Accessory protein-like is essential for IL-18-mediated signaling

IL-18 is an essential cytokine for both innate and adaptive immunity. Signaling by IL-18 requires IL-18Ralpha, which binds specifically to the ligand and contains sequence homology to IL-1R and TLRs. It is well established that IL-1R signaling requires an accessory cell surface protein, AcP. Other accessory proteins also exist with roles in regulating TLR signaling, but some have inhibitory functions. An AcP-like molecule (AcPL) has been identified with the ability to cooperate with IL-18Ralpha in vitro; however, the physiological function of AcPL remains unknown. In this study, we demonstrate that IL-18 signals are abolished in AcPL-deficient mice and cells. Splenocytes from mutant mice fail to respond to IL-18-induced proliferation and IFN-gamma production. In particular, Th1 cells lacking AcPL fail to produce IFN-gamma in response to IL-18. AcPL-deficient neutrophils also fail to respond to IL-18-induced activation and cytokine production. Furthermore, AcPL is required for NK-mediated cytotoxicity induced by in vivo IL-18 stimulation. However, AcPL is dispensable for the activation or inhibition of IL-1R and the various TLR signals that we have examined. These results suggest that AcPL is a critical and specific cell surface receptor that is required for IL-18 signaling.     

IRAK4 kinase activity is redundant for interleukin-1 (IL-1) receptor-associated kinase phosphorylation and IL-1 responsiveness

Interleukin-1 (IL-1) stimulation leads to the recruitment of interleukin-1 receptor-associated kinase (IRAK) to the IL-1 receptor, where IRAK is phosphorylated, ubiquitinated, and eventually degraded. Kinase-inactive mutant IRAK is still phosphorylated in response to IL-1 stimulation when it is transfected into IRAK-deficient cells, suggesting that there must be an IRAK kinase in the pathway. The fact that IRAK4, another IRAK family member necessary for the IL-1 pathway, is able to phosphorylate IRAK in vitro suggests that IRAK4 might be the IRAK kinase. However, we now found that the IRAK4 kinase-inactive mutant had the same ability as the wild-type IRAK4 in restoring IL-1-mediated signaling in human IRAK4-deficient cells, including NFkappaB-dependent reporter gene expression, the activation of NFkappaB and JNK, and endogenous IL-8 gene expression. These results strongly indicate that the kinase activity of human IRAK4 is not necessary for IL-1 signaling. Furthermore, we showed that the kinase activity of IRAK4 was not necessary for IL-1-induced IRAK phosphorylation, suggesting that IRAK phosphorylation can probably be achieved either by autophosphorylation or by trans-phosphorylation through IRAK4. In support of this, only the impairment of the kinase activity of both IRAK and IRAK4 efficiently abolished the IL-1 pathway, demonstrating that the kinase activity of IRAK and IRAK4 is redundant for IL-1-mediated signaling. Moreover, consistent with the fact that IRAK4 is a necessary component of the IL-1 pathway, we found that IRAK4 was required for the efficient recruitment of IRAK to the IL-1 receptor complex.     

Pellino 1 is required for interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptor-associated kinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6 (TRAF6) complex

The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NF kappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

IL-1 receptor-associated kinase 1 regulates susceptibility to organ-specific autoimmunity

Infections often precede the development of autoimmunity. Correlation between infection with a specific pathogen and a particular autoimmune disease ranges from moderately strong to quite weak. This lack of correspondence suggests that autoimmunity may result from microbial activation of a generic, as opposed to pathogen-specific host-defense response. The Toll-like receptors, essential to host recognition of microbial invasion, signal through a common, highly conserved pathway, activate innate immunity, and control adaptive immune responses. To determine the influence of Toll/IL-1 signaling on the development of autoimmunity, the responses of wild-type (WT) mice and IL-1R-associated kinase 1 (IRAK1)-deficient mice to induction of experimental autoimmune encephalomyelitis were compared. C57BL/6 and B6.IRAK1-deficient mice were immunized with MOG 35-55/CFA or MOG 35-55/CpG DNA/IFA. WT animals developed severe disease, whereas IRAK1-deficient mice were resistant to experimental autoimmune encephalomyelitis, exhibiting little or no CNS inflammation. IRAK1-deficient T cells also displayed impaired Th1 development, particularly during disease induction, despite normal TCR signaling. These results suggest that IRAK1 and the Toll/IL-1 pathway play an essential role in T cell priming, and demonstrate one means through which innate immunity can control subsequent development of autoimmunity. These findings may also help explain the association between antecedent infection and the development or exacerbations of some autoimmune diseases.     

IL-1 receptor-associated kinase modulates host responsiveness to endotoxin

Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock. Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer. The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction. LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells. We demonstrate that LPS activates IRAK in murine macrophages. IRAK-deficient macrophages, in contrast, are resistant to LPS. Deletion of IRAK disrupts several endotoxin-triggered signaling cascades. Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS. These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection.     

Cutting Edge: IL-1 receptor-associated kinase 4 structures reveal novel features and multiple conformations

IL-1R-associated kinase (IRAK)4 plays a central role in innate and adaptive immunity, and is a crucial component in IL-1/TLR signaling. We have determined the crystal structures of the apo and ligand-bound forms of human IRAK4 kinase domain. These structures reveal several features that provide opportunities for the design of selective IRAK4 inhibitors. The N-terminal lobe of the IRAK4 kinase domain is structurally distinctive due to a loop insertion after an extended N-terminal helix. The gatekeeper residue is a tyrosine, a unique feature of the IRAK family. The IRAK4 structures also provide insights into the regulation of its activity. In the apo structure, two conformations coexist, differing in the relative orientation of the two kinase lobes and the position of helix C. In the presence of an ATP analog only one conformation is observed, indicating that this is the active conformation.     

IL-1 receptor-associated kinase 1 mediates protection against Staphylococcus aureus infection

The interleukin-1 receptor-associated kinase-1 (IRAK-1) mediates signal transduction from Toll-like/IL-1/IL-18 receptors. Though a critical protective role against Staphylococcus aureus infection has been previously attributed to myeloid differentiation factor 88 (MyD88) and IRAK-4, both also involved in TLR/IL-1/IL-18 signaling, the role of IRAK-1 is unknown. IRAK-1-deficient (IRAK-1-/-) and wild-type mice were inoculated i.v. with 2 x 10(7) or 1 x 10(6) S. aureus per mouse to evaluate the role of IRAK-1 in S. aureus sepsis. Since IRAK-1 transduces IL-1R signals, IL-1R-/- mice were also included in experiments. IRAK-1-/- mice are susceptible to a high dose of S. aureus compared to wild-type controls. In contrast to the high mortality and extensive weight loss seen in IL-1R-deficient mice in response to 1 x 10(6) S. aureus, IRAK-1-/- mice are resistant to this low dose of S. aureus. Thus IRAK-1 plays an important role in the host response to staphylococcal sepsis.     

Dendritic cells mediate NK cell help for Th1 and CTL responses: two-signal requirement for the induction of NK cell helper function

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other's functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized "effector/memory" DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-gamma, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, "helper" function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.     

IL-12 is required for anti-OX40-mediated CD4 T cell survival

Engagement of OX40 greatly improves CD4 T cell function and survival. Previously, we showed that both OX40 engagement and CTLA-4 blockade led to enhanced CD4 T cell expansion, but only OX40 signaling increased survival. To identify pathways associated with OX40-mediated survival, the gene expression of Ag-activated CD4 T cells isolated from mice treated with anti-OX40 and -CTLA-4 was compared. This comparison revealed a potential role for IL-12 through increased expression of the IL-12R-signaling subunit (IL-12Rbeta2) on T cells activated 3 days previously with Ag and anti-OX40. The temporal expression of IL-12Rbeta2 on OX40-stimulated CD4 T cells was tightly regulated and peaked approximately 4-6 days after initial activation/expansion, but before the beginning of T cell contraction. IL-12 signaling, during this window of IL-12Rbeta2 expression, was required for enhanced T cell survival and survival was associated with STAT4-specific signaling. The findings from these observations were exploited in several different mouse tumor models where we found that the combination of anti-OX40 and IL-12 showed synergistic therapeutic efficacy. These results may lead to the elucidation of the molecular pathways involved with CD4 T cell survival that contribute to improved memory, and understanding of these pathways could lead to greater efficacy of immune stimulatory Abs in tumor-bearing individuals.     

The role of interleukin 1 receptor-associated kinase-4 (IRAK-4) kinase activity in IRAK-4-mediated signaling

Interleukin 1 receptor (IL-1R)-associated kinase-4 (IRAK-4) is required for various responses induced by IL-1R and Toll-like receptor signals. However, the molecular mechanism of IRAK-4 signaling and the role of its kinase activity have remained elusive. In this report, we demonstrate that IRAK-4 is recruited to the IL-1R complex upon IL-1 stimulation and is required for the recruitment of IRAK-1 and its subsequent activation/degradation. By reconstituting IRAK-4-deficient cells with wild type or kinase-inactive IRAK-4, we show that the kinase activity of IRAK-4 is required for the optimal transduction of IL-1-induced signals, including the activation of IRAK-1, NF-kappaB, and JNK, and the maximal induction of inflammatory cytokines. Interestingly, we also discover that the IRAK-4 kinase-inactive mutant is still capable of mediating some signals. These results suggest that IRAK-4 is an integral part of the IL-1R signaling cascade and is capable of transmitting signals both dependent on and independent of its kinase activity.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx ( approximately 1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H(2)O(2) treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H(2)O(2)-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.     

Th17 cells induce colitis and promote Th1 cell responses through IL-17 induction of innate IL-12 and IL-23 production

Both Th1 and Th17 cells have been implicated in the pathogenesis of inflammatory bowel disease and experimental colitis. However, the complex relationship between Th1 and Th17 cells and their relative contributions to the pathogenesis of inflammatory bowel disease have not been completely analyzed. Although it has been recently shown that Th17 cells can convert into Th1 cells, the underlying in vivo mechanisms and the role of Th1 cells converted from Th17 cells in the pathogenesis of colitis are still largely unknown. In this study, we report that Th17 cells from CBir1 TCR transgenic mice, which are specific for an immunodominant microbiota Ag, are more potent than Th1 cells in the induction of colitis, as Th17 cells induced severe colitis, whereas Th1 cells induced mild colitis when transferred into TCRβxδ(-/-) mice. High levels of IL-12 and IL-23 and substantial numbers of IFN-γ(+) Th1 cells emerged in the colons of Th17 cell recipients. Administration of anti-IL-17 mAb abrogated Th17 cell-induced colitis development, blocked colonic IL-12 and IL-23 production, and inhibited IFN-γ(+) Th1 cell induction. IL-17 promoted dendritic cell production of IL-12 and IL-23. Furthermore, conditioned media from colonic tissues of colitic Th17 cell recipients induced IFN-γ production by Th17 cells, which was inhibited by blockade of IL-12 and IL-23. Collectively, these data indicate that Th17 cells convert to Th1 cells through IL-17 induction of mucosal innate IL-12 and IL-23 production.     

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and toll-like receptor 7-mediated signaling and gene expression

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.