IRAK4 Kinase Activity Research Articles

Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Effect Of MYD88 Mutation In CLL On IRAK4 and BTK Inhibition In Vitro

Recent work in CLL and other lymphoproliferative disorders has established the importance of somatic mutations in the pathogenesis and prognosis of disease. MYD88 L265P mutations occur in approximately 30% of ABC diffuse large B cell lymphomas (ABC DLBCL) and 90% of Waldenstrom's macroglobulinemia (WM) cases and have been shown to be an activating mutation in both cancers. MYD88 is an important adaptor protein for IL-1 and many Toll-like receptors, which signal via dimerization followed by formation of a complex of MYD88 with IRAK4 and IRAK1, leading to activation of IRAK4 kinase activity, IRAK1 phosphorylation and downstream activation of NFκB. Previous work in CLL has demonstrated that MYD88 L265P mutations occur in a smaller subset of patients (2.9-6.25%) and although MYD88 mutation is thought to be a CLL driver mutation, its role in CLL is not clearly understood.We previously reported whole exome sequencing results from a cohort of 160 CLL patients, 10 of whom have MYD88 L265P mutation (6.25%). Eight of ten patients with MYD88 L265P mutation had mutated IGHV, consistent with prior reports of this association. However, patients with MYD88 L265P mutation did not have a younger age at diagnosis, possibly due to the overall younger median age of our patient cohort (54, range 34-77).RNA gene expression profiling of 146 CLL and 20 normal B cell samples revealed that normalized median expression of IRAK1, IRAK4 and NFKB1 appear lower in CLL samples versus normal B cells (IRAK1 p = 0.0001, IRAK4 p = 0.01, NFKB1 p = 0.0005). However, median expression levels of MYD88 appear higher in CLL samples versus normal B cells (p = 0.006). Although MYD88 L265P mutation has been shown to activate BTK in WM, mean BTK protein levels measured by flow cytometry were similar between CLL patients with and without MYD88 mutation (mean % cells positive for BTK, 98.3% in MYD88 L265P mutants vs. 98.16% in wild-type [WT], 3 patients/group), as were levels of phosphorylated BTK (mean % cells positive, 13.28% in MYD88 L265P vs. 14.35% in WT).Chronic BTK-mediated B cell receptor signaling is a hallmark of CLL and studies with the BTK inhibitor ibrutinib (PCI-32765) have demonstrated encouraging therapeutic results in vitro and in vivo. ND-2110 and ND-2158 are potent, highly selective IRAK4 kinase inhibitors which have single agent activity in vitro in ABC DLBCL cell lines as well as synergistic effects with BTK inhibitors. Given these findings, ND-2110 and ND-2158 were ideal candidates to test in CLL. As such, CLL samples with and without MYD88 L265P mutation (3 per group) were cultured in vitro in the presence of 5μM ND-2110 or ND-2158 as single agents or in combination with 2.5μM ibrutinib for 72 hours. A second, higher dose combination was also tested, using 25μM ND-2110 or ND-2158 and 3.75μM ibrutinib. Viability was assessed using Cell TiterGlo and normalized to DMSO treated cells. A general linear model was used to assess the association of % viability with MYD88 L265P mutation, IgM stimulation and combinations of IRAK4 inhibitor compounds and ibrutinib. MYD88 L265P was associated with higher viability, hence less cell killing (p = 0.002 5μM ND-2110 & 2.5μM ibrutinib; p < 0.0001 5μM ND-2158 & 2.5μM ibrutinib; p = 0.005 25μM ND-2158 & 3.75μM ibrutinib). Cells treated with 25μM ND-2158 + 3.75μM ibrutinib had significantly lower viability compared to cells treated with 25μM ND-2158 alone (p < 0.0001). IgM stimulation also resulted in lower viability compared to no stimulation, suggesting sensitization (p = 0.002 25μM ND-2100 + 3.75μM ibrutinib; p < 0.0001 all other combinations). Ibrutinib appeared to have less single agent activity in MYD88 L265P mutants, while the activity of single agent ND-2110 and ND-2158 was more equalized between groups. These preliminary results are currently being confirmed in additional patients.To further characterize the effects of these drugs, we have established a model system in which CLL cells are co-cultured with NIH3T3 cells expressing CD40L, with and without CpG stimulation. Preliminary results show that in one WT MYD88 CLL patient, these conditions resulted in significant induction of Ki67 expression, which was reduced by combination of either ND-2110 or 2158 with ibrutinib.These results suggest that IRAK4 inhibitors in combination with ibrutinib have interesting activity in CLL, which may differ based on MYD88 L265P mutation; experiments are in progress in additional patients to further characterize this effect. Disclosures:Chaudhary:NIMBUS Discovery: Employment. Romero:NIMBUS Discovery: Consultancy, Equity Ownership. Robinson:NIMBUS Discovery: Consultancy. Westlin:NIMBUS Discovery: Employment. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding.

IRAK4 kinase activity is not required for induction of endotoxin tolerance but contributes to TLR2-mediated tolerance

Prior exposure to LPS induces "endotoxin tolerance" that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance is thought to limit the excessive cytokine storm and prevent tissue damage during sepsis but renders the host immunocompromised and susceptible to secondary infections. Tolerance initiated via one TLR can affect cellular responses to challenge via the same TLR ("homotolerance") or through different TLRs ("heterotolerance"). IRAK4, an essential component of the MyD88-dependent pathway, functions as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in TLR4- and TLR2-induced tolerance using macrophages from WT versus IRAK4(KDKI) mice. Whereas IRAK4 kinase deficiency decreased LPS signaling, it did not prevent endotoxin tolerance, as endotoxin pretreatment of WT and IRAK4(KDKI) macrophages inhibited LPS-induced MAPK phosphorylation, degradation of IκB-α and recruitment of p65 to the TNF-α promoter, expression of proinflammatory cytokines, and increased levels of A20 and IRAK-M. Pretreatment of WT macrophages with Pam3Cys, a TLR2-TLR1 agonist, ablated p-p38 and p-JNK in response to challenge with Pam3Cys and LPS, whereas IRAK4(KDKI) macrophages exhibited attenuated TLR2-elicited homo- and heterotolerance at the level of MAPK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance but contributes significantly to TLR2-elicited homo- and heterotolerance.

IRAK4 kinase activity is not required for induction of endotoxin tolerance but significantly contributes to TLR2-mediated tolerance (P1224)

Abstract Prior exposure to LPS induces “endotoxin tolerance” that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance limits the excessive cytokine storm and prevent tissue damage during sepsis, but renders the host immunocompromised and susceptible to secondary infections. IRAK4 acts as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in the induction of TLR4 and TLR2 tolerance using macrophages from wild-type (WT) vs. IRAK4 kinase-dead knock-in (IRAK4KDKI) mice. Pretreatment of WT and IRAK4KDKI macrophages with LPS similarly inhibited LPS-induced degradation of IRAK1 and IκB-α, MAPK phosphorylation, induction of proinflammatory cytokines, and up-regulated expression of negative regulators of TLR signaling, A20 and IRAK-M. Prior exposure of WT macrophages with Pam3Cys, a TLR2-TLR1 agonist, ablated p38 and JNK phosphorylation in response to subsequent challenges with Pam3Cys or LPS. In contrast, IRAK4KDKI macrophages exhibited attenuated TLR2-elicited homo- and heterotolerance at the level p38 and JNK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance, but significantly contributes to TLR2-elicited homo- and heterotolerance.

Complete Dependence on IRAK4 Kinase Activity in TLR2, but Not TLR4, Signaling Pathways Underlies Decreased Cytokine Production and Increased Susceptibility to Streptococcus pneumoniae Infection in IRAK4 Kinase–Inactive Mice

IRAK4 is critical for MyD88-dependent TLR signaling, and patients with Irak4 mutations are extremely susceptible to recurrent bacterial infections. In these studies, mice homozygous for a mutant IRAK4 that lacks kinase activity (IRAK4(KDKI)) were used to address the role of IRAK4 in response to TLR agonists or bacterial infection. IRAK4(KDKI) macrophages exhibited diminished responsiveness to the TLR4 agonist LPS and little to no response to the TLR2 agonist Pam3Cys compared with wild-type macrophages as measured by cytokine mRNA, cytokine protein expression, and MAPK activation. Importantly, we identified two kinases downstream of the MAPKs, MNK1 and MSK1, whose phosphorylation is deficient in IRAK4(KDKI) macrophages stimulated through either TLR2 or TLR4, suggesting that IRAK4 contributes to TLR signaling beyond the initial phosphorylation of MAPKs. Additionally, IRAK4(KDKI) macrophages produced minimal cytokine mRNA expression in response to the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus compared with WT cells, and IRAK4(KDKI) mice exhibited increased susceptibility and decreased cytokine production in vivo upon S. pneumoniae infection. Treatment of infected mice with a complex of polyinosinic-polycytidylic acid with poly-L-lysine and carboxymethyl cellulose (Hiltonol), a potent TLR3 agonist, significantly improved survival of both WT and IRAK4(KDKI) mice, thereby providing a potential treatment strategy in both normal and immunocompromised patients.

IRAK4 Kinase As A Novel Therapeutic Target in the ABC Subtype of Diffuse Large B Cell Lymphoma

AbstractAbstract 62Activating mutations of MyD88, particularly L265P, occur in about 30% of activated B cell-like diffuse large B cell lymphomas (ABC DLBCLs), the more malignant molecular subtype of DLBCL that responds poorly to standard chemotherapy. We found that the oncogenic signal of mutant MyD88 is transduced predominantly through the Interleukin-1 Receptor-Associated Kinase (IRAK) cascade consisting of IRAK4 and IRAK1, resulting in constitutive activation of classical NF-κB signaling. Importantly, using knockdown-rescue experiments, we found that the kinase activity of IRAK4, but not IRAK1, is required for the oncogenic effect of mutant MyD88 in the survival of ABC DLBCL cell lines. As such, we have proposed that inhibitors of IRAK4 kinase activity could have a therapeutic impact in lymphomas with MyD88 mutations. We have identified two potent small molecule IRAK4 inhibitors, ND-2158 and ND-2110, that are highly selective across a panel of more than 300 kinases. When administered to rodents, both compounds lead to >90% suppression of LPS-induced TNF-α release in serum. Both inhibitors are universally toxic towards ABC DLBCL but not GCB DLBCL cell lines, consistent with the highly specific mechanism of action. The molecules demonstrate good pharmacologic drug-like properties and are expected to have a suitable safety profile for clinical evaluation. Mechanistic studies indicate that both inhibitors potently abrogate IRAK4-mediated phosphorylation of IRAK1 and NF-κB activity resulting from the MyD88 (L265P) mutation in ABC DLBCL. Additionally, these agents suppress secretion of the pro-inflammatory cytokines IL-6 and IL-10 by ABC DLBCL cells. A second, parallel survival pathway in ABC DLBCL is engaged by “chronic active” B cell receptor signaling, which can be blocked by inhibiting Bruton’s tyrosine kinase (BTK) either genetically or pharmacologically. Notably, the IRAK4 inhibitors strongly synergized with BTK knockdown in killing multiple ABC DLBCL cell lines. Our results provide a solid rationale for the further development of IRAK4 inhibitors for the therapy of ABC DLBCL and suggest that simultaneous inhibition of B cell receptor signaling may provide superior clinical responses. Disclosures:Romero:NIMBUS Discovery: Employment. Chaudhary:NIMBUS Discovery: Employment. Robinson:NIMBUS Discovery: Consultancy, Employment.

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.

Oncogenic MYD88 mutation drives Toll pathway to lymphoma

MYD88 is an adaptor protein through which most of the Toll-like receptors (TLRs) and receptors for interleukin (IL)-1 and IL-18 cytokines signal.1 MYD88 transduces signals to the nuclear factor κB (NFκB) transcription factors that, in turn, regulate the production of cytokines and anti-apoptotic molecules. In mice, MYD88 deficiency impairs immunity to a range of microorganisms, and in humans absence of MYD88 results in life-threatening recurrent pyogenic bacterial infections.2, 3 Given the central role of MYD88 and TLRs in immunity, overactivity of this pathway has been associated with autoimmune diseases and now, for the first time, TLR signalling is implicated in lymphomagenesis. In a recent Nature letter, Ngo et al.4 report that gain-of-function mutations in MYD88 drive a substantial percentage of B-cell lymphomas. This study demonstrates the need for developing pharmacological inhibitors targeting MYD88 and other components of the Toll pathway in lymphomas.

Induction of endotoxin tolerance in vivo inhibits LPS-inducible activation of IRAK4, p38 and NF-κB, decreases expression of pro-inflammatory cytokines and induces negative regulators of TLR4 signaling (116.14)

Abstract Toll-like receptors (TLRs) are conserved sensors of microbial pathogens and environmental or host-derived “danger” molecules. Development of TLR tolerance in septic patients re-programs monocyte responses to LPS, decreasing expression of pro-inflammatory cytokines without inhibiting anti-inflammatory and antimicrobial mediators, and increases a risk of secondary infections. To address the impact of endotoxin tolerization on activation of proximal TLR4 signaling pathways and expression of regulators of TLR4 signaling, we used an in vivo model of endotoxin tolerance. C57BL/6 mice were i.p. injected with LPS or PBC (used as a control), followed by in vitro challenge of isolated macrophages with medium or LPS and analyses of TLR4 signaling outputs and negative regulatory molecules. Compared to the control group, administration of LPS led to decreased TLR4-driven induction of IRAK4 kinase activity, degradation of IκB-α and phosphorylation of p38, and reduced expression of TNF-α, IL-6, and KC mRNA in peritoneal and splenic macrophages upon in vitro LPS challenge. In contrast, real-time PCR analyses revealed increased expression of IRAK-M, Tollip, SHIP, SARM, and SIKE in macrophages obtained from LPS-injected mice. We conclude that induction of endotoxin tolerance in vivo inhibits expression of pro-inflammatory cytokines and chemokines via impaired activation of IRAK4, p38 and NF-κB and up-regulation of negative regulators of MyD88- and TRIF-dependent pathways.

Oncogenically active MYD88 mutations in human lymphoma

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

IRAK-4 Inhibitors for Inflammation

Interleukin-1 receptor-associated kinases (IRAKs) are key components in the signal transduction pathways utilized by interleukin-1 receptor (IL-1R), interleukin-18 receptor (IL-18R), and Toll-like receptors (TLRs). Out of four members in the mammalian IRAK family, IRAK-4 is considered to be the “master IRAK”, the only family member indispensable for IL-1R/TLR signaling. In humans, mutations resulting in IRAK-4 deficiency have been linked to susceptibility to bacterial infections, especially recurrent pyogenic bacterial infections. Furthermore, knock-in experiments by several groups have clearly demonstrated that IRAK-4 requires its kinase activity for its function. Given the critical role of IRAK-4 in inflammatory processes, modulation of IRAK-4 kinase activity presents an attractive therapeutic approach for the treatment of immune and inflammatory diseases. The recent success in the determination of the 3-dimensional structure of the IRAK-4 kinase domain in complex with inhibitors has facilitated the understanding of the mechanistic role of IRAK-4 in immunity and inflammation as well as the development of specific IRAK-4 kinase inhibitors. In this article, we review the biological function of IRAK-4, the structural characteristics of the kinase domain, and the development of small molecule inhibitors targeting the kinase activity. We also review the key pharmacophores required for several classes of inhibitors as well as important features for optimal protein/inhibitor interactions. Lastly, we summarize how these insights can be translated into strategies to develop potent IRAK-4 inhibitors with desired properties as new anti-inflammatory therapeutic agents.

The kinase activities of interleukin-1 receptor associated kinase (IRAK)-1 and 4 are redundant in the control of inflammatory cytokine expression in human cells

IRAK-1 and IRAK-4 are protein kinases that mediate signaling by Toll/IL1/Plant R (TIR) domain-containing receptors including the IL-1, IL-18, and Toll-like receptors (TLRs). Although well studied in mouse systems, the mechanism by which they function in human systems is less clear. To extend our knowledge of how these proteins regulate inflammatory signaling in human cells, we genetically and pharmacologically manipulated IRAK-1 and IRAK-4 kinase activities in vitro. Ablation of IRAK-4 expression in human umbilical vein endothelial cells (HUVEC) with siRNA suppressed IL-1β induced IL-6 and IL-8 production whereas IRAK-1 siRNA suppressed TNFα induced but not IL-1β induced cytokine production. Complementation of IRAK-4-depleted cells with a kinase-inactive allele restored IL-1β induced cytokine gene expression suggesting that the IRAK-4 kinase activity is dispensable relative to its scaffolding function. Consistent with this finding, an IRAK-4 selective kinase inhibitor (RO6245) that inhibited IRAK-1 degradation failed to block IL-1β induced cytokine production. In contrast, an inhibitor of both IRAK-1 and IRAK-4 (RO0884) reduced IL-1β induced p38 MAP kinase, c-Jun N-terminal kinase activation, and IL-6 production in HUVEC. RO0884 also antagonized IL-1β, TNFα, and TLR-mediated cytokine production in human fibroblast-like synoviocytes and peripheral blood mononuclear cells. Therefore in human cells the non-kinase functions of IRAK-4 are essential, whereas the kinase activity of IRAK-4 appears redundant with that of IRAK-1. Pharmacologic inhibition of both kinases appears necessary to block pro-inflammatory cytokine production.

IRAK‐4 kinase activity‐dependent and ‐independent regulation of lipopolysaccharide‐inducible genes

IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.

IRAK‐4 kinase activity is required for IRAK‐4‐dependent innate and adaptive immune responses

Interleukin-1 receptor-associated kinase (IRAK)-4 is a serine-threonine kinase that plays an important role in innate and adaptive immune responses. While the requirement of IRAK-4 kinase activity has been studied in the context of IL-1R signaling, it is not clear whether IRAK-4 requires its kinase function for all of its roles in the immune system. IRAK-4 kinase-dead knock-in (IRAK-4KD/KD) mice were generated to further elucidate whether IRAK-4 kinase activity is required for IRAK-4 to induce cytokine production. IRAK-4KD/KD mice were impaired in their ability to produce cytokines in response to in vivo challenge with lipopolysaccharide (LPS), a potent TLR4 ligand. Cytokine production was also reduced in macrophages and dendritic cells from IRAK-4KD/KD mice in response to LPS and other TLR ligands. In addition, adaptive immune responses were impaired in IRAK-4KD/KD mice. Although in vitro T cell proliferation in response to TCR activation was unaffected in IRAK-4-deficient mice, in vivo T cell responses to lymphocytic choriomeningitits virus infection were significantly impaired in IRAK-4-knockout mice or mice expressing the kinase-dead mutant of IRAK-4. Collectively, these results indicate that IRAK-4 kinase activity is required for IRAK-4-dependent signaling in innate and adaptive immunity.

IRAK4 in TLR/IL‐1R signaling: Possible clinical applications

A member of the IL-1 receptor (IL-1R)-associated kinase (IRAK) family, IRAK4, has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. IRAK4 kinase-inactive knockin mice have been shown to be completely resistant to LPS- and CpG-induced shock, due to impaired TLR-mediated induction of pro-inflammatory cytokines and chemokines. A reduction of LPS-, R848- and IL-1-mediated mRNA stability contributes to the reduced cytokine and chemokine production in bone marrow (BM)-derived macrophages from IRAK4 kinase-inactive knockin mice: however, not all of the TLR/IL-1R signaling events are ablated in IRAK4 kinase-inactive knockin mice. A paper in this issue of the European Journal of Immunology shows that, while JNK activation is significantly impaired, NF-kappaB and IRF3 activation are retained in the absence of IRAK4 kinase activity. These residual TLR/IL-1R-induced signaling events allow the production of some cytokines and chemokines (including TNFalpha and CXCL1); at early times after the stimulation and induction of a group of TLR-mediated MyD88/IRAK4-independent genes in IRAK4 kinase-inactive knockin cells. Therefore, pharmacological blocking of IRAK4 kinase activity will retain some levels of host defence, while reducing the levels and duration of inflammatory responses, which should provide beneficial therapies for sepsis and chronic inflammatory diseases.

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation

Inflammation is critically involved in atherogenesis. Signaling from innate immunity receptors TLR2 and 4, IL-1 and IL-18 is mediated by MyD88 and further by interleukin-1 receptor activated kinases (IRAK) 4 and 1. We hypothesized that IRAK4 kinase activity is critical for development of atherosclerosis. IRAK4 kinase-inactive knock-in mouse was crossed with the ApoE−/− mouse. Lesion development was stimulated by carotid ligation. IRAK4 functional deficiency was associated with down-regulation of several pro-inflammatory genes, inhibition of macrophage infiltration, smooth muscle cell and lipid accumulation in vascular lesions. Reduction of plaque size and inhibition of outward remodeling were also observed. Similar effects were observed when ApoE−/− mice subjected to carotid ligation were treated with recombinant IL-1 receptor antagonist thereby validating the model in the relevant pathway context. Thus, IRAK4 functional deficiency inhibits vascular lesion formation in ApoE−/− mice, which further unravels mechanisms of vascular inflammation and identifies IRAK4 as a potential therapeutic target.

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.

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.

TLR8-mediated NF-κB and JNK Activation Are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.

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.