Inhibition of TLR activation and up-regulation of IL-1R-associated kinase-M expression by exogenous gangliosides

Gangliosides, sialic acid-containing glycosphingolipids present in the outer leaflet of plasma membranes, are produced at high levels by some tumors, are actively shed into the tumor microenvironment, and can be detected in high concentrations in the serum of cancer patients. These tumor-shed molecules are known to be immunosuppressive, although mechanisms remain to be fully elucidated. In this study, we show that membrane enrichment of human monocytes with purified exogenous gangliosides potently inhibits ligand-induced activation and proinflammatory cytokine production induced by a broad range of TLRs, including TLR2, TLR3, TLR6, and TLR7/8, in addition to a previously identified inhibitory effect on TLR4 and TLR5. Inhibition of TLR activation is reversible, with complete restoration of TLR signaling within 6-24 h of washout of exogenous gangliosides, and is selective for certain gangliosides (GM1, GD1a, and GD1b), whereas others (GM3) are inactive. To characterize the inhibition, we assessed the expression of the TLR signaling pathway inhibitor, IL-1 receptor associated kinase-M (IRAK-M). In response to ganglioside enrichment alone, we observed striking up-regulation of IRAK-M in monocytes, but without concomitant proinflammatory cytokine production. This contrasts with endotoxin tolerance, in which IRAK-M up-regulation follows proinflammatory cytokine expression caused by LPS exposure. We hypothesize that ganglioside treatment induces a state of tolerance to TLR signaling, leading to blunted activation of innate immune responses. In the tumor microenvironment, shed tumor ganglioside enrichment of APC membranes may likewise cause these cells to bypass the normal TLR signaling response and progress directly to the inhibitory state.     

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.     

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 ^−/−) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.     

Gangliosides inhibit flagellin signaling in the absence of an effect on flagellin binding to toll-like receptor 5

A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.     

Curcumin decreases toll-like receptor-2 gene expression and function in human monocytes and neutrophils

Toll-like receptor-2 (TLR2) is a pattern recognition receptor that senses many types of bacterial components and activates signaling pathways that induce inflammatory cytokines. A hyperresponsiveness to pathogens caused by increased expression of TLR2 triggers exaggeration of some inflammatory diseases. Here, we showed that curcumin, a well-known anti-inflammatory agent derived from the curry spice turmeric, inhibits TLR2 expression in various TLR2-expressing innate immune cell lines such as monocytic THP-1 cells, neutrophilic-differentiated HL-60 cells. Strong suppression of TLR2 gene expression was specifically observed at concentrations of curcumin in the range 40-100 μM. Consistent with decreased expression of TLR2 mRNA, protein expression and ligand-responsiveness of TLR2 were markedly reduced by curcumin treatment. Moreover, curcumin-dependent down-regulation of TLR2 expression and function was also observed in primary peripheral blood monocytes (MC) and polymorphonuclear neutrophils (PMN). Finally, we determined the importance of curcumin-dependent radical generation for the suppressive effect of curcumin on TLR2 expression. Thus, our data demonstrate that curcumin inhibits TLR2 gene expression and function possibly via an oxidative process.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Specific inhibition of MyD88-independent signaling pathways of TLR3 and TLR4 by resveratrol: molecular targets are TBK1 and RIP1 in TRIF complex

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-kappaB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-kappaB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-kappaB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-beta induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-kappaB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IkappaBalpha and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-kappaB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.     

Toll-like receptor 4 signaling plays a role in triggering periodontal infection

Toll-like receptors (TLRs) are a group of sensors on the surface of antigen-presenting cells, such as dendritic cells and macrophages, which recognize microbial pathogens and induce innate and adaptive immune responses. Periodontitis is an inflammatory disease characterized by the destruction of tooth-supporting structures. In order to address whether TLR4 signaling plays a role in periodontitis, we studied the gene expression change in human periodontal ligament cells (HPDLCs) in response to TLR4 ligand, lipopolysaccharide treatment by microarray analysis. Expression of TLR4 was detected in HPDLCs. Lipopolysaccharide treatment increased the expression of 12 genes (more than twofold), including TLR4, TLR5, TLR7, Pellino 1, colony stimulating factor 2 (CSF2) and IL-6. In addition, the expression of 15 genes (less than equal to twofold) was decreased, including Fos, LY64 and LY86. In addition, real-time PCR was used to confirm the change of gene expression of TLR4, IL-6 and Fos. We also showed that the upregulation of IL-6 by lipopolysaccharide treatment was TLR4-dependent. This pattern of gene expression indicates that pathogens may trigger TLR4 signaling and cause periodontitis. Manipulating TLR4 signaling may potentially become one of the recognized therapies for periodontitis.     

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

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, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.     

Membrane-anchored CD14 is required for LPS-induced TLR4 endocytosis in TLR4/MD-2/CD14 overexpressing CHO cells

Lipopolysaccharide (LPS) induces inflammatory activation through TLR4 (toll-like receptor-4)/MD-2 (myeloid differentiation-2)/CD14 (cluster of differentiation-14) complex. Although optimal LPS signaling is required to activate our innate immune systems against gram-negative bacterium, excessive amount of LPS signaling develops a detrimental inflammatory response in gram-negative bacterial infections. Downregulation of surface TLR4 expression is one of the critical mechanisms that can restrict LPS signaling. Here, we found that membrane-anchored CD14 is required for LPS-induced downregulation of TLR4 and MD-2 in CHO cells. Moreover, pretreatment of the cells with sterol-binding agent filipin reduced LPS-induced TLR4 downregulation, suggesting the involvement of caveolae-mediated endocytosis pathway. Involvement of caveolae in LPS-induced TLR4 endocytosis was further confirmed by immunoprecipitation. Thus, our data indicate that caveolae-dependent endocytosis pathway is involved in LPS-induced TLR4 downregulation and that this is dependent on membrane-anchored CD14 expression.     

Identification of the synthetic cannabinoid R(+)WIN55,212-2 as a novel regulator of IFN regulatory factor 3 activation and IFN-beta expression: relevance to therapeutic effects in models of multiple sclerosis

β-Interferons (IFN-βs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.     

Signaling pathways involved in the regulation of TNFα-induced toll-like receptor 2 expression in human gingival fibroblasts

Periodontitis is a chronic inflammatory disease characterized by a host inflammatory response against bacteria that leads to destruction of the supporting structures of the teeth. Bacterial components of pathogens in the periodontal pocket are recognized by toll-like receptors (TLRs) that trigger an inflammatory response. In this study, we investigated the effects of the pro-inflammatory cytokine tumor necrosis factor α (TNFα) on TLR2 expression in human gingival fibroblasts. In addition, we examined the signaling pathways involved in the regulation of TNFα-induced TLR2 expression. Our results showed that TNFα increased TLR2 mRNA and protein expression. Microarray analysis and the inhibition of specific signaling pathways demonstrated that c-Jun N-terminal kinases (JNK) and nuclear factor kappa B (NF-κB) were involved in the regulation of TNFα-induced TLR2 expression in gingival fibroblasts. Furthermore, the prostaglandin E(2) (PGE(2)) regulatory enzyme cytosolic phospholipase A(2) (cPLA(2)) and the anti-inflammatory prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), were found to regulate TLR2 mRNA expression stimulated by TNFα. Our findings suggest that these pathways and mediators, through the regulation of TLR2 expression in gingival fibroblasts, may be involved in the pathogenesis of periodontitis. The study provides new insights into the molecular mechanisms underlying the regulation of TLR2, implicated in the chronic inflammatory disease periodontitis.     

Human TOLLIP Regulates TLR2 and TLR4 Signaling and Its Polymorphisms Are Associated with Susceptibility to Tuberculosis

Tuberculosis, one of the leading causes of death worldwide, stimulates inflammatory responses with beneficial and pathologic consequences. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR-mediated innate immune response to M. tuberculosis could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using short hairpin RNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. In contrast, secretion of the anti-inflammatory cytokine IL-10 was induced by TOLLIP. We also discovered two common polymorphisms that are associated with either decreased levels of mRNA expression (rs3750920) or increased IL-6 production (rs5743899) in a sample of 56 healthy volunteers. Furthermore, in a case-population study in Vietnam with 760 cord blood samples and 671 TB case patients, we found that SNPs rs3750920 and rs5743899 were associated with susceptibility to tuberculosis (p = 7.03 × 10(-16) and 6.97 × 10(-7), respectively). These data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of tuberculosis. To our knowledge, these data also show the first associations of TOLLIP polymorphisms with any infectious disease. These data also implicate an unexpected mechanism of negative regulation of TLR signaling in human tuberculosis pathogenesis.     

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.     

Microglial Toll-like receptor 2 contributes to kainic acid-induced glial activation and hippocampal neuronal cell death

Recent studies indicate that Toll-like receptors (TLRs), originally identified as infectious agent receptors, also mediate sterile inflammatory responses during tissue damage. In this study, we investigated the role of TLR2 in excitotoxic hippocampal cell death using TLR2 knock-out (KO) mice. TLR2 expression was up-regulated in microglia in the ipsilateral hippocampus of kainic acid (KA)-injected mice. KA-mediated hippocampal cell death was significantly reduced in TLR2 KO mice compared with wild-type (WT) mice. Similarly, KA-induced glial activation and proinflammatory gene expression in the hippocampus were compromised in TLR2 KO mice. In addition, neurons in organotypic hippocampal slice cultures (OHSCs) from TLR2 KO mouse brains were less susceptible to KA excitotoxicity than WT OHSCs. This protection is partly attributed to decreased expression of proinflammatory genes, such as TNF-α and IL-1β in TLR2 KO mice OHSCs. These data demonstrate conclusively that TLR2 signaling in microglia contributes to KA-mediated innate immune responses and hippocampal excitotoxicity.