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.     

TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons

Toll-like receptor (TLR) 3 and 4 mediate the expression of many genes, including NF-kappaB- and interferon-regulatory factor (IRF)-3/interferon (IFN)-inducible genes, in macrophages and dendritic cells (DCs) in response to their ligand stimuli, polyI:C and lipopolysaccharide (LPS). Toll-IL-1 receptor homology domain (TIR)-containing adapter molecule 1 (TICAM-1) facilitates expression of IFN-inducible genes via TLR3. Although MyD88 and Mal/TIRAP adapters function downstream of TLR4, they barely induce IFN-beta. In addition, DC maturation as well as IFN-beta induction are largely independent of MyD88 and Mal/TIRAP. TICAM-1 is the functional adapter for both TLR3 and TLR4 that induces type 1 IFN and MyD88-independent DC maturation. In LPS-mediated TLR4 activation, a complex of TICAM-1 and an additional TLR4-binding adapter serves as the adapter. We named this TLR4-TICAM-1-bridging adapter TICAM-2. Our results reveal the details of MyD88-independent pathways which separately recruit the distinct adapters downstream of TLR3 and TLR4 and variations of the TLR output are in part regulated by the two additional adapters in DCs.     

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.     

Akt contributes to activation of the TRIF-dependent signaling pathways of TLRs by interacting with TANK-binding kinase 1

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.     

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.     

Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways

Toll-like receptor-3 is critically involved in host defense against viruses through induction of type I interferons (IFNs). Recent studies suggest that a Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and two protein kinases (TANK-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-epsilon) are critically involved in Toll-like receptor-3-mediated IFN-beta production through activation of IFN regulatory factor (IRF)-3 and IRF-7. In this study, we demonstrate that TRIF interacts with both IRF-7 and IRF-3. In addition to TBK1 and IKKepsilon, our results indicate that IKKbeta can also phosphorylate IRF-3 and activate the IFN-stimulated response element. TRIF-induced IRF-3 and IRF-7 activation was mediated by TBK1 and its downstream kinases IKKbeta and IKKepsilon. TRIF induced NF-kappaB activation through an IKKbeta- and tumor necrosis factor receptor-associated factor-6-dependent (but not TBK1- and IKKepsilon-dependent) pathway. In addition, TRIF also induced apoptosis through a RIP/FADD/caspase-8-dependent and mitochondrion-independent pathway. Furthermore, our results suggest that the TRIF-induced IFN-stimulated response element and NF-kappaB activation and apoptosis pathways are uncoupled and provide a molecular explanation for the divergent effects induced by the adapter protein TRIF.     

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.     

Lipopolysaccharide-mediated interferon regulatory factor activation involves TBK1-IKKepsilon-dependent Lys(63)-linked polyubiquitination and phosphorylation of TANK/I-TRAF

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKKepsilon-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/ITRAF is required for the TBK1- and IKKepsilon-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of double-stranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKKepsilon upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide- and TBK1-IKKepsilon-mediated Lys(63)-linked polyubiquitination, a mechanism that does not require TBK1-IKKepsilon kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKKepsilon complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys(63)-linked polyubiquitination of their scaffold protein, TANK.     

Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.     

CARD14/CARMA2sh and TANK differentially regulate poly(I:C)–induced inflammatory reaction in keratinocytes

CARD14/CARMA2sh (CARMA2sh) is a scaffold protein whose mutations are associated with the onset of human genetic psoriasis and other inflammatory skin disorders. Here we show that the immunomodulatory adapter protein TRAF family member-associated NF-κB activator (TANK) forms a complex with CARMA2sh and MALT1 in a human keratinocytic cell line. We also show that CARMA2 and TANK are individually required to activate the nuclear factor κB (NF-κB) response following exposure to polyinosinic-polycytidylic (poly [I:C]), an agonist of toll-like receptor 3. Finally, we present data indicating that TANK is essential for activation of the TBK1/IRF3 pathway following poly (I:C) stimulation, whereas CARMA2sh functions as a repressor of it. More important, we report that two CARMA2sh mutants associated with psoriasis bind less efficiently to TANK and are therefore less effective in suppressing the TBK1/IRF3 pathway. Overall, our data indicate that TANK and CARMA2sh regulate TLR3 signaling in human keratinocytes, which could play a role in the pathophysiology of psoriasis.