Transforming growth factor-beta differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional, potent anti-inflammatory cytokine produced by many cell types that regulates cell proliferation, apoptosis, and immune responses. Toll-like receptors (TLRs) recognize various pathogen-associated molecular patterns and are therefore a pivotal component of the innate immune system. In this study we show that TGF-beta1 blocks the NF-kappaB activation and cytokine release that is stimulated by ligands for TLRs 2, 4, and 5. We further show that TGF-beta1 can specifically interfere with TLR2, -4, or -5 ligand-induced responses involving the adaptor molecule MyD88 (myeloid differentiation factor 88) but not the TRAM/TRIF signaling pathway by decreasing MyD88 protein levels in a dose- and time-dependent manner without altering its mRNA expression. The proteasome inhibitor epoxomicin abolished the MyD88 degradation induced by TGF-beta1. Furthermore, TGF-beta1 resulted in ubiquitination of MyD88 protein, suggesting that TGF-beta1 facilitates ubiquitination and proteasomal degradation of MyD88 and thereby attenuates MyD88-dependent signaling by decreasing cellular levels of MyD88 protein. These findings importantly contribute to our understanding of molecular mechanisms mediating anti-inflammatory modulation of immune responses by TGF-beta1.     

MyD88-dependent and MyD88-independent pathways in synergy, priming, and tolerance between TLR agonists

TLRs sense components of microorganisms and are critical host mediators of inflammation during infection. Different TLR agonists can profoundly alter inflammatory effects of one another, and studies suggest that the sequence of exposure to TLR agonists may importantly impact on responses during infection. We tested the hypothesis that synergy, priming, and tolerance between TLR agonists follow a pattern that can be predicted based on differential engagement of the MyD88-dependent (D) and the MyD88-independent (I) intracellular signaling pathways. Inflammatory effects of combinations of D and I pathway agonists were quantified in vivo and in vitro. Experiments used several D-specific agonists, an I-specific agonist (poly(I:C)), and LPS, which acts through both the D and I pathways. D-specific agonists included: peptidoglycan-associated lipoprotein, Pam3Cys, flagellin, and CpG DNA, which act through TLR2 (peptidoglycan-associated lipoprotein and Pam3Cys), TLR5, and TLR9, respectively. D and I agonists were markedly synergistic in inducing cytokine production in vivo in mice. All of the D-specific agonists were synergistic with poly(I:C) in vitro in inducing TNF and IL-6 production by mouse bone marrow-derived macrophages. Pretreatment of bone marrow-derived macrophages with poly(I:C) led to a primed response to subsequent D-specific agonists and vice versa, as indicated by increased cytokine production, and increased NF-kappaB translocation. Pretreatment with a D-specific agonist augmented LPS-induced IFN-beta production. All D-specific agonists induced tolerance to one another. Thus, under the conditions studied here, simultaneous and sequential activation of both the D and I pathways causes synergy and priming, respectively, and tolerance is induced by agonists that act through the same pathway.     

Effects of possible endocrine disruptors on MyD88-independent TLR4 signaling

Endocrine disrupting chemicals (EDCs) may potentially worsen infectious diseases because EDCs disturb human immune function by interfering with endocrine balance. To evaluate the influence of EDCs on the innate immune function of macrophages, we investigated the effects of 37 possible EDCs on lipopolysaccharide-induced activation of the IFN-beta promoter. Alachlor, atrazine, benomyl, bisphenol A, carbaryl, diethyl phthalate, dipropyl phthalate, kelthane, kepone, malathion, methoxychlor, octachlorostyrene, pentachlorophenol, nonyl phenol, p-octylphenol, simazine and ziram all inhibited the activation. Kepone and ziram showed strong inhibitory effects. Aldicarb, amitrole, benzophenone, butyl benzyl phthalate, 2,4-dichlorophenoxy acetic acid, dibutyl phthalate, 2,4-dichlorophenol, dicyclohexyl phthalate, diethylhexyl adipate, diethylhexyl phthalate, dihexyl phthalate, di-n-pentyl phthalate, methomyl, metribuzin, nitrofen, 4-nitrotoluene, permethrin, trifluralin, 2,4,5-trichlorophenoxyacetic acid and vinclozolin had no significant effects at 100 muM. These results indicate that some agrochemicals and resin-related chemicals may potentially inhibit macrophage function, which suggests that endocrine disruptors may influence the development of infectious diseases.     

Clostridium butyricum activates TLR2-mediated MyD88-independent signaling pathway in HT-29 cells

Oral administration of Clostridium butyricum as probiotic is increasingly gaining importance in the treatment of diarrhea and the improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The objective of this study was to analyze the mechanisms involved in C. butyricum induction of the toll-like receptor (TLR) signaling. Knockdown of myeloid differentiation primary response protein 88 (MyD88) expression using small interfering RNA in this manner did not affect C. butyricum-induced elevated levels of nuclear factor κB (NF-κB), interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α), suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6, and TNF-α was evident in the absence of TLR2 expression, implying the need for TLR2 in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells, which adds to our understanding of the molecular mechanisms of this probiotic action on gut epithelium.     

Neohesperidin dihydrochalcone down-regulates MyD88-dependent and -independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts

Fulminant hepatic failure (FHF) is a lethal clinical syndrome characterized by the activation of macrophages and the increased production of inflammatory mediators. The purpose of this study was to investigate the effects of neohesperidin dihydrochalcone (NHDC), a widely-used low caloric artificial sweetener against FHF. An FHF experimental model was established in mice by intraperitoneal injection of D-galactosamine (d-GalN) (400 mg/kg)/lipopolysaccharides (LPS) (10 μg/kg). Mice were orally administered NHDC for 6 continuous days and at 1 h before d-GalN/LPS administration. RAW264.7 macrophages were used as an in vitro model. Cells were pre-treated with NHDC for 1 h before stimulation with LPS (10 μg/ml) for 6 h. d-GalN/LPS markedly increased the serum transaminase activities and levels of oxidative and inflammatory markers, which were significantly attenuated by NHDC. Mechanistic analysis indicated that NHDC inhibited LPS-induced myeloid differentiation factor 88 (MyD88) and TIR-containing adapter molecule (TRIF)-dependent signaling. Transient transfection of TLR4 or MyD88 siRNA inhibited the downstream inflammatory signaling. This effect could also be achieved by the pretreatment with NHDC. The fluorescence microscopy and flow cytometry results suggested that NHDC potently inhibited the binding of LPS to TLR4 in RAW264.7 macrophages. In addition, the inhibitory effect of NHDC on LPS-induced translocation of TLR4 into lipid raft domains played an important role in the amelioration of production of downstream pro-inflammatory molecules. Furthermore, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) by NHDC inhibited TLR4 signaling. In conclusion, our results suggest that NHDC attenuates d-GalN/LPS-induced FHF by inhibiting the TLR4-mediated inflammatory pathway, demonstrating a new application of NHDC as a hepatoprotective agent.     

IFN-alphabeta-mediated inflammatory responses and antiviral defense in liver is TLR9-independent but MyD88-dependent during murine cytomegalovirus infection

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

Role for MyD88-independent, TRIF pathway in lipid A/TLR4-induced endotoxin tolerance

Repeated exposure to low doses of endotoxin results in progressive hyporesponsiveness to subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance. In spite of its clinical significance in sepsis and characterization of the TLR4 signaling pathway as the principal endotoxin detection mechanism, the molecular determinants that induce tolerance remain obscure. We investigated the role of the TRIF/IFN-beta pathway in TLR4-induced endotoxin tolerance. Lipid A-induced homotolerance was characterized by the down-regulation of MyD88-dependent proinflammatory cytokines TNF-alpha and CCL3, but up-regulation of TRIF-dependent cytokine IFN-beta. This correlated with a molecular phenotype of defective NF-kappaB activation but a functional TRIF-dependent STAT1 signaling. Tolerance-induced suppression of TNF-alpha and CCL3 expression was significantly relieved by TRIF and IFN regulatory factor 3 deficiency, suggesting the involvement of the TRIF pathway in tolerance. Alternatively, selective activation of TRIF by poly(I:C)-induced tolerance to lipid A. Furthermore, pretreatment with rIFN-beta also induced tolerance, whereas addition of IFN-beta-neutralizing Ab during the tolerization partially alleviated tolerance to lipid A but not TLR2-induced endotoxin homo- or heterotolerance. Furthermore, IFNAR1-/- murine embryonal fibroblast and bone-marrow derived macrophages failed to induce tolerance. Together, these observations constitute evidence for a role of the TRIF/IFN-beta pathway in the regulation of lipid A/TLR4-mediated endotoxin homotolerance.     

大鼠海马神经元TLR4介导的MyD88依赖途径在神经炎症中的作用

目的:研究大鼠海马神经元是否有Toll样受体4(TLR4)介导的的髓样分化因子88(MyD88)依赖途径及该途径的激活在神经炎症中的作用。方法:采用体外培养7 d的新生大鼠海马神经元,细胞免疫荧光双标法鉴定海马神经元纯度。用TLR4配体脂多糖(LPS)或TLR4抗体预处理海马神经元,以激活或阻断TLR4的作用。实时定量PCR(RT-qPCR)方法检测海马神经元中MyD88、肿瘤坏死因子受体相关因子6(TRAF6)mRNA的表达;Westernblot方法测定海马神经元MyD88和TRAF6蛋白水平;细胞免疫荧光双标法观察海马神经元中核因子κB/P65(NF-κB/P65)的表达定位及TLR4激活或阻断后NF-κB/P65核易位情况;ELISA检测培养上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和一氧化氮(NO)的水平。结果:LPS能上调海马神经元MyD88和肿瘤坏死因子受体相关因子(TRAF6)mRNA水平;促使NF-κB/P65转位至核;增加MyD88和TRAF6蛋白的表达;增加海马神经元培养上清中TNF-α、IL-1β和NO含量;TLR4抗体预处理能减弱LPS对海马神经元NF-κB/P65核易位作用及降低培养上清中TNF-α、IL-1β和NO的水平。结论:大鼠海马神经元有TLR4介导的的MyD88依赖途径,该途径的激活能导致TNF-α、IL-lβ和NO含量的增加。海马神经元TLR4介导的MyD88依赖途径参与了神经炎症反应,神经元不是神经炎症反应中的被动者。     

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

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.     

Cutting edge: TNFR-associated factor (TRAF) 6 is essential for MyD88-dependent pathway but not toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent pathway in TLR signaling

Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-kappaB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-kappaB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-kappaB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling.     

MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88-/- mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2-/-, TLR4-/-, and TLR2/4-/- mice were similar to those of C57BL/6 mice; however, TLR4-/- and TLR2/4-/- mice had impaired antifungal responses. The phenotype of infected IL-1R1-/- mice was similar to that of MyD88-/- mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1-/- mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.     

Role for MyD88, TLR2 and TLR9 but not TLR1, TLR4 or TLR6 in experimental autoimmune encephalomyelitis

The potential roles of TLRs in the cause and pathogenesis of autoimmune CNS inflammation remain contentious. In this study, we examined the effects of targeted deletions of TLR1, TLR2, TLR4, TLR6, TLR9, and MyD88 on the induction of myelin oligodendrocyte glycoprotein 35-55 (MOG(35-55)) peptide/CFA/pertussis toxin-induced autoimmune encephalomyelitis. Although C57BL/6.Tlr1(-/-), C57BL/6.Tlr4(-/-) and C57BL/6.Tlr6(-/-) mice showed normal susceptibility to disease, signs were alleviated in female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice and C57BL/6.Tlr2/9(-/-) mice of both sexes. C57BL/6.Myd88(-/-) mice were completely protected. Lower clinical scores were associated with reduced leukocyte infiltrates. These results were confirmed by passive adoptive transfer of disease into female C57BL/6.Tlr2(-/-) and C57BL/6.Tlr9(-/-) mice, where protection in the absence of TLR2 was associated with fewer infiltrating CD4(+) cells in the CNS, reduced prevalence of detectable circulating IL-6, and increased proportions of central (CD62L(+)) CD4(+)CD25(+)Foxp3(+) regulatory T cells. These results provide a potential molecular mechanism for the observed effects of TLR signaling on the severity of autoimmune CNS inflammation.     

Cutting edge: TLR4 activation mediates liver ischemia/reperfusion inflammatory response via IFN regulatory factor 3-dependent MyD88-independent pathway

The triggering molecular mechanism of ischemia-reperfusion injury (IRI), which in clinical settings results in excessive and detrimental inflammatory responses, remains unclear. This study analyzes the role of the TLR system in an established murine model of liver warm ischemia followed by reperfusion. By contrasting in parallel TLR knockout mice with their wild-type counterparts, we found that TLR4, but not TLR2, was specifically required in initiating the IRI cascade, as manifested by liver function (serum alanine aminotransferase levels), pathology, and local induction of proinflammatory cytokines/chemokines (TNF-alpha, IL-6, IFN-inducible protein 10). We then investigated the downstream signaling pathway of TLR4 activation. Our results show that IFN regulatory factor 3, but not MyD88, mediated IRI-induced TLR4 activation leading to liver inflammation and hepatocellular damage. This study documents the selective usage of TLR in a clinically relevant noninfectious disease model, and identifies a triggering molecular mechanism in the pathophysiology of liver IRI.     

MyD88 but not TLR2, 4 or 9 is essential for IL-12 induction by lactic acid bacteria

Although lactic acid bacteria (LAB) affect the immune system, for example, having an anti-allergic effect, little is known about the actual mechanisms of immune modulation. Toll-like receptors (TLRs) recognize conserved microbial molecular patterns, and are presumed to be involved in the recognition of LAB. However, there are few detailed reports examining the relationships between TLR and LAB. We measured here production of IL-12, a cytokine considered to play an important role in anti-allergic effects, induced by Lactobacillus paracasei strain KW3110 and other typical LAB by cells from TLR2-, TLR4-, TLR9- and myeloid differentiation factor 88 (MyD88)-deficient mice. Unexpectedly, similar cytokine production from wild-type and TLR2-, 4- and 9-deficient mice was observed. In contrast, cells from MyD88-deficient mice failed to respond to stimulation with LAB. It is therefore concluded that although LAB, including strain KW3110, are not likely to be recognized by TLR2, 4 or 9, MyD88 is essential for the response to these bacteria.     

Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9

Bacterial pathogens are recognized by the innate immune system through pattern recognition receptors, such as Toll-like receptors (TLRs). Engagement of TLRs triggers signaling cascades that launch innate immune responses. Activation of MAPKs and NF-kappaB, elements of the major signaling pathways induced by TLRs, depends in most cases on the adaptor molecule MyD88. In addition, Gram-negative or intracellular bacteria elicit MyD88-independent signaling that results in production of type I interferon (IFN). Here we show that in mouse macrophages, the activation of MyD88-dependent signaling by the extracellular Gram-positive human pathogen group A streptococcus (GAS; Streptococcus pyogenes) does not require TLR2, a receptor implicated in sensing of Gram-positive bacteria, or TLR4 and TLR9. Redundant engagement of either of these TLR molecules was excluded by using TLR2/4/9 triple-deficient macrophages. We further demonstrate that infection of macrophages by GAS causes IRF3 (interferon-regulatory factor 3)-dependent, MyD88-independent production of IFN. Surprisingly, IFN is induced also by GAS lacking slo and sagA, the genes encoding cytolysins that were shown to be required for IFN production in response to other Gram-positive bacteria. Our data indicate that (i) GAS is recognized by a MyD88-dependent receptor other than any of those typically used by bacteria, and (ii) GAS as well as GAS mutants lacking cytolysin genes induce type I IFN production by similar mechanisms as bacteria requiring cytoplasmic escape and the function of cytolysins.     

TLR4 is the signaling but not the lipopolysaccharide uptake receptor

TLR4 is the primary recognition molecule for inflammatory responses initiated by bacterial LPS (endotoxin). Internalization of endotoxin by various cell types is an important step for its removal and detoxification. Because of its role as an LPS-signaling receptor, TLR4 has been suggested to be involved in cellular LPS uptake as well. LPS uptake was investigated in primary monocytes and endothelial cells derived from TLR4 and CD14 knockout C57BL/6 mice using tritiated and fluorescein-labeled LPS. Intracellular LPS distribution was investigated by deconvolution confocal microscopy. We could not observe any difference in LPS uptake and intracellular LPS distribution in either monocytes or endothelial cells between TLR4(-/-) and wild-type cells. As expected, CD14(-/-) monocytes showed a highly impaired LPS uptake, confirming CD14-dependent uptake in monocytes. Upon longer incubation periods, the CD14-deficient monocytes mimicked the LPS uptake pattern of endothelial cells. Endothelial cell LPS uptake is slower than monocyte uptake, LBP rather than CD14 dependent, and sensitive to polyanionic polymers, which have been shown to block scavenger receptor-dependent uptake mechanisms. We conclude that TLR4 is not involved in cellular LPS uptake mechanisms. In membrane CD14-positive cells, LPS is predominantly taken up via CD14-mediated pathways, whereas in the CD14-negative endothelial cells, there is a role for scavenger receptor-dependent pathways.