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.     

Parthenolide inhibits TRIF-dependent signaling pathway of toll-like receptors in RAW264.7 macrophages

Toll-like receptors (TLRs) play an important role in induction of innate immune responses for host defense against invading microbial pathogens. Microbial component engagement of TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. Parthenolide, an active ingredient of feverfew (Tanacetum parthenium), has been used for centuries to treat many chronic diseases. Parthenolide inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-κB kinase. However, it is not known whether parthenolide inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of parthenolide, its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly [I:C]) was examined. Parthenolide inhibited nuclear factor-κB and interferon regulatory factor 3 activation induced by LPS or poly[I:C], and the LPS-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10. These results suggest that parthenolide can modulate TRIF-dependent signaling pathways of TLRs, and may be the basis of effective therapeutics for chronic inflammatory diseases.     

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.     

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.     

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.     

Overlapping and distinct roles of GRK5 in TLR2-, and TLR3-induced inflammatory response in vivo

G-protein coupled receptor kinase-5 (GRK5) is a recently described NFκB regulator in TLR4 signaling pathway. To determine whether the role of GRK5 is MyD88- or TRIF-dependent, we injected wild type and GRK5 knockout mice with Pam3CSK4 (MyD88-dependent TLR1/2 ligand) and Poly(I:C) (TRIF-dependent TLR3 ligand) and examined the in vivo systemic inflammatory response. Our results demonstrate that GRK5 regulates IL-12p40 and G-CSF via a mechanism that is common to both MyD88 and TRIF. However, GRK5 regulates IL-5 and MCP-1 in a MyD88-dependent but TNFα in a TRIF-dependent manner. Together, our results demonstrate multiple roles of GRK5 in TLR signaling.     

Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.     

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.     

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.     

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.     

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.     

Suppression of homodimerization of toll-like receptor 4 by isoliquiritigenin

Toll-like receptors (TLRs) play important inductive roles in innate immune responses for host defense against invading microbial pathogens. Activation of TLR4 by lipopolysaccharide (LPS) induces dimerization of TLR4 and, subsequently, activation of downstream signaling pathways including nuclear factor-kappa B and interferon regulatory factor 3. TLR4 dimerization may be an early regulatory event in activating signaling pathways induced by LPS. Here, biochemical evidence is reported that isoliquiritigenin, one of the major ingredients derived from licorice root, inhibits LPS-induced TLR4 dimerization resulting in inhibition of nuclear factor-kappa B and interferon regulatory factor 3 activation, and cyclooxygenase-2 and inducible nitric oxide synthase expression. These results suggest that isoliquiritigenin modulates TLR-mediated signaling pathways at the receptor level. Furthermore, these results suggest that TLRs themselves may be important targets for the prevention of chronic inflammatory diseases.     

Toll-like receptor downstream signaling

The family of Toll-like receptors (TLRs) senses conserved structures found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. The signal transduction is initiated from the Toll/interleukin-1 receptor (TIR) domain of TLRs after pathogen recognition. Almost all TLRs use a TIR-containing adapter MyD88 to activate a common signaling pathway that results in the activation of NF-kappaB to express cytokine genes relevant to inflammation. Recently, three further TIR-containing adapters have been identified and shown to selectively interact with several TLRs. In particular, activation of the TRIF-dependent pathway confers antiviral responses by inducing anti-viral genes including that encoding interferon-beta. Taken together, these results indicate that the interaction between individual TLRs and the different combinations of adapters directs appropriate responses against distinct pathogens.     

Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A(2) (cPLA(2)) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA(2) activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA(2) phosphorylation and cPLA(2)-hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA(2) activation and lipid release. cPLA(2) phosphorylation and cPLA(2)-hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA(2) activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88- and TRIF-dependent MAPK pathways result in cPLA(2) activation and production of pro-inflammatory lipid mediators.     

Toll-like receptor downstream signaling

The family of Toll-like receptors (TLRs) senses conserved structures found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. The signal transduction is initiated from the Toll/interleukin-1 receptor (TIR) domain of TLRs after pathogen recognition. Almost all TLRs use a TIR-containing adapter MyD88 to activate a common signaling pathway that results in the activation of NF-κB to express cytokine genes relevant to inflammation. Recently, three further TIR-containing adapters have been identified and shown to selectively interact with several TLRs. In particular, activation of the TRIF-dependent pathway confers antiviral responses by inducing anti-viral genes including that encoding interferon-β. Taken together, these results indicate that the interaction between individual TLRs and the different combinations of adapters directs appropriate responses against distinct pathogens.     

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Toll receptors and pathogen resistance

Toll receptors in insects, mammals and plants are key players that sense the invasion of pathogens. Toll-like receptors (TLRs) in mammals have been established to detect specific components of bacterial and fungal pathogens. Furthermore, recent evidence indicates that TLRs are involved in the recognition of viral invasion. Signalling pathways via TLRs originate from the conserved Toll/IL-1 receptor (TIR) domain. The TIR domain-containing MyD88 acts as a common adaptor that induces inflammatory cytokines; however, there exists a MyD88-independent pathway that induces type I IFNs in TLR4 and TLR3 signalling. Another TIR domain-containing adaptor, TIRAP/Mal has recently been shown to mediate the MyD88-dependent activation in the TLR4 and TLR2 signalling pathway. Thus, individual TLRs may have their own signalling systems that characterize their specific activities.