Wen Qian

A critical role for IRAK4 kinase activity in Toll-like receptor–mediated innate immunity

IRAK4 is a member of IL-1 receptor (IL-1R)–associated kinase (IRAK) family and has been shown to play an essential role in Toll-like receptor (TLR)–mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase–inactive knock-in mice were completely resistant to lipopolysaccharide (LPS)- and CpG-induced shock, due to impaired TLR-mediated induction of proinflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1R–mediated nuclear factor κB activation, a reduction of LPS-, R848-, and IL-1–mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow–derived macrophages from IRAK4 kinase–inactive knock-in mice. Both TLR7- and TLR9-mediated type I interferon production was abolished in plasmacytoid dendritic cells isolated from IRAK4 knock-in mice. In addition, influenza virus–induced production of interferons in plasmacytoid DCs was also dependent on IRAK4 kinase activity. Collectively, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.

Astrocyte-Restricted Ablation of Interleukin-17-Induced Act1-Mediated Signaling Ameliorates Autoimmune Encephalomyelitis

Interleukin-17 (IL-17) secreted by T helper 17 (Th17) cells is essential in the development of experimental autoimmune encephalomyelitis (EAE). However, it remains unclear how IL-17-mediated signaling in different cellular compartments participates in the central nervous system (CNS) inflammatory process. We examined CNS inflammation in mice with specific deletion of Act1, a critical component required for IL-17 signaling, in endothelial cells, macrophages and microglia, and neuroectoderm (neurons, astrocytes, and oligodendrocytes). In Act1-deficient mice, Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils, and macrophages. Act1 deficiency in endothelial cells or in macrophages and microglia did not substantially impact the development of EAE. However, targeted Act1 deficiency in neuroectoderm-derived CNS-resident cells resulted in markedly reduced severity in EAE. Specifically, Act1-deficient astrocytes showed impaired IL-17-mediated inflammatory gene induction. Thus, astroctyes are critical in IL-17-Act1-mediated leukocyte recruitment during autoimmune-induced inflammation of the CNS.

Act1, a U-box E3 ubiquitin ligase for IL-17 signaling.

The adaptor protein Act1 functions as a ubiquitin ligase to mediate interleukin-17 receptor–dependent activation of nuclear factor-κB. Acting Out a New Role Interleukin-17A (IL-17A) is the founding member of a family of proinflammatory cytokines implicated in the development of autoimmunity and the fight against microbial infection. IL-17 signaling, which is mediated by a number of IL-17 receptor (IL-17R) complexes (see the Perspective by Levin), depends on TRAF6, a scaffold protein and E3 ubiquitin ligase, and activates the transcription factor NF-κB. TRAF6 is recruited to IL-17Rs through another adaptor protein, Act1. Liu et al. now show that Act1 is also a member of the U-box family of E3 enzymes and that its ubiquitin ligase activity is required for IL-17R signaling. In response to IL-17, TRAF6 was recruited to Act1 and underwent Lys63-mediated ubiquitination, a process that was required for the activation of NF-κB. A mutant form of TRAF6 lacking the residue targeted by ubiquitin failed to support IL-17–mediated activation of NF-κB in TRAF6-deficient cells. Together, these data identify TRAF6 as a substrate of Act1 and suggest that sequential ubiquitination events regulate IL-17–induced inflammation. Interleukin-17 (IL-17), a proinflammatory cytokine mainly produced by cells of the T helper 17 (TH17) lineage, is required for host defense against bacterial and fungal infections and plays a critical role in the pathogenesis of inflammatory and autoimmune diseases. Act1 is an essential adaptor molecule in IL-17–mediated signaling and is recruited to the IL-17 receptor (IL-17R) upon IL-17 stimulation through an interaction between its SEFIR domain and that of the IL-17R. Here, we report that Act1 is a U-box E3 ubiquitin ligase and that its activity is essential for IL-17–mediated signaling pathways. Through the use of the Ubc13-Uev1A E2 complex, Act1 mediated the lysine-63–linked ubiquitination of tumor necrosis factor receptor–associated factor 6 (TRAF6), a component of IL-17–mediated signaling. Deletion and point mutations of the Act1 U-box abolished Act1-mediated ubiquitination of TRAF6 and impaired the ability of Act1 to restore IL-17–dependent signaling and expression of target genes in Act1−/− mouse embryonic fibroblasts. We also showed that the lysine-124 residue of TRAF6 was critical for efficient Act1-mediated ubiquitination of TRAF6 and for the ability of TRAF6 to mediate IL-17–induced activation of nuclear factor κB. Thus, we propose that Act1 mediates IL-17–induced signaling pathways through its E3 ubiquitin ligase activity and that TRAF6 is a critical substrate of Act1, which indicates the importance of protein ubiquitination in the IL-17–dependent inflammatory response.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1–TRAF6–transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFκB Activation Pathways Bifurcate at IL-1 Receptor-associated Kinase Modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131–144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFκB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKα/β phosphorylation and IKKβ activation, resulting in classical NFκB activation through IκBα phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKγ phosphorylation and IKKα activation, resulting in NFκB activation through IκBα phosphorylation and subsequent dissociation from NFκB but without IκBα degradation. These results provide significant insight to our further understanding of NFκB activation pathways.

Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48- and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.

Pellino 3b negatively regulates IL-1-induced TAK1-dependent NFκB activation

IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48- and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.

HuR Is Required for IL-17–Induced Act1-Mediated CXCL1 and CXCL5 mRNA Stabilization

IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17–induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17–induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17–induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17–induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17–induced inflammation.

Cutting Edge: TNF Receptor-Associated Factor 4 Restricts IL-17–Mediated Pathology and Signaling Processes

The effector T cell subset, Th17, plays a significant role in the pathogenesis of multiple sclerosis and of other autoimmune diseases. The signature cytokine, IL-17, engages the IL-17R and recruits the E3-ligase NF-κB activator 1 (Act1) upon stimulation. In this study, we examined the role of TNFR-associated factor (TRAF)4 in IL-17 signaling and Th17-mediated autoimmune encephalomyelitis. Primary cells from TRAF4-deficient mice displayed markedly enhanced IL-17–activated signaling pathways and induction of chemokine mRNA. Adoptive transfer of MOG35–55 specific wild-type Th17 cells into TRAF4-deficient recipient mice induced an earlier onset of disease. Mechanistically, we found that TRAF4 and TRAF6 used the same TRAF binding sites on Act1, allowing the competition of TRAF4 with TRAF6 for the interaction with Act1. Taken together, the results of this study reveal the necessity of a unique role of TRAF4 in restricting the effects of IL-17 signaling and Th17-mediated disease.

A CC′ Loop Decoy Peptide Blocks the Interaction Between Act1 and IL-17RA to Attenuate IL-17– and IL-25–Induced Inflammation

A cell-permeable peptide derived from an adaptor protein blocks cytokine receptor signaling and reduces inflammation. Quelling Inflammation with a Peptide Members of the interleukin-17 (IL-17) family of cytokines, including IL-17A and IL-25, are implicated in the pathogenesis of various inflammatory conditions, including allergen-induced pulmonary inflammation. Indeed, the concentrations of IL-17A and IL-25 are higher in the lungs of asthmatic patients than in those of unaffected individuals. IL-17A and IL-25 share a common receptor subunit, IL-17RA, to which is recruited the adaptor protein Act1 through interactions between its SEFIR domain and the SEFIR domain of the receptor. Liu et al. identified the CC′ loop region within the SEFIR domain of Act1 as critical for receptor-adaptor protein interactions. A cell-permeable peptide based on this region inhibited IL-17– and IL-25–dependent signaling in vitro and reduced disease in a mouse model of pulmonary inflammation, suggesting that such a strategy may provide therapies to treat IL-17–mediated inflammatory diseases in humans. Interleukin-17 (IL-17) and IL-25 signaling induce the expression of genes encoding inflammatory factors and are implicated in the pathology of various inflammatory diseases. Nuclear factor κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-17 or IL-25, and it is recruited to their receptors (IL-17R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to fibroblast growth factor genes) and IL-17R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll–IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB′ loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB′ loop from Act1 or IL-17RA (a common subunit of both IL-17R and IL-25R) did not affect Act1–IL-17RA interactions; rather, deletion of the CC′ loop from Act1 or IL-17RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC′ loop of Act1 bound directly to IL-17RA. A cell-permeable decoy peptide based on the CC′ loop sequence inhibited IL-17– or IL-25–mediated signaling in vitro, as well as IL-17– and IL-25–induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC′ loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-17 or IL-25 signaling.