Inhibition of homodimerization of toll-like receptor 4 by 6-shogaol

Toll-like receptors (TLRs) play a critical role in sensing microbial components and inducing innate immune and inflammatory responses by recognizing invading microbial pathogens. Lipopolysaccharide-induced dimerization of TLR4 is required for the activation of downstream signaling pathways including nuclear factor-kappa B (NF-κB). Therefore, TLR4 dimerization may be an early regulatory event in activating ligand-induced signaling pathways and induction of subsequent immune responses. Here, we report biochemical evidence that 6-shogaol, the most bioactive component of ginger, inhibits lipopolysaccharide-induced dimerization of TLR4 resulting in the inhibition of NF-κB activation and the expression of cyclooxygenase-2. Furthermore, we demonstrate that 6-shogaol can directly inhibit TLR-mediated signaling pathways at the receptor level. These results suggest that 6-shogaol can modulate TLR-mediated inflammatory responses, which may influence the risk of chronic inflammatory diseases.     

Suppression of the TRIF-dependent signaling pathway of toll-like receptors by isoliquiritigenin in RAW264.7 macrophages

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens and initiating innate immune responses. The stimulation of TLRs by microbial components triggers the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. Isoliquiritigen in (ILG), an active ingredient of Licorice, has been used for centuries to treat many chronic diseases. ILG inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether ILG inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of ILG, we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by several agonists. ILG inhibited nuclear factor-κB and interferon regulatory factor 3 activation induced by lipopolysaccharide or polyinosinic-polycytidylic acid. ILG inhibited the lipopolysaccharide-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10, and regulated activation of normal T-cell expressed and secreted (RANTES). These results suggest that ILG can modulate TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.     

Acrolein with an alpha, beta-unsaturated carbonyl group inhibits LPS-induced homodimerization of toll-like receptor 4

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde present in a number of environmental sources, especially cigarette smoke. It reacts strongly with the thiol groups of cysteine residues by Michael addition and has been reported to inhibit nuclear factor-kappaB (NF-kappaB) activation by lipopolysaccharide (LPS). The mechanism by which it inhibits NF-kappaB is not clear. Toll-like receptors (TLRs) play a key role in sensing microbial components and inducing innate immune responses, and LPS-induced dimerization of TLR4 is required for activation of downstream signaling pathways. Thus, dimerization of TLR4 may be one of the first events involved in activating TLR4-mediated signaling pathways. Stimulation of TLR4 by LPS activates both myeloid differential factor 88 (MyD88)- and TIR domain-containing adapter inducing IFNbeta(TRIF)-dependent signaling pathways leading to activation of NF-kappaB and IFN-regulatory factor 3 (IRF3). Acrolein inhibited NF-kappaB and IRF3 activation by LPS, but it did not inhibit NF-kappaB or IRF3 activation by MyD88, inhibitor kappaB kinase (IKK)beta, TRIF, or TNF-receptor-associated factor family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1). Acrolein inhibited LPS-induced dimerization of TLR4, which resulted in the down-regulation of NF-kappaB and IRF3 activation. These results suggest that activation of TLRs and subsequent immune/inflammatory responses induced by endogenous molecules or chronic infection can be modulated by certain chemicals with a structural motif that enables Michael addition.     

Alteration of Toll-like receptor 4 activation by 4-hydroxy-2-nonenal mediated by the suppression of receptor homodimerization

Toll-like receptors (TLRs) detect invading microbial pathogens and initiate immune responses as part of host defense mechanisms. They also respond to host-derived substances released from injured cells and tissues to ensure wound healing and tissue homeostasis. Dysregulation of TLRs increases the risk of chronic inflammatory diseases and immune disorders. Inflammatory events are often accompanied by oxidative stress, which generates lipid peroxidation products such as 4-hydroxy-2-nonenal (4-HNE). Therefore, we investigated if 4-HNE affects TLR activation. We found that 4-HNE blocked LPS (a TLR4 agonist)-induced activation of NFκB and IRF3 as well as expression of IFNβ, IP-10, RANTES, and TNFα. To investigate the mechanism of inhibition by 4-HNE, we examined its effects on TLR4 dimerization, one of the initial steps in TLR4 activation. 4-HNE suppressed both ligand-induced and ligand-independent receptor dimerization. The thiol donors, DTT and NAC, prevented the inhibitory effects of 4-HNE on TLR4 dimerization, and LC–MS/MS analysis showed that 4-HNE formed adducts with cysteine residues of synthetic peptides derived from TLR4. These observations suggest that the reactivity of 4-HNE with sulfhydryl moieties is implicated in the inhibition of TLR4 activation. Furthermore, inhibition of TLR4 activation by 4-HNE resulted in down-regulation of the phagocytic activity of macrophages. Collectively, these results demonstrate that 4-HNE blocks TLR4-mediated macrophage activation, gene expression, and phagocytic functions, at least partly by suppressing receptor dimerization. They further suggest that 4-HNE influences innate immune responses at sites of infection and inflammation by inhibiting TLR4 activation.     

Comparative fine maps of bovine toll-like receptor 4 and toll-like receptor 2 regions

Abstract Toll-like receptors are cell-surface receptors that activate innate and adaptive immune responses. We have used a 5000-rad, whole-genome radiation hybrid panel to map Toll-like receptor 4 (TLR4) to the distal end of bovine Chromosome (Chr) 8, and Toll-like receptor 2 (TLR2) to the proximal end of bovine Chr 17. To facilitate comparative mapping and contig construction, we have also used 5000- and 12,000-rad, whole-genome radiation hybrid panels to produce fine maps of the regions surrounding these genes in cattle. These fine maps triple the number of available markers in the TLR4 region and more than double the number of available markers in the TLR2 region. Comparative analyses show gene order conservation between the bovine Chr 8 region and human Chr 9, and between the bovine Chr 17 region and human Chr 4. In addition, the bovine Chr 8 region refines an evolutionary chromosomal breakpoint from a 10-megabase region to a 2.5-megabase region, and the bovine Chr 17 map suggests a new evolutionary chromosomal breakpoint.     

Functional consequences of toll-like receptor 4 polymorphisms

Toll-like receptor 4 (TLR4) is an important pathogen recognition receptor that recognizes mainly lipopolysaccharide (LPS) of Gram-negative bacteria, but also structures from fungal and mycobacterial pathogens, as well as endogenous ligands. Two nonsynonymous polymorphisms of TLR4, Asp299Gly and Thr399Ile, have been suggested to alter the function of the receptor. Some, but not all, studies have proposed that these polymorphisms lead to reduced cytokine response and increased susceptibility to Gram-negative infections. In this review, we compare studies that assessed the effect of the Asp299Gly and Thr399Ile polymorphisms on susceptibility to Gram-negative infections and examine the phenotypic consequences of these polymorphisms. In addition, we review the geographical distribution of TLR4 polymorphisms and present a model for evolutionary pressures on the TLR4 genetic make-up.     

Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages

Thymosin beta-4 (Tβ4) is an actin-binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The mechanism of Tβ4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tβ4 which also participates in wound healing process. The objective of this study was to find how Tβ4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tβ4 was studied. A naturally transformed chicken macrophage cell line HTC was treated with different TLR agonists and the cellular Tβ4 changes was determined at 6 and 24 h after stimulations using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry. Real time PCR was used to determine changes in gene expression. The results showed that TLR agonists such as peptidoglycan (PGN) or lipopolysacharide (LPS) caused depletions in cellular Tβ4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1β, -6, and nitric oxide synthase genes at 6 h but failed to modulate Tβ4 gene at that time point indicating that the Tβ4 externalization was not associated with its production. To find whether Tβ4 externalization was associated with cell death, we measured the lactate dehydrogenase (LDH) activity of the conditioned media as an indicator of cell damage. The results showed that the TLR agonists which induced depletion of intracellular Tβ4 at 24 h also increased the LDH content of the conditioned media, suggesting that the Tβ4 in the extracellular media most likely originated from dying macrophages.     

Cytoplasmic domain-mediated dimerizations of toll-like receptor 4 observed by beta-lactamase enzyme fragment complementation

Toll-like receptors (TLRs) detect the presence of microbial challenge and initiate innate immune defensive responses. In this work we have explored the mechanism and role of TLR dimerization in signal transduction using the newly developed technique of beta-lactamase protein fragment complementation, among others. We observed that TLR4 interactions with itself, with MyD88, or with TLR2 were accurately reported by the enzyme complementation technique. That technique, as well as co-immunoprecipitation, transfection-initiated cell activation, and site-directed mutagenesis all suggest an important role for TLR intracellular domains in receptor dimerization. These findings broaden our understanding of TLR self-interactions as well as heterodimer formation.     

Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4

Cystic fibrosis is characterised in the lungs by high levels of neutrophil elastase (NE). NE induces interleukin-8 (IL-8) expression via an IL-1 receptor-associated kinase signalling pathway. Here, we show that these events involve the cell surface membrane bound toll-like receptor 4 (TLR4). We demonstrate that human embryonic kidney (HEK)293 cells transfected with a TLR4 cDNA (HEK-TLR4) express TLR4 mRNA and protein and induce IL-8 promoter activity in response to NE. Treatment of both HEK-TLR4 and human bronchial epithelial cells with NE decreases TLR4 protein expression. Furthermore, a TLR4 neutralising antibody abrogates NE-induced IL-8 production, and induces tolerance to a secondary lipopolysaccharide stimulus. These data implicate TLR4 in NE induced IL-8 expression in bronchial epithelium.     

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.     

miRNA-200b improves hepatic fibrosis induced by CCL4 by regulating toll-like receptor 4 in mice

To study the effect of miRNA-200b on hepatic fibrosis induced by CCl₄ in mice. The C59BL/6 mice were randomly divided into three groups (normal control [NC], CCLR model [Model], and CCl ₄ + miRNA-200b [miRNA]). The hepatic fibrosis was induced by CCl ₄ injected subcutaneously twice per week in Model and miRNA groups. After 6 weeks building model, the mice of miRNA group were injected the miRNA-200b from caudal vein twice per week. The mice of Model and miRNA groups were continuously fed for 3 weeks. The IL-1β, IL-6, and TNF-α concentrations of serum were measured by enzyme-linked immunosorbent assay. The hepatic tissues of difference groups were observed by hematoxylin and eosin (H&E) staining, sirius red staining, Masson staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay and measured toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) proteins expressions by western blot assay. The correlation between miRNA-200b and TLR4 were analyzed by dual luciferase target assay. Compared with NC group, the interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) concentrations of Model group were significantly upregulated (P < 0.05, respectively). With miRNA-200b overexpression, the IL-1β, IL-6, and TNF-α concentrations were significantly suppressed (P < 0.05, respectively). The pathologies were improved by H&E staining, sirius red staining, and Masson staining; meanwhile, the hepatic cell apoptosis rate was significantly suppressed (P < 0.05). The TLR4 and NF-κB protein expressions of miRNA group were significantly suppressed compared with the Model group (P < 0.05, respectively). By dual luciferase target assay, the TLR4 was a target gene of miRNA-200b. The miRNA-200b upregulation improved hepatic fibrosis induced by CCl ₄ via regulation of TLR4 in vivo.     

Characterization of the homodimerization interface and functional hotspots of the CXCR4 chemokine receptor

The recent crystallographic structures of the human chemokine CXC Receptor 4 (CXCR4) provide experimental evidence of a human G Protein-Coupled Receptor (GPCR) dimer in atomic detail. The CXCR4 homodimers reveal an unexpected dimerization mode involving transmembrane helices TM5 and TM6, which is examined here using all-atom molecular dynamics (MD) simulations in the physiological environment of a lipid bilayer. The bacteriophage T4 lysozyme (T4L), which was fused to the crystallized protein but absent in our simulations, is found to slightly affect the observed relative position of the protomers in the two dimers studied here, and consequently some rearrangements of the dimerization interface are proposed. In addition, the simulations provide further evidence about the role of the two stabilizing single point mutations introduced to crystallize the receptor. Finally, this work analyzes the structural and dynamic role of key residues involved both in ligand binding and in the infection process of HIV. In particular, the different side chain conformations of His113(3.39) are found to influence the dynamics of the surrounding functional hotspot region being evaluated both in the presence and in the absence of the co-crystallized ligand IT1t. The analysis reported here adds valuable knowledge for future structure-based drug design (SBDD) efforts on this pharmacological target.     

Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.     

Polymorphisms of toll-like receptor 2 and 4 genes in Chagas disease

The aim of this study was to test the possible implication of toll-like receptor 2 (TLR2) and TLR4 gene polymorphisms in determining the susceptibility to Chagas' disease. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 475 individuals from Colombia, 143 seropositive with chagasic cardiomyopathy, 132 seropositive asymptomatic and 200 seronegative. The TLR2 arginine to glutamine substitution at residue 753(Arg753Gln) polymorphism was absent in the groups analyzed. The TLR4 Asp299Gly and Thr399Ile polymorphisms are in linkage disequilibrium and we observed a very low frequency of these polymorphisms in our study population (2.6% and 1.8% respectively). The overall TLR2 and TLR4 alleles and genotype distribution in seronegative and seropositive were not significantly different. We compared the frequencies between asymptomatic patients and those with chagasic cardiomyopathy and we did not observe any significant differences in the distribution of alleles or genotypes. In summary, this study corroborates the low frequency of TLR2 and TLR4 polymorphisms observed in other populations and suggest that these do not play an important role in Chagas' disease. The validation of these findings in independent cohorts is needed to firmly establish a role for TLR2 and TLR4 variants in Chagas' disease.     

Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia

Chronic infection and inflammation are among the most important factors contributing to cancer development and growth. Toll-like receptors (TLRs) are important families of pattern recognition receptors, which recognize conserved components of microbes and trigger the immune response against invading microorganisms. TLR4 is the signaling receptor for lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Recent studies demonstrate that TLRs are expressed in some tumor cells, and that the expression of TLRs in these cells is associated with tumorigenesis. Cervical intraepithelial neoplasia (CIN) is a key stage in the development of cervical cancer and human papillomavirus (HPV) infection is an essential factor in cervical carcinogenesis. As the cervix is in constant contact with bacteria, especially Gram-negative bacteria, we hypothesize that TLR4-mediated bacterial stimulation may be involved in the tumorigenesis of cervical cancer. In the present study, the expression and distribution of TLR4 in CIN and cervical squamous carcinoma were investigated by immunohistochemistry. To our surprise, we observed a decrease in the expression of TLR4 during the progression of cervical neoplasia and this down-regulation of TLR4 appeared to be associated with the expression of \textP ^{1 6\textINK4A} , {\text{P}}^{{ 1 6^{\text{INK4A}} }} , which is a crucial marker of HPV integration into host cells. These data offer further insight regarding the association of HPV infection and TLR signaling during the carcinogenesis of cervical cancer.     

Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14(+) cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.     

Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease.

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. Here, we show that a spontaneous loss-of-function mutation in the Tlr4 gene strongly inhibits microglial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFalpha and nitric oxide. Treatment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neurotoxicity. In addition, stimulation experiments in transfected HEK293 cells allowed to define a tri-molecular receptor complex consisting of TLR4, MD-2 and CD14 necessary for full cellular activation by aggregated amyloid peptide. A clinical relevance of these findings is supported by a marked upregulation of Tlr4 mRNA in APP transgenic mice and by an increased expression of TLR4 in Alzheimer's disease brain tissue associated with amyloid plaque deposition. Together, these observations provide the first evidence for a role of the key innate immune receptor, TLR4, in neuroinflammation in Alzheimer's disease.     

Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.