in the management of depression, insomnia, epilepsy, Alzheimer's disease (AD), diabetes, obesity, alopecia, migraine, cancer, and immune and cardiac disorders. In this review, we discuss the mechanism of action of melatonin in these disorders, which could aid in the design of novel melatonin receptor ligands. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Polymorphisms in the Inflammatory Pathway Genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG Are Associated with Susceptibility of Inflammatory Bowel Disease in a Danish Cohort

    DEFF Research Database (Denmark)

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

    2014-01-01

    associated with risk of CD and the polymorphisms TLR2 (rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs352139), LY96 (rs11465996), NFKBIA (rs696), TNFA (rs1800629), TNFRSF1A (rs4149570), IL10 (rs3024505), IL23R (rs11209026), PTPN22 (rs2476601) and PPARG (rs1801282) were associated with risk of UC. When...... including all patients (IBD) the polymorphisms TLR2 (rs4696480 and rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs187084), TNFRSF1A (rs4149570), IL6R (rs4537545), IL10 (rs3024505), IL23R (rs11209026) and PTPN22 (rs2476601) were associated with risk. After Bonferroni correction for multiple testing...

  20. A New Manganese Dinuclear Complex with Phenolate Ligands and a Single Unsupported Oxo Bridge. Storage of Two Positive Charges within Less than 500 mV. Relevance to Photosynthesis.

    Science.gov (United States)

    Horner, Olivier; Anxolabéhère-Mallart, Elodie; Charlot, Marie-France; Tchertanov, Lyuba; Guilhem, Jean; Mattioli, Tony A.; Boussac, Alain; Girerd, Jean-Jacques

    1999-03-22

    The compound [Mn(III)(2)OL(2)](ClO(4))(2).2.23CHCl(3).0.65CH(2)Cl(2) where L(-) is the monoanionic N,N-bis(2-pyridylmethyl)-N'-salicyliden-1,2-diaminoethane ligand, has been synthesized. The complex dication [Mn(III)(2)OL(2)](2+) contains a linear Mn(III)-O-Mn(III) unit with a Mn-Mn distance of 3.516 Å. The pentadentate ligand L(-) wraps around the Mn(III) ion. Electrochemically, it is possible to prepare the one electron oxidized trication [Mn(2)OL(2)](3+) which crystallizes as [Mn(2)OL(2)](ClO(4))(2.37)(PF(6))(0.63).1.5CH(3)CN. The complex trication [Mn(2)OL(2)](3+) contains a Mn(III)-O-Mn(IV) unit with a Mn-Mn distance of 3.524 Å and a Mn-O-Mn angle of 178.7(2) degrees. The contraction of the coordination sphere around the Mn(IV) is clearly observed. The [Mn(2)OL(2)](2+) dication possesses a S = 0 electronic ground state with J = -216 cm(-)(1) (H = -JS(1)().S(2)()), whereas the [Mn(2)OL(2)](3+) trication shows a S = (1)/(2) ground state with J = -353 cm(-)(1). The UV-visible spectrum of [Mn(2)OL(2)](3+) exhibits an intense absorption band (epsilon = 3040 M(-)(1) cm(-)(1)) centered at 570 nm assigned to a phenolate --> Mn(IV) charge-transfer transition. The potentials of the redox couples determined by cyclic voltammetry are E degrees ([Mn(2)OL(2)](3+)/[Mn(2)OL(2)](2+)) = 0.54 V/saturated calomel electrode (SCE) and E degrees ([Mn(2)OL(2)](4+)/[Mn(2)OL(2)](3+)) = 0.99 V/SCE. Upon oxidation at 1.3 V/SCE, the band at 570 nm shifts to 710 nm (epsilon = 2500 M(-)(1) cm(-)(1)) and a well-defined band appears at 400 nm which suggests the formation of a phenoxyl radical. The [Mn(2)OL(2)](3+)( )()complex exhibits a 18-line X-band electron paramagnetic resonance (EPR) spectrum which has been simulated with rhombic tensors |A(1)(x)()| = 160 x 10(-)(4) cm(-)(1); |A(1)(y)()| = 130 x 10(-)(4) cm(-)(1); |A(1)(z)()| = 91 x 10(-)(4) cm(-)(1); |A(2)(x)()| = 62 x 10(-)(4) cm(-)(1); |A(2)(y)()| = 59 x 10(-)(4) cm(-)(1); |A(2)(z)()| = 62 x 10(-)(4) cm(-)(1) and g(x)() = 2.006; g

  1. 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.

  2. 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.

  3. 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.

  4. Gut microbiota is a key modulator of insulin resistance in TLR 2 knockout mice.

    Directory of Open Access Journals (Sweden)

    Andréa M Caricilli

    2011-12-01

    Full Text Available Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics, the metabolic characteristics, and insulin signaling in TLR2 knockout (KO mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes

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

    Energy Technology Data Exchange (ETDEWEB)

    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. 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.

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

    Science.gov (United States)

    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.

  8. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

    Science.gov (United States)

    Liu, Mingfu; Douthwaite, Stephen

    2002-01-01

    The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit. PMID:12417742

  9. Database of ligand-induced domain movements in enzymes

    Directory of Open Access Journals (Sweden)

    Hayward Steven

    2009-03-01

    Full Text Available Abstract Background Conformational change induced by the binding of a substrate or coenzyme is a poorly understood stage in the process of enzyme catalysed reactions. For enzymes that exhibit a domain movement, the conformational change can be clearly characterized and therefore the opportunity exists to gain an understanding of the mechanisms involved. The development of the non-redundant database of protein domain movements contains examples of ligand-induced domain movements in enzymes, but this valuable data has remained unexploited. Description The domain movements in the non-redundant database of protein domain movements are those found by applying the DynDom program to pairs of crystallographic structures contained in Protein Data Bank files. For each pair of structures cross-checking ligands in their Protein Data Bank files with the KEGG-LIGAND database and using methods that search for ligands that contact the enzyme in one conformation but not the other, the non-redundant database of protein domain movements was refined down to a set of 203 enzymes where a domain movement is apparently triggered by the binding of a functional ligand. For these cases, ligand binding information, including hydrogen bonds and salt-bridges between the ligand and specific residues on the enzyme is presented in the context of dynamical information such as the regions that form the dynamic domains, the hinge bending residues, and the hinge axes. Conclusion The presentation at a single website of data on interactions between a ligand and specific residues on the enzyme alongside data on the movement that these interactions induce, should lead to new insights into the mechanisms of these enzymes in particular, and help in trying to understand the general process of ligand-induced domain closure in enzymes. The website can be found at: http://www.cmp.uea.ac.uk/dyndom/enzymeList.do

  10. 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.

  11. Syntheses, crystal structures and magnetic properties of two mixed-valence Co(iii)Co(ii) compounds derived from Schiff base ligands: field-supported single-ion-magnet behavior with easy-plane anisotropy.

    Science.gov (United States)

    Mandal, Shuvankar; Mondal, Suraj; Rajnák, Cyril; Titiš, Ján; Boča, Roman; Mohanta, Sasankasekhar

    2017-10-14

    Two μ-phenoxo-μ 1,1 -azide dinuclear Co III Co II complexes [Co III (N 3 ) 2 L 1 (μ 1,1 -N 3 )Co II (N 3 )]·MeOH (1) and [Co III (N 3 ) 2 L 2 (μ 1,1 -N 3 )Co II (N 3 )]·MeOH (2) (HL 1 and HL 2 are two Schiff base ligands having N 2 O-N 2 O compartments) both possess one hexacoordinate Co(iii) and one pentacoordinate Co(ii) center. DC magnetic susceptibility and magnetization measurements show an appreciable amount of positive magnetic anisotropy (D/hc∼ 40 cm -1 ) that is also confirmed by ab initio CASSCF calculations. AC susceptibility measurements of 1 reveal that it exhibits a slow magnetic relaxation with two relaxation channels. The external magnetic field supports the low-frequency (LF) channel that escapes on heating more progressively than the high-frequency (HF) branch. The relaxation time is as slow as τ = 255 ms at T = 1.9 K and B DC = 0.6 T, where the LF mole fraction is 69%. The complex 2 also displays similar field-supported slow magnetic relaxation with two relaxation channels.

  12. TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis

    Directory of Open Access Journals (Sweden)

    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.

  13. Increased TLR4 expression in murine placentas after oral infection with periodontal pathogens

    Science.gov (United States)

    Arce, R.M.; Barros, S.P.; Wacker, B.; Peters, B.; Moss, K.; Offenbacher, S.

    2009-01-01

    Maternal periodontitis has emerged as a putative risk factor for preterm births in humans. The periodontitis-associated dental biofilm is thought to serve as an important source of oral bacteria and related virulence factors that hematogenously disseminate and affect the fetoplacental unit; however the underlying biological mechanisms are yet to be fully elucidated. This study hypothesized that an oral infection with the human periodontal pathogens Campylobacter rectus and Porphyromonas gingivalis is able to induce fetal growth restriction, placental inflammation and enhance Toll-like receptors type 4 (TLR4) expression in a murine pregnancy model. Female Balb/C mice (n=40) were orally infected with C. rectus and/or P. gingivalis over a 16-week period and mated once per week. Pregnant mice were sacrificed at embryonic day (E) 16.5 and placentas were collected and analyzed for TLR4 mRNA levels and qualitative protein expression by real time PCR and immunofluorescence. TLR4 mRNA expression was found to be increased in C. rectus-infected group (1.98±0.886 fold difference, Pperiodontal pathogens. The TLR4 pathway has been implicated in the pathogenesis of preterm births; therefore the abnormal regulation of placental TLR4 may give new insights into how maternal periodontitis and periodontal pathogens might be linked to placental inflammation and preterm birth pathogenesis. PMID:19101032

  14. Andrographolide inhibits multiple myeloma cells by inhibiting the TLR4/NF-κB signaling pathway.

    Science.gov (United States)

    Gao, Hui; Wang, Jianrong

    2016-02-01

    Andrographolide is an active component from the extract of Andrographis paniculata [(Burm.f) Nees], a medicinal plant from the Acanthaceae family. Pharmacological studies have revealed that andrographolide possesses anti-bacterial, anti-inflammatory, anti-viral, immune regulatory and hepatoprotective properties, and is efficacious in the treatment of cardiovascular diseases, while exhibiting low toxicity and low cost. The present study aimed to determine the inhibitory effects of andrographolide on the growth of multiple myeloma (MM) cells and its possible impact on the Toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling pathway. Cell proliferation was detected using an MTT assay, cellular apoptosis was measured using flow cytometry, and caspase-9/3 activation were assessed using colorimetric assay kits. Furthermore, TLR4 and NF-κB protein expression was determined by western blot analysis. The results revealed that andrographolide reduced the proliferation, while increasing cellular apoptosis and caspase-9/3 activation of MM cells, in addition to downregulating the expression of TLR4 and NF-κB protein. Of note, TLR4- or NF-κB-targeting small-interfering (si)RNA enhanced the andrographolide-induced inhibition of cell proliferation and induction of apoptosis of MM cells. The results of the present study therefore suggested that andrographolide inhibited multiple myeloma cells via the TLR4/NF-κB signaling pathway.

  15. Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

    Directory of Open Access Journals (Sweden)

    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.

  16. IGF-1R Inhibitor Ameliorates Diabetic Nephropathy with Suppressed HMGN1/TLR4 Pathway.

    Science.gov (United States)

    Jiali, Yu; Jingjing, Da; Rong, Dong; Yi, Sun; Yingjie, Nie; Fuxun, Yu; Lima, Zuo; Yan, Zha

    2018-01-30

    contribution of high mobility group nucleosome-binding protein 1 (HMGN1)/ Toll-like receptor 4 (TLR4) pathway in diabetic nephropathy (DN). And as an intervention of the potential mechanism above, the insulin growth factor 1 receptor (IGF-1R) inhibitor was examined for its therapeutic effect in the diabetic mice. Male C57BL/6J mice were administered streptozotocin(STZ) to induce diabetes and thus divided into 5 groups: the untreated group (DN group), the benazepril-treated group (BEN-DN group), the insulin-treated group (INS-DN group) and the IGF-1R inhibitor-treated group (IGF-DN group). Immunohistochemistry and in situ hybrization were performed to detect the expression of HMGN1 and TLR4 in renal tissue. To evaluate the effect of IGF-1R inhibitor, levels of blood glucose and kidney/body weight (KW/BW) were measured. And morphological changes and mesangial matrix expansion in kidneys were also detected. Increased expression of HMGN1 and TLR4 in renal tissue of STZ-induced type1 diabetic mellitus (T1DM) mice models was observed. IGF-1R inhibitor attenuate the established nephropathy with reduced expression of TLR4 protein, as revealed by a decrease in mesangial index. IGF-1R inhibitor might have therapeutic potential in DN through inhibition of HMGN1/TLR4 pathway. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Song Min

    2011-08-01

    Full Text Available Abstract Background Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD, are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs, a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aβ deposits and buffer-soluble Aβ in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aβ deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aβ deposits start. Methods Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. Results While no difference was found in cerebral Aβ load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg was less than that in a TLR4 wild-type AD model (TLR4W Tg. At 9 months, TLR4M Tg mice had increased Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes. Conclusion This is the first demonstration of TLR4

  18. Relationship of intestinal flora disorder with TLR/NK-kB and inflammatory mediator expression in patients with ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Ma Tao

    2017-07-01

    Full Text Available Objective: To study the relationship of intestinal flora disorder with TLR/NK-kB and inflammatory mediator expression in patients with ulcerative colitis (UC. Methods: Patients who were diagnosed with UC in Yan’an People’s Hospital between May 2015 and February 2017 were selected, patients with active UC and patients with remission UC were included in the AUC and RUC group; patients who were diagnosed with colonic polyps in Yan’an People’s Hospital through physical examination during the same period were selected as the control group. Feces was collected to test the flora number, mucosa lesion was collected to determine the expression of TLR/NK-kB and inflammatory mediators, and serum was collected to detect the levels of inflammatory mediators. Results: Bifidobacterium, lactobacillus acidophilus and bacteroides number in feces of AUC group and RUC group were significantly lower than those of control group while escherichia coli and enterococcus number in feces, TLR2, TLR4, TLR5, TLR9, NK-kB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were significantly higher than those of control group; bifidobacterium, lactobacillus acidophilus and bacteroides number in feces of AUC group were significantly lower than those of RUC group while escherichia coli and enterococcus number in feces, TLR2, TLR4, TLR5, TLR9, NKkB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were significantly higher than those of RUC group; TLR2, TLR4, TLR5, TLR9, NK-kB, HMGB1, TNF-α, IL-6 and IL-17 mRNA expression in intestinal mucosa lesion as well as HMGB1, TNF-α, IL-6 and IL-17 levels in serum were negatively correlated with bifidobacteria, lactobacillus acidophilus and bacteroides number, and positively correlated with escherichia coli and enterococcus number. Conclusion: The intestinal flora

  19. Four new single nucleotide polymorphisms (SNPs) of toll-like ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... innate immunity against pathogen infection and bridge the innate and adaptive immunity. It had been shown that in mammals, TLR7 could be an important recognizing receptor of the single-stranded RNA viruses and activated the innate immune responses through a signal transduction pathway (Kadowaki ...

  20. Puerarin attenuates cisplatin-induced rat nephrotoxicity: The involvement of TLR4/NF-κB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Xu Ma

    Full Text Available Puerarin was a major isoflavonoid derived from the Chinese medical herb radix puerariae (Gegen. In present study effect of puerarin on cisplatin nephrotoxicity was evaluated. Rat model of nephrotoxicity was established by a single intraperitoneal injection of cisplatin (7mg/kg. Puerarin was administrated through caudal vein injection once per day at the dose of 10mg/kg, 30mg/kg and 50mg/kg. Biochemical assays showed that after cisplatin treatment the serum urea and creatinine increased significantly compared with control (P<0.05. Cisplatin treatment significantly increased xanthine oxidase (XO activity and malondialdehyde (MDA formation, and significantly decreased the levels and /or activities of enzymatic and non-enzymatic antioxidants (GSH, GPx, GST, GR, SOD, CAT, in the kidney tissues. Renal levels of TNF-α and IL-6, two important inflammatory cytokines, were also upregulated by cisplatin. Histopathological examination indicated that cisplatin treatment resulted in severe necrosis and degeneration, hyaline casts in the tubules, intertubular hemorrhage, congestion and swelling in glomerulus and leukocytes infiltration in the kidney tissues. Western blot results demonstrated that cisplatin increased TLR4 and NF-κB protein expression in the kidney tissues. However, all these changes induced by cisplatin were significantly attenuated by puerarin treatment in dose-dependent manner, which indicated the renal protective effect of puerarin. Cell culture experiments illustrated that puerarin alone treatment concentration-dependently inhibited COLO205 and HeLa tumor cell growth and dose-dependently promoted the antitumor activity of cisplatin in COLO205 and HeLa tumor cells. The promotion effects might be attributed to suppression of cisplatin-increased NF-κB p65 expression by puerarin. Taken together, findings in this study suggested that puerarin exhibited renal protection against cisplatin nephrotoxicity via inhibiting TLR4/NF-κB signaling

  1. Puerarin attenuates cisplatin-induced rat nephrotoxicity: The involvement of TLR4/NF-κB signaling pathway

    Science.gov (United States)

    Ma, Xu; Yan, Lei; Zhu, Qing; Shao, Fengmin

    2017-01-01

    Puerarin was a major isoflavonoid derived from the Chinese medical herb radix puerariae (Gegen). In present study effect of puerarin on cisplatin nephrotoxicity was evaluated. Rat model of nephrotoxicity was established by a single intraperitoneal injection of cisplatin (7mg/kg). Puerarin was administrated through caudal vein injection once per day at the dose of 10mg/kg, 30mg/kg and 50mg/kg. Biochemical assays showed that after cisplatin treatment the serum urea and creatinine increased significantly compared with control (PCisplatin treatment significantly increased xanthine oxidase (XO) activity and malondialdehyde (MDA) formation, and significantly decreased the levels and /or activities of enzymatic and non-enzymatic antioxidants (GSH, GPx, GST, GR, SOD, CAT), in the kidney tissues. Renal levels of TNF-α and IL-6, two important inflammatory cytokines, were also upregulated by cisplatin. Histopathological examination indicated that cisplatin treatment resulted in severe necrosis and degeneration, hyaline casts in the tubules, intertubular hemorrhage, congestion and swelling in glomerulus and leukocytes infiltration in the kidney tissues. Western blot results demonstrated that cisplatin increased TLR4 and NF-κB protein expression in the kidney tissues. However, all these changes induced by cisplatin were significantly attenuated by puerarin treatment in dose-dependent manner, which indicated the renal protective effect of puerarin. Cell culture experiments illustrated that puerarin alone treatment concentration-dependently inhibited COLO205 and HeLa tumor cell growth and dose-dependently promoted the antitumor activity of cisplatin in COLO205 and HeLa tumor cells. The promotion effects might be attributed to suppression of cisplatin-increased NF-κB p65 expression by puerarin. Taken together, findings in this study suggested that puerarin exhibited renal protection against cisplatin nephrotoxicity via inhibiting TLR4/NF-κB signaling, with no inhibition but

  2. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA...... or slightly lower potencies than (S)-AA [e.g., EC(50) = 76 microM for (2S,4S)-4-methyl-AA (5a) as compared to EC(50) = 35 microM for (S)-AA]. The position of the methyl substituent had a profound effect on the observed pharmacology, whereas the absolute stereochemistry at the methylated carbon atom had a very......) analogs, and the synthesis, stereochemistry, and enantiopharmacology of 3-methyl-AA (4a-d), 4-methyl-AA (5a-d), 5-methyl-AA (6a-d), and (E)-Delta(4)-5-methyl-AA (7a and 7b) are reported. The compounds were resolved using chiral HPLC and the configurational assignments of the enantiomers were based on X...

  3. 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.

  4. 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

  5. Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk.

    Science.gov (United States)

    Feng, Mingye; Chen, James Y; Weissman-Tsukamoto, Rachel; Volkmer, Jens-Peter; Ho, Po Yi; McKenna, Kelly M; Cheshier, Samuel; Zhang, Michael; Guo, Nan; Gip, Phung; Mitra, Siddhartha S; Weissman, Irving L

    2015-02-17

    Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

  6. A method for determining customer requirement weights based on TFMF and TLR

    Science.gov (United States)

    Ai, Qingsong; Shu, Ting; Liu, Quan; Zhou, Zude; Xiao, Zheng

    2013-11-01

    'Customer requirements' (CRs) management plays an important role in enterprise systems (ESs) by processing customer-focused information. Quality function deployment (QFD) is one of the main CRs analysis methods. Because CR weights are crucial for the input of QFD, we developed a method for determining CR weights based on trapezoidal fuzzy membership function (TFMF) and 2-tuple linguistic representation (TLR). To improve the accuracy of CR weights, we propose to apply TFMF to describe CR weights so that they can be appropriately represented. Because the fuzzy logic is not capable of aggregating information without loss, TLR model is adopted as well. We first describe the basic concepts of TFMF and TLR and then introduce an approach to compute CR weights. Finally, an example is provided to explain and verify the proposed method.

  7. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    DEFF Research Database (Denmark)

    Nguyen, E.D.; Meiler, J.; Norn, C.

    2013-01-01

    is increased by one order of magnitude for every 10 residues known to contact the ligand. Additionally, in the case of DOR, knowledge of a single specific ligand-protein contact improved sampling efficiency 7 fold. These findings offer specific guidelines which may lead to increased success in determining...

  8. Redox-​Active Ligand-​Induced Homolytic Bond Activation

    NARCIS (Netherlands)

    Broere, D.L.J.; Metz, L.L.; de Bruin, B.; Reek, J.N.H.; Siegler, M.A.; van der Vlugt, J.I.

    2015-01-01

    Coordination of the novel redox-​active phosphine-​appended aminophenol pincer ligand (PNOH2) to PdII generates a paramagnetic complex with a persistent ligand-​centered radical. The complex undergoes fully reversible single-​electron oxidn. and redn. Homolytic bond activation of diphenyldisulfide

  9. Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

    Science.gov (United States)

    Matysiak, Stefan; Hellmuth, Klaus; El-Sagheer, Afaf H; Shivalingam, Arun; Ariyurek, Yavuz; de Jong, Marco; Hollestelle, Martine J; Out, Ruud; Brown, Tom

    2017-12-19

    DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

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

    Lifescience Database Archive (English)

    Full Text Available 18031251 Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation inrespiratory dise...l) (.csml) Show Toll-like receptor (TLR)-based networks regulate neutrophilic inflammation inrespiratory dise...utrophilic inflammation inrespiratory disease. Authors Sabroe I, Whyte MK. Publication Biochem Soc Trans. 20

  11. 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

  12. DMPD: Transcriptional signaling by double-stranded RNA: role of TLR3. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15733829 Transcriptional signaling by double-stranded RNA: role of TLR3. Sen GC, Sa...rkar SN. Cytokine Growth Factor Rev. 2005 Feb;16(1):1-14. (.png) (.svg) (.html) (.csml) Show Transcriptional sign...aling by double-stranded RNA: role of TLR3. PubmedID 15733829 Title Transcriptional signaling by double

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

    Directory of Open Access Journals (Sweden)

    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.

  14. DMPD: Pellino proteins: novel players in TLR and IL-1R signalling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17635639 Pellino proteins: novel players in TLR and IL-1R signalling. Schauvliege R..., Janssens S, Beyaert R. J Cell Mol Med. 2007 May-Jun;11(3):453-61. (.png) (.svg) (.html) (.csml) Show Pellino proteins...: novel players in TLR and IL-1R signalling. PubmedID 17635639 Title Pellino proteins: novel play

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Peng Zhou

    2016-01-01

    Full Text Available Aims. This work was conducted to establish an in vitro Parkinson’s disease (PD model by exposing BV-2 cells to 1-methyl-4-phenylpyridinium (MPP+ and exploring the roles of TLR2/TLR4/TLR9 in inflammatory responses to MPP+. Methods/Results. MTT assay showed that cell viability of BV-2 cells was 84.78 ± 0.86% and 81.18 ± 0.99% of the control after incubation with 0.1 mM MPP+ for 12 hours and 24 hours, respectively. Viability was not significantly different from the control group. With immunofluorescence technique, we found that MPP+ incubation at 0.1 mM for 12 hours was the best condition to activate BV-2 cells. In this condition, the levels of TNF-α, IL-1β, and iNOS protein were statistically increased compared to the control according to ELISA tests. Real time RT-PCR and western blot measurements showed that TLR4 was statistically increased after 0.1 mM MPP+ incubation for 12 hours. Furthermore, after siRNA interference of TLR4 mRNA, NF-κB activation and the levels of TNF-α, IL-1β, and iNOS were all statistically decreased in this cell model. Conclusion. MPP+ incubation at the concentration of 0.1 mM for 12 hours is the best condition to activate BV-2 cells for mimicking PD inflammation in BV-2 cells. TLR4 signalling plays a critical role in the activation of BV-2 cells and the induction of inflammation in this cell model.

  17. TLR9 polymorphisms and systemic lupus erythematosus risk: an update meta-analysis study.

    Science.gov (United States)

    Wang, Duan; Zhang, Chao; Zhou, Zongke; Pei, Fuxing

    2016-04-01

    It has been reported that the Toll-like receptor 9 (TLR9) gene polymorphisms may be associated with systemic lupus erythematosus (SLE) risk. However, some studies yielded conflicting results. Therefore, a comprehensive meta-analysis was performed to assess the precise association between TLR9 polymorphisms and SLE susceptibility. We performed a systematic search in PubMed, Embase (Ovid), China National Knowledge Internet, and Wanfang databases up to July 15, 2015. Odds ratio (OR) and 95 % confidence interval (CI) were used to pool the effect size. Statistical analyses were performed with STATA 11.0 software. In total, 21 studies from nineteen articles with 10,273 subjects were included in this meta-analysis. The overall results suggested that there was a statistically significant association between TLR9 rs187084 polymorphism and SLE risk observed in recessive model (TT vs. TC + CC: OR 1.17, 95 % CI 1.05-1.30, P = 0.005), codominant model (TT vs. CC: OR 1.22, 95 % CI 1.03-1.43, P = 0.019), and allele model (T vs. C: OR 1.15, 95 % CI 1.02-1.30, P = 0.020) in Asians. However, we found that there may be no significant association between the other three TLR9 polymorphisms and SLE risk in either Asians or non-Asians. In conclusion, the meta-analysis results suggested that TLR9 rs187084 polymorphism may increase the risk of SLE in Asians. However, no significant association between TLR9 SNPs (rs352139, rs352140, and rs5743836) and SLE risk was identified.

  18. Mixed-ligand binuclear copper(II) complex of 5 ...

    Indian Academy of Sciences (India)

    carbon, saturated Ag/AgCl and platinum wire are used as the working, reference and auxiliary electrodes, ... was mounted on a glass fibre for diffraction experiment. X-ray single crystal data were collected on a Kappa ..... with the covalent nature of the metal–ligand bond. 3.4 DNA binding studies. 3.4a Absorption spectral ...

  19. Single crystal EPR of the mixed-ligand complex of copper(II) with L-glutamic acid and 1,10-phenanthroline: a study on the narrowing of the hyperfine structure by exchange.

    Science.gov (United States)

    Neuman, Nicolás I; Franco, Vanina G; Ferroni, Felix M; Baggio, Ricardo; Passeggi, Mario C G; Rizzi, Alberto C; Brondino, Carlos D

    2012-12-20

    We report an EPR study at X- and Q-bands of polycrystalline and single crystal samples of the mixed copper(II) complex with L-glutamic acid (glu) and 1,10-phenantroline (phen), [Cu(glu)(phen)(H(2)O)](+) NO(3)(-)·2(H(2)O). The polycrystalline sample spectrum at Q-band showed well resolved g(∥ )and g(⊥) features and partially solved hyperfine structure at g(∥), typical for weakly exchange coupled systems. This is confirmed from the angular variation of the EPR spectra which shows for certain magnetic field orientations a partially solved hyperfine structure characteristic of weak exchange, whereas a single Lorentzian line corresponding to strong exchange is observed for others. Analysis and simulation of the single crystal EPR spectra were performed using the random frequency modulation model of Anderson. Numerical simulations of the angular variation of the EPR spectra showed that the narrowing of the hyperfine structure is due to an exchange-mediated mechanism in which transitions between any pair of lines are equally likely. The exchange interaction responsible for this process is mediated by hydrophobic interactions between two phen molecules and a mixed chemical path of the type CuA-O(ap)H···O-C-O(eq)-CuB, for which we evaluated an average isotropic exchange parameter |J| ≈ 25 × 10(-4) cm(-1).

  20. Ligand-guided receptor optimization.

    Science.gov (United States)

    Katritch, Vsevolod; Rueda, Manuel; Abagyan, Ruben

    2012-01-01

    Receptor models generated by homology or even obtained by crystallography often have their binding pockets suboptimal for ligand docking and virtual screening applications due to insufficient accuracy or induced fit bias. Knowledge of previously discovered receptor ligands provides key information that can be used for improving docking and screening performance of the receptor. Here, we present a comprehensive ligand-guided receptor optimization (LiBERO) algorithm that exploits ligand information for selecting the best performing protein models from an ensemble. The energetically feasible protein conformers are generated through normal mode analysis and Monte Carlo conformational sampling. The algorithm allows iteration of the conformer generation and selection steps until convergence of a specially developed fitness function which quantifies the conformer's ability to select known ligands from decoys in a small-scale virtual screening test. Because of the requirement for a large number of computationally intensive docking calculations, the automated algorithm has been implemented to use Linux clusters allowing easy parallel scaling. Here, we will discuss the setup of LiBERO calculations, selection of parameters, and a range of possible uses of the algorithm which has already proven itself in several practical applications to binding pocket optimization and prospective virtual ligand screening.

  1. 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

  2. DMPD: TLR signalling and activation of IRFs: revisiting old friends from the NF-kappaBpathway. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16006187 TLR signalling and activation of IRFs: revisiting old friends from the NF-...kappaBpathway. Moynagh PN. Trends Immunol. 2005 Sep;26(9):469-76. (.png) (.svg) (.html) (.csml) Show TLR sign...alling and activation of IRFs: revisiting old friends from the NF-kappaBpathway. PubmedID 16006187 Title TLR sign

  3. An Analysis of Central Residues Between Ligand-Bound and Ligand-Free Protein Structures Based on Network Approach.

    Science.gov (United States)

    Amala, Arumugam; Emerson, Isacc Arnold

    2017-08-01

    Depiction of protein structures as networks of interacting residues has enabled us to understand the structure and function of the protein. Previous investigations on closeness centrality have identified protein functional sites from three- dimensional structures. It is well recognized that ligand binding to a receptor protein induces a wide range of structural changes. An interesting question is how central residues function during conformational changes triggered during ligand binding? The aim of this study is to comprehend at what extent central residues change during ligand binding to receptor proteins. To determine this, we examined 37 pairs of protein structures consisting of ligand-bound and ligand-free forms. These protein structures were modelled as an undirected network and significant central residues were obtained using residue centrality measures. In addition to these, the basic network parameters were also analysed. On analysing the residue centrality measures, we observed that 60% of central residues were common in both the ligand-bound and ligand-free states. The geometry of the central residues revealed that they were situated closer to the protein center of the mass. Finally, we demonstrated the effectiveness of central residues in amino acids substitutions and in the evolution itself. The closeness centrality was also analyzed among different protein domain sizes and the values gradually declined from single-domains to multi-domain proteins suggesting that the network has potential for hierarchical organization. Betweenness centrality measure was also used to determine the central residues and 31% of these residues were common between the holo/apo states. Findings reveal that central residues play a significant role in determining the functional properties of proteins. These results have implications in predicting binding/active site residues, specifically in the context of drug designing, if additional information concerning ligand binding is

  4. 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

  5. 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.

  6. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-01-01

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

  8. 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.

  9. Novel olfactory ligands via terpene synthases.

    Science.gov (United States)

    Touchet, Sabrina; Chamberlain, Keith; Woodcock, Christine M; Miller, David J; Birkett, Michael A; Pickett, John A; Allemann, Rudolf K

    2015-05-01

    A synthetic biology approach to the rational design of analogues of olfactory ligands by providing unnatural substrates for the enzyme synthesising (S)-germacrene D, an olfactory ligand acting as a plant derived insect repellent, to produce novel ligands is described as a viable alternative to largely unsuccessful ligand docking studies. (S)-14,15-Dimethylgermacrene D shows an unexpected reversal in behavioural activity.

  10. Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA.

    Science.gov (United States)

    Karasik, Ellen; Kwiatkowski, Laura D; Hui, Hilda L; Colby, Judith F; Noble, Robert W

    2004-06-22

    Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.

  11. Diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the TLR4/NF-κB pathway.

    Science.gov (United States)

    Barcelos, Rômulo Pillon; Bresciani, Guilherme; Cuevas, Maria José; Martínez-Flórez, Susana; Soares, Félix Alexandre Antunes; González-Gallego, Javier

    2017-07-01

    Nonsteroidal anti-inflammatory drugs, such as diclofenac, are widely used to treat inflammation and pain in several conditions, including sports injuries. This study analyzes the influence of diclofenac on the toll-like receptor-nuclear factor kappa B (TLR-NF-κB) pathway in skeletal muscle of rats submitted to acute eccentric exercise. Twenty male Wistar rats were divided into 4 groups: control-saline, control-diclofenac, exercise-saline, and exercise-diclofenac. Diclofenac or saline were administered for 7 days prior to an acute eccentric exercise bout. The inflammatory status was evaluated through mRNA levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α), and protein content of COX-2, IL-6, and TNF-α in vastus lateralis muscle. Data obtained showed that a single bout of eccentric exercise significantly increased COX-2 gene expression. Similarly, mRNA expression and protein content of other inflammation-related genes also increased after the acute exercise. However, these effects were attenuated in the exercise + diclofenac group. TLR4, myeloid differentiation primary response gene 88 (MyD88), and p65 were also upregulated after the acute eccentric bout and the effect was blunted by the anti-inflammatory drug. These findings suggest that pretreatment with diclofenac may represent an effective tool to ameliorate the pro-inflammatory status induced by acute exercise in rat skeletal muscle possibly through an attenuation of the TLR4-NF-κB signaling pathway.

  12. 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 gen