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

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Nives Hörmann

Full Text Available The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs. Here, we report that colonization of germ-free (GF Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2 and protein-kinase B (AKT induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

Mutations in the TLR3 signaling pathway and beyond in adult patients with herpes simplex encephalitis.

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Mørk, N; Kofod-Olsen, E; Sørensen, K B; Bach, E; Ørntoft, T F; Østergaard, L; Paludan, S R; Christiansen, M; Mogensen, T H

2015-12-01

Herpes simplex encephalitis (HSE) in children has previously been linked to defects in type I interferon production downstream of Toll-like receptor (TLR)3. In the present study, we used whole-exome sequencing to investigate the genetic profile of 16 adult patients with a history of HSE. We identified novel mutations in IRF3, TYK2 and MAVS, molecules involved in generating innate antiviral immune responses, which have not previously been associated with HSE. Moreover, data revealed mutations in TLR3, TRIF, TBK1 and STAT1 known to be associated with HSE in children but not previously described in adults. All discovered mutations were heterozygous missense mutations, the majority of which were associated with significantly decreased antiviral responses to HSV-1 infection and/or the TLR3 agonist poly(I:C) in patient peripheral blood mononuclear cells compared with controls. Altogether, this study demonstrates novel mutations in the TLR3 signaling pathway in molecules previously identified in children, suggesting that impaired innate immunity to HSV-1 may also increase susceptibility to HSE in adults. Importantly, the identification of mutations in innate signaling molecules not directly involved in TLR3 signaling suggests the existence of innate immunodeficiencies predisposing to HSE beyond the TLR3 pathway.

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages

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Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Yang, Mi-So [Department of Microbiology, Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Song, Du-Sub [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); School of life sciences and Biotechnology, Korea University 5-ka, Anam-Dong, Sungbuk-ku, Seoul 136-701 (Korea, Republic of); Kim, Jae-Kyung; Park, Jong-Heum; Song, Beom-Seok; Park, Sang-Hyun; Lee, Ju-Woon [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Park, Hyun-Jin [School of life sciences and Biotechnology, Korea University 5-ka, Anam-Dong, Sungbuk-ku, Seoul 136-701 (Korea, Republic of); Kim, Jae-Hun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Baek, E-mail: ebbyun80@kaeri.re.kr [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@kongju.ac.k [Department of Food Science and Technology, Kongju National University, Yesan 340-800 (Korea, Republic of)

2013-08-16

Highlights: •Pro B2 elevated the expression of IRAK-M, a negative regulator of TLR signaling. •LPS-induced expression of cell surface molecules was inhibited by Pro B2. •LPS-induced production of pro-inflammatory cytokines was inhibited by Pro B2. •Pro B2 inhibited LPS-induced activation of MAPKs and NF-κB through IRAK-M. •Pro B2 inactivated naïve T cells by inhibiting LPS-induced cytokines via IRAK-M. -- Abstract: Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development

DMPD: TLR signaling. [Dynamic Macrophage Pathway CSML Database

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Full Text Available 2007 Feb 1. (.png) (.svg) (.html) (.csml) Show TLR signaling. PubmedID 17275323 Title TLR signaling. Author...s Kawai T, Akira S. Publication Semin Immunol. 2007 Feb;19(1):24-32. Epub 2007 Feb 1. Pathway - PNG File (.png) SVG File (.svg) HTML... File (.html) CSML File (.csml) Open .csml file with CIOP

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

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Yin-Ping Jia

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

The GABAA Receptor α2 Subunit Activates a Neuronal TLR4 Signal in the Ventral Tegmental Area that Regulates Alcohol and Nicotine Abuse

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Irina Balan

2018-04-01

Full Text Available Alcoholism initiates with episodes of excessive alcohol drinking, known as binge drinking, which is one form of excessive drinking (NIAAA Newsletter, 2004 that is related to impulsivity and anxiety (Ducci et al., 2007; Edenberg et al., 2004 and is also predictive of smoking status. The predisposition of non-alcohol exposed subjects to initiate binge drinking is controlled by neuroimmune signaling that includes an innately activated neuronal Toll-like receptor 4 (TLR4 signal. This signal also regulates cognitive impulsivity, a heritable trait that defines drug abuse initiation. However, the mechanism of signal activation, its function in dopaminergic (TH+ neurons within the reward circuitry implicated in drug-seeking behavior [viz. the ventral tegmental area (VTA], and its contribution to nicotine co-abuse are still poorly understood. We report that the γ-aminobutyric acidA receptor (GABAAR α2 subunit activates the TLR4 signal in neurons, culminating in the activation (phosphorylation/nuclear translocation of cyclic AMP response element binding (CREB but not NF-kB transcription factors and the upregulation of corticotropin-releasing factor (CRF and tyrosine hydroxylase (TH. The signal is activated through α2/TLR4 interaction, as evidenced by co-immunoprecipitation, and it is present in the VTA from drug-untreated alcohol-preferring P rats. VTA infusion of neurotropic herpes simplex virus (HSV vectors for α2 (pHSVsiLA2 or TLR4 (pHSVsiTLR4 but not scrambled (pHSVsiNC siRNA inhibits signal activation and both binge alcohol drinking and nicotine sensitization, suggesting that the α2-activated TLR4 signal contributes to the regulation of both alcohol and nicotine abuse.

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

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

2015-02-13

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

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.

Modulation of IL-33/ST2-TIR and TLR signalling pathway by fingolimod and analogues in immune cells.

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Rüger, K; Ottenlinger, F; Schröder, M; Zivković, A; Stark, H; Pfeilschifter, J M; Radeke, H H

2014-12-01

For the immune modulatory drug fingolimod (FTY720), lymphocyte sequestration has been extensively studied and accepted as mode of action. Further, direct effects on immune cell signalling are incompletely understood. Herein, we used the parent drug and newly synthesized analogues to investigate their effects on dendritic cell (DC) calcium signalling and on Th1, Th2 and Th17 responses. DC calcium signalling was determined with a single cell-based confocal assay and IL-33/ST2-TIR Th2-like response with ST2-transduced EL4-6.1 thymoma cells. The Th1/Th17 responses were examined with a LPS/TLR-enhanced antigen presentation assay with OVA-TCRtg CD4 and CD8 spleen cells. Our results revealed a comparable influence of fingolimod and S1P on intracellular calcium level in DC, while an oxy-derivative of fingolimod exhibited an EC50 of 3.3 nm, being 14 times more potent than FTY720-P. The IL-33/ST2-TIR Th2-like response in ST2-EL4 cells was inhibited by fingolimod and analogues at varying degrees. Using the OVA-TCRtg LPS/TLR-enhanced spleen cell assay, we found that fingolimod inhibited both IL-17 and IFN-γ production. In contrast, fingolimod phosphate failed to decrease Th1 cytokines. Interestingly, the effects of the parent compound fingolimod were modulated by the PP2A inhibitor okadaic acid, thus suggesting PP2A as relevant intracellular target. These studies describe detailed immune-modulating properties of fingolimod, including interference with a prototypical Th2 response via IL-33/ST2-TIR. Moreover, differential effects of fingolimod versus its phosphorylated derivative on TLR-activated and antigen-dependent Th1 activation suggest PP2A as an additional target of fingolimod immune therapy. Together with the analogues tested, these data may guide the development of more specific fingolimod derivatives. © 2014 John Wiley & Sons Ltd.

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

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Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

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.

In Vivo Role of TLR2 and MyD88 Signaling in Eliciting Innate Immune Responses in Staphylococcal Endophthalmitis

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Talreja, Deepa; Singh, Pawan Kumar; Kumar, Ashok

2015-01-01

Purpose. The purpose of this study was to investigate the protective mechanisms evoked by TLR2 and MyD88 signaling in bacterial endophthalmitis in vivo. Methods. Endophthalmitis was induced in wild-type (WT), TLR2−/−, MyD88−/−, and Cnlp−/− mice by intravitreal injections of a laboratory strain (RN6390) and two endophthalmitis isolates of Staphylococcus aureus. Disease progression was monitored by assessing corneal and vitreous haze, bacterial burden, and retinal tissue damage. Levels of inflammatory cytokines/chemokines were determined using quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to assess neutrophil infiltration. Cathelicidin-related antimicrobial peptide (CRAMP) expression was determined by immunostaining and dot blot. Results. Eyes infected with either laboratory or clinical isolates exhibited higher levels of inflammatory mediators at the early stages of infection (≤24 hours) in WT mice than in TLR2−/− or MyD88−/− mice. However, their levels surpassed that of WT mice at the later stages of infection (>48 hours), coinciding with increased bacterial burden and retinal damage. Both TLR2−/− and MyD88−/− retinas produced reduced levels of CRAMP, and its deficiency (Cnlp−/−) rendered the mice susceptible to increased bacterial burden and retinal tissue damage as early as 1 day post infection. Analyses of inflammatory mediators and neutrophil levels in WT versus Cnlp−/− mice showed a trend similar to that observed in TLR2 and MyD88 KO mice. Furthermore, we observed that even a 10-fold lower infective dose of S. aureus was sufficient to cause endophthalmitis in TLR2−/− and MyD88−/− mice. Conclusions. TLR2 and MyD88 signaling plays an important role in protecting the retina from staphylococcal endophthalmitis by production of the antimicrobial peptide CRAMP. PMID:25678692

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

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

2016-09-01

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

P-MAPA immunotherapy potentiates the effect of cisplatin on serous ovarian carcinoma through targeting TLR4 signaling.

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de Almeida Chuffa, Luiz Gustavo; de Moura Ferreira, Grazielle; Lupi, Luiz Antonio; da Silva Nunes, Iseu; Fávaro, Wagner José

2018-01-17

Toll-like receptors (TLRs) are transmembrane proteins expressed on the surface of ovarian cancer (OC) and immune cells. Identifying the specific roles of the TLR-mediated signaling pathways in OC cells is important to guide new treatments. Because immunotherapies have emerged as the adjuvant treatment for patients with OC, we investigated the effect of a promising immunotherapeutic strategy based on protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) combined with cisplatin (CIS) on the TLR2 and TLR4 signaling pathways via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) in an in vivo model of OC. Tumors were chemically induced by a single injection of 100 μg of 7,12-dimethylbenz(a)anthracene (DMBA) directly under the left ovarian bursa in Fischer 344 rats. After the rats developed serous papillary OC, they were given P-MAPA, CIS or the combination P-MAPA+CIS as therapies. To understand the effects of the treatments, we assessed the tumor size, histopathology, and the TLR2- and TLR4-mediated inflammatory responses. Although CIS therapy was more effective than P-MAPA in reducing the tumor size, P-MAPA immunotherapy significantly increased the expressions of TLR2 and TLR4. More importantly, the combination of P-MAPA with CIS showed a greater survival rate compared to CIS alone, and exhibited a significant reduction in tumor volume compared to P-MAPA alone. The combination therapy also promoted the increase in the levels of the following OC-related proteins: TLR4, MyD88, TRIF, inhibitor of phosphorylated NF-kB alpha (p-IkBα), and nuclear factor kappa B (NF-kB p65) in both cytoplasmic and nuclear sites. While P-MAPA had no apparent effect on tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, it seems to increase interferon-γ (IFN-γ), which may induce the Thelper (Th1)-mediated immune response. Collectively, our results suggest that P-MAPA immunotherapy combined with cisplatin

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

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

2010-04-13

Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence. Coexpression of alphaT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with alphaT2ib indicated interaction of alphaT2ib with its cognate antigen within cells. alphaT2ib inhibited NF-kappaB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding alphaT2ib into HEK293 cells demonstrated high efficiency of the TLR2-alphaT2ib interaction. The alphaT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-alphaT2ib. Transduction with AdValphaT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFalpha mRNA accumulation, as

Hyaluronan signaling during ozone-induced lung injury requires TLR4, MyD88, and TIRAP.

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Zhuowei Li

Full Text Available Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4. In this study, we further investigated the role of TLR4 adaptors in ozone-induced airway hyperresponsiveness (AHR and the direct response to hyaluronan fragments (HA. Using a murine model of AHR, C57BL/6J, TLR4-/-, MyD88-/-, and TIRAP-/- mice were characterized for AHR after exposure to either ozone (1 ppm × 3 h or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4-/-, MyD88-/-, or TIRAP-/- mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4-/-, MyD88-/-, or TIRAP-/- mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway.

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

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Lindenmaier Werner

2010-04-01

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

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

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Jincheng Chen

2015-07-01

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

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

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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.

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

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

2014-11-01

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

A novel CCR-2/TLR-2 triggered signaling in murine peritoneal macrophages intensifies bacterial (Staphylococcus aureus) killing by reactive oxygen species through TNF-R1.

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Nandi, Ajeya; Bishayi, Biswadev

2017-10-01

Macrophages are remarkably versatile in their ability to recognize and respond to a wide range of stimuli by expressing a variety of surface and intracellular receptors and triggering multiple signal transduction pathways. The onset of microbial infection is primarily determined by the initial contacts made by the microbes with the host macrophages. Although there prevail a relationship between the chemokine receptor and Toll like receptors during disease, particularly TLR-2 and CCR-2 signaling interdependence on each other has not been yet investigated during acute staphylococcal infection. Thus, the present study was aimed to trace possible interaction between CCR-2 and TLR-2 in peritoneal macrophages during acute Staphylococcus aureus infection. We found that neutralization of CCR-2 attenuates TLR-2 expression and restricts S. aureus burden but TLR-2 neutralization augments CCR-2 expression in macrophages, along with compromised host-derived reactive oxygen species production. S. aureus infection to CCR-2 intact but TLR-2 neutralized macrophages triggered production of IL-1β, TNF-α, IL-6, IFN-γ, MCP-1 and expression of iNOS, TNFR-1 and GPx with concomitant decrease in IL-10 production. Further, study with NG-monomethyl-l-arginine (L-NMMA) [iNOS blocker] and buthionine sulfoximine (BSO) [GPx blocker] revealed that S. aureus infection enhanced TLR-2 expression in CCR-2 intact and TLR-2 neutralized macrophages possibly via iNOS and TNFR-1 up regulation and GPx down regulation. Overall, our data indicate that targeting CCR-2 with neutralizing antibody in the early phase of S. aureus infection could restrict excessive inflammation with less compromised bacterial killing. It certainly would be a therapeutic strategy in S. aureus induced inflammatory and infective diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

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

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

2017-08-07

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

Role of Extracellular RNA and TLR3‐Trif Signaling in Myocardial Ischemia–Reperfusion Injury

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Chen, Chan; Feng, Yan; Zou, Lin; Wang, Larry; Chen, Howard H.; Cai, Jia‐Yan; Xu, Jun‐Mei; Sosnovik, David E.; Chao, Wei

2014-01-01

Background Toll‐like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia‐reperfusion (I/R) injury. Methods and Results Wild‐type (WT), TLR3−/−, Trif−/−, and interferon (IFN) α/β receptor‐1 deficient (IFNAR1−/−) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3−/− or Trif−/− mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)‐MyD88 signaling, lack of TLR3‐Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif−/− mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose‐dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3‐deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. Conclusion TLR3‐Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction. PMID:24390148

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

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

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Eliran Moshe Reuven

2014-08-01

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

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.

Constitutive MHC class I molecules negatively regulate TLR-triggered inflammatory responses via the Fps-SHP-2 pathway.

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Xu, Sheng; Liu, Xingguang; Bao, Yan; Zhu, Xuhui; Han, Chaofeng; Zhang, Peng; Zhang, Xuemin; Li, Weihua; Cao, Xuetao

2012-04-22

The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions. Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in crosstalk with TLR signaling via the Fps-SHP-2 pathway and control TLR-triggered innate inflammatory responses.

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.

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.

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

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

2010-01-01

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

A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling.

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Kitagawa, Yoshinori; Yamaguchi, Mayu; Zhou, Min; Komatsu, Takayuki; Nishio, Machiko; Sugiyama, Tsuyoshi; Takeuchi, Kenji; Itoh, Masae; Gotoh, Bin

2011-05-01

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

Antitumor Activity of Portulaca Oleracea L. Polysaccharide on HeLa Cells Through Inducing TLR4/NF-κB Signaling.

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Zhao, Rui; Zhang, Tao; Ma, Baoling; Li, Xing

2017-01-01

Abstarct We have previously shown that Portulaca oleracea L. polysaccharide (POL-P3b) possesses the ability to inhibit cervical cancer cell growth in vitro and in vivo. In this study, we explored how toll-like receptor 4 (TLR4) signaling correlated with the antitumor mechanism of POL-P3b. Western blotting was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using enzyme-linked immunosorbent assay (ELISA) kits. The effects of POL-P3b on the proliferation and apoptosis in HeLa cells were determined by WST-8 assay and Hoechst 33342/propidium iodide (PI) assay. Our results demonstrated that lipopolysaccharide (LPS) binding to TLR4 on tumor cells could enhance HeLa cell proliferation and increase the expression of TLR4 and the downstream molecules. Treating HeLa cells with POL-P3b could decrease the proliferation of HeLa cells, and upregulate Bax level and downregulate Bcl-2 level in a concentration-dependent manner. In addition, POL-P3b inhibited the protein expression levels of TLR4, MyD88, TRAF6, Activator Protein-1 (AP-1) and nuclear factor-κB (NF-κB) subunit P65 in HeLa cells. Furthermore, POL-P3b also reduced the production of cytokine/chemokine. Taken together, the present work suggested the antitumor mechanism of POL-P3b by downregulating TLR4 downstream signaling pathway and inducing cell apoptosis. Our results may provide direct evidence to suggest that POL-P3b should be considered as a potent nutrient supplement for oncotherapy.

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

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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.

TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation.

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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.

BAD-LAMP controls TLR9 trafficking and signalling in human plasmacytoid dendritic cells.

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Combes, Alexis; Camosseto, Voahirana; N'Guessan, Prudence; Argüello, Rafael J; Mussard, Julie; Caux, Christophe; Bendriss-Vermare, Nathalie; Pierre, Philippe; Gatti, Evelina

2017-10-13

Toll-like receptors (TLR) are essential components of the innate immune system. Several accessory proteins, such as UNC93B1, are required for transport and activation of nucleic acid sensing Toll-like receptors in endosomes. Here, we show that BAD-LAMP (LAMP5) controls TLR9 trafficking to LAMP1 + late endosomes in human plasmacytoid dendritic cells (pDC), leading to NF-κB activation and TNF production upon DNA detection. An inducible VAMP3 +/ LAMP2 +/ LAMP1 - endolysosome compartment exists in pDCs from which TLR9 activation triggers type I interferon expression. BAD-LAMP-silencing enhances TLR9 retention in this compartment and consequent downstream signalling events. Conversely, sustained BAD-LAMP expression in pDCs contributes to their lack of type I interferon production after exposure to a TGF-β-positive microenvironment or isolation from human breast tumours. Hence, BAD-LAMP limits interferon expression in pDCs indirectly, by promoting TLR9 sorting to late endosome compartments at steady state and in response to immunomodulatory cues.TLR9 is highly expressed by plasmacytoid dendritic cells and detects nucleic acids, but to discriminate between host and microbial nucleic acids TLR9 is sorted into different endosomal compartments. Here the authors show that BAD-LAMP limits type 1 interferon responses by sorting TLR9 to late endosomal compartments.

Dioscorin isolated from Dioscorea alata activates TLR4-signaling pathways and induces cytokine expression in macrophages.

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Fu, Shu-Ling; Hsu, Ya-Hui; Lee, Pei-Yeh; Hou, Wen-Chi; Hung, Ling-Chien; Lin, Chao-Hsiung; Chen, Chiu-Ming; Huang, Yu-Jing

2006-01-06

The Toll-like receptor 4 (TLR4)-signaling pathway is crucial for activating both innate and adaptive immunity. TLR4 is a promising molecular target for immune-modulating drugs, and TLR4 agonists are of therapeutic potential for treating immune diseases and cancers. Several medicinal herb-derived components have recently been reported to act via TLR4-dependent pathways, suggesting that medicinal plants are potential resources for identifying TLR4 activators. We have applied a screening procedure to systematically identify herbal constituents that activate TLR4. To exclude possible LPS contamination in these plant-derived components, a LPS inhibitor, polymyxin B, was added during screening. One of the plant components we identified from the screening was dioscorin, the glycoprotein isolated from Dioscorea alata. It induced TLR4-downstream cytokine expression in bone marrow cells isolated from TLR4-functional C3H/HeN mice but not from TLR4-defective C3H/HeJ mice. Dioscorin also stimulated multiple signaling molecules (NF-kappaB, ERK, JNK, and p38) and induced the expression of cytokines (TNF-alpha, IL-1beta, and IL-6) in murine RAW 264.7 macrophages. Furthermore, the ERK, p38, JNK, and NF-kappaB-mediated pathways are all involved in dioscorin-mediated TNF-alpha production. In summary, our results demonstrate that dioscorin is a novel TLR4 activator and induces macrophage activation via typical TLR4-signaling pathways.

DMPD: TLR signaling. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

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

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

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

2015-01-01

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

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

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Anne Månsson Kvarnhammar

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

Retinal astrocytes pretreated with NOD2 and TLR2 ligands activate uveitogenic T cells.

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Guomin Jiang

Full Text Available On entering the tissues, infiltrating autoreactive T cells must be reactivated locally to gain pathogenic activity. We have previously reported that, when activated by Toll-like receptor 3 (TLR3 and TLR4 ligands, retinal astrocytes (RACs are able to function as antigen-presenting cells to re-activate uveitogenic T cells and allow responder T cells to induce uveitis in mice. In the present study, we found that, although the triggering of TLR2 or nucleotide-binding oligomerization domain receptor 2 (NOD2 alone did not activate RACs, their combined triggering induced RACs with the phenotypes required to efficiently re-activate interphotoreceptor retinoid-binding protein (IRBP-specific T cells. The synergistic effect of TLR2 and NOD2 ligands on RAC activation might be explained by the observations that bacterial lipoprotein (BLP, a TLR2 ligand was able to upregulate NOD2 expression and the combination of BLP and muramyldipeptide (MDP, a NOD2 ligand enhanced the expression of RICK (Rip2, the signaling molecule of NOD2. Moreover, the synergistic effect of MDP and BLP on RACs was lost when the RACs were derived from NOD2 knockout mice or were pre-treated with Rip2 antagonist. Thus, our data suggest that exogenous or endogenous molecules acting on both TLR2 and NOD2 on RACs might have an enhancing effect on susceptibility to autoimmune uveitis.

Implication of TLR- but not of NOD2-signaling pathways in dendritic cell activation by group B Streptococcus serotypes III and V.

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Paul Lemire

Full Text Available Group B Streptococcus (GBS is an important agent of life-threatening invasive infection. It has been previously shown that encapsulated type III GBS is easily internalized by dendritic cells (DCs, and that this internalization had an impact on cytokine production. The receptors underlying these processes are poorly characterized. Knowledge on the mechanisms used by type V GBS to activate DCs is minimal. In this work, we investigated the role of Toll-like receptor (TLR/MyD88 signaling pathway, the particular involvement of TLR2, and that of the intracellular sensing receptor NOD2 in the activation of DCs by types III and V GBS. The role of capsular polysaccharide (CPS, one of the most important GBS virulence factors in bacterial-DC interactions was evaluated using non-encapsulated mutants. Despite differences in the role of CPS between types III and V GBS in bacterial internalization and intracellular survival, no major differences were observed in their capacity to modulate release of cytokines by DC. For both serotypes, CPS had a minor role in this response. Production of cytokines by DCs was shown to strongly rely on MyD88-dependent signaling pathways, suggesting that DCs recognize GBS and become activated mostly through TLR signaling. Yet, GBS-infected TLR2-/- DCs only showed a partial reduction in the production of IL-6 and CXCL1 compared to control DCs. Surprisingly, CXCL10 release by type III or type V GBS-infected DCs was MyD88-independent. No differences in DC activation were observed between NOD2-/- and control DCs. These results demonstrate the involvement of various receptors and the complexity of the cytokine production pathways activated by GBS upon DC infection.

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

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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

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

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Shweta Jain

Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

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Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

2011-01-01

Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage

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Lin Sen

2012-03-01

Full Text Available Abstract Background Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4 plays a role in inflammatory damage caused by brain disorders. Methods In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics. Results Compared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH. Conclusions Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately

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

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

2016-01-01

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

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

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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...

The role of TLR2 and bacterial lipoprotein in enhancing airway inflammation and immunity

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Amit A Lugade

2011-04-01

Full Text Available Non-typeable Haemophilus influenzae (NTHI colonizes the lower respiratory tract of patients with chronic obstructive pulmonary disease (COPD and also causes exacerbations of the disease. The 16-kDa lipoprotein P6 has been widely studied as a potential vaccine antigen due to its highly conserved expression amongst NTHI strains. Although P6 is known to induce potent inflammatory responses, its role in the pathogenesis of NTHI infection in vivo has not been examined. Additionally, the presence of an amino-terminal lipid motif on P6 serves to activate host TLR2 signaling. The role of host Toll-like receptor 2 (TLR2 and NTHI expression of the lipoprotein P6 on the induction of airway inflammation and generation of adaptive immune responses following chronic NTHI stimulation was evaluated with TLR2-deficient mice and a P6-deficient NTHI strain. Absence of either host TLR2 or bacterial P6 resulted in diminished levels of immune cell infiltration within lungs of mice exposed to NTHI. Pro-inflammatory cytokine secretion was also reduced in lungs that did not express TLR2 or were exposed to NTHI devoid of P6. Induction of specific antibodies to P6 was severely limited in TLR2-deficient mice. Although mice exposed to the P6-deficient NTHI strain were capable of generating antibodies to other surface antigens of NTHI, these levels were lower compared to those observed in mice exposed to P6-expressing NTHI. Therefore, cognate interaction between host TLR2 and bacterial P6 serves to enhance lung inflammation and elicit robust adaptive immune responses during NTHI exposure. Strategies to limit NTHI inflammation while simultaneously promoting robust immune responses may benefit from targeting the TLR2:P6 signaling axis.

Orphan Nuclear Receptor ERRα Controls Macrophage Metabolic Signaling and A20 Expression to Negatively Regulate TLR-Induced Inflammation.

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Yuk, Jae-Min; Kim, Tae Sung; Kim, Soo Yeon; Lee, Hye-Mi; Han, Jeongsu; Dufour, Catherine Rosa; Kim, Jin Kyung; Jin, Hyo Sun; Yang, Chul-Su; Park, Ki-Sun; Lee, Chul-Ho; Kim, Jin-Man; Kweon, Gi Ryang; Choi, Hueng-Sik; Vanacker, Jean-Marc; Moore, David D; Giguère, Vincent; Jo, Eun-Kyeong

2015-07-21

The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming. Copyright © 2015 Elsevier Inc. All rights reserved.

Innate immune response of alveolar macrophage to house dust mite allergen is mediated through TLR2/-4 co-activation.

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Chia-Fang Liu

Full Text Available House dust mite, Dermatophagoides pteronyssinus (Der p, is one of the major allergens responsible for allergic asthma. However, the putative receptors involved in the signalization of Der p to the innate immune cells are still poorly defined as well as the impact of their activation on the outcome of the allergen-induced cell response. We previously reported that the HDM activation of mouse alveolar macrophages (AM involves the TLR4/CD14 cell surface receptor complex. Here using a TLR ligand screening essay, we demonstrate that HDM protein extract engages the TLR2, in addition to the TLR4, in engineered TLR-transfected HEK cells but also in the MH-S mouse alveolar macrophage cell line model. Moreover we found that the concomitant recruitment of the MH-S cell's TLR2 and TLR4 receptors by the HDM extract activates the MyD88-dependent signaling pathway and leads to the secretion of the NF-κB regulated pro-inflammatory factors NO and TNF-α. However unlike with the canonical TLR4 ligand (i.e. the bacterial LPS mobilization of TLR4 by the HDM extract induces a reduced production of the IL-12 pro-inflammatory cytokine and fails to trigger the expression of the T-bet transcription factor. Finally we demonstrated that HDM extract down-regulates LPS induced IL-12 and T-bet expression through a TLR2 dependent mechanism. Therefore, we propose that the simultaneous engagement of the TLR2 and TLR4 receptors by the HDM extract results in a cross regulated original activation pattern of the AM which may contribute to the Th2 polarization of the allergen-induced immune response. The deciphering of these cross-regulation networks is of prime importance to open the way for original therapeutic strategies taking advantage of these receptors and their associated signaling pathways to treat allergic asthma.

Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

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Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

2018-01-01

Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

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Zheng Lu

2018-05-01

Full Text Available Arctigenin (ARG, one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21 and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α and transforming growth factor beta1 (TGF-β1 in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126. Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway.

Science.gov (United States)

Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

2018-01-01

Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L. , has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis

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Nikolay N. Kuzmich

2017-10-01

Full Text Available Toll-Like Receptor 4 (TLR4 signal pathway plays an important role in initiating the innate immune response and its activation by bacterial endotoxin is responsible for chronic and acute inflammatory disorders that are becoming more and more frequent in developed countries. Modulation of the TLR4 pathway is a potential strategy to specifically target these pathologies. Among the diseases caused by TLR4 abnormal activation by bacterial endotoxin, sepsis is the most dangerous one because it is a life-threatening acute system inflammatory condition that still lacks specific pharmacological treatment. Here, we review molecules at a preclinical or clinical phase of development, that are active in inhibiting the TLR4-MyD88 and TLR4-TRIF pathways in animal models. These are low-molecular weight compounds of natural and synthetic origin that can be considered leads for drug development. The results of in vivo studies in the sepsis model and the mechanisms of action of drug leads are presented and critically discussed, evidencing the differences in treatment results from rodents to humans.

Interactions of Notch1 and TLR4 signaling pathways in DRG neurons of in vivo and in vitro models of diabetic neuropathy.

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Chen, Tianhua; Li, Hao; Yin, Yiting; Zhang, Yuanpin; Liu, Zhen; Liu, Huaxiang

2017-11-02

Understanding the interactions between Notch1 and toll-like receptor 4 (TLR4) signaling pathways in the development of diabetic peripheral neuropathy may lead to interpretation of the mechanisms and novel approaches for preventing diabetic neuropathic pain. In the present study, the interactions between Notch1 and TLR4 signaling pathways were investigated by using dorsal root ganglion (DRG) from diabetic neuropathic pain rats and cultured DRG neurons under high glucose challenge. The results showed that high glucose induced not only Notch1 mRNA, HES1 mRNA, and TLR4 mRNA expression, but also Notch1 intracellular domain (NICD1) and TLR4 protein expression in DRG neurons. The proportion of NICD1-immunoreactive (IR) and TLR4-IR neurons in DRG cultures was also increased after high glucose challenge. The above alterations could be partially reversed by inhibition of either Notch1 or TLR4 signaling pathway. Inhibition of either Notch1 or TLR4 signaling pathway could improve mechanical allodynia and thermal hyperalgesia thresholds. Inhibition of Notch1 or TLR4 signaling also decreased tumor necrosis factor-α (TNF-α) levels in DRG from diabetic neuropathic rats. These data imply that the interaction between Notch1 and TLR4 signaling pathways is one of the important mechanisms in the development or progression of diabetic neuropathy.

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

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

2018-04-01

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

Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

International Nuclear Information System (INIS)

Ivičak-Kocjan, Karolina; Panter, Gabriela; Benčina, Mojca; Jerala, Roman

2013-01-01

Highlights: •The chimeric protein fusing flagellin to the TLR5 ectodomain is constitutively active. •Mutation P736H within the BB-loop of TLR5 TIR domain renders the receptor inactive. •The R90D mutation in flagellin inactivated autoactivation of the chimeric protein. •The 2:2 stoichiometry of the TLR5:flagellin complex is physiologically relevant. -- Abstract: Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation

Determination of the physiological 2:2 TLR5:flagellin activation stoichiometry revealed by the activity of a fusion receptor

Energy Technology Data Exchange (ETDEWEB)

Ivičak-Kocjan, Karolina; Panter, Gabriela; Benčina, Mojca [Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana (Slovenia); The Centre of Excellence EN-FIST, 1000 Ljubljana (Slovenia); Jerala, Roman, E-mail: roman.jerala@ki.si [Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana (Slovenia); The Centre of Excellence EN-FIST, 1000 Ljubljana (Slovenia); The Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana (Slovenia)

2013-05-24

Highlights: •The chimeric protein fusing flagellin to the TLR5 ectodomain is constitutively active. •Mutation P736H within the BB-loop of TLR5 TIR domain renders the receptor inactive. •The R90D mutation in flagellin inactivated autoactivation of the chimeric protein. •The 2:2 stoichiometry of the TLR5:flagellin complex is physiologically relevant. -- Abstract: Toll-like receptor 5 (TLR5) recognizes flagellin of most flagellated bacteria, enabling activation of the MyD88-dependent signaling pathway. The recently published crystal structure of a truncated zebrafish TLR5 ectodomain in complex with an inactive flagellin fragment indicated binding of two flagellin molecules to a TLR5 homodimer, however this complex did not dimerize in solution. In the present study, we aimed to determine the physiological stoichiometry of TLR5:flagellin activation by the use of a chimeric protein composed of an active flagellin fragment linked to the N-terminus of human TLR5 (SF-TLR5). This construct was constitutively active. Inactivation by the R90D mutation within flagellin demonstrated that autoactivation of the chimeric protein depended solely on the specific interaction between TLR5 and flagellin. Addition of wild-type hTLR5 substantially lowered autoactivation of SF-TLR5 in a concentration dependent manner, an effect which was reversible by the addition of exogenous Salmonella typhimurium flagellin, indicating the biological activity of a TLR5:flagellin complex with a 2:2 stoichiometry. These results, in addition to the combinations of inactive P736H mutation within the BB-loop of the TIR domain of TLR5 and SF-TLR5, further confirm the mechanism of TLR5 activation.

MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2.

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Gu, Xing; Gao, Yan; Mu, De-Guang; Fu, En-Qing

2017-05-15

Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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Lauryn Samelko

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

TLR2−/− Mice Display Decreased Severity of Giardiasis via Enhanced Proinflammatory Cytokines Production Dependent on AKT Signal Pathway

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Xin Li

2017-09-01

Full Text Available Giardia infection is one of the most common causes of waterborne diarrheal disease in a wide array of mammalian hosts, including humans globally. Although numerous studies have indicated that adaptive immune responses are important for Giardia defense, however, whether the host innate immune system such as TLRs recognizes Giardia remains poorly understood. TLR2 plays a crucial role in pathogen recognition, innate immunity activation, and the eventual pathogen elimination. In this study, we investigated the role of TLR2 as a non-protective inflammatory response on controlling the severity of giardiasis. RT-PCR analysis suggested that TLR2 expression was increased in vitro. We demonstrated that Giardia lamblia-induced cytokines expression by the activation of p38 and ERK pathways via TLR2. Interestingly, the expression of IL-12 p40, TNF-α, and IL-6, but not IFN-γ, was enhanced in TLR2-blocked and TLR2−/− mouse macrophages exposed to G. lamblia trophozoites compared with wild-type (WT mouse macrophages. Further analysis demonstrated that G. lamblia trophozoites reduced cytokines secretion by activating AKT pathway in WT mouse macrophages. Immunohistochemical staining in G. lamblia cysts infected TLR2−/− and WT mice showed that TLR2 was highly expressed in duodenum in infected WT mice. Also, infected TLR2−/− and AKT-blocked mice showed an increased production of IL-12 p40 and IFN-γ compared with infected WT mice at the early stage during infection. Interestingly, infected TLR2−/− and AKT-blocked mice displayed a decreased parasite burden, an increased weight gain rate, and short parasite persistence. Histological morphometry showed shortened villus length, hyperplastic crypt and decreased ratio of villus height/crypt depth in infected WT mice compared with in infected TLR2−/− and AKT-blocked mice. Together, our results suggested that TLR2 deficiency leads to alleviation of giardiasis and reduction of parasite burden through

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

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Taslima T. Lina

2016-07-01

Full Text Available Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40% were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2 and p38 mitogen-activated protein kinase (MAPK pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4 expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

TLR5 signaling enhances the proliferation of human allogeneic CD40-activated B cell induced CD4hiCD25+ regulatory T cells.

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Ping-Lung Chan

Full Text Available Although diverse functions of different toll-like receptors (TLR on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4(hiCD25(+ regulatory T cells from naïve CD4(+CD25(- T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4(hiCD25(+ regulatory T cells. It was found that induced CD4(hiCD25(+ regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4(hiCD25(+ regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4(hiCD25(+ regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4(hiCD25(+ regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4(hiCD25(+ regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.

TLR4 and Insulin Resistance

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Jane J. Kim

2010-01-01

Full Text Available Chronic inflammation is a key feature of insulin resistance and obesity. Toll-Like Receptor 4 (TLR4, involved in modulating innate immunity, is an important mediator of insulin resistance and its comorbidities. TLR4 contributes to the development of insulin resistance and inflammation through its activation by elevated exogenous ligands (e.g., dietary fatty acids and enteric lipopolysaccharide and endogenous ligands (e.g., free fatty acids which are elevated in obese states. TLR4, expressed in insulin target tissues, activates proinflammatory kinases JNK, IKK, and p38 that impair insulin signal transduction directly through inhibitory phosphorylation of insulin receptor substrate (IRS on serine residues. TLR4 activation also leads to increased transcription of pro-inflammatory genes, resulting in elevation of cytokine, chemokine, reactive oxygen species, and eicosanoid levels that promote further insulin-desensitization within the target cell itself and in other cells via paracrine and systemic effects. Increased understanding of cell type-specific TLR4-mediated effects on insulin action present the opportunity and challenge of developing related therapeutic approaches for improving insulin sensitivity while preserving innate immunity.

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.

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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

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

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Jin Young-Hee

2011-12-01

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

Stimulants of Toll-like receptor (TLR)-2 and TLR-4 are abundant in certain minimally-processed vegetables.

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Erridge, Clett

2011-06-01

Stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 have been shown to promote insulin resistance and atherosclerosis in animal models of these diseases. As minimally processed vegetables (MPV) can contain a relatively large bacterial load compared to other foodstuffs, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in MPV using a transfection-based bioassay calibrated with Escherichia coli LPS and the synthetic lipopeptide Pam(3)CSK(4). Of 5 classes of MPV and 3 classes of related vegetable products considered to be likely to contain a high microbial load, diced onion and bean sprouts contained the highest levels of stimulants of TLR2 (up to 18.5 μg Pam(3)CSK(4)-equivalents per g) and TLR4 (up to 11.4 μg LPS-equivalents per g). By contrast, the majority of fresh whole vegetables examined reproducibly contained minimal or undetectable levels of TLR2- or TLR4-stimulants. The accumulation of TLR-stimulants in MPVs correlated well with growth of enterobacterial spoilage organisms. In conclusion, the modern trend towards eating minimally processed vegetables rather than whole foods is likely to be associated with increased oral exposure to stimulants of TLR2 and TLR4. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cellular Decision Making by Non-Integrative Processing of TLR Inputs

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Ryan A. Kellogg

2017-04-01

Full Text Available Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs that converge on the nuclear factor κB (NF-κB pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term “non-integrative” processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback.

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

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Lena Seifert

2015-12-01

Full Text Available Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1–/– mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

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

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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.

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

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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

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

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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.

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

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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

The TLR2 Antagonist Staphylococcal Superantigen-Like Protein 3 Acts as a Virulence Factor to Promote Bacterial Pathogenicity in vivo.

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Koymans, Kirsten J; Goldmann, Oliver; Karlsson, Christofer A Q; Sital, Wiedjai; Thänert, Robert; Bisschop, Adinda; Vrieling, Manouk; Malmström, Johan; van Kessel, Kok P M; de Haas, Carla J C; van Strijp, Jos A G; Medina, Eva

2017-01-01

Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by Staphylococcus aureus. Many studies have demonstrated the importance of TLR2 in murine S. aureus infection. S. aureus evades TLR2 activation by secreting two proteins, staphylococcal superantigen-like protein 3 (SSL3) and 4 (SSL4). In this study, we demonstrate that antibodies against SSL3 and SSL4 are found in healthy individuals, indicating that humans are exposed to these proteins during S. aureus colonization or infection. To investigate the TLR2-antagonistic properties of SSL3 and SSL4, we compared the infection with wild-type and SSL3/4 knockout S. aureus strains in an intravenous murine infection model. Direct evaluation of the contribution of SSL3/4 to infection pathogenesis was hindered by the fact that the SSLs were not expressed in the murine system. To circumvent this limitation, an SSL3-overproducing strain (pLukM-SSL3) was generated, resulting in constitutive expression of SSL3. pLukM-SSL3 exhibited increased virulence compared to the parental strain in a murine model that was found to be TLR2 dependent. Altogether, these data indicate that SSL3 contributes to S. aureus virulence in vivo. © 2017 S. Karger AG, Basel.

Mycobacterium tuberculosis Upregulates TNF-α Expression via TLR2/ERK Signaling and Induces MMP-1 and MMP-9 Production in Human Pleural Mesothelial Cells.

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Wei-Lin Chen

Full Text Available Tumor necrosis factor (TNF-α and matrix metalloproteinases (MMPs are elevated in pleural fluids of tuberculous pleuritis (TBP where pleural mesothelial cells (PMCs conduct the first-line defense against Mycobacterium tuberculosis (MTB. However, the clinical implication of TNF-α and MMPs in TBP and the response of PMCs to MTB infection remain unclear.We measured pleural fluid levels of TNF-α and MMPs in patients with TBP (n = 18 or heart failure (n = 18 as controls. Radiological scores for initial effusion amount and residual pleural fibrosis at 6-month follow-up were assessed. In vitro human PMC experiments were performed to assess the effect of heat-killed M. tuberculosis H37Ra (MTBRa on the expression of TNF-α and MMPs.As compared with controls, the effusion levels of TNF-α, MMP-1 and MMP-9 were significantly higher and correlated positively with initial effusion amount in patients with TBP, while TNF-α and MMP-1, but not MMP-9, were positively associated with residual pleural fibrosis of TBP. Moreover, effusion levels of TNF-α had positive correlation with those of MMP-1 and MMP-9 in TBP. In cultured PMCs, MTBRa enhanced TLR2 and TLR4 expression, activated ERK signaling, and upregulated TNF-α mRNA and protein expression. Furthermore, knockdown of TLR2, but not TLR4, significantly inhibited ERK phosphorylation and TNF-α expression. Additionally, both MTBRa and TNF-α markedly induced MMP-1 and MMP-9 synthesis in human PMCs, and TNF-α neutralization substantially reduced the production of MMP-1, but not MMP-9, in response to MTBRa stimulation.MTBRa activates TLR2/ERK signalings to induce TNF-α and elicit MMP-1 and MMP-9 in human PMCs, which are associated with effusion volume and pleural fibrosis and may contribute to pathogenesis of TBP. Further investigation of manipulation of TNF-α and MMP expression in pleural mesothelium may provide new insights into the mechanisms and rational treatment strategies for TBP.

Electroacupuncture Improved Hippocampal Neurogenesis following Traumatic Brain Injury in Mice through Inhibition of TLR4 Signaling Pathway

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Yuqin Ye

2017-01-01

Full Text Available The protective role of electroacupuncture (EA treatment in diverse neurological diseases such as ischemic stroke is well acknowledged. However, whether and how EA act on hippocampal neurogenesis following traumatic brain injury (TBI remains poorly understood. This study aims to investigate the effect of EA on hippocampal neurogenesis and neurological functions, as well as its underlying association with toll-like receptor 4 (TLR4 signaling in TBI mice. BrdU/NeuN immunofluorescence was performed to label newborn neurons in the hippocampus after EA treatment. Water maze test and neurological severity score were used to evaluate neurological function posttrauma. The hippocampal level of TLR4 and downstream molecules and inflammatory cytokines were, respectively, detected by Western blot and enzyme-linked immunosorbent assay. EA enhanced hippocampal neurogenesis and inhibited TLR4 expression at 21, 28, and 35 days after TBI, but the beneficial effects of EA on posttraumatic neurogenesis and neurological functions were attenuated by lipopolysaccharide-induced TLR4 activation. In addition, EA exerted an inhibitory effect on both TLR4/Myd88/NF-κB and TLR4/TRIF/NF-κB pathways, as well as the inflammatory cytokine expression in the hippocampus following TBI. In conclusion, EA promoted hippocampal neurogenesis and neurological recovery through inhibition of TLR4 signaling pathway posttrauma, which may be a potential approach to improve the outcome of TBI.

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

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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.

EGCG Maintains Th1/Th2 Balance and Mitigates Ulcerative Colitis Induced by Dextran Sulfate Sodium through TLR4/MyD88/NF-κB Signaling Pathway in Rats

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Xue Bing

2017-01-01

Full Text Available Objective. To observe the protective effect of epigallocatechin gallate (EGCG on dextran sulfate sodium- (DSS- induced ulcerative colitis in rats and to explore the roles of TLR4/MyD88/NF-κB signaling pathway. Methods. Rat models of ulcerative colitis were established by giving DSS. EGCG (50 mg/kg/d was given to assess disease activity index. HE staining was applied to observe histological changes. ELISA and qPCR detected the expression of inflammatory factors. Flow cytometry was used to measure the percentage of CD4+IFN-γ+ and CD4+IL-4+ in the spleen and colon. TLR4 antagonist E5564 was given in each group. Flow cytometry was utilized to detect CD4+IFN-γ+ and CD4+IL-4+ cells. Immunohistochemistry, qPCR, and western blot assay were applied to measure the expression of TLR4, MyD88, and NF-κB. Results. EGCG improved the intestinal mucosal injury in rats, inhibited production of inflammatory factors, maintained the balance of Th1/Th2, and reduced the expression of TLR4, MyD88, and NF-κB. After TLR4 antagonism, the protective effect of EGCG on intestinal mucosal injury was weakened in rats with ulcerative colitis, and the expressions of inflammatory factors were upregulated. Conclusion. EGCG can inhibit the intestinal inflammatory response by reducing the severity of ulcerative colitis and maintaining the Th1/Th2 balance through the TLR4/MyD88/NF-κB signaling pathway.

Matrine pretreatment improves cardiac function in rats with diabetic cardiomyopathy via suppressing ROS/TLR-4 signaling pathway.

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Liu, Zhong-wei; Wang, Jun-kui; Qiu, Chuan; Guan, Gong-chang; Liu, Xin-hong; Li, Shang-jian; Deng, Zheng-rong

2015-03-01

Matrine is an alkaloid from Sophora alopecuroides L, which has shown a variety of pharmacological activities and potential therapeutic value in cardiovascular diseases. In this study we examined the protective effects of matrine against diabetic cardiomyopathy (DCM) in rats. Male SD rats were injected with streptozotocin (STZ) to induce DCM. One group of DCM rats was pretreated with matrine (200 mg·kg(-1)·d(-1), po) for 10 consecutive days before STZ injection. Left ventricular function was evaluated using invasive hemodynamic examination, and myocardiac apoptosis was assessed. Primary rat myocytes were used for in vitro experiments. Intracellular ROS generation, MDA content and GPx activity were determined. Real-time PCR and Western blotting were performed to detect the expression of relevant mRNAs and proteins. DCM rats exhibited abnormally elevated non-fasting blood glucose levels at 4 weeks after STZ injection, and LV function impairment at 16 weeks. The cardiac tissues of DCM rats showed markedly increased apoptosis, excessive ROS production, and activation of TLR-4/MyD-88/caspase-8/caspase-3 signaling. Pretreatment with matrine significantly decreased non-fasting blood glucose levels and improved LV function in DCM rats, which were associated with reducing apoptosis and ROS production, and suppressing TLR-4/MyD-88/caspase-8/caspase-3 signaling in cardiac tissues. Incubation in a high-glucose medium induced oxidative stress and activation of TLR-4/MyD-88 signaling in cultured myocytes in vitro, which were significantly attenuated by pretreatment with N-acetylcysteine. Excessive ROS production in DCM activates the TLR-4/MyD-88 signaling, resulting in cardiomyocyte apoptosis, whereas pretreatment with matrine improves cardiac function via suppressing ROS/TLR-4 signaling pathway.

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

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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.

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.

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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.

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

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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.

Postnatal TLR2 activation impairs learning and memory in adulthood.

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

2015-08-01

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

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

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Carlos Wong-Baeza

2015-01-01

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

2'-O-methyl-modified RNAs act as TLR7 antagonists.

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Robbins, Marjorie; Judge, Adam; Liang, Lisa; McClintock, Kevin; Yaworski, Ed; MacLachlan, Ian

2007-09-01

RNA molecules such as single-stranded RNA (ssRNA) and small interfering RNA (siRNA) duplexes induce Toll-like receptor (TLR)-mediated immune stimulation after intracellular delivery. We have previously shown that selective incorporation of 2'-O-methyl (2'OMe) residues into siRNA abrogates cytokine production without reduction of gene silencing activity. Here we show that 2'OMe-modified RNA acts as a potent inhibitor of RNA-mediated cytokine induction in both human and murine systems. This activity does not require the direct incorporation of 2'OMe nucleotides into the immunostimulatory RNA or that the 2'OMe nucleotide-containing RNA be annealed as a complementary strand to form a duplex. Our results indicate that 2'OMe RNA acts as a potent antagonist of immunostimulatory RNA. We further show that 2'OMe RNA is able significantly to reduce both interferon-alpha (IFN-alpha) and interleukin-6 (IL-6) induction by the small-molecule TLR7 agonist loxoribine in human peripheral blood mononuclear cells (human PBMCs), in murine Flt3L dendritic cells (Flt3L DCs), and in vivo in mice. These results indicate that 2'OMe-modified RNA may have utility as an inhibitor of TLR7 with potential applications in the treatment of inflammatory and autoimmune diseases that involve TLR7-mediated immune stimulation.

HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

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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

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways.

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Seifert, Lena; Deutsch, Michael; Alothman, Sara; Alqunaibit, Dalia; Werba, Gregor; Pansari, Mridul; Pergamo, Matthew; Ochi, Atsuo; Torres-Hernandez, Alejandro; Levie, Elliot; Tippens, Daniel; Greco, Stephanie H; Tiwari, Shaun; Ly, Nancy Ngoc Giao; Eisenthal, Andrew; van Heerden, Eliza; Avanzi, Antonina; Barilla, Rocky; Zambirinis, Constantinos P; Rendon, Mauricio; Daley, Donnele; Pachter, H Leon; Hajdu, Cristina; Miller, George

2015-12-01

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

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Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

2015-08-01

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

NOD2 and TLR2 ligands trigger the activation of basophils and eosinophils by interacting with dermal fibroblasts in atopic dermatitis-like skin inflammation

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Jiao, Delong; Wong, Chun-Kwok; Qiu, Huai-Na; Dong, Jie; Cai, Zhe; Chu, Man; Hon, Kam-Lun; Tsang, Miranda Sin-Man; Lam, Christopher Wai-Kei

2016-01-01

The skin of patients with atopic dermatitis (AD) has a unique predisposition for colonization by Staphylococcus aureus (S. aureus), which contributes to the inflammation and grim prognosis of AD. Although the mechanism underlying the S. aureus-induced exacerbation of AD remains unclear, recent studies have found a pivotal role for pattern recognition receptors in regulating the inflammatory responses in S. aureus infection. In the present study, we used a typical mouse model of AD-like skin inflammation and found that S. aureus-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and toll-like receptor 2 (TLR2) ligands exacerbated AD-like symptoms, which were further deteriorated by the in vivo expansion of basophils and eosinophils. Subsequent histological analyses revealed that dermal fibroblasts were pervasive in the AD-like skin lesions. Co-culture of human dermal fibroblasts with basophils and eosinophils resulted in a vigorous cytokine/chemokine response to the NOD2/TLR2 ligands and the enhanced expression of intercellular adhesion molecule-1 on the dermal fibroblasts. Basophils and eosinophils were primarily responsible for the AD-related cytokine/chemokine expression in the co-cultures. Direct intercellular contact was necessary for the crosstalk between basophils and dermal fibroblasts, while soluble mediators were sufficient to mediate the eosinophil–fibroblast interactions. Moreover, the intracellular p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and nuclear factor-kappa B signaling pathways were essential for NOD2/TLR2 ligand-mediated activation of basophils, eosinophils, and dermal fibroblasts in AD-related inflammation. This study provides the evidence of NOD2/TLR2-mediated exacerbation of AD through activation of innate immune cells and therefore sheds light on a novel mechanistic pathway by which S. aureus contributes to the pathophysiology of AD. PMID:26388234

TLR-dependent control of Francisella tularensis infection and host inflammatory responses.

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Allison L Abplanalp

2009-11-01

Full Text Available Francisella tularensis is the causative agent of tularemia and is classified as a Category A select agent. Recent studies have implicated TLR2 as a critical element in the host protective response to F. tularensis infection, but questions remain about whether TLR2 signaling dominates the response in all circumstances and with all species of Francisella and whether F. tularensis PAMPs are predominantly recognized by TLR2/TLR1 or TLR2/TLR6. To address these questions, we have explored the role of Toll-like receptors (TLRs in the host response to infections with F. tularensis Live Vaccine Strain (LVS and F. tularensis subspecies (subsp. novicida in vivo.C57BL/6 (B6 control mice and TLR- or MyD88-deficient mice were infected intranasally (i.n. or intradermally (i.d. with F. tularensis LVS or with F. tularensis subsp. novicida. B6 mice survived >21 days following infection with LVS by both routes and survival of TLR1(-/-, TLR4(-/-, and TLR6(-/- mice infected i.n. with LVS was equivalent to controls. Survival of TLR2(-/- and MyD88(-/- mice, however, was significantly reduced compared to B6 mice, regardless of the route of infection or the subspecies of F. tularensis. TLR2(-/- and MyD88(-/- mice also showed increased bacterial burdens in lungs, liver, and spleen compared to controls following i.n. infection. Primary macrophages from MyD88(-/- and TLR2(-/- mice were significantly impaired in the ability to secrete TNF and other pro-inflammatory cytokines upon ex vivo infection with LVS. TNF expression was also impaired in vivo as demonstrated by analysis of bronchoalveolar lavage fluid and by in situ immunofluorescent staining.We conclude from these studies that TLR2 and MyD88, but not TLR4, play critical roles in the innate immune response to F. tularensis infection regardless of the route of infection or the subspecies. Moreover, signaling through TLR2 does not depend exclusively on TLR1 or TLR6 during F. tularensis LVS infection.

TLR2-dependent inhibition of macrophage responses to IFN-gamma is mediated by distinct, gene-specific mechanisms.

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Sarah A Benson

2009-07-01

Full Text Available Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-gamma through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam(3CSK(4 and assayed responses to IFN-gamma. Pam(3CSK(4 stimulation prior to IFN-gamma inhibited transcription of the unrelated IFN-gamma-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-gamma-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-kappaB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

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

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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.

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

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Yun Jeong Kim

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

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

Reprogramming of murine macrophages through TLR2 confers viral resistance via TRAF3-mediated, enhanced interferon production.

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Darren J Perkins

Full Text Available The cell surface/endosomal Toll-like Receptors (TLRs are instrumental in initiating immune responses to both bacteria and viruses. With the exception of TLR2, all TLRs and cytosolic RIG-I-like receptors (RLRs with known virus-derived ligands induce type I interferons (IFNs in macrophages or dendritic cells. Herein, we report that prior ligation of TLR2, an event previously shown to induce "homo" or "hetero" tolerance, strongly "primes" macrophages for increased Type I IFN production in response to subsequent TLR/RLR signaling. This occurs by increasing activation of the transcription factor, IFN Regulatory Factor-3 (IRF-3 that, in turn, leads to enhanced induction of IFN-β, while expression of other pro-inflammatory genes are suppressed (tolerized. In vitro or in vivo "priming" of murine macrophages with TLR2 ligands increase virus-mediated IFN induction and resistance to infection. This priming effect of TLR2 is mediated by the selective upregulation of the K63 ubiquitin ligase, TRAF3. Thus, we provide a mechanistic explanation for the observed antiviral actions of MyD88-dependent TLR2 and further define the role of TRAF3 in viral innate immunity.

Mycobacterial Phenolic Glycolipids Selectively Disable TRIF-Dependent TLR4 Signaling in Macrophages

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Reid Oldenburg

2018-01-01

Full Text Available Phenolic glycolipids (PGLs are cell wall components of a subset of pathogenic mycobacteria, with immunomodulatory properties. Here, we show that in addition, PGLs exert antibactericidal activity by limiting the production of nitric oxide synthase (iNOS in mycobacteria-infected macrophages. PGL-mediated downregulation of iNOS was complement receptor 3-dependent and comparably induced by bacterial and purified PGLs. Using Mycobacterium leprae PGL-1 as a model, we found that PGLs dampen the toll-like receptor (TLR4 signaling pathway, with macrophage exposure to PGLs leading to significant reduction in TIR-domain-containing adapter-inducing interferon-β (TRIF protein level. PGL-driven decrease in TRIF operated posttranscriptionally and independently of Src-family tyrosine kinases, lysosomal and proteasomal degradation. It resulted in the defective production of TRIF-dependent IFN-β and CXCL10 in TLR4-stimulated macrophages, in addition to iNOS. Our results unravel a mechanism by which PGLs hijack both the bactericidal and inflammatory responses of host macrophages. Moreover, they identify TRIF as a critical node in the crosstalk between CR3 and TLR4.

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

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Cristiane Beatriz de Oliveira

2015-02-01

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

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

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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...

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

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Zheng Pang

Full Text Available Toll-like receptors (TLRs recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88 and/or TIR-domain-containing adapter-inducing interferon-β (TRIF pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1, a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2, whereas TRIF-interferon-stimulated response elements (ISRE-mediated cytokine production (IFNβ, RANTES and IP-10 was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

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

toward HSV-2 infection. After a systemic infection, the cytokine serum response was markedly reduced in the double knockout mice, but only partly affected in either strain of the single knockout mice. This was supported by in vitro data showing that HSV-induced cytokine expression relayed on TLR2 and TLR...... stimulate innate antiviral activities, thereby protecting against HSV infection in the brain....

Andrographolide Suppress Tumor Growth by Inhibiting TLR4/NF-κB Signaling Activation in Insulinoma

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Zhang, Qian-Qian; Ding, Yi; Lei, Yan; Qi, Cui-Ling; He, Xiao-Dong; Lan, Tian; Li, Jiang-Chao; Gong, Ping; Yang, Xuesong; Geng, Jian-Guo; Wang, Li-Jing

2014-01-01

Insulinomas are rare tumors, and approximately 10% of insulinomas are malignant. Accumulating evidence has implicated that we still lack effective therapy to treat the patients who are diagnosed with rare malignant insulinoma. Previous studies have reported that Andrographolide (Andro) could inhibit cell cycle progression, reduce cell invasion and induce cell apoptosis in many common cancer cells. However, the effects of andro are cell type-dependent. So we emplored the β-TC-6 cells and the RIP1-Tag2 transgenic mouse model of endogenously growing insulinoma model to elucidate the possible anti-cancer effect of Andro on insulinoma, an uncommon type of malignant cancers in this study. Our experiments revealed that Andro significantly inhibited tumor growth at both the early-stage and the advanced-stage of insulinoma through targeting the TLR4/NF-κB signaling pathway. This work initially provides the evidence that the TLR4/NF-κB signaling pathway might be vital as a potential therapeutic target, and also indispensable in Andro-mediated anti-cancer effect in insulinoma. PMID:24719558

The TLR2 Antagonist Staphylococcal Superantigen-Like Protein 3 Acts as a Virulence Factor to Promote Bacterial Pathogenicity in vivo

NARCIS (Netherlands)

Koymans, Kirsten J.; Goldmann, Oliver; Karlsson, Christofer A.Q.; Sital, Wiedjai; Thänert, Robert; Bisschop, Adinda; Vrieling, Manouk; Malmström, Johan; van Kessel, Kok P M; De Haas, Carla J.C.; Van Strijp, Jos A.G.; Medina, Eva

2017-01-01

Toll-like receptor (TLR) signaling is important in the initiation of immune responses and subsequent instigation of adaptive immunity. TLR2 recognizes bacterial lipoproteins and plays a central role in the host defense against bacterial infections, including those caused by Staphylococcus aureus.

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

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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

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 player...s in TLR and IL-1R signalling. PubmedID 17635639 Title Pellino proteins: novel player...tml) CSML File (.csml) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

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

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Aurelie Le Mandat Schultz

Full Text Available BACKGROUND: The etiology of necrotizing enterocolitis (NEC results from a combination of several risk factors that act synergistically and occurs in the same circumstances as those which lead to innate immunity activation. Pattern recognition molecules could be an important player in the initiation of an exaggerated inflammatory response leading to intestinal injury in NEC. METHODOLOGY/PRINCIPAL FINDINGS: We specifically evaluated intestinal epithelial cell (IEC expression of Toll-like receptor 2 (TLR-2, TLR-4, NOD2 and phosphorylated NF-kappaB (pNF-kappaB after mucosal injury in a rat model of NEC induced by prematurity, systemic hypoxia, and a rich protein formula. In the control group (group 1, neonatal rats were full-term and breast-fed; in the experimental groups, rat pups were preterm at day 21 of gestation and rat-milk fed (group 2 or hand-gavaged with a protein rich formula after a hypoxia-reoxygenation procedure (group 3. Morphological mucosal changes in the small bowel were scored on hematoxylin- and eosin-stained sections. Immunohistochemistry was performed on frozen tissue sections using anti TLR-2 and active pNF-kappaB p65 antibodies. Real-time RT-PCR was performed to assess mRNA expression of NOD2, TLR-2 and TLR-4. Proliferation and apoptosis were studied in paraffin sections using anti Ki-67 and caspase-3 antibodies, respectively. The combination of immaturity, protein rich formula and a hypoxia-reoxygenation procedure induces pathological mucosal damage consistent with NEC. There was an overexpression of TLR-2, and pNF-kappaB in IECs that was correlated with the severity of mucosal damage, together with an increase of apoptotic IECs and markedly impaired proliferation. In addition, these immunological alterations appeared before severe mucosal damage. TLR-2 mRNA were also increased in NEC together with TLR-4 mRNA using real-time RT-PCR whereas NOD2 expression was unchanged. CONCLUSIONS/SIGNIFICANCE: These results show that this

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

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

2017-05-01

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

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

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

2013-04-01

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

TLR3 and TLR4 expression in healthy and diseased human endometrium

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Kimmig Rainer

2008-09-01

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

Bruton's tyrosine kinase mediates the synergistic signalling between TLR9 and the B cell receptor by regulating calcium and calmodulin.

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Elaine F Kenny

Full Text Available B cells signal through both the B cell receptor (BCR which binds antigens and Toll-like receptors (TLRs including TLR9 which recognises CpG DNA. Activation of TLR9 synergises with BCR signalling when the BCR and TLR9 co-localise within an auto-phagosome-like compartment. Here we report that Bruton's tyrosine kinase (BTK is required for synergistic IL6 production and up-regulation of surface expression of MHC-class-II, CD69 and CD86 in primary murine and human B cells. We show that BTK is essential for co-localisation of the BCR and TLR9 within a potential auto-phagosome-like compartment in the Namalwa human B cell line. Downstream of BTK we find that calcium acting via calmodulin is required for this process. These data provide new insights into the role of BTK, an important target for autoimmune diseases, in B cell activation.

The Effects of Agaricus blazei Murill Polysaccharides on Cadmium-Induced Apoptosis and the TLR4 Signaling Pathway of Peripheral Blood Lymphocytes in Chicken.

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Liu, Wenjing; Ge, Ming; Hu, Xuequan; Lv, Ai; Ma, Dexing; Huang, Xiaodan; Zhang, Ruili

2017-11-01

In this study, we investigated the effects of Agaricus blazei Murill polysaccharides (ABP) on cadmium (Cd)-induced apoptosis and the TLR4 signaling pathway of chicken peripheral blood lymphocytes (PBLs). Seven-day-old healthy chickens were randomly divided into four groups, and each group contained 20 males. The cadmium-supplemented diet group (Cd group) was fed daily with full feed that contained 140 mg cadmium chloride (CdCl 2 )/kg and 0.2 mL saline. The A. blazei Murill polysaccharide diet group (ABP group) was fed daily with full feed with 0.2 mL ABP solution (30 mg/mL) by oral gavage. The cadmium-supplemented plus A. blazei Murill polysaccharide diet group (Cd + ABP group) was fed daily with full feed containing 140 mg CdCl 2 /kg and 0.2 mL ABP solution (30 mg/mL) by gavage. The control group was fed daily with full feed with 0.2 mL saline per day. We measured the apoptosis rate and messenger RNA (mRNA) levels of apoptosis genes (caspase-3, Bax, and Bcl-2), the mRNA levels of TLR4 and TLR4 signaling pathway-related factors (MyD88, TRIF, NF-κB, and IRF3), the TLR4 protein expression, and the concentrations of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in chicken PBLs. The results showed that the PBL apoptosis rate was significantly increased, the mRNA levels of caspase-3 and Bax were significantly increased, while that of Bcl-2 was significantly reduced. The Bax/Bcl-2 ratio was significantly increased in the Cd group at 20, 40, and 60 days after treatment compared with that in the control group. After treatment with ABP, the above changes were clearly suppressed. At the same time, ABP reduced the concentrations of IL-1β, IL-6, and TNF-α induced by Cd. We also found that ABP inhibited the TLR4 mRNA level and protein expression and inhibited the mRNA levels of MyD88, TRIF, NF-κB, and IRF3. The results demonstrated that Cd could induce apoptosis, activate the TLR4 signaling pathway, and induce the expression of inflammatory cytokines in

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

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Adeline M Hajjar

Full Text Available Although lipopolysaccharide (LPS stimulation through the Toll-like receptor (TLR-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

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

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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.

Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling.

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Ye, Kai; Chen, Qi-Wei; Sun, Ya-Feng; Lin, Jian-An; Xu, Jian-Hua

2018-02-01

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment created by immune cells facilitates tumor migration. Epithelial-mesenchymal transition (EMT) is involved in the progression of cancer invasion and metastasis in an inflammatory microenvironment. B-lymphoma Moloney murine leukemia virus insertion region 1 (BMI-1) acts as an oncogene in various tumors. Ectopic expression of Bmi-1 have an effect on EMT and invasiveness. The purpose of this study was to investigate the efficacy of BMI-1 on inflammation-induced tumor migration and EMT and the underlying mechanism. We observed that the expression of BMI-1, TNF-α, and IL-1β was significantly increased in HT29 and HCT116 cells after THP-1 Conditioned-Medium (THP-1-CM) stimulation. Additionally, inhibition of BMI-1 impeded cell invasion induced by THP-1-CM-stimulation in both HT29 and HCT116 cells. BMI-1 knockdown remarkably repressed THP-1-CM-induced EMT by regulating the expression of EMT biomarkers with an increase in E-cadherin accompanied by decrease in N-cadherin and vimentin. Furthermore, downregulation of BMI-1 dramatically impeded THP-1-CM-triggered Toll-like receptor 4(TLR4)/myeloid differentiation protein 2(MD-2)/myeloid differentiation factor 88(MyD88) activity by repressing the expression of the TLR4/MD-2 complex and MyD88. Further data demonstrated that knockout of BMI-1 also dampened NF-κB THP-1-CM-triggered activity. Taken all data together, our findings established that BMI-1 modulated TLR4/MD-2/MyD88 complex-mediated NF-κB signaling involved in inflammation-induced cancer cells invasion and EMT, and therefore, could be a potential chemopreventive agent against inflammation-associated colorectal cancer. Establishment of an inflammatory microenvironment. Suppression of BMI-1 reverses THP-1-CM-induced inflammatory cytokine production in CRC. Loss of BMI-1 suppressed TLR4/MD-2/MyD88 complex-mediated NF-κB signaling. © 2017 Wiley

Wogonin improves histological and functional outcomes, and reduces activation of TLR4/NF-κB signaling after experimental traumatic brain injury.

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Chien-Cheng Chen

Full Text Available Traumatic brain injury (TBI initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. This study was undertaken to investigate the effects of wogonin, a flavonoid with potent anti-inflammatory properties, on functional and histological outcomes, brain edema, and toll-like receptor 4 (TLR4- and nuclear factor kappa B (NF-κB-related signaling pathways in mice following TBI.Mice subjected to controlled cortical impact injury were injected with wogonin (20, 40, or 50 mg·kg(-1 or vehicle 10 min after injury. Behavioral studies, histology analysis, and measurement of blood-brain barrier (BBB permeability and brain water content were carried out to assess the effects of wogonin. Levels of TLR4/NF-κB-related inflammatory mediators were also examined. Treatment with 40 mg·kg(-1 wogonin significantly improved functional recovery and reduced contusion volumes up to post-injury day 28. Wogonin also significantly reduced neuronal death, BBB permeability, and brain edema beginning at day 1. These changes were associated with a marked reduction in leukocyte infiltration, microglial activation, TLR4 expression, NF-κB translocation to nucleus and its DNA binding activity, matrix metalloproteinase-9 activity, and expression of inflammatory mediators, including interleukin-1β, interleukin-6, macrophage inflammatory protein-2, and cyclooxygenase-2.Our results show that post-injury wogonin treatment improved long-term functional and histological outcomes, reduced brain edema, and attenuated the TLR4/NF-κB-mediated inflammatory response in mouse TBI. The neuroprotective effects of wogonin may be related to modulation of the TLR4/NF-κB signaling pathway.

Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2

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Li, Ling; Li, Xin; Gong, Pengtao; Zhang, Xichen; Yang, Zhengtao; Yang, Ju; Li, Jianhua

2018-01-01

Trichomoniasis, caused by Trichomonas vaginalis infection, is the most prevalent sexually transmitted disease in female and male globally. However, the mechanisms by innate immunity against T. vaginalis infection have not been fully elucidated. Toll-like receptor2 (TLR2) has been shown to be involved in pathogen recognition, innate immunity activation, and inflammatory response to the pathogens. Nonetheless, the function of TLR2 against T. vaginalis remains unclear. In the present study, we investigated the role of TLR2 in mouse macrophages against T. vaginalis. RT-qPCR analysis revealed that T. vaginalis stimulation increased the gene expression of TLR2 in wild-type (WT) mouse macrophages. T. vaginalis also induced the secretion of IL-6, TNF-α, and IFN-γ in WT mouse macrophages, and the expression of these cytokines significantly decreased in TLR2-/- mouse macrophages and in WT mouse macrophages pretreated with MAPK inhibitors SB203580 (p38) and PD98059 (ERK). Western blot analysis demonstrated that T. vaginalis stimulation induced the activation of p38, ERK, and p65 NF-κB signal pathways in WT mouse macrophages, and the phosphorylation of p38, ERK, and p65 NF-κB significantly decreased in TLR2-/- mouse macrophages. Taken together, our data suggested that T. vaginalis may regulates proinflammatory cytokines production by activation of p38, ERK, and NF-κB p65 signal pathways via TLR2 in mouse macrophages. TLR2 might be involved in the defense and elimination of T. vaginalis infection. PMID:29692771

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

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

2017-11-01

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

Extracellular vesicles modulate host-microbe responses by altering TLR2 activity and phagocytosis.

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Jeroen van Bergenhenegouwen

Full Text Available Oral delivery of Gram positive bacteria, often derived from the genera Lactobacillus or Bifidobacterium, can modulate immune function. Although the exact mechanisms remain unclear, immunomodulatory effects may be elicited through the direct interaction of these bacteria with the intestinal epithelium or resident dendritic cell (DC populations. We analyzed the immune activation properties of Lactobacilli and Bifidobacterium species and made the surprising observation that cellular responses in vitro were differentially influenced by the presence of serum, specifically the extracellular vesicle (EV fraction. In contrast to the tested Lactobacilli species, tested Bifidobacterium species induce TLR2/6 activity which is inhibited by the presence of EVs. Using specific TLR ligands, EVs were found to enhance cellular TLR2/1 and TLR4 responses while TLR2/6 responses were suppressed. No effect could be observed on cellular TLR5 responses. We determined that EVs play a role in bacterial aggregation, suggesting that EVs interact with bacterial surfaces. EVs were found to slightly enhance DC phagocytosis of Bifidobacterium breve whereas phagocytosis of Lactobacillus rhamnosus was virtually absent upon serum EV depletion. DC uptake of a non-microbial substance (dextran was not affected by the different serum fractions suggesting that EVs do not interfere with DC phagocytic capacity but rather modify the DC-microbe interaction. Depending on the microbe, combined effects of EVs on TLR activity and phagocytosis result in a differential proinflammatory DC cytokine release. Overall, these data suggest that EVs play a yet unrecognized role in host-microbe responses, not by interfering in recipient cellular responses but via attachment to, or scavenging of, microbe-associated molecular patterns. EVs can be found in any tissue or bodily fluid, therefore insights into EV-microbe interactions are important in understanding the mechanism of action of potential

Atorvastatin attenuates experimental contrast-induced acute kidney injury: a role for TLR4/MyD88 signaling pathway.

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Yue, Rongzheng; Zuo, Chuan; Zeng, Jing; Su, Baihai; Tao, Ye; Huang, Songmin; Zeng, Rui

2017-11-01

To investigate the protective effect of different atorvastatin doses on contrast-induced acute kidney injury and the related mechanism. Healthy male Sprague-Dawley (SD) rats were randomly divided into the blank control group, experimental control group and different-dose atorvastatin groups. A rat model of contrast-induced acute kidney injury was established. We detected changes in serum creatinine (Scr) and blood urea nitrogen (BUN) before and after model establishment, observed and scored renal tubular injury, analyzed rat renal cell apoptosis, and measure the expression of signal pathway proteins and downstream inflammatory factors. After contrast agent injection, the Scr and BUN levels of the experimental control group were significantly increased, the different doses applied in the atorvastatin group significantly reduced the Scr and BUN levels (p atorvastatin doses have protective effects on contrast-induced acute renal tubular injury in rats, possibly by targeting TLR4, suppressing TLR4 expression, regulating the TLR4/Myd88 signaling pathway, and inhibiting the expression of downstream inflammatory factors.

Purinergic signaling to terminate TLR responses in macrophages

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Kajal eHamidzadeh

2016-03-01

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

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

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Simon Blankley

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

Live Faecalibacterium prausnitzii induces greater TLR2 and TLR2/6 activation than the dead bacterium in an apical anaerobic co-culture system.

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Maier, Eva; Anderson, Rachel C; Altermann, Eric; Roy, Nicole C

2018-02-01

Inappropriate activation of intestinal innate immune receptors, such as toll-like receptors (TLRs), by pathogenic bacteria is linked to chronic inflammation. In contrast, a "tonic" level of TLR activation by commensal bacteria is required for intestinal homeostasis. A technical challenge when studying this activation in vitro is the co-culturing of oxygen-requiring mammalian cells with obligate anaerobic commensal bacteria. To overcome this, we used a novel apical anaerobic co-culture system to successfully adapt a TLR activation assay to be conducted in conditions optimised for both cell types. Live Faecalibacterium prausnitzii, an abundant obligate anaerobe of the colonic microbiota, induced higher TLR2 and TLR2/6 activation than the dead bacterium. This enhanced TLR induction by live F. prausnitzii, which until now has not previously been described, may contribute to maintenance of gastrointestinal homeostasis. This highlights the importance of using physiologically relevant co-culture systems to decipher the mechanisms of action of live obligate anaerobes. © 2017 John Wiley & Sons Ltd.

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

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Sander eDe Kivit

2014-02-01

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

Boxb mediate BALB/c mice corneal inflammation through a TLR4/MyD88-dependent signaling pathway in Aspergillus fumigatus keratitis

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Min Liu

2018-04-01

Full Text Available AIM: To investigate whether high-mobility group box 1 (HMGB1 Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the Toll-like receptor 4 (TLR4/myeloid differentiation primary response 88 (MyD88-dependent signaling pathway in Aspergillus fumigatus (A. fumigatus keratitis. METHODS: The mice corneas were pretreated with phosphate buffer saline (PBS, Boxb before A. fumigatus infection. The abdominal cavity extracted macrophages were pretreated with PBS, Boxb, TLR4 inhibitor (CLI-095, Dimethyl sulfoxide (DMSO separately before A. fumigatus hyphae stimulation. HMGB1 was detected in normal and infected mice corneas and macrophages by real-time reverse transcriptase polymerase chain reaction (RT-PCR, the TLR4, MyD88, interleukin-1β (IL-1β, tumor necrosis factor-α (TNF-α were detected by Western blot and PCR. RESULTS: In BALB/c mice corneas, the expressions of TLR4, HMGB1, IL-1β, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α compared with PBS control after infection. In BALB/c mice abdominal cavity extracted macrophages, pretreatment with Boxb increased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α. CONCLUSION: In A. fumigatus keratitis, Boxb play a pro-inflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-MyD88 signal pathway.

TLR4 and NKT cell synergy in immunotherapy against visceral leishmaniasis.

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Subir Karmakar

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

MicroRNA-129-5p inhibits the development of autoimmune encephalomyelitis-related epilepsy by targeting HMGB1 through the TLR4/NF-kB signaling pathway.

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Liu, Ai-Hua; Wu, Ya-Ting; Wang, Yu-Ping

2017-06-01

The study aimed to explore the effects of microRNA-129-5p (miR-129-5p) on the development of autoimmune encephalomyelitis (AE)-related epilepsy by targeting HMGB1 through the TLR4/NF-kB signaling pathway in a rat model. AE-related epilepsy models were established. Sprague-Dawley (SD) rats were randomly divided into control, model, miR-129-5p mimics, miR-129-5p inhibitor, HMGB1 shRNA, TLR4/NF-kB (TLR4/NF-kB signaling pathway was inhibited) and miR-129-5p mimics+HMGB1 shRNA groups respectively. Latency to a first epilepsy seizure attack was recorded. Neuronal injuries in the hippocampus regions were detected using HE, Nissl and FJB staining methods 24h following model establishment. Microglial cells were detected by OX-42 immunohistochemistry. Expressions of miR-129-5p, HMGB1 and TLR4/NF-kB signaling pathway-related proteins were detected by qRT-PCR. Protein expressions of HMGB1 and TLR4/NF-kB signaling pathway-related proteins were detected by Western blotting. Dual luciferase reporter gene assay showed that miR-129-5p was negatively targeting HMGB1. Neurons of hippocampal tissues in rats were heavily injured by an injection of lithium chloride. Compared with the model and control groups, neuronal injury of the hippocampus and AE-related epilepsy decreased and microglial cells increased in the miR-129-5p mimics, HMGB1 shRNA and TLR4/NF-kB groups; however, in the miR-129-5p inhibitor group, miR-129-5p expression decreased, HMGB1 expression increased, TLR4/NF-kB signaling pathway was activated, latency to a first epilepsy seizure attack was shortened, and neuronal injury increased. This study provides evidence that miR-129-5p inhibits the development of AE-related epilepsy by suppressing HMGB1 expression and inhibiting TLR4/NF-kB signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

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Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

2014-01-01

Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo. © 2014 S. Karger AG, Basel.

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

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Camille Aubry

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

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

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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

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.

Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus.

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Janardhanan, Jeshina; Joseph Martin, Sherry; Astrup, Elisabeth; Veeramanikandan, R; Aukrust, Pål; Abraham, Ooriapadickal C; Varghese, George M

2013-11-01

Scrub typhus is a highly prevalent bacterial infection in India and South Asia that is caused by Orientia tsutsugamushi. The innate immune response to infections is modulated by Toll-like receptors (TLRs) and heat shock proteins (HSPs). This study was done to assess the prevalence and possible association of TLR and HSP polymorphisms in scrub typhus. TLR4 Asp299Gly, TLR4 Thr399Ile, TLR2 Arg753Gln and HSP70-2 A1267G are single-nucleotide polymorphisms (SNPs) that may modulate their activities, and these SNPs were assessed in 137 scrub typhus patients and 134 controls by PCR restriction fragment length polymorphism. We found that the two TLR4 mutations, TLR4 D299G and TLR4T399I, were present in 19.5% and 22% of the study population, respectively, and was in significant linkage disequilibrium with a D' of 0.8. The TLR2 mutation was found to be rare, whereas the HSP A>G mutation was very common (77.5%). Compared with the controls, the prevalence of heterozygous genotype of the TLR4D299G SNP, but not any of the other SNPs, was significantly higher among scrub typhus patients. Further studies using a larger sample size and more candidate genes may better enable in determining the role of these associations in susceptibility and severity of scrub typhus.

IL-27 Induced by Select Candida spp. via TLR7/NOD2 Signaling and IFN-β Production Inhibits Fungal Clearance

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Patin, Emmanuel C.; Jones, Adam V.; Thompson, Aiysha; Clement, Mathew; Liao, Chia-Te; Griffiths, James S.; Wallace, Leah E.; Bryant, Clare E.; Lang, Roland; Rosenstiel, Philip; Humphreys, Ian R.; Taylor, Philip R.

2016-01-01

Candida spp. elicit cytokine production downstream of various pathogen recognition receptors, including C-type lectin-like receptors, TLRs, and nucleotide oligomerization domain (NOD)–like receptors. IL-12 family members IL-12p70 and IL-23 are important for host immunity against Candida spp. In this article, we show that IL-27, another IL-12 family member, is produced by myeloid cells in response to selected Candida spp. We demonstrate a novel mechanism for Candida parapsilosis–mediated induction of IL-27 in a TLR7-, MyD88-, and NOD2-dependent manner. Our data revealed that IFN-β is induced by C. parapsilosis, which in turn signals through the IFN-α/β receptor and STAT1/2 to induce IL-27. Moreover, IL-27R (WSX-1)–deficient mice systemically infected with C. parapsilosis displayed enhanced pathogen clearance compared with wild-type mice. This was associated with increased levels of proinflammatory cytokines in the serum and increased IFN-γ and IL-17 responses in the spleens of IL-27R–deficient mice. Thus, our data define a novel link between C. parapsilosis, TLR7, NOD2, IFN-β, and IL-27, and we have identified an important role for IL-27 in the immune response against C. parapsilosis. Overall, these findings demonstrate an important mechanism for the suppression of protective immune responses during infection with C. parapsilosis, which has potential relevance for infections with other fungal pathogens. PMID:27259855

The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane.

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Tanimura, Natsuko; Saitoh, Shin-Ichiroh; Ohto, Umeharu; Akashi-Takamura, Sachiko; Fujimoto, Yukari; Fukase, Koichi; Shimizu, Toshiyuki; Miyake, Kensuke

2014-06-01

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane. © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress

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Sardar Sindhu

2018-01-01

Full Text Available Background/Aims: Metabolic diseases such as obesity and type-2 diabetes (T2D are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. Methods: TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC, or primary monocytes were treated with hydrogen peroxide (H2O2 for induction of reactive oxygen species (ROS-mediated oxidative stress. Superoxide dismutase (SOD activity was measured using a commercial kit. Data (mean±SEM were compared using unpaired student’s t-test and P<0.05 was considered significant. Results: The adipose tissue TLR10 gene/protein expression was found to be significantly upregulated in obesity as well as T2D which correlated with body mass index (BMI. ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB/mitogen activated protein kinase (MAPK signaling as well as endoplasmic reticulum (ER stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. Conclusion: Oxidative stress

Structure-Activity Relationship in TLR4 Mutations: Atomistic Molecular Dynamics Simulations and Residue Interaction Network Analysis

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Anwar, Muhammad Ayaz; Choi, Sangdun

2017-03-01

Toll-like receptor 4 (TLR4), a vital innate immune receptor present on cell surfaces, initiates a signaling cascade during danger and bacterial intrusion. TLR4 needs to form a stable hexamer complex, which is necessary to dimerize the cytoplasmic domain. However, D299G and T399I polymorphism may abrogate the stability of the complex, leading to compromised TLR4 signaling. Crystallography provides valuable insights into the structural aspects of the TLR4 ectodomain; however, the dynamic behavior of polymorphic TLR4 is still unclear. Here, we employed molecular dynamics simulations (MDS), as well as principal component and residue network analyses, to decipher the structural aspects and signaling propagation associated with mutations in TLR4. The mutated complexes were less cohesive, displayed local and global variation in the secondary structure, and anomalous decay in rotational correlation function. Principal component analysis indicated that the mutated complexes also exhibited distinct low-frequency motions, which may be correlated to the differential behaviors of these TLR4 variants. Moreover, residue interaction networks (RIN) revealed that the mutated TLR4/myeloid differentiation factor (MD) 2 complex may perpetuate abnormal signaling pathways. Cumulatively, the MDS and RIN analyses elucidated the mutant-specific conformational alterations, which may help in deciphering the mechanism of loss-of-function mutations.

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

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Enda Shevlin

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

TLR9 ligation in pancreatic stellate cells promotes tumorigenesis.

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

2015-11-16

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

Bifidobacterium breve C50 secretes lipoprotein with CHAP domain recognized in aggregated form by TLR2.

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Scuotto, Angelo; Djorie, Serge; Colavizza, Michel; Romond, Pierre-Charles; Romond, Marie-Bénédicte

2014-12-01

Extracellular components secreted by Bifidobacterium breve C50 can induce maturation, high IL-10 production and prolonged survival of dendritic cells via a TLR2 pathway. In this study, the components were isolated from the supernatant by gel filtration chromatography. Antibodies raised against the major compounds with molecular weight above 600 kDa (Bb C50BC) also recognized compounds of lower molecular weight (200–600 kDa). TLR2 and TLR6 bound to the components already recognized by the antibodies. Trypsin digestion of Bb C50BC released three major peptides whose sequences displayed close similarities to a putative secreted protein with a CHAP amidase domain from B. breve. The 1300-bp genomic region corresponding to the hypothetical protein was amplified by PCR. The deduced polypeptide started with an N-terminal signal sequence of 45 amino acids, containing the lipobox motif (LAAC) with the cysteine in position 25, and 2 positively charged residues within the first 14 residues of the signal sequence. Lipid detection in Bb C50BC by GC/MS further supported the implication of a lipoprotein. Sugars were also detected in Bb C50BC. Close similarity with the glucan-binding protein B from Bifidobacterium animalis of two released peptides from Bb C50BC protein suggested that glucose moieties, possibly in glucan form, could be bound to the lipoprotein. Finally, heating at 100 °C for 5 min led to the breakdown of Bb C50BC in compounds of molecular weight below 67 kDa, which suggested that Bb C50BC was an aggregate. One might assume that a basic unit was formed by the lipoprotein bound putatively to glucan. Besides the other sugars and hexosamines recognized by galectin 1 were localized at the surface of the Bb C50BC aggregate. In conclusion, the extracellular components secreted by B. breve C50 were constituted of a lipoprotein putatively associated with glucose moieties and acting in an aggregating form as an agonist of TLR2/TLR6.

TLR-4 polymorphisms and leukocyte TLR-4 expression in febrile UTI and renal scarring.

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Bayram, Meral Torun; Soylu, Alper; Ateş, Halil; Kızıldağ, Sefa; Kavukçu, Salih

2013-09-01

In this study, we aimed to determine the relation of TLR-4 Asp299Gly and Thr399Ile polymorphisms and monocyte/neutrophil TLR-4 expression to febrile urinary tract infection (UTI) and renal scar development in children. The study was performed in children with a history of febrile UTI. Patients with and without renal scarring were classified as group 1 and group 2, respectively, while the control cases in our previous study were used as the control group (group 3). All three groups were compared for the rate of TLR-4 Asp299Gly and Thr399Ile polymorphisms, and for basal and lipopolysaccharide-stimulated monocyte/neutrophil TLR-4 expression levels. There were 168 patients (86 in group 1, 82 in group 2) and 120 control cases. Monocyte/neutrophil TLR-4 expression levels were similar in groups 1 and 2. However, both groups had lower TLR-4 expression than group 3. The rate of TLR-4 Asp299Gly polymorphism was not different in all groups. TLR-4 Thr399Ile polymorphism was higher in groups 1 and 2 than in group 3 (14.0, 12.2, and 2.0 %, respectively), while group 1 and group 2 were not different. Furthermore, monocyte TLR-4 expression level was lower in those having TLR-4 Thr399Ile polymorphism than in those without this polymorphism. Patients with febrile UTI had more frequent TLR-4 Thr399Ile polymorphism and lower monocyte/neutrophil TLR-4 expression. These findings indicate that children carrying TLR-4 Thr399Ile polymorphism and/or having low level of monocyte/neutrophil TLR-4 expression have a tendency to develop febrile UTI. However, we could not show the association of TLR-4 polymorphisms and of TLR-4 expression level to renal scarring.

Taurine Attenuates Hepatic Inflammation in Chronic Alcohol-Fed Rats Through Inhibition of TLR4/MyD88 Signaling.

Science.gov (United States)

Lin, Chao-Jen; Chiu, Chun-Ching; Chen, Yi-Chen; Chen, Mu-Lin; Hsu, Tsai-Ching; Tzang, Bor-Show

2015-12-01

Accumulating evidence indicates that overconsumption of ethanol contributes in many ways to the pathogenesis of hepatic injury. Although studies indicate that taurine decreases lipogenesis, oxidative stress, and inflammatory cytokines, the protective effect of taurine against alcohol-induced liver injury is still unclear. To clarify the precise signaling involved in the beneficial effect of taurine on alcohol-induced liver injury, rats were randomly divided into four treatment groups: (1) control (Ctl), (2) alcohol (Alc), (3) Alc+taurine (Tau), and (4) Alc+silymarin (Sil). The Tau and Sil groups had lower lymphocyte infiltration and significantly lower TLR-4/MyD88 and IκB/NFκB compared to the Alc group. The inducible nitric oxide synthase (iNOS), C-reactive protein (CRP), tumor necrosis factors (TNF)-α, interleukin (IL)-6, and IL-1β were also significantly lower in the Tau and Sil groups than in the Alc group. The experimental results indicated that hepatoprotection against alcohol-induced inflammation may be mediated by decreased TLR-4/MyD88 signaling.

Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

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Frazer, Lauren C; Darville, Toni; Chandra-Kuntal, Kumar; Andrews, Charles W; Zurenski, Matthew; Mintus, Margaret; AbdelRahman, Yasser M; Belland, Robert J; Ingalls, Robin R; O'Connell, Catherine M

2012-01-01

Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

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Lauren C Frazer

Full Text Available Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL in C. caviae. However, 2-deoxyglucose (2DG treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

Increased Expression of the Innate Immune Receptor TLR10 in Obesity and Type-2 Diabetes: Association with ROS-Mediated Oxidative Stress.

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Sindhu, Sardar; Akhter, Nadeem; Kochumon, Shihab; Thomas, Reeby; Wilson, Ajit; Shenouda, Steve; Tuomilehto, Jaakko; Ahmad, Rasheed

2018-01-01

Metabolic diseases such as obesity and type-2 diabetes (T2D) are known to be associated with chronic low-grade inflammation called metabolic inflammation together with an oxidative stress milieu found in the expanding adipose tissue. The innate immune Toll-like receptors (TLR) such as TLR2 and TLR4 have emerged as key players in metabolic inflammation; nonetheless, TLR10 expression in the adipose tissue and its significance in obesity/T2D remain unclear. TLR10 gene expression was determined in the adipose tissue samples from healthy non-diabetic and T2D individuals, 13 each, using real-time RT-PCR. TLR10 protein expression was determined by immunohistochemistry, confocal microscopy, and flow cytometry. Regarding in vitro studies, THP-1 cells, peripheral blood mononuclear cells (PBMC), or primary monocytes were treated with hydrogen peroxide (H2O2) for induction of reactive oxygen species (ROS)-mediated oxidative stress. Superoxide dismutase (SOD) activity was measured using a commercial kit. Data (mean±SEM) were compared using unpaired student's t-test and Pobesity as well as T2D which correlated with body mass index (BMI). ROS-mediated oxidative stress induced high levels of TLR10 gene/protein expression in monocytic cells and PBMC. In these cells, oxidative stress induced a time-dependent increase in SOD activity. Pre-treatment of cells with anti-oxidants/ROS scavengers diminished the expression of TLR10. ROS-induced TLR10 expression involved the nuclear factor-kappaB (NF-κB)/mitogen activated protein kinase (MAPK) signaling as well as endoplasmic reticulum (ER) stress. H2O2-induced oxidative stress interacted synergistically with palmitate to trigger the expression of TLR10 which associated with enhanced expression of proinflammatory cytokines/chemokine. Oxidative stress induces the expression of TLR10 which may represent an immune marker for metabolic inflammation. © 2018 The Author(s). Published by S. Karger AG, Basel.

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

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Paola eMassari

2014-08-01

Full Text Available AbstractToll-Like Receptor (TLR signaling represents one of the best studied pathways to implement defense mechanisms against invading microbes in humans as well as in animals. TLRs respond to specific microbial ligands and to danger signals produced by the host during infection, and initiate downstream cascades that activate both innate and adaptive immunity. TLRs are expressed by professional immune cells and by the large majority of non-hematopoietic cells, including epithelial cells. In epithelial tissues, TLR functions are particularly important because these sites are constantly exposed to microorganisms, due to their location at the host interface with the environment. While at these sites, specific defense mechanisms and inflammatory responses are initiated via TLR signaling against pathogens, suppression or lack of TLR activation is also observed in response to the commensal microbiota. The mechanisms by which TLR signaling is regulated in mucosal epithelial cells include differential expression and levels of TLRs (and their signaling partners, their cellular localization and positioning within the tissue in a fashion that favors responses to pathogens while dampening responses to commensals and maintaining tissue homeostasis in physiologic conditions. In this review, the expression and activation of TLRs in mucosal epithelial cells of several sites of the human body are examined. Specifically, the oral cavity, the ear canal and eye, the airways, the gut and the reproductive tract are discussed, along with how site-specific host defense mechanisms are implemented via TLR signaling.

TLR2 deficiency aggravates lung injury caused by mechanical ventilation

NARCIS (Netherlands)

Kuipers, Maria Theresa; Jongsma, Geartsje; Hegeman, Maria A; Tuip-de Boer, Anita M; Wolthuis, Esther K; Choi, Goda; Bresser, Paul; van der Poll, Tom; Schultz, Marcus J; Wieland, Catharina W

Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene

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...

Baicalin inhibits toll-like receptor 2/4 expression and downstream signaling in rat experimental periodontitis.

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Sun, Jun-Yi; Li, Dong-Ling; Dong, Yan; Zhu, Chun-Hui; Liu, Jin; Li, Jue-Dan; Zhou, Tao; Gou, Jian-Zhong; Li, Ang; Zang, Wei-Jin

2016-07-01

Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis (P. gingivalis) for 4weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200mg/kg/day) or vehicle for 4weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1β, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis. Copyright © 2016 Elsevier B.V. All rights reserved.

Early decreased TLR2 expression on monocytes is associated with their reduced phagocytic activity and impaired maturation in a porcine polytrauma model

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Schimunek, Lukas; Serve, Rafael; Teuben, Michel P. J.; Störmann, Philipp; Auner, Birgit; Woschek, Mathias; Pfeifer, Roman; Horst, Klemens; Simon, Tim-P.; Kalbitz, Miriam; Sturm, Ramona; Pape, Hans-C.; Hildebrand, Frank; Marzi, Ingo

2017-01-01

In their post-traumatic course, trauma patients suffering from multiple injuries have a high risk for immune dysregulation, which may contribute to post-injury complications and late mortality. Monocytes as specific effector cells of the innate immunity play a crucial role in inflammation. Using their Pattern Recognition Receptors (PRRs), notably Toll-Like Receptors (TLR), the monocytes recognize pathogens and/or pathogen-associated molecular patterns (PAMPs) and organize their clearance. TLR2 is the major receptor for particles of gram-positive bacteria, and initiates their phagocytosis. Here, we investigated the phagocytizing capability of monocytes in a long-term porcine severe trauma model (polytrauma, PT) with regard to their TLR2 expression. Polytrauma consisted of femur fracture, unilateral lung contusion, liver laceration, hemorrhagic shock with subsequent resuscitation and surgical fracture fixation. After induction of PT, peripheral blood was withdrawn before (-1 h) and directly after trauma (0 h), as well as 3.5 h, 5.5 h, 24 h and 72 h later. CD14+ monocytes were identified and the expression levels of H(S)LA-DR and TLR2 were investigated by flow cytometry. Additionally, the phagocytizing activity of monocytes by applying S. aureus particles labelled with pHrodo fluorescent reagent was also assessed by flow cytometry. Furthermore, blood samples from 10 healthy pigs were exposed to a TLR2-neutralizing antibody and subsequently to S. aureus particles. Using flow cytometry, phagocytizing activity was determined. P below 0.05 was considered significant. The number of CD14+ monocytes of all circulating leukocytes remained constant during the observational time period, while the percentage of CD14+H(S)LA-DR+ monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of TLR2+ expressing cells out of all monocytes significantly decreased directly, 3.5 h and 5.5 h after trauma. The percentage of phagocytizing monocytes decreased

TLR4-HMGB1 signaling pathway affects the inflammatory reaction of autoimmune myositis by regulating MHC-I.

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Wan, Zemin; Zhang, Xiujuan; Peng, Anping; He, Min; Lei, Zhenhua; Wang, Yunxiu

2016-12-01

To analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-α, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis. We built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-γ on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-γ, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-α and IL-6. The expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all Pmyositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFβ, and IDO1

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Moyer, Benjamin J. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Rojas, Itzel Y. [Department of Medicine, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Department of Pharmacology & Toxicology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Kerley-Hamilton, Joanna S. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Hazlett, Haley F. [Department of Pharmacology & Toxicology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Department of Immunology & Microbiology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Nemani, Krishnamurthy V. [Department of Radiology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Trask, Heidi W.; West, Rachel J. [Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Lupien, Leslie E. [Department of Medicine, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); Collins, Alan J. [Department of Immunology & Microbiology, Geisel School of Medicine at Dartmouth, Dartmouth Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 (United States); and others

2016-06-01

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding

Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFβ, and IDO1

International Nuclear Information System (INIS)

Moyer, Benjamin J.; Rojas, Itzel Y.; Kerley-Hamilton, Joanna S.; Hazlett, Haley F.; Nemani, Krishnamurthy V.; Trask, Heidi W.; West, Rachel J.; Lupien, Leslie E.; Collins, Alan J.

2016-01-01

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding

Vitamin K2 can suppress the expression of Toll-like receptor 2 (TLR2) and TLR4, and inhibit calcification of aortic intima in ApoE-/- mice as well as smooth muscle cells.

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Wang, Zhaojun; Wang, Zhongqun; Zhu, Jie; Long, Xinguang; Yan, Jinchuan

2018-02-01

Background and objectives Vascular calcification is a common complication in atherosclerosis. Accumulating evidence showed that Toll-like receptors (TLRs) mediate pro-inflammatory and atherosclerosis. Recent studies demonstrated that vascular calcification is one of the detrimental effects of vitamin K (Vit K) antagonists. However, the effects of Vit K on the expression of TLR2 and 4 and intimal calcification in artery remained unidentified. Methods and results Eighteen ApoE -/- mice were randomly divided into model group, Vit K-treated group, and control group. The mice of model and Vit K-treated group were fed with high-fat diet, while control group mice were fed with normal diet. Mice of Vit K-treated group were administered orally with vitamin K2 (40 mg.kg -1 .day -1 ) for 12 weeks. Twelve weeks later the aortic sections of mice were acquired and stained with hematoxylin and eosin and von Kossa, respectively. Calcium content and activity of alkaline phosphatase (ALP) at aortic tissues were measured. The expression levels of TLR2 and TLR4 in aorta sections were detected by immunohistochemisty and RT-PCR, respectively. The effects of Vit K on cellular calcification were further studied in A7r5 SMCs. Results demonstrated that high-fat diet induced typical atherosclerosis with intimal calcification in ApoE -/- mice, while in Vit K-treated group atherosclerosis and calcium deposits were not serious; Vit K2 also inhibited cellular calcification in A7r5 SMCs. Quantitative analysis showed that calcium and ALP activity at aortic tissues in the Vit K-treated mice were significantly lower than that of the model group ( P < 0.01); Compared to the control group, the expression levels of TLR2 and TLR4 in the model group were significantly higher ( P < 0.05), while in Vit K-treated group the levels of TLR2 and 4 were significantly lower than that in the model group. Furthermore, the content of calcium was positively related to the expression levels of TLR2 and TLR4

Hyperin protects against LPS-induced acute kidney injury by inhibiting TLR4 and NLRP3 signaling pathways

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Chunzhi, Gong; Zunfeng, Li; Chengwei, Qin; Xiangmei, Bu; Jingui, Yu

2016-01-01

Hyperin is a flavonoid compound derived from Ericaceae, Guttifera, and Celastraceae that has been shown to have various biological effects, such as anti-inflammatory and anti-oxidant effects. However, there is no evidence to show the protective effects of hyperin on lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Therefore, we investigated the protective effects and mechanism of hyperin on LPS-induced AKI in mice. The levels of TNF-α, IL-6, and IL-1β were tested by ELISA. The effects of hyperin on blood urea nitrogen (BUN) and serum creatinine were also detected. In addition, the expression of TLR4, NF-κB, and NLRP3 were detected by western blot analysis. The results showed that hyperin significantly inhibited LPS-induced TNF-α, IL-6, and IL-1β production. The levels of BUN and creatinine were also suppressed by hyperin. Furthermore, LPS-induced TLR4 expression and NF-κB activation were also inhibited by hyperin. In addition, treatment of hyperin dose-dependently inhibited LPS-induced NLRP3 signaling pathway. In conclusion, the results showed that hyperin inhibited LPS-induced inflammatory response by inhibiting TLR4 and NLRP3 signaling pathways. Hyperin has potential application prospects in the treatment of sepsis-induced AKI. PMID:27813491

Can the TLR-4-Mediated Signaling Pathway Be “A Key Inflammatory Promoter for Sporadic TAA”?

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Giovanni Ruvolo

2014-01-01

Full Text Available Thoracic aorta shows with advancing age various changes and a progressive deterioration in structure and function. As a result, vascular remodeling (VR and medial degeneration (MD occur as pathological entities responsible principally for the sporadic TAA onset. Little is known about their genetic, molecular, and cellular mechanisms. Recent evidence is proposing the strong role of a chronic immune/inflammatory process in their evocation and progression. Thus, we evaluated the potential role of Toll like receptor- (TLR- 4-mediated signaling pathway and its polymorphisms in sporadic TAA. Genetic, immunohistochemical, and biochemical analyses were assessed. Interestingly, the rs4986790 TLR4 polymorphism confers a higher susceptibility for sporadic TAA (OR=14.4, P=0.0008 and it represents, together with rs1799752 ACE, rs3918242 MMP-9, and rs2285053 MMP-2 SNPs, an independent sporadic TAA risk factor. In consistency with these data, a significant association was observed between their combined risk genotype and sporadic TAA. Cases bearing this risk genotype showed higher systemic inflammatory mediator levels, significant inflammatory/immune infiltrate, a typical MD phenotype, lower telomere length, and positive correlations with histopatological abnormalities, hypertension, smoking, and ageing. Thus, TLR4 pathway should seem to have a key role in sporadic TAA. It might represent a potential useful tool for preventing and monitoring sporadic TAA and developing personalized treatments.

Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.

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Anna Martirosyan

Full Text Available Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+ T and CD8(+ T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

Uncarinic Acid C Isolated from Uncaria rhynchophylla Induces Differentiation of Th1-Promoting Dendritic Cells Through TLR4 Signaling.

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Kim, Kyu Sik; Pham, Thanh Nhan Nguyen; Jin, Chun-Ji; Umeyama, Akemi; Shoji, Noboru; Hashimoto, Toshihiro; Lee, Je-Jung; Takei, Masao

2011-02-28

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. DC might be a potential target for URC. We demonstrate that URC activates human DC as documented by phenotypic and functional maturation, and altered cytokine production. The expression of CD1a, CD38, CD40, CD54, CD80, CD83, CD86, HLA-DR and CCR7 on URC-primed DC was enhanced. The production of IL-12p70 by URC-primed DC was higher than that of lipopolysaccharide (LPS)-primed DC. The production of IL-12p70 by URC-primed DC was inhibited by the anti-Toll-like receptor 4 (TLR4) monoclonal antibody (mAb), but partially abolished by anti-TLR2 mAb. mRNA coding for TLR2 and TLR4 was expressed in URC-primed DC. URC-primed DC induced the NF-κB transcription factor. Naïve T cells co-cultured with URC-primed DC turned into typical Th1 cells that produced large quantities of IFN-γ depending on IL-12 secretion. URC enhanced the T cell stimulatory capacity in an allo MLR. In the cytotoxic T-lymphocyte assay (CTL) assay, DNA fragmentation assay and (51)Cr release on URC-primed DC were more augmented than that of TNF-α-primed DC. DC matured with URC had an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that URC modulates DC function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR4 signaling, and may be used on DC-based vaccine for cancer immunotherapy.

Genetic Diversity of IFγ, IL1β, TLR2, and TLR8 Loci in Pulmonary Tuberculosis in Kazakhstan

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Dauren Yerezhepov

2014-12-01

Full Text Available Introduction. Tuberculosis (TB is caused by bacterium Mycobacterium tuberculosis (MTB, and according to the WHO, up to 30% of world population is infected with latent TB. Pathogenesis of TB is multifactorial, and its development depends on environmental, social, microbial, and genetic factors of both the bacterium and the host. The number of TB cases in Kazakhstan has decreased in the past decade, but multidrug-resistant (MDR TB cases are dramatically increasing. Polymorphisms in genes responsible for immune response have been associated with TB susceptibility. The objective of this study was to investigate the risk of developing pulmonary TB (PTB associated with polymorphisms in several inflammatory pathway genes among Kazakhstani population.Methods. 703 participants from 3 regions of Kazakhstan were recruited for a case-control study. 251 participants had pulmonary TB (PTB, and 452 were healthy controls (HC. Males and females represented 42.39% and 57.61%, respectively. Of all participants, 67.4% were Kazakhs, 22.8% Russians, 3.4% Ukrainians, and 6.4% were of other origins. Clinical and epidemiological data were collected from medical records, interviews, and questionnaires. DNA samples were genotyped using TaqMan assay on 4 polymorphisms: IFNγ (rs2430561 and IL1β (rs16944, TLR2 (rs5743708 and TLR8 (rs3764880. Statistical data was analyzed using SPSS 19.Results. Genotyping by IFγ, IL1β, TLR2 showed no significant association with PTB susceptibility (p > 0.05. TLR8 genotype A/G was significantly higher in females (F/M – 41.5%/1.3% and G/G in males (M/F – 49%/20.7% (χ2=161.43, p < 0.001. A significantly increased risk of PTB development was observed for TLR A/G with an adjusted OR of 1.48 (95%, CI: 0.96 - 2.28, and a protective feature was revealed for TLR8 G/G genotype (OR: 0.81, 95%, CI: 0.56 - 1.16, p = 0.024. Additional grouping by gender revealed that TLR8 G/G contributes as protective genotype (OR: 1.83, 95%, CI: 1.18 - 2.83, p

TLR4 activates NF-κB in human ovarian granulosa tumor cells

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Woods, Dori C.; White, Yvonne A.R.; Dau, Caroline; Johnson, A.L.

2011-01-01

Highlights: → TLR4 is expressed in human ovarian granulosa tumor cells. → Acting through TLR4, LPS and HSP60 induce a NFκB signaling cascade in human ovarian granulosa tumor cells. → NFκB 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-κB) 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 IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFα plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB 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-κB does not sensitize GCTs to TRAIL or cisplatin.

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

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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.

Glutamate alleviates muscle protein loss by modulating TLR4, NODs, Akt/FOXO and mTOR signaling pathways in LPS-challenged piglets.

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Ping Kang

Full Text Available The experiment was conducted to study the effect of the glutamate (Glu on muscle protein loss through toll-like receptor 4 (TLR4, nucleotide-binding oligomerization domain proteins (NODs, Akt/Forkhead Box O (Akt/FOXO and mammalian target of rapamycin (mTOR signaling pathways in LPS-challenged piglets. Twenty-four weaned piglets were assigned into four treatments: (1 Control; (2 LPS+0% Glu; (3 LPS + 1.0% Glu; (4 LPS + 2.0% Glu. The experiment was lasted for 28 days. On d 28, the piglets in the LPS challenged groups were injected with LPS on 100 μg/kg body weight (BW, and the piglets in the control group were injected with the same volume of 0.9% NaCl solution. After 4 h LPS or saline injection, the piglets were slaughtered and the muscle samples were collected. Glu supplementation increased the protein/DNA ratio in gastrocnemius muscle, and the protein content in longissimus dorsi (LD muscle after LPS challenge (P<0.05. In addition, Glu supplementation decreased TLR4, IL-1 receptor-associated kinase (IRAK 1, receptor-interacting serine/threonine-protein kinase (RIPK 2, and nuclear factor-κB (NF-κB mRNA expression in gastrocnemius muscle (P<0.05, MyD88 mRNA expression in LD muscle, and FOXO1 mRNA expression in LD muscle (P<0.05. Moreover, Glu supplementation increased p-Akt/t-Akt ratio (P<0.05 in gastrocnemius muscle, and p-4EBP1/t-4EBP1 ratio in both gastrocnemius and LD muscles (P<0.05. Glu supplementation in the piglets' diets might be an effective strategy to alleviate LPS-induced muscle protein loss, which might be due to suppressing the mRNA expression of TLR4 and NODs signaling-related genes, and modulating Akt/FOXO and mTOR signaling pathways.

Tyrosine Phosphorylation in Toll-Like Receptor Signaling

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Chattopadhyay, Saurabh; Sen, Ganes C.

2014-01-01

There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

RP105 Protects Against Apoptosis in Ischemia/Reperfusion-Induced Myocardial Damage in Rats by Suppressing TLR4-Mediated Signaling Pathways

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Jun Yang

2015-07-01

Full Text Available Background: Myocardial apoptosis is heavily implicated in the myocardial damage caused by ischemia-reperfusion (I/R. Toll-like receptor 4 (TLR4 is a potent inducer of these apoptotic cascades. In contrast, the radioprotective 105 kDa protein (RP105 is a specific negative regulator of TLR4 signaling pathways. However, the precise mechanisms by which RP105 inhibits myocardium apoptosis via TLR4-associated pathways during I/R is not fully understood. Methods: We utilized a rat model of myocardial ischemic reperfusion injury (MIRI. Animals were pre-treated with Ad-EGFP adenovirus, Ad-EGFP-RP105 adenovirus, saline, or nothing (sham. After three days, rats underwent a 30min left anterior descending coronary artery occlusion and a 4h reperfusion. Mycardial tissue was assessed by immunohistochemistry, TUNEL-staining, Western blot, quantitative RT-PCR, and a morphometric assay. Results: RP105 overexpression resulted in a reduction in infarct size, fewer TUNEL-positive cardiomyocytes, and a reduction in mitochondrial-associated apoptosis cascade activity. Further, RP105 overexpression repressed I/R-induced myocardial injury by attenuating myocardial apoptosis. This was mediated by inhibiting TLR4 activation and the phosphorylation of P38MAPK and the downstream transcription factor AP-1. Conclusion: RP105 overexpression leads to the de-activation of TLR4, P38MAPK, and AP-1 signaling pathways, and subsequently represses apoptotic cascades and ensuing damage of myocardial ischemic reperfusion. These findings may become the basis of a novel therapeutic approach for reducing of cardiac damage caused by MIRI.

Signatures of selection acting on the innate immunity gene Toll-like receptor 2 (TLR2) during the evolutionary history of rodents.

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Tschirren, B; Råberg, L; Westerdahl, H

2011-06-01

Patterns of selection acting on immune defence genes have recently been the focus of considerable interest. Yet, when it comes to vertebrates, studies have mainly focused on the acquired branch of the immune system. Consequently, the direction and strength of selection acting on genes of the vertebrate innate immune defence remain poorly understood. Here, we present a molecular analysis of selection on an important receptor of the innate immune system of vertebrates, the Toll-like receptor 2 (TLR2), across 17 rodent species. Although purifying selection was the prevalent evolutionary force acting on most parts of the rodent TLR2, we found that codons in close proximity to pathogen-binding and TLR2-TLR1 heterodimerization sites have been subject to positive selection. This indicates that parasite-mediated selection is not restricted to acquired immune system genes like the major histocompatibility complex, but also affects innate defence genes. To obtain a comprehensive understanding of evolutionary processes in host-parasite systems, both innate and acquired immunity thus need to be considered. © 2011 The Authors. Journal of Evolutionary Biology © 2011 European Society For Evolutionary Biology.

Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

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Byun, Eui-Baek; Choi, Han-Gyu; Sung, Nak-Yun; Byun, Eui-Hong

2012-01-01

Highlights: ► Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. ► EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. ► EGCG-treated DCs inhibited MAPKs activation and NF-κB p65 translocation via 67LR. ► EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

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Byun, Eui-Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Choi, Han-Gyu [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of); Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@gmail.com [Department of Microbiology and Research Institute for Medical Sciences, College of Medicine, Chungnam National University, Daejeon 301-747 (Korea, Republic of)

2012-10-05

Highlights: Black-Right-Pointing-Pointer Expressions of CD80, CD86, and MHC class I/II were inhibited by EGCG via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited LPS-induced pro-inflammatory cytokines via 67LR. Black-Right-Pointing-Pointer EGCG-treated DCs inhibited MAPKs activation and NF-{kappa}B p65 translocation via 67LR. Black-Right-Pointing-Pointer EGCG elevated the expression of the Tollip protein through 67LR in DCs. -- Abstract: Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-{alpha}, interleukin [IL]-1{beta}, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor {kappa}B (NF-{kappa}B) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.

The Probiotic Lactobacillus Prevents Citrobacter rodentium-Induced Murine Colitis in a TLR2-Dependent Manner.

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Ryu, Seung-Hyun; Park, Jong-Hyung; Choi, Soo-Young; Jeon, Hee-Yeon; Park, Jin-Il; Kim, Jun-Young; Ham, Seung-Hoon; Choi, Yang-Kyu

2016-07-28

The main objective of this study was to investigate whether Lactobacillus rhamnosus GG (LGG) ameliorated the effects of Citrobactor rodentium infection in Toll-like receptor 2 (TLR2) knockout (KO) and TLR4 KO mice, as well as in wild-type C57BL/6 (B6) mice. TLR2 KO, TLR4 KO, and B6 mice were divided into three groups per each strain. Each group had an uninfected control group (n = 5), C. rodentium-infected group (n = 8), and LGG-pretreated C. rodentium-infected group (n = 8). The survival rate of B6 mice infected with C. rodentium was higher when pretreated with LGG. Pretreatment with LGG ameliorated C. rodentium-induced mucosal hyperplasia in B6 and TLR4 KO mice. However, in C-rodentium-infected TLR2 KO mice, mucosal hyperplasia persisted, regardless of pretreatment with LGG. In addition, LGG-pretreated B6 and TLR4 KO mice showed a decrease in spleen weight and downregulation of tumor necrosis factor alpha, interferon gamma, and monocyte chemotactic protein 1 mRNA expression compared with the non-pretreated group. In contrast, such changes were not observed in TLR2 KO mice, regardless of pretreatment with LGG. From the above results, we conclude that pretreatment with LGG ameliorates C. rodentium-induced colitis in B6 and TLR4 KO mice, but not in TLR2 KO mice. Therefore, LGG protects mice from C. rodentium-induced colitis in a TLR2-dependent manner.

Effect of experimental ileitis on expression of tlr-2 in lymphfocytes of small intestine

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A. S. Zherebiatiev

2013-08-01

Full Text Available Introduction Crohn’s disease and ulcerative colitis are the main clinical phenotypes of inflammatory bowel disease (IBD. Both forms of IBD can increase the incidence of gastrointestinal and colon cancers, and both ones are associated with significant morbidity and mortality worldwide. In addition, they can begin early in life and persist for long periods. The pathogenesis of IBD is complex and multifactorial. Differential alteration of Toll-like receptor (TLR expression in inflammatory bowel disease was first described 15 years ago. Studies have led to the current concept that TLRs represent key mediators of innate host defense in the intestine, and they are involved in mucosa maintaining as well as commensal homeostasis. Recent findings in diverse murine models of ileitis have helped to reveal the importance of TLR dysfunction mechanisms in IBD pathogenesis. The aim of research The aim of this study was to investigate the effect of acute ileitis on expression intensity of TLR-2 in lymphocytes of small intestine. Materials and methods Male Wistar rats with weight 200–250 g were housed in standard wire-mesh bottom cages at constant temperature of 25°C and 12/12 h light/dark cycles. The rats were given water and standard laboratory diet with no restriction prior to indomethacin injection. A total number of 20 rats were examined, including control group (n = 10. For induction of acute ileitis, rats received one subcutaneous dose of indomethacin (Sigma, 15 mg/kg. Tissue was examined on the fifth day. For histological examination sections were colored with haematoxylin and eosin. The TLR-2+cells were determined using a direct immunofluorescence technique with using monoclonal rat anti-TLR-2 antibodies. Images were taken by using a fluorescence microscope PrimoStar (ZEISS, Germany with a computer-assisted video system AxioCam 5c (ZEISS, Germany including the NIH-Image software (NIH Image version 1·46. All statistical analyses were performed

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

Andrographolide suppresses proliferation of human colon cancer SW620 cells through the TLR4/NF-κB/MMP-9 signaling pathway.

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Zhang, Rui; Zhao, Jian; Xu, Jian; Jiao, De-Xin; Wang, Jian; Gong, Zhi-Qiang; Jia, Jian-Hui

2017-10-01

Modern pharmacological research has revealed that andrographolide has various functions, including anti-bacterial, anti-inflammatory and anti-viral effects, immunoregulation, treating cardiovascular and cerebrovascular diseases, and prevention and treatment of alcoholic liver injury. The present study investigated whether andrographolide suppresses the proliferation of human colon cancer cell through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB/matrix metalloproteinase-9 (MMP-9) signaling pathway. The MTT assay and lactate dehydrogenase assay were used to evaluate the anticancer effects of andrographolide on cell proliferation and cytotoxicity in human colon cancer SW620 cells. Flow cytometry was used to analyze the anticancer effects of andrographolide on apoptosis by Annexin V-fluorescein isothiocyanate/propidium iodide kit. The effects of andrographolide on the activity of caspase-3/9 were measured using ELISA. Western blot analysis was also used to analyze the protein expression of TLR4, myeloid differentiation primary response gene 88 (MyD88), NF-κB-p65 and MMP-9. In the present study, it was found that andrographolide suppressed the cell proliferation, augmented cytotoxicity, evoked cell apoptosis and activated caspase-3/9 activities in human colon cancer SW620 cells. The results revealed that the anti-proliferation effects of andrographolide on the SW620 cells was associated with the inhibition of TLR4, MyD88, NF-κB-p65 and MMP-9 signaling activation. The results suggest that andrographolide is a promising drug for treatment of human colon cancer via suppression of the TLR4/NF-κB/MMP-9 signaling pathway.

Elevated muscle TLR4 expression and metabolic endotoxemia in human aging.

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Ghosh, Sangeeta; Lertwattanarak, Raweewan; Garduño, Jose de Jesus; Galeana, Joaquin Joya; Li, Jinqi; Zamarripa, Frank; Lancaster, Jack L; Mohan, Sumathy; Hussey, Sophie; Musi, Nicolas

2015-02-01

Aging is associated with alterations in glucose metabolism and sarcopenia that jointly contribute to a higher risk of developing type 2 diabetes. Because aging is considered as a state of low-grade inflammation, in this study we examined whether older, healthy (lean, community-dwelling) participants have altered signaling flux through toll-like receptor 4 (TLR4), a key mediator of innate and adaptive immune responses. We also examined whether a 4-month aerobic exercise program would have an anti-inflammatory effect by reducing TLR4 expression and signaling. At baseline, muscle TLR4, nuclear factor κB p50 and nuclear factor κB p65 protein content, and c-Jun N-terminal kinase phosphorylation were significantly elevated in older versus young participants. The plasma concentration of the TLR4 agonist lipopolysaccharide and its binding protein also were significantly elevated in older participants, indicative of metabolic endotoxemia, which is a recently described phenomenon of increased plasma endotoxin level in metabolic disease. These alterations in older participants were accompanied by decreased insulin sensitivity, quadriceps muscle volume, and muscle strength. The exercise training program increased insulin sensitivity, without affecting quadriceps muscle volume or strength. Muscle TLR4, nuclear factor κB, and c-Jun N-terminal kinase, and plasma lipopolysaccharide and lipopolysaccharide binding protein were not changed by exercise. In conclusion, insulin resistance and sarcopenia of aging are associated with increased TLR4 expression/signaling, which may be secondary to metabolic endotoxemia. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rice Bran Feruloylated Oligosaccharides Activate Dendritic Cells via Toll-Like Receptor 2 and 4 Signaling

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Chi Chen Lin

2014-04-01

Full Text Available This work presents the effects of feruloylated oligosaccharides (FOs of rice bran on murine bone marrow-derived dendritic cells (BMDCs and the potential pathway through which the effects are mediated. We found that FOs induced phenotypic maturation of DCs, as shown by the increased expression of CD40, CD80/CD86 and MHC-I/II molecules. FOs efficiently induced maturation of DCs generated from C3H/HeN or C57BL/6 mice with normal toll-like receptor 4 (TLR-4 or TLR-2 but not DCs from mice with mutated TLR4 or TLR2. The mechanism of action of FOs may be mediated by increased phosphorylation of ERK, p38 and JNK mitogen-activated protein kinase (MAPKs and increased NF-kB activity, which are important signaling molecules downstream of TLR-4 and TLR-2. These data suggest that FOs induce DCs maturation through TLR-4 and/or TLR-2 and that FOs might have potential efficacy against tumor or virus infection or represent a candidate-adjuvant approach for application in immunotherapy and vaccination.

Role of innate immune receptors TLR2 and TLR4 as mediators of the inflammatory reaction in human visceral adipose tissue.

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Fusaru, Ana Marina; Stănciulescu, Camelia Elena; Surlin, V; Taisescu, C; Bold, Adriana; Pop, O T; Baniţă, Ileana Monica; Crăiţoiu, Stefania; Pisoschi, Cătălina Gabriela

2012-01-01

White adipose tissue from different locations is characterized by significant differences in the structure of adipocyte "secretoma". Fat accumulation in the central-visceral depots is usually associated with a chronic inflammatory state, which is complicated by the metabolic syndrome. Recently, the adipose tissue was emerged to have an essential role in the innate immunity, adipocytes being considered effector cells due to the presence of the Toll-like receptors (TLRs). In this study, we compared the expression of TNF-α, TLR2 and TLR4 in peripheral-subcutaneous and central-peritoneal adipose depots in three different conditions - lean, obese and obese diabetic - using immunohistochemistry. Our results suggest a correlation between the incidence of the stromal vascular cells and adipocytes TNF-α and TLR4 in the visceral depots in strong correlation with adipose tissue expansion. TLR2 positive cells were seen in the peripheral depots from all groups without any association with fat accumulation. These results focus on the existence of a new pathogenic pathway, the activation of TLR4, for the involvement of visceral adipose tissue in the activation and maintenance of the inflammatory cascade in obesity.

The potent suppressive effect of β-d-mannuronic acid (M2000) on molecular expression of the TLR/NF-kB Signaling Pathway in ankylosing spondylitis patients.

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Roozbehkia, Maryam; Mahmoudi, Mahdi; Aletaha, Somaye; Rezaei, Nima; Fattahi, Mohammad Javad; Jafarnezhad-Ansariha, Fahimeh; Barati, Anis; Mirshafiey, Abbas

2017-11-01

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease characterized by the inflammation of sacroiliac joints and axial skeleton. A combination of genetic, environmental and immunological factors are involved in AS's pathogenesis. TLRs are type I transmembrane glycoproteins that play a crucial role in the innate immune responses against invading pathogens. Observational studies have demonstrated a possible association between TLR dysregulation and AS. The β-d-mannuronic acid (M2000), as a novel NSAID with immunosuppressive property, has shown an inhibitory effect on Toll-like receptor (TLR) 2, 4 signaling in HEK293 cells. In the present study, we investigated the gene expression of Myd88, IKB-alpha, NF-kB and MAPK14 (genes of the TLR/NF-kB Signaling Pathway) in AS patients in comparison to healthy subjects and also the effect of β-d-mannuronic acid on disease activity and mRNA expression of these molecules in affected patients. We showed for the first time that the gene expression level of Myd88, IKB-alpha, NF-kB and MAPK14 was higher in AS patients in comparison to healthy subjects. Moreover we confirmed that the β-d-mannuronic acid not just reduced significantly the disease activity of AS individuals compared to placebo, but also it could significantly decrease the expression level of genes associated with TLR/NF-kB Signaling Pathway in treated patients with M2000. These results may provide a new therapeutic approach to attenuate inflammatory responses in AS patients, (Identified; IRCT 2013062213739N1). Copyright © 2017. Published by Elsevier B.V.

TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

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Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani

2006-01-01

CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-κB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis

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

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Dool-Ri Oh

2014-01-01

Full Text Available Toll-like receptor (TLR ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF-κB-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF-κB transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF-κB activation and IL-8 production in both TLR5− and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

Altered Expression of TLR2 and TLR4 on Peripheral CD14+ Blood Monocytes in Children with Urinary Tract Infection.

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Karananou, Panagiota; Fleva, Alexandra; Tramma, Despoina; Alataki, Anastasia; Pavlitou-Tsiontsi, Aikaterini; Emporiadou-Peticopoulou, Maria; Papadopoulou-Alataki, Efimia

2016-01-01

Urinary tract infection (UTI) is the second most common bacterial infection, after otitis media, in infants and children. The mechanisms of disease susceptibility and the role of immunity in the pathogenesis of UTI in children have been evaluated. In recent years, Toll-Like Receptors (TLRs) have been recognized as specific components of the innate immune system constituting important mediators in host immune recognition. The aim of the present study was to determine ΤLR2 and TLR4 expression during the acute phase of UTI in infants and children by measuring the CD14/TLR2 and CD14/TLR4 expression on monocytes. We also attempted to compare the TLRs expression with the immunological status of the patients to healthy children. The study group consisted of 60 children (36 females and 24 males) and the control group included 60 age-matched pediatric subjects (27 females and 33 males). In our study, no antibody deficiency was found either in the children with UTI or in healthy subjects. There might be a connection between low IgA, IgG, and IgG subclasses serum levels and UTI as there was a statistically significant difference between patients and healthy children. A higher expression of CD14/TLR2 was revealed in patients (90,07%) compared to controls (85,48%) as well as CD14/TLR4 in patients (90,53%) compared to controls (87,25%) (statistically significant difference, p UTIs' pathogenesis in children.

Toll-like receptor 2 signaling protects mice from tumor development in a mouse model of colitis-induced cancer.

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Emily L Lowe

2010-09-01

Full Text Available Inflammatory bowel disease (IBD is a disorder of chronic inflammation with increased susceptibility to colorectal cancer. The etiology of IBD is unclear but thought to result from a dysregulated adaptive and innate immune response to microbial products in a genetically susceptible host. Toll-like receptor (TLR signaling induced by intestinal commensal bacteria plays a crucial role in maintaining intestinal homeostasis, innate immunity and the enhancement of intestinal epithelial cell (IEC integrity. However, the role of TLR2 in the development of colorectal cancer has not been studied. We utilized the AOM-DSS model for colitis-associated colorectal cancer (CAC in wild type (WT and TLR2(-/- mice. Colons harvested from WT and TLR2(-/- mice were used for histopathology, immunohistochemistry, immunofluorescence and cytokine analysis. Mice deficient in TLR2 developed significantly more and larger colorectal tumors than their WT controls. We provide evidence that colonic epithelium of TLR2(-/- mice have altered immune responses and dysregulated proliferation under steady-state conditions and during colitis, which lead to inflammatory growth signals and predisposition to accelerated neoplastic growth. At the earliest time-points assessed, TLR2(-/- colons exhibited a significant increase in aberrant crypt foci (ACF, resulting in tumors that developed earlier and grew larger. In addition, the intestinal microenvironment revealed significantly higher levels of IL-6 and IL-17A concomitant with increased phospho-STAT3 within ACF. These observations indicate that in colitis, TLR2 plays a protective role against the development of CAC.

Dysregulation of toll-like receptor (TLR) 2 expression on monocytes and upregulation of the frequency of T cells expressing TLR2 in patients with chronic hepatitis C virus infection

DEFF Research Database (Denmark)

Ronit, Andreas; Salem, Mohammad; Hartling, Hans J

2013-01-01

Toll-like receptors (TLRs) initiate inflammatory responses that may play a role in disease progression in patients infected with hepatitis C virus (HCV). TLR2 and TLR4 surface expression were assessed on CD14(+) monocytes, CD4(+) and CD8(+) T cells in treatment naïve patients with chronic HCV...... infection with fibrosis, without fibrosis, co-infected with human immunodeficiency virus (HIV), and in healthy controls. Increased expression of TLR2 was found on monocytes in HCV-infected patients with fibrosis (p...

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

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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.

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 ...

Gene conversion limits divergence of mammalian TLR1 and TLR6

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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.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

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Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

2017-01-01

Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κB-p53/p21 signaling.

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Feng, Guijuan; Zheng, Ke; Cao, Tong; Zhang, Jinlong; Lian, Min; Huang, Dan; Wei, Changbo; Gu, Zhifeng; Feng, Xingmei

2018-02-26

Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The β-galactosidase (SA-β-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-β-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells.

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Ciaran Skerry

Full Text Available Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA. In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA. Exposure of human peripheral blood mononuclear cells (PBMC to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01, LprH (p<0.05, LprI (p<0.05, LprP (p<0.001, LprQ (p<0.005, MPT83 (p<0.005, or PhoS1 (p<0.05, resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05. These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells.

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Skerry, Ciaran; Klinkenberg, Lee G; Page, Kathleen R; Karakousis, Petros C

2016-01-01

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

Detailed qualitative dynamic knowledge representation using a BioNetGen model of TLR-4 signaling and preconditioning.

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An, Gary C; Faeder, James R

2009-01-01

preconditioning behavior with increasing LPS at 10, 100, 1000 and 10,000 and a secondary dose of LPS at 10,000 administered at approximately 27h of simulated time. Simulations of 'knockout' versions of the model allowed further examination of the interactions within the signaling cascade. The model demonstrated a dose-dependent TNF response curve to increasing stimulus by LPS. Preconditioning simulations demonstrated a similar dose-dependency of preconditioning doses leading to attenuation of response to subsequent LPS challenge - a 'tolerance' dynamic. These responses match dynamics reported in the literature. Furthermore, the simulated 'knockout' results suggested the existence and need for dual negative feedback control mechanisms, represented by the zinc ring-finger protein A20 and inhibitor kappa B proteins (IkappaB), in order for both effective attenuation of the initial stimulus signal and subsequent preconditioned 'tolerant' behavior. We present an example of detailed, qualitative dynamic knowledge representation using the TLR-4 signaling pathway, its control mechanisms and overall behavior with respect to preconditioning. The intent of this approach is to demonstrate a method of translating the extensive mechanistic knowledge being generated at the basic science level into an executable framework that can provide a means of 'conceptual model verification.' This allows for both the 'checking' of the dynamic consequences of a mechanistic hypothesis and the creation of a modular component of an overall model directed at the engineering goal of biomedical research. It is hoped that this paper will increase the use of knowledge representation and communication in this fashion, and facilitate the concatenation and integration of community-wide knowledge.

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor*

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Paramo, Teresa; Piggot, Thomas J.; Bryant, Clare E.; Bond, Peter J.

2013-01-01

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling. PMID:24178299

The structural basis for endotoxin-induced allosteric regulation of the Toll-like receptor 4 (TLR4) innate immune receptor.

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Paramo, Teresa; Piggot, Thomas J; Bryant, Clare E; Bond, Peter J

2013-12-20

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The "clamshell-like" motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the "molecular switch" in endotoxic signaling.

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

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Kun Chen

2017-01-01

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

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

OpenAIRE

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

2016-01-01

Cobalt alloy debris has been implicated as causative in the early failure of some designs of current total joint implants. The ability of implant debris to cause excessive inflammation via danger signaling (NLRP3 inflammasome) vs. pathogen associated pattern recognition receptors (e.g. Toll-like receptors; TLRs) remains controversial. Recently, specific non-conserved histidines on human TLR4 have been shown activated by cobalt and nickel ions in solution. However, whether this TLR activation ...

Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

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Rajsekhar Bhowmick

Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

Flaxseed Oil Attenuates Intestinal Damage and Inflammation by Regulating Necroptosis and TLR4/NOD Signaling Pathways Following Lipopolysaccharide Challenge in a Piglet Model.

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Zhu, Huiling; Wang, Haibo; Wang, Shuhui; Tu, Zhixiao; Zhang, Lin; Wang, Xiuying; Hou, Yongqing; Wang, Chunwei; Chen, Jie; Liu, Yulan

2018-05-01

Flaxseed oil is a rich source of α-linolenic acid (ALA), which is the precursor of the long-chain n-3 polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). This study investigates the protective effect of flaxseed oil against intestinal injury induced by lipopolysaccharide (LPS). Twenty-four weaned pigs were used in a 2 × 2 factorial experiment with dietary treatment (5% corn oil vs 5% flaxseed oil) and LPS challenge (saline vs LPS). On day 21 of the experiment, pigs were administrated with LPS or saline. At 2 h and 4 h post-administration, blood samples were collected. After the blood harvest at 4 h, all piglets were slaughtered and intestinal samples were collected. Flaxseed oil supplementation led to the enrichment of ALA, EPA, and total n-3 PUFAs in intestine. Flaxseed oil improved intestinal morphology, jejunal lactase activity, and claudin-1 protein expression. Flaxseed oil downregulated the mRNA expression of intestinal necroptotic signals. Flaxseed oil also downregulated the mRNA expression of intestinal toll-like receptors 4 (TLR4) and its downstream signals myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB), and nucleotide-binding oligomerization domain proteins 1, 2 (NOD1, NOD2) and its adapter molecule, receptor-interacting protein kinase 2 (RIPK2). These results suggest that dietary addition of flaxseed oil enhances intestinal integrity and barrier function, which is involved in modulating necroptosis and TLR4/NOD signaling pathways. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

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Tomlinson, Gillian; Chimalapati, Suneeta; Pollard, Tracey; Lapp, Thabo; Cohen, Jonathan; Camberlein, Emilie; Stafford, Sian; Periselneris, Jimstan; Aldridge, Christine; Vollmer, Waldemar; Picard, Capucine; Casanova, Jean-Laurent; Noursadeghi, Mahdad; Brown, Jeremy

2014-10-01

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host-pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB-regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2-associated responses were preserved. Experiments using leukocytes from IL-1R-associated kinase-4-deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R-associated kinase-4-mediated inflammatory responses to S. pneumoniae. Copyright © 2014 The Authors.

Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway in experimental traumatic brain injury

Science.gov (United States)

2014-01-01

neuron co-culture following treatment with curcumin after LPS stimulation. LPS increased TLR4 immunoreactivity and morphological activation in microglia and increased neuronal apoptosis, whereas curcumin normalized this upregulation. The increased protein levels of TLR4, MyD88 and NF-κB in microglia were attenuated by curcumin treatment. Conclusions Our results suggest that post-injury, curcumin administration may improve patient outcome by reducing acute activation of microglia/macrophages and neuronal apoptosis through a mechanism involving the TLR4/MyD88/NF-κB signaling pathway in microglia/macrophages in TBI. PMID:24669820

Cold-inducible RNA-binding protein mediates cold air inducible airway mucin production through TLR4/NF-κB signaling pathway.

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Chen, Lingxiu; Ran, Danhua; Xie, Wenyue; Xu, Qing; Zhou, Xiangdong

2016-10-01

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases and cold air stimulation has been shown to be associated with the severity of these diseases. However, the regulatory mechanisms that mediate excessive mucin production under cold stress remain elusive. Recently, the cold-inducible RNA-binding protein (CIRP) has been shown to be markedly induced after exposure to cold air. In this study, we sought to explore the expression of CIRP within bronchial biopsy specimens, the effect on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in cold air stimulation process. We found that CIRP protein expression was significantly increased in patients with COPD and in mice treated with cold air. Moreover, cold air stimulation induced MUC5AC expression in wild-type mice but not in CIRP(-/-) mice. In vitro, cold air stress significantly elevated the transcriptional and protein expression levels of MUC5AC in human bronchial epithelial cells. CIRP, toll-like receptor 4 (TLR4) and phosphorylated NF-κB p65 (p-p65) increased significantly in response to cold stress and CIRP siRNA, TLR4 - neutralizing Ab and a specific inhibitor of NF-κB could attenuated cold stress inducible MUC5AC expression. In addition, CIRP siRNA could hindered the expression levels of TLR4 and p-p65 both induced by cold stress. Taken together, these results suggest that airway epithelial cells constitutively express CIRP in vitro and in vivo. CIRP is responsible for cold-inducible MUC5AC expression by activating TLR4/NF-κB signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Andrographolide Ameliorates Liver Fibrosis in Mice: Involvement of TLR4/NF-κB and TGF-β1/Smad2 Signaling Pathways

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Liteng Lin

2018-01-01

Full Text Available Liver fibrosis is characterized by activated hepatic stellate cells (HSC and extracellular matrix accumulation. Blocking the activation of HSC and the inflammation response are two major effective therapeutic strategies for liver fibrosis. In addition to the long history of using andrographolide (Andro for inflammatory disorders, we aimed at elucidating the pharmacological effects and potential mechanism of Andro on liver fibrosis. In this study, liver fibrosis was induced by carbon tetrachloride (CCl4 and the mice were intraperitoneally injected with Andro for 6 weeks. HSC cell line (LX-2 and primary HSC were also treated with Andro in vitro. Treatment of CCl4-induced mice with Andro decreased the levels of alanine aminotransferase (ALT and aspartate aminotransferase (AST, Sirius red staining as well as the expression of α smooth muscle actin (α-SMA and transforming growth factor- (TGF- β1. Furthermore, the expression of Toll-like receptor (TLR4 and NF-κB p50 was also inhibited by Andro. Additionally, in vitro data confirmed that Andro treatment not only attenuated the expression of profibrotic and proinflammatory factors but also blocked the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways. These results demonstrate that Andro prevents liver inflammation and fibrosis, which is in correlation with the inhibition of the TGF-β1/Smad2 and TLR4/NF-κB p50 pathways, highlighting Andro as a potential therapeutic strategy for liver fibrosis.

Modulation of neonatal microbial recognition: TLR-mediated innate immune responses are specifically and differentially modulated by human milk.

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LeBouder, Emmanuel; Rey-Nores, Julia E; Raby, Anne-Catherine; Affolter, Michael; Vidal, Karine; Thornton, Catherine A; Labéta, Mario O

2006-03-15

The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1-5) of breast milk on ligand-induced cell stimulation via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS. Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation, suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including those coding for signal intermediates and regulators. A proteinaceous milk component of > or =80 kDa was found to be responsible for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms regulating TLR activation.

Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects.

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Reyna, Sara M; Ghosh, Sangeeta; Tantiwong, Puntip; Meka, C S Reddy; Eagan, Phyllis; Jenkinson, Christopher P; Cersosimo, Eugenio; Defronzo, Ralph A; Coletta, Dawn K; Sriwijitkamol, Apiradee; Musi, Nicolas

2008-10-01

OBJECTIVE- Tall-like receptor (TLR)4 has been implicated in the pathogenesis of free fatty acid (FFA)-induced insulin resistance by activating inflammatory pathways, including inhibitor of kappaB (IkappaB)/nuclear factor kappaB (NFkappaB). However, it is not known whether insulin-resistant subjects have abnormal TLR4 signaling. We examined whether insulin-resistant subjects have abnormal TLR4 expression and TLR4-driven (IkappaB/NFkappaB) signaling in skeletal muscle. RESEARCH DESIGN AND METHODS- TLR4 gene expression and protein content were measured in muscle biopsies in 7 lean, 8 obese, and 14 type 2 diabetic subjects. A primary human myotube culture system was used to examine whether FFAs stimulate IkappaB/NFkappaB via TLR4 and whether FFAs increase TLR4 expression/content in muscle. RESULTS- Obese and type 2 diabetic subjects had significantly elevated TLR4 gene expression and protein content in muscle. TLR4 muscle protein content correlated with the severity of insulin resistance. Obese and type 2 diabetic subjects also had lower IkappaBalpha content, an indication of elevated IkappaB/NFkappaB signaling. The increase in TLR4 and NFkappaB signaling was accompanied by elevated expression of the NFkappaB-regulated genes interleukin (IL)-6 and superoxide dismutase (SOD)2. In primary human myotubes, acute palmitate treatment stimulated IkappaB/NFkappaB, and blockade of TLR4 prevented the ability of palmitate to stimulate the IkappaB/NFkappaB pathway. Increased TLR4 content and gene expression observed in muscle from insulin-resistant subjects were reproduced by treating myotubes from lean, normal-glucose-tolerant subjects with palmitate. Palmitate also increased IL-6 and SOD2 gene expression, and this effect was prevented by inhibiting NFkappaB. CONCLUSIONS- Abnormal TLR4 expression and signaling, possibly caused by elevated plasma FFA levels, may contribute to the pathogenesis of insulin resistance in humans.

Anti-influenza A virus activity of rhein through regulating oxidative stress, TLR4, Akt, MAPK, and NF-κB signal pathways.

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Qian-Wen Wang

Full Text Available Rhein, an anthraquinone compound existing in many traditional herbal medicines, has anti-inflammatory, antioxidant, antitumor, antiviral, hepatoprotective, and nephroprotective activities, but its anti-influenza A virus (IAV activity is ambiguous. In the present study, through plaque inhibition assay, time-of-addition assay, antioxidant assay, qRT-PCR, ELISA, and western blotting assays, we investigated the anti-IAV effect and mechanism of action of rhein in vitro and in vivo. The results showed that rhein could significantly inhibit IAV adsorption and replication, decrease IAV-induced oxidative stress, activations of TLR4, Akt, p38, JNK MAPK, and NF-κB pathways, and production of inflammatory cytokines and matrix metalloproteinases in vitro. Oxidant H2O2 and agonists of TLR4, Akt, p38/JNK and IKK/NF-κB could significantly antagonize the inhibitory effects of rhein on IAV-induced cytopathic effect (CPE and IAV replication. Through an in vivo test in mice, we also found that rhein could significantly improve the survival rate, lung index, pulmonary cytokines, and pulmonary histopathological changes. Rhein also significantly decreased pulmonary viral load at a high dose. In conclusion, rhein can inhibit IAV adsorption and replication, and the mechanism of action to inhibit IAV replication may be due to its ability to suppress IAV-induced oxidative stress and activations of TLR4, Akt, p38, JNK MAPK, and NF-κB signal pathways.

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.

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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.

Mycobacterium abscessus glycopeptidolipid prevents respiratory epithelial TLR2 signaling as measured by HβD2 gene expression and IL-8 release.

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Lisa B Davidson

Full Text Available Mycobacterium abscessus has emerged as an important cause of lung infection, particularly in patients with bronchiectasis. Innate immune responses must be highly effective at preventing infection with M. abscessus because it is a ubiquitous environmental saprophyte and normal hosts are not commonly infected. M. abscessus exists as either a glycopeptidolipid (GPL expressing variant (smooth phenotype in which GPL masks underlying bioactive cell wall lipids, or as a variant lacking GPL which is immunostimulatory and invasive in macrophage infection models. Respiratory epithelium has been increasingly recognized as playing an important role in the innate immune response to pulmonary pathogens. Respiratory epithelial cells express toll-like receptors (TLRs which mediate the innate immune response to pulmonary pathogens. Both interleukin-8 (IL-8 and human β-defensin 2 (HβD2 are expressed by respiratory epithelial cells in response to toll-like receptor 2 (TLR2 receptor stimulation. In this study, we demonstrate that respiratory epithelial cells respond to M. abscessus variants lacking GPL with expression of IL-8 and HβD2. Furthermore, we demonstrate that this interaction is mediated through TLR2. Conversely, M. abscessus expressing GPL does not stimulate expression of IL-8 or HβD2 by respiratory epithelial cells which is consistent with "masking" of underlying bioactive cell wall lipids by GPL. Because GPL-expressing smooth variants are the predominant phenotype existing in the environment, this provides an explanation whereby initial M. abscessus colonization of abnormal lung airways escapes detection by the innate immune system.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

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Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

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

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

2017-01-25

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

[Fisetin alleviates hypoxia/reoxygenation injury in rat hepatocytes via modulation of TLR4/NF-κB signaling pathway].

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Pu, Junliang; Wan, Lei; Zheng, Daofeng; Wei, Xufu; Wu, Zhongjun; Tang, Chengyong

2017-07-01

Objective To investigate the protective effect of fisetin (FIS) against hypoxia/reoxygenation (H/R) injury in rat hepatocytes and its mechanism. Methods H/R injury model of BRL-3A cells was established and the cells were pretreated with FIS. Survival rate was detected by CCK-8 assay. Cell apoptosis was measured by flow cytometry. The levels of ALT and AST were determined by microplate assay. The production of TNF-α and IL-1β were detected by ELISA. The mRNA and protein levels of TLR4 and NF-κBp65 were analyzed by quantitative real-time PCR and Western blotting, respectively. Results After subjected to H/R, cell survival rate decreased and the apoptosis level increased. The levels of ALT and AST in cell supernatant were elevated, so were the production of TNF-α and IL-1β. FIS pretreatment increased the cell survival rate and inhibited apoptosis. The levels of ALT, AST and the production of TNF-α and IL-1β were reduced significantly. Moreover, FIS inhibited the increasing expression levels of TLR4 and NF-κBp65 induced by H/R. Conclusion FIS alleviates the hepatocyte injury induced by H/R via modulation of TLR4/NF-κB signaling pathway.

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

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Min Du

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

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

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Lin Qu

2011-09-01

Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

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Michiel van Gent

2014-02-01

Full Text Available Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR. TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpesviruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs. The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

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.

Hepatitis C Virus NS3 Mediated Microglial Inflammation via TLR2/TLR6 MyD88/NF-κB Pathway and Toll Like Receptor Ligand Treatment Furnished Immune Tolerance.

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Ayilam Ramachandran Rajalakshmy

Full Text Available Recent evidence suggests the neurotrophic potential of hepatitis C virus (HCV. HCV NS3 protein is one of the potent antigens of this virus mediating inflammatory response in different cell types. Microglia being the immune surveillance cells in the central nervous system (CNS, the inflammatory potential of NS3 on microglia was studied. Role of toll like receptor (TLR ligands Pam2CSK3 and Pam3CSK4 in controlling the NS3 mediated microglial inflammation was studied using microglial cell line CHME3.IL (Interleukin-8, IL-6, TNF-α (Tumor nicrosis factor alpha and IL-1β gene expressions were measured by semi quantitative RT-PCR (reverse transcription-PCR. ELISA was performed to detect IL-8, IL-6, TNF-α, IL-1β and IL-10 secretion. FACS (Flourescent activated cell sorting was performed to quantify TLR1, TLR2, TLR6, MyD88 (Myeloid differntiation factor 88, IkB-α (I kappaB alpha and pNF-κB (phosphorylated nuclear factor kappaB expression. Immunofluorescence staining was performed for MyD88, TLR6 and NF-κB (Nuclear factor kappaB. Student's t-test or One way analysis of variance with Bonferoni post hoc test was performed and p < 0.05 was considered significant.Microglia responded to NS3 by secreting IL-8, IL-6, TNF-α and IL-1β via TLR2 or TLR6 mediated MyD88/NF-κB pathway. Transcription factor NF-κB was involved in activating the cytokine gene expression and the resultant inflammatory response was controlled by NF-κB inhibitor, Ro106-9920, which is known to down regulate pro-inflammatory cytokine secretion. Activation of the microglia by TLR agonists Pam3CSK4 and Pam2CSK4 induced immune tolerance against NS3. TLR ligand treatment significantly down regulated pro-inflammatory cytokine secretion in the microglia. IL-10 secretion was suggested as the possible mechanism by which TLR agonists induced immune tolerance. NS3 as such was not capable of self-inducing immune tolerance in microglia.In conclusion, NS3 protein was capable of activating

Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

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Latty, Sarah Louise; Sakai, Jiro; Hopkins, Lee; Verstak, Brett; Paramo, Teresa; Berglund, Nils A; Cammorota, Eugenia; Cicuta, Pietro; Gay, Nicholas J; Bond, Peter J; Klenerman, David; Bryant, Clare E

2018-01-24

Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling. © 2018, Latty et al.

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

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

2017-01-01

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

Fisetin Alleviates Lipopolysaccharide-Induced Acute Lung Injury via TLR4-Mediated NF-κB Signaling Pathway in Rats.

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Feng, Guang; Jiang, Ze-Yu; Sun, Bo; Fu, Jie; Li, Tian-Zuo

2016-02-01

Acute lung injury (ALI), a common component of systemic inflammatory disease, is a life-threatening condition without many effective treatments. Fisetin, a natural flavonoid from fruits and vegetables, was reported to have wide pharmacological properties such as anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was to detect the effects of fisetin on lipopolysaccharide (LPS)-induced acute lung injury and investigate the potential mechanism. Fisetin was injected (1, 2, and 4 mg/kg, i.v.) 30 min before LPS administration (5 mg/kg, i.v.). Our results showed that fisetin effectively reduced the inflammatory cytokine release and total protein in bronchoalveolar lavage fluids (BALF), decreased the lung wet/dry ratios, and obviously improved the pulmonary histology in LPS-induced ALI. Furthermore, fisetin inhibited LPS-induced increases of neutrophils and macrophage infiltration and attenuated MPO activity in lung tissues. Additionally, fisetin could significantly inhibit the Toll-like receptor 4 (TLR4) expression and the activation of NF-κB in lung tissues. Our data indicates that fisetin has a protective effect against LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways, and fisetin may be a promising candidate for LPS-induced ALI treatment.

Basophils activated via TLR signaling may contribute to pathophysiology of type 1 autoimmune pancreatitis.

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Yanagawa, Masato; Uchida, Kazushige; Ando, Yugo; Tomiyama, Takashi; Yamaguchi, Takashi; Ikeura, Tsukasa; Fukui, Toshiro; Nishio, Akiyoshi; Uemura, Yoshiko; Miyara, Takayuki; Okamoto, Hiroyuki; Satoi, Souhei; Okazaki, Kazuichi

2018-03-01

Pathophysiology of type 1 autoimmune pancreatitis (AIP) is still unclear. We previously reported that M2 macrophages might play an important role in type 1 AIP. Recently, it has been reported that basophils regulate differentiation to M2 macrophages. In this study, we investigated basophils from the pancreatic tissue and peripheral blood of individuals with type 1 AIP. By using immunohistochemistry, we investigated basophils in pancreatic tissue from 13 patients with type 1 AIP and examined expression of toll-like receptors (TLRs) by these cells. Additionally, we obtained peripheral blood samples from 27 healthy subjects, 40 patients with type 1 AIP, 8 patients with alcoholic chronic pancreatitis, 10 patients with bronchial asthma, and 10 patients with atopic dermatitis, and analyzed activation of basophils by stimulating them with ligands of TLR1-9. We also compared TLR expression in basophils from the tissue and blood samples. Basophils were detected in pancreatic tissues from 10 of 13 patients with type 1 AIP. Flow cytometric analysis revealed that the ratios of basophils activated by TLR4 stimulation in type 1 AIP (9.875 ± 1.148%) and atopic dermatitis (11.768 ± 1.899%) were significantly higher than those in healthy subjects (5.051 ± 0.730%; P pathophysiology of type 1 AIP.

Pretreatment of Sialic Acid Efficiently Prevents Lipopolysaccharide-Induced Acute Renal Failure and Suppresses TLR4/gp91-Mediated Apoptotic Signaling

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Shih-Ping Hsu

2016-05-01

Full Text Available Background/Aims: Lipopolysaccharides (LPS binding to Toll-like receptor 4 (TLR4 activate NADPH oxidase gp91 subunit-mediated inflammation and oxidative damage. Recognizing the high binding affinity of sialic acid (SA with LPS, we further explored the preventive potential of SA pretreatment on LPS-evoked acute renal failure (ARF. Methods: We determined the effect of intravenous SA 30 min before LPS-induced injury in urethane-anesthetized female Wistar rats by evaluating kidney reactive oxygen species (ROS responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. Results: LPS time-dependently reduced arterial blood pressure, renal microcirculation, and increased blood urea nitrogen and creatinine in the rats. LPS enhanced monocyte/macrophage infiltration and ROS production, and subsequently impaired kidneys with the enhancement of TLR4/NADPH oxidase gp91/Caspase 3/poly-(ADP-ribose-polymerase (PARP-mediated apoptosis in the kidneys. SA pretreatment effectively alleviated LPS-induced ARF. The levels of LPS-increased ED-1 infiltration and ROS production in the kidney were significantly depressed by SA pretreatment. Furthermore, SA pretreatment significantly depressed TLR4 activation, gp91 expression, and Caspase 3/PARP induced apoptosis in the kidneys. Conclusion: We suggest that pretreatment of SA significantly and preventively attenuated LPS-induced detrimental effects on systemic and renal hemodynamics, renal ROS production and renal function, as well as, LPS-activated TLR4/gp91/Caspase3 mediated apoptosis signaling.

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

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Xu MX

2017-01-01

Full Text Available Min-Xuan Xu,1,2,* Ming Wang,3,* Wei-Wei Yang4 1Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing, 2College of Engineering and Applied Sciences, Nanjing University, Nanjing, 3Department of Urology, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, 4Department of Nephrology, Huai’an First People’s Hospital, Nanjing Medical University, Jiangsu, People’s Republic of China *These authors contributed equally to this work Abstract: High-fat diet-induced metabolic syndrome followed by chronic kidney disease caused by intestinal endotoxemia have received extensive attention. Toll-like receptor 4 (TLR4/nuclear factor-kappa B (NF-κB and oxidative stress-related Nrf2/Keap1 were regarded as the key target points involved in metabolic inflammation and kidney injury. However, the molecular mechanism of interaction between TLR4/NF-κB and Nrf2 activation in high-fat diet-induced renal injury is not absolutely understood. Quercetin, a natural product, has been reported to possess antitumor and anti-inflammatory effects. In this regard, this study attempted to prepare poly(d,l-lactide-co-glycolide-loaded gold nanoparticles precipitated with quercetin (GQ to investigate the anti-inflammatory and anti-oxidative stress effects in high-fat diet-induced kidney failure. For this study, C57BL/6 mice fed fat-rich fodder were used as the metabolic syndrome model to evaluate the protective effects of GQ on kidney injury and to determine whether TLR4/NF-κB and Nrf2 pathways were associated with the process. Moreover, histological examinations, enzyme-linked immunosorbent assay, Western blot, and basic blood tests and systemic inflammation-related indicators were used to investigate the inhibitory effects of GQ and underlying molecular mechanism by which it may reduce renal injury. Of note, podocyte

Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

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Fanton d'Andon, Martine; Quellard, Nathalie; Fernandez, Béatrice; Ratet, Gwenn; Lacroix-Lamandé, Sonia; Vandewalle, Alain; Boneca, Ivo G.; Goujon, Jean-Michel; Werts, Catherine

2014-01-01

Background Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process. Methodology/principal findings Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis. Conclusion/significance To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors

Pattern-recognition receptors: signaling pathways and dysregulation in canine chronic enteropathies-brief review.

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Heilmann, Romy M; Allenspach, Karin

2017-11-01

Pattern-recognition receptors (PRRs) are expressed by innate immune cells and recognize pathogen-associated molecular patterns (PAMPs) as well as endogenous damage-associated molecular pattern (DAMP) molecules. With a large potential for synergism or convergence between their signaling pathways, PRRs orchestrate a complex interplay of cellular mediators and transcription factors, and thus play a central role in homeostasis and host defense. Aberrant activation of PRR signaling, mutations of the receptors and/or their downstream signaling molecules, and/or DAMP/PAMP complex-mediated receptor signaling can potentially lead to chronic auto-inflammatory diseases or development of cancer. PRR signaling pathways appear to also present an interesting new avenue for the modulation of inflammatory responses and to serve as potential novel therapeutic targets. Evidence for a dysregulation of the PRR toll-like receptor (TLR)2, TLR4, TLR5, and TLR9, nucleotide-binding oligomerization domain-containing protein (NOD)2, and the receptor of advanced glycation end products (RAGE) exists in dogs with chronic enteropathies. We describe the TLR, NOD2, and RAGE signaling pathways and evaluate the current veterinary literature-in comparison to human medicine-to determine the role of TLRs, NOD2, and RAGE in canine chronic enteropathies.

Polymorphisms at the innate immune receptor TLR2 are associated with Borrelia infection in a wild rodent population.

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Tschirren, Barbara; Andersson, Martin; Scherman, Kristin; Westerdahl, Helena; Mittl, Peer R E; Råberg, Lars

2013-05-22

The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture-mark-recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.

Interactions of TLR4 and PPARγ, Dependent on AMPK Signalling Pathway Contribute to Anti-Inflammatory Effects of Vaccariae Hypaphorine in Endothelial Cells

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Haijian Sun

2017-07-01

Full Text Available Background /Aims: Accumulating evidence indicates that endothelial inflammation is one of the critical determinants in pathogenesis of atherosclerotic cardiovascular disease. Our previous studies had demonstrated that Vaccariae prevented high glucose or oxidative stress-triggered endothelial dysfunction in vitro. Very little is known about the potential effects of hypaphorine from Vaccariae seed on inflammatory response in endothelial cells. Methods: In the present study, we evaluated the anti-inflammatory effects of Vaccariae hypaphorine (VH on lipopolysaccharide (LPS-challenged endothelial EA.hy926 cells. The inflammatory cytokines including tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, monocyte chemoattractant protein 1 (MCP-1 and vascular cellular adhesion molecule-1 (VCAM-1 were measured by real-time PCR (RT-PCR. The expressions of adenosine monophosphate-activated protein kinase (AMPK, acetyl-CoA carboxylase (ACC, toll-like receptor 4 (TLR4, peroxisome proliferator-activated receptor γ (PPARγ were detected by Western blotting or immunofluorescence. Results: We showed that LPS stimulated the expressions of TNF-α, IL-1β, MCP-1, VCAM-1 and TLR4, but attenuated the phosphorylation of AMPK and ACC as well as PPARγ protein levels, which were reversed by VH pretreatment. Moreover, we observed that LPS-upregulated TLR4 protein expressions were inhibited by PPARγ agonist pioglitazone, and the downregulated PPARγ expressions in response to LPS were partially restored by knockdown of TLR4. The negative regulation loop between TLR4 and PPARγ response to LPS was modulated by AMPK agonist AICAR (5-Aminoimidazole-4-carboxamide riboside or acadesine or A769662. Conclusions: Taken together, our results suggested that VH ameliorated LPS-induced inflammatory cytokines production in endothelial cells via inhibition of TLR4 and activation of PPARγ, dependent on AMPK signalling pathway.

Polymorphisms in the TLR4 and TLR5 gene are significantly associated with inflammatory bowel disease in German shepherd dogs.

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Kathrani, Aarti; House, Arthur; Catchpole, Brian; Murphy, Angela; German, Alex; Werling, Dirk; Allenspach, Karin

2010-12-23

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease.

Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

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Yang, Jinju; Qin, Nannan; Zhang, Hongwei; Yang, Rui; Xiang, Benqiong; Wei, Qun

2016-04-19

Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy.

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Santana, Nadja de Lima; Rêgo, Jamile Leão; Oliveira, Joyce Moura; Almeida, Lucas Frederico de; Braz, Marcos; Machado, Lídia Maria Medeiros; Machado, Paulo Roberto Lima; Castellucci, Léa Cristina

2017-04-01

Leprosy or hansen's disease is a spectral disease whose clinical forms mostly depends on host's immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host's production of key cytokines and chemokines involved in the pathogenesis of this disease.

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

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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.

Sonic hedgehog-dependent induction of microRNA 31 and microRNA 150 regulates Mycobacterium bovis BCG-driven toll-like receptor 2 signaling.

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Ghorpade, Devram Sampat; Holla, Sahana; Kaveri, Srini V; Bayry, Jagadeesh; Patil, Shripad A; Balaji, Kithiganahalli Narayanaswamy

2013-02-01

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α(-/-) macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.

Stress-induced neuroinflammation is mediated by GSK3-dependent TLR4 signaling that promotes susceptibility to depression-like behavior.

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Cheng, Yuyan; Pardo, Marta; Armini, Rubia de Souza; Martinez, Ana; Mouhsine, Hadley; Zagury, Jean-Francois; Jope, Richard S; Beurel, Eleonore

2016-03-01

Most psychiatric and neurological diseases are exacerbated by stress. Because this may partially result from stress-induced inflammation, we examined factors involved in this stress response. After a paradigm of inescapable foot shock stress that causes learned helplessness depression-like behavior, eighteen cytokines and chemokines increased in mouse hippocampus, peaking 6-12h after stress. A 24h prior pre-conditioning stress accelerated the rate of stress-induced hippocampal cytokine and chemokine increases, with most reaching peak levels after 1-3h, often without altering the maximal levels. Toll-like receptor 4 (TLR4) was involved in this response because most stress-induced hippocampal cytokines and chemokines were attenuated in TLR4 knockout mice. Stress activated glycogen synthase kinase-3 (GSK3) in wild-type mouse hippocampus, but not in TLR4 knockout mice. Administration of the antidepressant fluoxetine or the GSK3 inhibitor TDZD-8 reduced the stress-induced increases of most hippocampal cytokines and chemokines. Stress increased hippocampal levels of the danger-associated molecular pattern (DAMP) protein high mobility group box 1 (HMGB1), activated the inflammatory transcription factor NF-κB, and the NLRP3 inflammasome. Knockdown of HMGB1 blocked the acceleration of cytokine and chemokine increases in the hippocampus caused by two successive stresses. Fluoxetine treatment blocked stress-induced up-regulation of HMGB1 and subsequent NF-κB activation, whereas TDZD-8 administration attenuated NF-κB activation downstream of HMGB1. To test if stress-induced cytokines and chemokines contribute to depression-like behavior, the learned helplessness model was assessed. Antagonism of TNFα modestly reduced susceptibility to learned helplessness induction, whereas TLR4 knockout mice were resistant to learned helplessness. Thus, stress-induces a broad inflammatory response in mouse hippocampus that involves TLR4, GSK3, and downstream inflammatory signaling, and

Stress-induced neuroinflammation is mediated by GSK3-dependent TLR4 signaling that promotes susceptibility to depression-like behavior

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Cheng, Yuyan; Pardo, Marta; de Souza Armini, Rubia; Martinez, Ana; Mouhsine, Hadley; Zagury, Jean-Francois; Jope, Richard S.; Beurel, Eleonore

2016-01-01

Most psychiatric and neurological diseases are exacerbated by stress. Because this may partially result from stress-induced inflammation, we examined factors involved in this stress response. After a paradigm of inescapable foot shock stress that causes learned helplessness depression-like behavior, eighteen cytokines and chemokines increased in mouse hippocampus, peaking 6 to 12 hr after stress. A 24 hr prior pre-conditioning stress accelerated the rate of stress-induced hippocampal cytokine and chemokine increases, with most reaching peak levels after 1 to 3 hr, often without altering the maximal levels. Toll-like receptor 4 (TLR4) was involved in this response because most stress-induced hippocampal cytokines and chemokines were attenuated in TLR4 knockout mice. Stress activated glycogen synthase kinase-3 (GSK3) in wild-type mouse hippocampus, but not in TLR4 knockout mice. Administration of the antidepressant fluoxetine or the GSK3 inhibitor TDZD-8 reduced the stress-induced increases of most hippocampal cytokines and chemokines. Stress increased hippocampal levels of the danger-associated molecular pattern (DAMP) protein high mobility group box 1 (HMGB1), activated the inflammatory transcription factor NF-κB, and the NLRP3 inflammasome. Knockdown of HMGB1 blocked the acceleration of cytokine and chemokine increases in the hippocampus caused by two successive stresses. Fluoxetine treatment blocked stress-induced up-regulation of HMGB1 and subsequent NF-κB activation, whereas TDZD-8 administration attenuated NF-κB activation downstream of HMGB1. To test if stress-induced cytokines and chemokines contribute to depression-like behavior, the learned helplessness model was assessed. Antagonism of TNFα modestly reduced susceptibility to learned helplessness induction, whereas TLR4 knockout mice were resistant to learned helplessness. Thus, stress-induces a broad inflammatory response in mouse hippocampus that involves TLR4, GSK3, and downstream inflammatory

Immunomodulatory effect of APS and PSP is mediated by Ca2+-cAMP and TLR4/NF-κB signaling pathway in macrophage.

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Wang, Zhixue; Liu, Zijing; Zhou, Lijng; Long, Tingting; Zhou, Xing; Bao, Yixi

2017-01-01

This study is to investigate the role of second messengers and TLR4/NF-κB signaling pathway in the immunomodulatory activities of Astragalus polysaccharide (APS) and Polysaccharopeptide (PSP) in macrophages. RAW 264.7 macrophage cells were treated with APS, PSP, lipopolysaccharide (LPS), or NiCl 2 . Power-spectral method was used to detect protein kinase C (PKC) and Griess reaction to detect nitric oxide (NO). ELISA was conducted to detect cyclic adenosine monophosphate (cAMP), diglycerides (DAG), inositol 1, 4, 5-triphosphate (IP3), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Confocal laser scanning microscopy was performed to detect calcium level. qRT-PCR and Western blot was used to detect mRNA and protein expression of NF-κB. APS and PSP significantly increased the concentrations of intracellular second messengers (NO, cAMP, DAG, IP3, Ca 2+ ) and the activity of PKC in macrophages (pAPS and PSP (pAPS and PSP mediated immunomodulatory activities in macrophages. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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Georgina L Hold

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

TLR4/MyD88/NF-κB signaling and PPAR-γ within the paraventricular nucleus are involved in the effects of telmisartan in hypertension

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Li, Hong-Bao; Li, Xiang; Huo, Chan-Juan; Su, Qing; Guo, Jing [Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an Jiaotong University Cardiovascular Research Center, Xi' an 710061 (China); Yuan, Zu-Yi [Department of Cardiovascular Medicine, First Affiliated Hospital of Medical College of Xi' an Jiaotong University, Xi' an 710061 (China); Zhu, Guo-Qing [Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Physiology, Nanjing Medical University, Nanjing 210029 (China); Shi, Xiao-Lian, E-mail: shxl@mail.xjtu.edu.cn [Department of Pharmacology, School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an 710061 (China); Liu, Jin-Jun, E-mail: jupet@163.com [Department of Physiology and Pathophysiology, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an 710061 (China); Kang, Yu-Ming, E-mail: ykang@mail.xjtu.edu.cn [Department of Physiology and Pathophysiology, Key Laboratory of Environment and Genes Related to Diseases, Xi' an Jiaotong University School of Basic Medical Sciences, Xi' an Jiaotong University Health Science Center, Xi' an Jiaotong University Cardiovascular Research Center, Xi' an 710061 (China)

2016-08-15

Previous findings from our laboratory and others indicate that the main therapeutic effect of angiotensin II type 1 receptor (AT1-R) antagonists is to decrease blood pressure and exert anti-inflammatory effects in the cardiovascular system. In this study, we determined whether AT1-R antagonist telmisartan within the hypothalamic paraventricular nucleus (PVN) attenuates hypertension and hypothalamic inflammation via both the TLR4/MyD88/NF-κB signaling pathway and peroxisome proliferator-activated receptor-γ (PPAR-γ) in the PVN in hypertensive rats. Spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were treated for 4 weeks through bilateral PVN infusion with the AT1-R antagonist telmisartan (TEL, 10 μg/h), or losartan (LOS, 20 μg/h), or the PPAR-γ antagonist GW9662 (GW, 100 μg/h), or vehicle via osmotic minipump. Mean arterial pressure (MAP) was recorded by a tail-cuff occlusion method. PVN tissue and blood were collected for the measurement of AT1-R, PPAR-γ, pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6), inducible nitric oxide synthase (iNOS), TLR4, MyD88, nuclear factor-kappa B (NF-κB) activity and plasma norepinephrine (NE), respectively. Hypertensive rats exhibited significantly higher level of AT1-R and lower level of PPAR-γ in the PVN. PVN treatment with TEL attenuated MAP, improved cardiac hypertrophy, reduced TNF-α, IL-1β, IL-6, iNOS levels, and plasma NE in SHR but not in WKY rats. These results were associated with reduced TLR4, MyD88 and NF-κB levels and increased PPAR-γ level in the PVN of hypertensive rats. Our findings suggest that TLR4/MyD88/NF-κB signaling and PPAR-γ within the PVN are involved in the beneficial effects of telmisartan in hypertension. - Highlights: • PVN infusion of TEL in spontaneously hypertensive rats is reported. • PVN infusion of TEL attenuates hypertension and proinflammatory cytokines in PVN. • PVN blockade of AT1-R attenuates

The Emerging Role of TLR and Innate Immunity in Cardiovascular Disease

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Rolf Spirig

2012-01-01

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

Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.

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Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing

2017-08-01

Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Polymorphisms in the Tlr4 and Tlr5 Gene Are Significantly Associated with Inflammatory Bowel Disease in German Shepherd Dogs

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Kathrani, Aarti; House, Arthur; Catchpole, Brian; Murphy, Angela; German, Alex; Werling, Dirk; Allenspach, Karin

2010-01-01

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease. PMID:21203467

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

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Yang Zhang

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

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy

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Nadja de Lima Santana

Full Text Available BACKGROUND Leprosy or hansen’s disease is a spectral disease whose clinical forms mostly depends on host’s immune and genetic factors. Different Toll-like receptors (TLR variants have been described associated with leprosy, but with some lack of replication across different populations. OBJECTIVES To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. METHODS Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551, TLR2 (rs7656411, rs3804099 and TLR4 (rs1927914, rs1927911. A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA test in the serum of a subgroup of patients with and without leprosy reactions. FINDINGS Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2. Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. MAIN CONCLUSIONS All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host’s production of key cytokines and chemokines involved in the pathogenesis of this disease.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy

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Santana, Nadja de Lima; Rêgo, Jamile Leão; Oliveira, Joyce Moura; de Almeida, Lucas Frederico; Braz, Marcos; Machado, Lídia Maria Medeiros; Machado, Paulo Roberto Lima; Castellucci, Léa Cristina

2017-01-01

BACKGROUND Leprosy or hansen’s disease is a spectral disease whose clinical forms mostly depends on host’s immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. OBJECTIVES To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. METHODS Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. FINDINGS Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. MAIN CONCLUSIONS All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host’s production of key cytokines and chemokines involved in the pathogenesis of this disease. PMID:28327786

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.

Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways.

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Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

2016-10-10

The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases.

Polymorphisms within the Toll-Like Receptor (TLR-2, -4, and -6 Genes in Cattle

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Marco Mariotti

2009-07-01

Full Text Available In mammals, members of the TLR gene family play a primary role in the recognition of pathogen-associated molecular patterns from bacteria, viruses, protozoa and fungi. Recently, cattle TLR genes have been mapped to chromosomes using a radiation hybrid panel. Nucleotide sequences of bovine TLR2, TLR4 and TLR6 genes were screened to identify novel SNPs that can be used in studies of cattle resistance to diseases. In total, 8 SNPs were identified and were submitted to the NCBI dbSNP database. The frequencies of the SNPs were assessed in 16 different bovine European cattle breeds and a phylogenetic analysis carried out to describe the relationships between the breeds. Even if from our analysis the SNPs do not appear located in loci under selection, a deviation of three SNPs from Hardy Weinberg equilibrium was observed, and we hypothesize that some of the polymorphisms may be fixated since many generations. The described variations in immune function related genes will contribute to research on disease response in cattle. In fact, the SNPs can be used in association studies between polymorphisms and cattle resistance to diseases.

Staphylococcus epidermidis polysaccharide intercellular adhesin induces IL-8 expression in human astrocytes via a mechanism involving TLR2.

LENUS (Irish Health Repository)

Stevens, Niall T

2009-03-01

Staphylococcus epidermidis is an opportunistic biofilm-forming pathogen associated with neurosurgical device-related meningitis. Expression of the polysaccharide intercellular adhesin (PIA) on its surface promotes S. epidermidis biofilm formation. Here we investigated the pro-inflammatory properties of PIA against primary and transformed human astrocytes. PIA induced IL-8 expression in a dose- and\\/or time-dependent manner from U373 MG cells and primary normal human astrocytes. This effect was inhibited by depletion of N-acetyl-beta-d-glucosamine polymer from the PIA preparation with Lycopersicon esculentum lectin or sodium meta-periodate. Expression of dominant-negative versions of the TLR2 and TLR4 adaptor proteins MyD88 and Mal in U373 MG cells inhibited PIA-induced IL-8 production. Blocking IL-1 had no effect. PIA failed to induce IL-8 production from HEK293 cells stably expressing TLR4. However, in U373 MG cells which express TLR2, neutralization of TLR2 impaired PIA-induced IL-8 production. In addition to IL-8, PIA also induced expression of other cytokines from U373 MG cells including IL-6 and MCP-1. These data implicate PIA as an important immunogenic component of the S. epidermidis biofilm that can regulate pro-inflammatory cytokine production from human astrocytes, in part, via TLR2.

Sodium Octanoate Modulates the Innate Immune Response of Bovine Mammary Epithelial Cells through the TLR2/P38/JNK/ERK1/2 Pathway: Implications during Staphylococcus aureus Internalization.

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Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2017-01-01

Bovine mammary epithelial cells (bMECs) contribute to mammary gland defense against invading pathogens, such as Staphylococcus aureus (intracellular facultative), which is recognized by TLR2. In a previous report, we showed that sodium octanoate [NaO, a medium chain fatty acid (C8)] induces (0.25 mM) or inhibits (1 mM) S. aureus internalization into bMECs and differentially regulates the innate immune response (IIR). However, the molecular mechanisms have not been described, which was the aim of this study. The results showed that α5β1 integrin membrane abundance (MA) was increased in 0.25 mM NaO-treated cells, but TLR2 or CD36 MA was not modified. When these receptors were blocked individually, 0.25 mM NaO-increased S. aureus internalization was notably reduced. Interestingly, in this condition, the IIR of the bMECs was impaired because MAPK (p38, JNK, and ERK1/2) phosphorylation and the activation of transcription factors related to these pathways were decreased. In addition, the 1 mM NaO treatment induced TLR2 MA, but neither the integrin nor CD36 MA was modified. The reduction in S. aureus internalization induced by 1 mM NaO was increased further when TLR2 was blocked. In addition, the phosphorylation levels of the MAPKs increased, and 13 transcriptional factors related to the IIR were slightly activated (CBF, CDP, c-Myb, AP-1, Ets-1/Pea-3, FAST-1, GAS/ISRE, AP-2, NFAT-1, OCT-1, RAR/DR-5, RXR/DR-1, and Stat-3). Moreover, the 1 mM NaO treatment up-regulated gene expression of IL-8 and RANTES and secretion of IL-1β. Notably, when 1 mM NaO-treated bMECs were challenged with S. aureus , the gene expression of IL-8 and IL-10 increased, while IL-1β secretion was reduced. In conclusion, our results showed that α5β1 integrin, TLR2 and CD36 are involved in 0.25 mM NaO-increased S. aureus internalization in bMECs. In addition, 1 mM NaO activates bMECs via the TLR2 signaling pathways (p38, JNK, and ERK1/2), which improves IIR before S. aureus invasion. Additionally

Structural Change in Microbiota by a Probiotic Cocktail Enhances the Gut Barrier and Reduces Cancer via TLR2 Signaling in a Rat Model of Colon Cancer.

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Kuugbee, Eugene Dogkotenge; Shang, Xueqi; Gamallat, Yaser; Bamba, Djibril; Awadasseid, Annoor; Suliman, Mohammed Ahmed; Zang, Shizhu; Ma, Yufang; Chiwala, Gift; Xin, Yi; Shang, Dong

2016-10-01

Structural change in the gut microbiota is implicated in cancer. The beneficial modulation of the microbiota composition with probiotics and prebiotics prevents diseases. We investigated the effect of oligofructose-maltodextrin-enriched Lactobacillus acidophilus, Bifidobacteria bifidum, and Bifidobacteria infantum (LBB), on the gut microbiota composition and progression of colorectal cancer. Sprague Dawley rats were acclimatized, given ampicillin (75 mg/kg), and treated as follows; GCO: normal control; GPR: LBB only; GPC: LBB+ 1,2-dimethylhydrazine dihydrochloride (DMH); and GCA: DMH only (cancer control). 16S V4 Pyrosequencing for gut microbiota analysis, tumor studies, and the expression of MUC2, ZO-1, occludin, TLR2, TLR4, caspase 3, COX-2, and β-catenin were conducted at the end of experiment. Probiotic LBB treatment altered the gut microbiota. The relative abundance of genera Pseudomonas, Congregibacter, Clostridium, Candidactus spp., Phaeobacter, Escherichia, Helicobacter, and HTCC was decreased (P cancer control. The altered gut microbiota was associated with decreased tumor incidence (80 % in GPC vs. 100 % in GCA, P = 0.0001), tumor volume (GPC 84.23 (42.75-188.4) mm(3) vs. GCA 243 (175.5-344.5) mm(3), P cancer control GCA (P colon cancer development by decreasing tumor incidence, multiplicity/count, and volume via enhanced TLR2-improved gut mucosa epithelial barrier integrity and suppression of apoptosis and inflammation.

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

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Landry Blanc

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

The role of Ca(2+) mediated signaling pathways on the effect of taurine against Streptococcus uberis infection.

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Dai, Bin; Zhang, Jinqiu; Liu, Ming; Lu, Jinye; Zhang, Yuanshu; Xu, Yuanyuan; Miao, Jinfeng; Yin, Yulong

2016-08-30

To provide insight into the mechanisms of taurine attenuation of pro-inflammatory response in mouse mammary epithelial cell line (EpH4-Ev, purchased by ATCC, USA) after Streptococcus uberis (S. uberis, 0140J) challenge, we infected MECs with S. uberis (2.5×10(7)cfumL(-1), MOI=10) for 3h and quantified changes in TLR-2 and calcium (Ca(2+)) mediated signaling pathways. The results indicate that S. uberis infection significantly increases the expression of TLR-2, intracellular Ca(2+) levels, PLC-γ1 and PKC-α, the activities of transcription factors NF-κB and NFAT, and related cytokines (TNF-α, IL-1β, IL-6, G-CSF, IL-2, KC, IL-15, FasL, MCP-1, and LIX) in culture supernatants. Taurine administration downregulated all these indices, the activities of NF-κB and NFAT. Cytokine secretions were similar using special PKC inhibitor Go 6983 and NFAT inhibitor VIVIT. Our data indicate that S. uberis infection induces pro-inflammatory response of MECs through a TLR-2 mediated signaling pathway. In addition, taurine can prevent MEC damage by affecting both PLC-γ1-Ca(2+)-PKC-α-NF-κB and PLC-γ1-Ca(2+)-NFATs signaling pathways. This is the first report to demonstrate the mechanisms of taurine attenuated pro-inflammatory response in MECs after S. uberis challenge. Copyright © 2016 Elsevier B.V. All rights reserved.

Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma.

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Li, Zhigang; Fan, Erica K; Liu, Jinghua; Scott, Melanie J; Li, Yuehua; Li, Song; Xie, Wen; Billiar, Timothy R; Wilson, Mark A; Jiang, Yong; Wang, Ping; Fan, Jie

2017-05-11

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mφ) direct trauma-induced inflammation, and Mφ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mφ and the subsequent regulation of Mφ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mφ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mφ. However, autophagy activation also suppressed Mφ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mφ homeostasis in response to trauma.

Downregulation of toll-like receptor-mediated signalling pathways in oral lichen planus.

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Sinon, Suraya H; Rich, Alison M; Parachuru, Venkata P B; Firth, Fiona A; Milne, Trudy; Seymour, Gregory J

2016-01-01

The objective of this study was to investigate the expression of Toll-like receptors (TLR) and TLR-associated signalling pathway genes in oral lichen planus (OLP). Initially, immunohistochemistry was used to determine TLR expression in 12 formalin-fixed archival OLP tissues with 12 non-specifically inflamed oral tissues as controls. RNA was isolated from further fresh samples of OLP and non-specifically inflamed oral tissue controls (n = 6 for both groups) and used in qRT(2)-PCR focused arrays to determine the expression of TLRs and associated signalling pathway genes. Genes with a statistical significance of ±two-fold regulation (FR) and a P-value < 0.05 were considered as significantly regulated. Significantly more TLR4(+) cells were present in the inflammatory infiltrate in OLP compared with the control tissues (P < 0.05). There was no statistically significant difference in the numbers of TLR2(+) and TLR8(+) cells between the groups. TLR3 was significantly downregulated in OLP (P < 0.01). TLR8 was upregulated in OLP, but the difference between the groups was not statistically significant. The TLR-mediated signalling-associated protein genes MyD88 and TIRAP were significantly downregulated (P < 0.01 and P < 0.05), as were IRAK1 (P < 0.05), MAPK8 (P < 0.01), MAP3K1 (P < 0.05), MAP4K4 (P < 0.05), REL (P < 0.01) and RELA (P < 0.01). Stress proteins HMGB1 and the heat shock protein D1 were significantly downregulated in OLP (P < 0.01). These findings suggest a downregulation of TLR-mediated signalling pathways in OLP lesions. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

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Eric D. Abston

2012-01-01

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

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

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Jacco C Karper

Full Text Available BACKGROUND: RP105 (CD180 is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/- mice resulting in a higher proliferation rate. In RP105(-/- mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2 vs.1947 ± 278 µm(2,p = 0.0014. Local LPS application augmented neointima formation in both groups, but in RP105(-/- mice this effect was more pronounced (10316±1243 µm(2 vs.4208 ± 555 µm(2,p = 0.0002, suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2,p<0.05, whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.

Inflammation induced mTORC2-Akt-mTORC1 signaling promotes macrophage foam cell formation.

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Banerjee, Dipanjan; Sinha, Archana; Saikia, Sudeshna; Gogoi, Bhaskarjyoti; Rathore, Arvind K; Das, Anindhya Sundar; Pal, Durba; Buragohain, Alak K; Dasgupta, Suman

2018-06-05

The transformation of macrophages into lipid loaded foam cells is a critical and early event in the pathogenesis of atherosclerosis. Several recent reports highlighted that induction of TLR4 signaling promotes macrophage foam cell formation; however, the underlying molecular mechanisms have not been clearly elucidated. Here, we found that the TLR4 mediated inflammatory signaling communicated with mTORC2-Akt-mTORC1 metabolic cascade in macrophage and thereby promoting lipid uptake and foam cell formation. Mechanistically, LPS treatment markedly upregulates TLR4 mediated inflammatory pathway which by activating mTORC2 induces Akt phosphorylation at serine 473 and that aggravate mTORC1 dependent scavenger receptors expression and consequent lipid accumulation in THP-1 macrophages. Inhibition of mTORC2 either by silencing Rictor expression or inhibiting its association with mTOR notably prevents LPS induced Akt activation, scavenger receptors expression and macrophage lipid accumulation. Although suppression of mTORC1 expression by genetic knockdown of Raptor did not produce any significant change in Akt S473 phosphorylation, however, incubation with Akt activator in Rictor silenced cells failed to promote scavenger receptors expression and macrophage foam cell formation. Thus, present research explored the signaling pathway involved in inflammation induced macrophage foam cells formation and therefore, targeting this pathway might be useful for preventing macrophage foam cell formation. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

PAFR activation of NF-?B p65 or p105 precursor dictates pro- and anti-inflammatory responses during TLR activation in murine macrophages

OpenAIRE

Ishizuka, Edson K.; Filgueiras, Luciano Ribeiro; Rios, Francisco J.; Serezani, Carlos H.; Jancar, Sonia

2016-01-01

Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammat...

Decreased Secondary Lesion Growth and Attenuated Immune Response after Traumatic Brain Injury in Tlr2/4−/− Mice

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Sandro M. Krieg

2017-08-01

Full Text Available Danger-associated molecular patterns are released by damaged cells and trigger neuroinflammation through activation of non-specific pattern recognition receptors, e.g., toll-like receptors (TLRs. Since the role of TLR2 and 4 after traumatic brain injury (TBI is still unclear, we examined the outcome and the expression of pro-inflammatory mediators after experimental TBI in Tlr2/4−/− and wild-type (WT mice. Tlr2/4−/− and WT mice were subjected to controlled cortical injury and contusion volume and brain edema formation were assessed 24 h thereafter. Expression of inflammatory markers in brain tissue was measured by quantitative PCR 15 min, 3 h, 6 h, 12 h, and 24 h after controlled cortical impact (CCI. Contusion volume was significantly attenuated in Tlr2/4−/− mice (29.7 ± 0.7 mm3 as compared to 33.5 ± 0.8 mm3 in WT; p < 0.05 after CCI while brain edema was not affected. Only interleukin (IL-1β gene expression was increased after CCI in the Tlr2/4−/− relative to WT mice. Inducible nitric oxide synthetase, TNF, IL-6, and COX-2 were similar in injured WT and Tlr2/4−/− mice, while the increase in high-mobility group box 1 was attenuated at 6 h. TLR2 and 4 are consequently shown to potentially promote secondary brain injury after experimental CCI via neuroinflammation and may therefore represent a novel therapeutic target for the treatment of TBI.

Novel glycolipid TLR2 ligands of the type Pam2Cys-α-Gal: synthesis and biological properties.

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Thomann, Jean-Sébastien; Monneaux, Fanny; Creusat, Gaëlle; Spanedda, Maria Vittoria; Heurtault, Béatrice; Habermacher, Chloé; Schuber, Francis; Bourel-Bonnet, Line; Frisch, Benoît

2012-05-01

A more complete understanding of the mechanism of action of TLR agonists has fueled the investigation of new synthetic immunoadjuvants. In this context, we designed and synthesized glycolipids of the type Pam(2)Cys-α-Galactose as novel immunoadjuvants. Their synthesis required modifying a hydrophobic tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety, i.e. the minimal structure required for TLR2 agonist activity, by addition of a hydrophilic head, either an α-Galactosylpyranose or an α-Galactosylfuranose to gain respectively Pam(2)CGalp and Pam(2)CGalf. While preparing a carbohydrate building block, an unexpected stereoselectivity was observed during a halide ion-catalytic process on a protected galactofuranose: the alpha anomer was obtained with surprisingly high selectivity (α/β ratio>9) and with good isolated yield (51%). The TLR2 binding properties of Pam(2)CGalp and Pam(2)CGalf were then fully evaluated. Their efficiency in triggering the proliferation of BALB/c mouse splenocytes was also compared to that of Pam(2)CAG and Pam(3)CAG, two well-established ligands of TLRs. Moreover, the maturation state of murine dendritic cells previously incubated with either Pam(2)CGalp or Pam(2)CGalf was monitored by flow cytometry and compared to that induced by lipopolysaccharide. Pam(2)CGalp and Pam(2)CGalf were found to be equivalent TLR2 agonists, and induced splenocyte proliferation and DC maturation. With very similar activity, Pam(2)CGalp and Pam(2)CGalf were also 10-fold to 100-fold better than Pam(2)CAG and Pam(3)CAG at inducing B cell proliferation. This represents the first time a glucidic head has been added to the tBoc-[2,3-bispalmitoyloxy-(2R)-propyl]-R-cysteinyl moiety whilst maintaining the immunomodulating activity. This should greatly enrich the data available on Pam(2)C structure/activity relationships. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

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.

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

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Triantafilou, Martha; De Glanville, Benjamin; Aboklaish, Ali F; Spiller, O Brad; Kotecha, Sailesh; Triantafilou, Kathy

2013-01-01

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

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

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Martha Triantafilou

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

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...... 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....../-651 promoter polymorphisms are associated with atopy prevalence among young adults exposed to farm environments. Udgivelsesdato: 2007-Nov...

Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways.

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Kang, Hyeon Hui; Kim, In Kyoung; Lee, Hye In; Joo, Hyonsoo; Lim, Jeong Uk; Lee, Jongmin; Lee, Sang Haak; Moon, Hwa Sik

2017-08-19

Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-β (TRIF), I-κB, NF-κB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, α1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-κB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-κB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

TLR5 signaling, commensal microbiota and systemic tumor promoting inflammation: the three parcae of malignant progression.

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Rutkowski, Melanie R; Conejo-Garcia, Jose R

2015-08-01

We have reported that TLR5-mediated recognition of commensal microbiota modulates systemic tumor-promoting inflammation and malignant progression of tumors at distal locations. Approximately 7-10% of the general population harbors a deleterious single nucleotide polymorphism in TLR5, implicating a novel role for genetic variation during the initiation and progression of cancer.

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

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Diego C Reino

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

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

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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

Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro

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Zhengtu Li

2017-01-01

Full Text Available Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2 and avian influenza viruses (H6N2 and H9N2 in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6 and chemokines (IP-10, MIG, and CCL-5 stimulated by A/PR/8/34 (H1N1 at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL; however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

Ursolic acid isolated from Uncaria rhynchophylla activates human dendritic cells via TLR2 and/or TLR4 and induces the production of IFN-gamma by CD4+ naïve T cells.

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Jung, Tae-Young; Pham, Thanh Nhan Nguyen; Umeyama, Akemi; Shoji, Noboru; Hashimoto, Toshihiro; Lee, Je-Jung; Takei, Masao

2010-09-25

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-gamma), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy. 2010 Elsevier B.V. All rights reserved.

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

Role of ESAT-6 in renal injury by regulating microRNA-155 expression via TLR4/MyD88 signaling pathway in mice with Mycobacterium tuberculosis infection.

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Zhou, Zhong-Qi; Wang, Zhi-Kui; Zhang, Lei; Ren, Yue-Qin; Ma, Zhong-Wei; Zhao, Nan; Sun, Fu-Yun

2017-08-31

The study aims to investigate the underlying mechanism involved in the early secretory antigenic target-6 (ESAT-6) in renal injury through regulation of the expression of miR-155 through the oll-like receptor (TLR)-4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway in Mycobacterium tuberculosis (MTB)-infected mice. Sixty C57BL/6 mice with MTB-induced renal injury were randomly assigned into control, MTB, mimic, inhibitor, inhibitor + ESAT6, and inhibitor + ESAT6 + TAK242 groups. Body weight, the ratio of kidney weight to body weight (Kw/Bw), blood urea nitrogen (BUN), and serum creatinine (Scr) of mice were measured. Flow cytometry was used to detect renal activation in mice. Expressions of miR-155 and ESAT6 were detected by quantitative real-time PCR (qRT-PCR), and Western blotting was used to examine the expressions of ESAT6, TLR4, and MyD88. Expressions of tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), and interferon-γ (IFN-γ) were measured by qRT-PCR and ELISA. Compared with the control group, the BUN and Scr levels as well as the expression levels of miR-155 , TLR4, MyD88, TNF-α, IL-17, and IFN-γ increased, while Kw/Bw decreased in the MTB and mimic groups. In comparison with the MTB group, the above indexes except Kw/Bw were elevated in the mimic group, but were reduced in the inhibitor group, while the Kw/Bw dropped in the mimic group but increased in the inhibitor group. Compared with the inhibitor group, the Kw/Bw decreased while the rest of the indexes increased in the inhibitor + ESAT6 group. ESAT6 may induce renal injury by promoting miR-155 expression through the TLR-4/MyD88 signaling pathway in MTB-infected mice. © 2017 The Author(s).

Dexmedetomidine reduces ventilator-induced lung injury (VILI by inhibiting Toll-like receptor 4 (TLR4/nuclear factor (NF-κB signaling pathway

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Hongli Chen

2018-02-01

Full Text Available Mechanical ventilation (MV may lead to ventilator-induced lung injury (VILI. Previous research has shown that dexmedetomidine attenuates pulmonary inflammation caused by MV, but the underlying mechanisms remain unclear. Our study aims to test whether dexmedetomidine has a protective effect against VILI and to explore the possible molecular mechanisms using the rat model. Thirty adult male Wistar rats weighing 200-250 g were randomly assigned to 5 groups (n = 6: control, low tidal volume MV (LMV, high tidal volume (HVT MV (HMV, HVT MV + dexmedetomidine (DEX, HVT MV + dexmedetomidine + yohimbine (DEX+Y. Rats were euthanized after being ventilated for 4 hours. Pathological changes, lung wet/dry (W/D weight ratio, lung myeloperoxidase (MPO activity, levels of inflammatory cytokines (i.e., interleukin [IL]-1β, tumor necrosis factor alpha [TNF-α], and IL-6 in the bronchoalveolar lavage fluid (BALF and lung tissues, expression of Toll-like receptor 4 (TLR4 and nuclear factor (NF-κB, and activation of NF-κB in lung tissues were measured. Compared with HMV, DEX group showed fewer pathological changes, lower W/D ratios and decreased MPO activity of the lung tissues and lower concentrations of the inflammatory cytokines in the BALF and lung tissues. Dexmedetomidine significantly inhibited the expression of TLR4 and NF-κB and activation of NF-κB. Yohimbine partly alleviated the effects of dexmedetomidine. Dexmedetomidine reduced the inflammatory response to HVT-MV and had a protective effect against VILI, with the inhibition of the TLR4/NF-κB signaling pathway, at least partly via α2-adrenoceptors.

TRAM is involved in IL-18 signaling and functions as a sorting adaptor for MyD88.

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Hidenori Ohnishi

Full Text Available MyD88, a Toll/interleukin-1 receptor homology (TIR domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.

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

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

2012-01-01

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

Pepsin Digest of Wheat Gliadin Fraction Increases Production of IL-1β via TLR4/MyD88/TRIF/MAPK/NF-κB Signaling Pathway and an NLRP3 Inflammasome Activation

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Palová-Jelínková, Lenka; Dáňová, Klára; Drašarová, Hana; Dvořák, Miloš; Funda, David P.; Fundová, Petra; Kotrbová-Kozak, Anna; Černá, Marie; Kamanová, Jana; Martin, Stefan F.; Freudenberg, Marina; Tučková, Ludmila

2013-01-01

Celiac disease (CD) is a gluten-responsive, chronic inflammatory enteropathy. IL-1 cytokine family members IL-1β and IL-18 have been associated with the inflammatory conditions in CD patients. However, the mechanisms of IL-1 molecule activation in CD have not yet been elucidated. We show in this study that peripheral blood mononuclear cells (PBMC) and monocytes from celiac patients responded to pepsin digest of wheat gliadin fraction (PDWGF) by a robust secretion of IL-1β and IL-1α and a slightly elevated production of IL-18. The analysis of the upstream mechanisms underlying PDWGF-induced IL-1β production in celiac PBMC show that PDWGF-induced de novo pro-IL-1β synthesis, followed by a caspase-1 dependent processing and the secretion of mature IL-1β. This was promoted by K+ efflux and oxidative stress, and was independent of P2X7 receptor signaling. The PDWGF-induced IL-1β release was dependent on Nod-like receptor family containing pyrin domain 3 (NLRP3) and apoptosis-associated speck like protein (ASC) as shown by stimulation of bone marrow derived dendritic cells (BMDC) from NLRP3−/− and ASC−/− knockout mice. Moreover, treatment of human PBMC as well as MyD88−/− and Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)−/− BMDC illustrated that prior to the activation of caspase-1, the PDWGF-triggered signal constitutes the activation of the MyD88/TRIF/MAPK/NF-κB pathway. Moreover, our results indicate that the combined action of TLR2 and TLR4 may be required for optimal induction of IL-1β in response to PDWGF. Thus, innate immune pathways, such as TLR2/4/MyD88/TRIF/MAPK/NF-κB and an NLRP3 inflammasome activation are involved in wheat proteins signaling and may play an important role in the pathogenesis of CD. PMID:23658628

PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

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S. V. Zubova

2012-01-01

Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

Tissue Factor and Toll Like Receptor (TLR)4 in Hyperglycemia-Hyperinsulinemia: Effects in Healthy Subjects, and Type 1 and Type 2 Diabetes Mellitus

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Singh, Anamika; Boden, Guenther; Rao, A. Koneti

2015-01-01

SUMMARY Background Diabetes mellitus (DM) patients have increased cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycemia (HG), hyperinsulinemia (HI) and combined HG+HI (induced using 24 hr infusion clamps) increases TF-PCA in healthy and T2DM subjects, but not in T1DM subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. Objectives and Methods We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24 hr HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. Results In vivo, in healthy subjects, 24 hr HG + HI infusion increased TLR4 6-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hr). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. Conclusions This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects. PMID:25653143

Contrasting patterns of diversity and population differentiation at the innate immunity gene toll-like receptor 2 (TLR2) in two sympatric rodent species.

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Tschirren, Barbara; Andersson, Martin; Scherman, Kristin; Westerdahl, Helena; Råberg, Lars

2012-03-01

Comparing patterns of diversity and divergence between populations at immune genes and neutral markers can give insights into the nature and geographic scale of parasite-mediated selection. To date, studies investigating such patterns of selection in vertebrates have primarily focused on the acquired branch of the immune system, whereas it remains largely unknown how parasite-mediated selection shapes innate immune genes both within and across vertebrate populations. Here, we present a study on the diversity and population differentiation at the innate immune gene Toll-like receptor 2 (TLR2) across nine populations of yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) in southern Sweden. In yellow-necked mice, TLR2 diversity was very low, as was TLR2 population differentiation compared to neutral loci. In contrast, several TLR2 haplotypes co-occurred at intermediate frequencies within and across bank vole populations, and pronounced isolation by distance between populations was observed. The diversity and differentiation at neutral loci was similar in the two species. These results indicate that parasite-mediated selection has been acting in dramatically different ways on a given immune gene in ecologically similar and sympatric species. Furthermore, the finding of TLR2 population differentiation at a small geographical scale in bank voles highlights that vertebrate innate immune defense may be evolutionarily more dynamic than has previously been appreciated. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

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...

Serum Amyloid A Induces Toll-Like Receptor 2-Dependent Inflammatory Cytokine Expression and Atrophy in C2C12 Skeletal Muscle Myotubes.

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Passey, Samantha L; Bozinovski, Steven; Vlahos, Ross; Anderson, Gary P; Hansen, Michelle J

2016-01-01

Skeletal muscle wasting is an important comorbidity of Chronic Obstructive Pulmonary Disease (COPD) and is strongly correlated with morbidity and mortality. Patients who experience frequent acute exacerbations of COPD (AECOPD) have more severe muscle wasting and reduced recovery of muscle mass and function after each exacerbation. Serum levels of the pro-inflammatory acute phase protein Serum Amyloid A (SAA) can rise more than 1000-fold in AECOPD and are predictively correlated with exacerbation severity. The direct effects of SAA on skeletal muscle are poorly understood. Here we have examined SAA effects on pro-inflammatory cachectic cytokine expression (IL-6 and TNFα) and atrophy in C2C12 myotubes. SAA increased IL-6 (31-fold) and TNFα (6.5-fold) mRNA levels compared to control untreated cells after 3h of SAA treatment, and increased secreted IL-6 protein at 24h. OxPAPC, a dual TLR2 and TLR4 inhibitor, reduced the response to SAA by approximately 84% compared to SAA alone, and the TLR2 neutralising antibody T2.5 abolished SAA-induced expression of IL-6, indicating that SAA signalling in C2C12 myotubes is primarily via TLR2. SAA also reduced myotube width by 10-13% and induced a 2.5-fold increase in the expression of the muscle atrophy gene Atrogin-1, suggesting direct effects of SAA on muscle wasting. Blocking of TLR2 inhibited the SAA-induced decrease in myotube width and Atrogin-1 gene expression, indicating that SAA induces atrophy through TLR2. These data demonstrate that SAA stimulates a robust pro-inflammatory response in skeletal muscle myotubes via the TLR2-dependent release of IL-6 and TNFα. Furthermore, the observed atrophy effects indicate that SAA could also be directly contributing to the wasting and poor recovery of muscle mass. Therapeutic strategies targeting this SAA-TLR2 axis may therefore ameliorate muscle wasting in AECOPD and a range of other inflammatory conditions associated with loss of muscle mass.

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

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

2008-01-01

the TLR7 and TLR8 genes. METHODS: We investigated the involvement of TLR7 and TLR8 in the aetiology of asthma and related disorders by a family based association analysis of two independently ascertained family samples comprising 540 and 424 individuals from 135 and 100 families, respectively. Ten...

Upregulated TLR3 Promotes Neuropathic Pain by Regulating Autophagy in Rat With L5 Spinal Nerve Ligation Model.

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Chen, Weijia; Lu, Zhijun

2017-02-01

Microglia, rapidly activated following peripheral nerve injury (PNI), accumulate within the spinal cord and adopt inflammation that contributes to development and maintenance of neuropathic pain. Microglia express functional Toll-like receptors (TLRs), which play pivotal roles in regulating inflammatory processes. However, little is known about the role of TLR3 in regulating neuropathic pain after PNI. Here TLR3 expression and autophagy activation was assayed in dorsal root ganglions and in microglia following PNI by using realtime PCR, western blot and immunohistochemistry. The role of TLR3/autophagy signaling in regulating tactile allodynia was evaluated by assaying paw mechanical withdrawal threshold and cold allodynia after intrathecal administration of Poly (I:C) and 3-methyladenine (3-MA). We found that L5 spinal nerve ligation (SNL) induces the expression of TLR3 in dorsal root ganglions and in primary rat microglia at the mRNA and protein level. Meanwhile, L5 SNL results in an increased activation of autophagy, which contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of Poly (I:C), a TLR3 agonist, significantly increases the activation of microglial autophagy, whereas TLR3 knockdown markedly inhibits L5 SNL-induced microglial autophagy. Poly (I:C) treatment promotes the expression of proinflammatory mediators, whereas 3-MA (a specific inhibitor of autophagy) suppresses Poly (I:C)-induced secretion of proinflammatory cytokines. Autophagy inhibition further inhibits TLR3-mediated mechanical and cold hypersensitivity following SNL. These results suggest that inhibition of TLR3/autophagy signaling contributes to alleviate neurophathic pain triggered by SNL.

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

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Pan, Hong; Wu, Xinyi

2012-01-01

Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion

The influence of TLR4 agonist lipopolysaccharides on hepatocellular carcinoma cells and the feasibility of its application in treating liver cancer.

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Gu, Junsheng; Sun, Ranran; Shen, Shen; Yu, Zujiang

2015-01-01

This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on liver cancer cell and the feasibility to perform liver cancer adjuvant therapy. Human liver cancer cell lines HepG2, H7402, and PLC/PRF/5 were taken as models, and the expression of TLRs mRNA was detected by real time-polymerase chain reaction method semiquantitatively. WST-1 method was used to detect the influence of LPS on the proliferation ability of liver cancer cells; propidium iodide (PI) single staining and Annexin V/PI double staining were used to test the influence of LPS on the cell cycle and apoptosis, respectively, on human liver cancer cell line H7402. Fluorescent quantitative polymerase chain reaction and Western blot method were used to determine the change of expression of Cyclin D1. The results demonstrated that most TLRs were expressed in liver cancer cells; stimulating TLR4 by LPS could upregulate TLR4 mRNA and the protein level, activate NF-κB signaling pathway downstream of TLR4, and mediate the generation of inflammatory factors IL-6, IL-8, and TNF-α; LPS was found to be able to strengthen the proliferation ability of liver cancer cells, especially H7402 cells; the expression of Cyclin D1 rose and H7402 cells were promoted to transit from G1 stage to S stage under the stimulation of LPS, but cell apoptosis was not affected. It was also found that LPS was able to activate signal transducer and activator of transcription -3 (STAT3) signaling pathway in H7402 cells and meanwhile significantly increase the initiation activity of STAT3; proliferation promoting effect of LPS to liver cancer cells remarkably lowered once STAT3 was blocked or inhibited. Thus, TLR4 agonist LPS is proved to be able to induce liver cancer cells to express inflammation factors and mediate liver cancer cell proliferation and generation of multidrug resistance by activating the cyclooxygenase-2/prostaglandin signal axis as well as the STAT3 pathway.

[Effect of resveratrol on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment].

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Lv, Jia-Shu; Jiang, Xue-Wei; Zhang, Yan; Zhen, Lei

2017-02-01

Through a study of the molecular mechanism of the effect of resveratrol(RSV) on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment, the therapeutic effect and molecular mechanism of resveratrol on periodontitis in patients with diabetes mellitus was investigated. Gingival epithelial cells were cultured in vitro; according to the way of action, the cultured cells were divided into control group, high glucose group(HG) and HG+RSV group. The mRNA expression of TLR4 was detected by PCR; The third generation of gingival epithelial cells were pre-treated with or without RSV for 24 h under high glucose conditions, and subsequently treated with LPS at 100 ng/mL for 2 h. ELISA was used to detect the secretion of IL-1 beta, IL-6, IL-8 and TNF- alpha; the activation of TLR4 downstream signaling molecules NF-κB p65, p38 MAPK, and STAT3 was determined by Western blot. SPSS17.0 software package was used for statistical analysis. RSV could reverse the increase of TLR4 level in gingival epithelial cells in high glucose medium.LPS markedly increased the expression and secretion of IL-1β, IL-6, IL-8, and TNF-α in GECs cultured in high glucose medium, which was partly blocked in the presence of RSV. Furthermore, Western blot results showed that RSV significantly suppressed the phosphorylation of TLR4 downstream factors NF-κB p65, p38MAPK, and STAT3. RSV reduces inflammatory cytokine secretion in gingival epithelial cells, through negative regulation of TLR4 signaling pathway.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway

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Weijie Liang

2017-02-01

Full Text Available Background/Aims: Hyperglycemia activates multiple signaling molecules, including reactive oxygen species (ROS, toll-like receptor 4 (TLR4, receptor-interacting protein 3 (RIP3, a kinase promoting necroptosis, which mediate hyperglycemia-induced cardiac injury. This study explored whether inhibition of ROS-TLR4-necroptosis pathway contributed to the protection of ATP-sensitive K+ (KATP channel opening against high glucose-induced cardiac injury and inflammation. Methods: H9c2 cardiac cells were treated with 35 mM glucose (HG to establish a model of HG-induced insults. The expression of RIP3 and TLR4 were tested by western blot. Generation of ROS, cell viability, mitochondrial membrane potential (MMP and secretion of inflammatory cytokines were measured as injury indexes. Results: HG increased the expression of TLR4 and RIP3. Necrostatin-1 (Nec-1, an inhibitor of necroptosis or TAK-242 (an inhibitor of TLR4 co-treatment attenuated HG-induced up-regulation of RIP3. Diazoxide (DZ, a mitochondrial KATP channel opener or pinacidil (Pin, a non-selective KATP channel opener or N-acetyl-L-cysteine (NAC, a ROS scavenger pre-treatment blocked the up-regulation of TLR4 and RIP3. Furthermore, pre-treatment with DZ or Pin or NAC, or co-treatment with TAK-242 or Nec-1 attenuated HG-induced a decrease in cell viability, and increases in ROS generation, MMP loss and inflammatory cytokines secretion. However, 5-hydroxy decanoic acid (5-HD, a mitochondrial KATP channel blocker or glibenclamide (Gli, a non-selective KATP channel blocker pre-treatment did not aggravate HG-induced injury and inflammation. Conclusion: KATP channel opening protects H9c2 cells against HG-induced injury and inflammation by inhibiting ROS-TLR4-necroptosis pathway.

Pimecrolimus enhances TLR2/6-induced expression of antimicrobial peptides in keratinocytes.

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Büchau, Amanda S; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Gallo, Richard L

2008-11-01

Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrolimus enhances distinct expression of cathelicidin, CD14, and human beta-defensin-2 and beta-defensin-3 in response to TLR2/6 ligands. Some of these responses were further enhanced by 1,25 vitamin D3. Pimecrolimus also increased the functional capacity of keratinocytes to inhibit growth of Staphylococcus aureus and decreased TLR2/6-induced expression of IL-10 and IL-1beta. Furthermore, pimecrolimus inhibited nuclear translocation of NFAT and NF-kappaB in keratinocytes. These observations uncover a previously unreported function for pimecrolimus in cutaneous innate host defense.

Syncytin-1, an endogenous retroviral protein, triggers the activation of CRP via TLR3 signal cascade in glial cells.

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Wang, Xiuling; Liu, Zhongchun; Wang, Peigang; Li, Shan; Zeng, Jie; Tu, Xiaoning; Yan, Qiujin; Xiao, Zheman; Pan, Mengxian; Zhu, Fan

2018-01-01

Schizophrenia is a devastating psychiatric disorder that impacts on social functioning and quality of life, and there is accumulating evidence that inflammation is a potential pathogenic mechanism of schizophrenia. However, the mechanism of inflammation possibly occurred in schizophrenia has not been well understood. The endogenous retroviral protein syncytin-1 and inflammatory marker CRP are both abnormally expressed in schizophrenia patients. CRP is one of the markers of bacterial infection generally. Less clear is whether virus or viral protein can trigger the activation of CRP. Here, we detected a robust increase of the levels of syncytin-1 and CRP in schizophrenia patients, and displayed a positive correlation and marked consistency between expressions of syncytin-1 and CRP in schizophrenia patients. Furthermore, overexpression of syncytin-1 significantly elevated the levels of CRP, TLR3, and IL-6 in both human microglia and astrocytes. TLR3 deficiency impaired the expressions of CRP and IL-6 induced by syncytin-1. Importantly, we observed a cellular co-localization and a direct interaction between syncytin-1 and TLR3. Additionally, knockdown of IL-6 inhibited the syncytin-1-induced CRP expression. Thus, the totality of these results showed that viral protein syncytin-1 could trigger the activation of CRP, which might explain the elevated CRP in sterile inflammation and exhibit a novel mechanism for regulation of inflammation by syncytin-1 in schizophrenia. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual Role of the Tyrosine Kinase Syk in Regulation of Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells.

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Besma Aouar

Full Text Available Crosslinking of regulatory immunoreceptors (RR, such as BDCA-2 (CD303 or ILT7 (CD85g, of plasmacytoid dendritic cells (pDCs efficiently suppresses production of type-I interferon (IFN-α/β and other cytokines in response to Toll-like receptor (TLR 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function-IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.

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

Effects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7 Pathway in Influenza Virus Infected Mouse Lungs in a Cold Environment.

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Qin, Hong-Qiong; Shi, Shan-Shan; Fu, Ying-Jie; Yan, Yu-Qi; Wu, Sha; Tang, Xiao-Long; Chen, Xiao-Yin; Hou, Guang-Hui; Jiang, Zhen-You

2018-01-01

We wished to investigate the effects of the traditional Chinese medicine Gui Zhi Ma Huang Ge Ban Tang on controlling influenza A virus (IAV) infection and improving inflammation in mouse lungs. Mice were maintained in normal and cold environments and infected with IAV by intranasal application, respectively. Real-time quantitative polymerase chain reaction was used to measure mRNA expression of TLR7, myeloid differentiation primary response 88 (MyD88), and nuclear factor-kappa B (NF- κ B)p65 in the TLR7 signaling pathway and virus replication in lungs. Western blotting was used to measure expression levels of TLR7, MyD88, and NF- κ B p65 proteins. Flow cytometry was used to detect the proportion of T-helper (Th)1/Th2 and Th17/T-regulatory (Treg) cells. Application of Gui Zhi Ma Huang Ge Ban Tang in influenza-infected mice in a cold environment showed (i) downregulation of TLR7, MyD88, and NF- κ Bp65; (ii) inhibition of transcriptional activities of promoters coding for TLR7, MyD88, and NF- κ Bp65; (iii) reduction in the proportion of Th1/Th2 and Th17/Treg cells. Gui Zhi Ma Huang Ge Ban Tang had a good therapeutic effect on mice infected with IAV, especially in the cold environment. It could reduce lung inflammation in mice significantly and elicit an anti-influenza effect by downregulating expression of the key factors in TLR7 signaling pathway.

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

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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.

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.

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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.