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.     

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

Calcineurin negatively regulates TLR-mediated activation pathways

In innate immunity, microbial components stimulate macrophages to produce antimicrobial substances, cytokines, other proinflammatory mediators, and IFNs via TLRs, which trigger signaling pathways activating NF-kappaB, MAPKs, and IFN response factors. We show in this study that, in contrast to its activating role in T cells, in macrophages the protein phosphatase calcineurin negatively regulates NF-kappaB, MAPKs, and IFN response factor activation by inhibiting the TLR-mediated signaling pathways. Evidence for this novel role for calcineurin was provided by the findings that these signaling pathways are activated when calcineurin is inhibited either by the inhibitors cyclosporin A or FK506 or by small interfering RNA-targeting calcineurin, and that activation of these pathways by TLR ligands is inhibited by the overexpression of a constitutively active form of calcineurin. We further found that IkappaB-alpha degradation, MAPK activation, and TNF-alpha production by FK506 were reduced in macrophages from mice deficient in MyD88, Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF), TLR2, or TLR4, whereas macrophages from TLR3-deficient or TLR9 mutant mice showed the same responses to FK506 as those of wild-type cells. Biochemical studies indicate that calcineurin interacts with MyD88, TRIF, TLR2, and TLR4, but not with TLR3 or TLR9. Collectively, these results suggest that calcineurin negatively regulates TLR-mediated activation pathways in macrophages by inhibiting the adaptor proteins MyD88 and TRIF, and a subset of TLRs.     

Chlamydia pneumoniae-induced foam cell formation requires MyD88-dependent and -independent signaling and is reciprocally modulated by liver X receptor activation

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.     

TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta

Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-kappaB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-beta genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-beta induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-kappaB and the IFN-beta promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-beta, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.     

The Asp299Gly polymorphism alters TLR4 signaling by interfering with recruitment of MyD88 and TRIF

Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-β (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-β mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-β mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.     

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.     

Garlic (Allium sativum) extract inhibits lipopolysaccharide-induced Toll-like receptor 4 dimerization

Garlic has long been used as a folk medicine. Numerous studies have demonstrated that a garlic extract and its sulfur-containing compounds inhibited nuclear factor kappa B (NF-kappaB) activation induced by various receptor agonists including lipopolysaccharide (LPS). Toll-like receptors (TLRs) play a key role in sensing diverse microbial products and inducing innate immune responses. The dimerization of TLR4 is required for the activation of downstream signaling pathways, including NF-kappaB. Therefore, TLR4 dimerization may be one of the first lines of regulation in activating LPS-induced signaling pathways. We report here biochemical evidence that the ethyl acetate fraction of garlic inhibited the LPS-induced dimerization of TLR4, resulting in the inhibition of NF-kappaB activation and the expression of cyclooxygenase 2 and inducible nitric oxide synthase. Our results demonstrate for the first time that a garlic extract can directly inhibit the TLRs-mediated signaling pathway at the receptor level. These results shed a new insight into understanding how garlic modulates the immune responses that could modify the risk of many chronic diseases.     

Signaling of apoptosis through TLRs critically involves toll/IL-1 receptor domain-containing adapter inducing IFN-beta, but not MyD88, in bacteria-infected murine macrophages

TLRs are important sensors of the innate immune system that serve to identify conserved microbial components to mount a protective immune response. They furthermore control the survival of the challenged cell by governing the induction of pro- and antiapoptotic signaling pathways. Pathogenic Yersinia spp. uncouple the balance of life and death signals in infected macrophages, which compels the macrophage to undergo apoptosis. The initiation of apoptosis by Yersinia infection specifically involves TLR4 signaling, although Yersinia can activate TLR2 and TLR4. In this study we characterized the roles of downstream TLR adapter proteins in the induction of TLR-responsive apoptosis. Experiments using murine macrophages defective for MyD88 or Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF) revealed that deficiency of TRIF, but not of MyD88, provides protection against Yersinia-mediated cell death. Similarly, apoptosis provoked by treatment of macrophages with the TLR4 agonist LPS in the presence of a proteasome inhibitor was inhibited in TRIF-defective, but not in MyD88-negative, cells. The transfection of macrophages with TRIF furthermore potently promoted macrophage apoptosis, a process that involved activation of a Fas-associated death domain- and caspase-8-dependent apoptotic pathway. These data indicate a crucial function of TRIF as proapoptotic signal transducer in bacteria-infected murine macrophages, an activity that is not prominent for MyD88. The ability to elicit TRIF-dependent apoptosis was not restricted to TLR4 activation, but was also demonstrated for TLR3 agonists. Together, these results argue for a specific proapoptotic activity of TRIF as part of the host innate immune response to bacterial or viral infection.     

Absence of TRIF signaling in lipopolysaccharide-stimulated murine mast cells

In macrophages, two signaling pathways, dependent on MyD88 or TIR domain-containing adaptor-inducing IFN-β (TRIF) signaling, emanate from the LPS receptor TLR4/MD-2. In this study, we show that in murine bone marrow-derived mast cells (BMMCs), only the MyD88-dependent pathway is activated by LPS. The TRIF signaling branch leading both to NF-κB activation and enhanced proinflammatory cytokine production, as well as to IRF3 activation and subsequent IFN-β production, is absent in LPS-stimulated BMMCs. IRF3 activation is also absent in peritoneal mast cells from LPS-injected mice. We observed strongly diminished TRAM expression in BMMCs, but overexpression of TRAM only moderately enhanced IL-6 and did not boost IFN-β responses to LPS in these cells. A combination of very low levels of TRAM and TLR4/MD-2 with the known absence of membrane-bound CD14 are expected to contribute to the defective TRIF signaling in mast cells. We also show that, unlike in macrophages, in BMMCs the TRIF-dependent and -independent IFN-αβ responses to other recognized IFN inducers (dsRNA, adenovirus, and B-DNA) are absent. These results show how the response to the same microbial ligand using the same receptor can be regulated in different cell types of the innate immune system.     

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.     

Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes.

Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4, a member of the TLR family that participates in pathogen recognition. TLRs recruit a cytoplasmic protein, MyD88, upon pathogen recognition, mediating its function for immune responses. Two major pathways for LPS have been suggested in recent studies, which are referred to as MyD88-dependent and -independent pathways. We report in this study the characterization of the MyD88-independent pathway via TLR4. MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS, whereas they responded to LPS by activating IFN-regulatory factor 3 as well as inducing the genes containing IFN-stimulated regulatory elements such as IP-10. In contrast, a lipopeptide that activates TLR2 had no ability to activate IFN-regulatory factor 3. The MyD88-independent pathway was also activated in cells lacking both MyD88 and TNFR-associated factor 6. Thus, TLR4 signaling is composed of at least two distinct pathways, a MyD88-dependent pathway that is critical to the induction of inflammatory cytokines and a MyD88/TNFR-associated factor 6-independent pathway that regulates induction of IP-10.     

WDFY1 mediates TLR3/4 signaling by recruiting TRIF

Toll-like receptors (TLRs) are pattern recognition receptors that sense a variety of pathogens, initiate innate immune responses, and direct adaptive immunity. All TLRs except TLR3 recruit the adaptor MyD88 to ultimately elicit inflammatory gene expression, whereas TLR3 and internalized TLR4 use TIR-domain-containing adaptor TRIF for the induction of type I interferon and inflammatory cytokines. Here, we identify the WD repeat and FYVE-domain-containing protein WDFY1 as a crucial adaptor protein in the TLR3/4 signaling pathway. Overexpression of WDFY1 potentiates TLR3- and TLR4-mediated activation of NF-κB, interferon regulatory factor 3 (IRF3), and production of type I interferons and inflammatory cytokines. WDFY1 depletion has the opposite effect. WDFY1 interacts with TLR3 and TLR4 and mediates the recruitment of TRIF to these receptors. Our findings suggest a crucial role for WDFY1 in bridging the TLR–TRIF interaction, which is necessary for TLR signaling.     

Dual signaling of MyD88 and TRIF is critical for maximal TLR4-induced dendritic cell maturation

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.