Construction of IRAK4 inhibitor activity prediction model based on machine learning.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a crucial serine/threonine protein kinase that belongs to the IRAK family and plays a pivotal role in Toll-like receptor (TLR) and Interleukin-1 receptor (IL-1R) signaling pathways. Due to IRAK4's significant role in immunity, inflammation, and malignancies, it has become an intriguing target for discovering and developing potent small-molecule inhibitors. Consequently, there is a pressing need for rapid and accurate prediction of IRAK4 inhibitor activity. Leveraging a comprehensive dataset encompassing activity data for 1628 IRAK4 inhibitors, we constructed a prediction model using the LightGBM algorithm and molecular fingerprints. This model achieved an R2 of 0.829, an MAE of 0.317, and an RMSE of 0.460 in independent testing. To further validate the model's generalization ability, we tested it on 90 IRAK4 inhibitors collected in 2023. Subsequently, we applied the model to predict the activity of 13,268 compounds with docking scores less than - 9.503 kcal/mol. These compounds were initially screened from a pool of 1.6 million molecules in the chemdiv database through high-throughput molecular docking. Among these, 259 compounds with predicted pIC50 values greater than or equal to 8.00 were identified. We then performed ADMET predictions on these selected compounds. Finally, through a rigorous screening process, we identified 34 compounds that adhere to the four complementary drug-likeness rules, making them promising candidates for further investigation. Additionally, molecular dynamics simulations confirmed the stable binding of the screened compounds to the IRAK4 protein. Overall, this work presents a machine learning model for accurate prediction of IRAK4 inhibitor activity and offers new insights for subsequent structure-guided design of novel IRAK4 inhibitors.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Understanding the immune profile of sudden infant death syndrome - proteomic perspectives.

AIM: The aim of this study was to investigate a panel of immune proteins in cases of sudden infant death syndrome (SIDS). It was hypothesised that, in at least a subset of SIDS, a dysregulated immune response may be a contributing factor leading to death. METHODS: The subjects included 46 SIDS cases and 41 controls autopsied at the Department of Forensic Sciences, Norway. The causes of death in the controls were accidents/trauma. Samples of cerebrospinal fluid (CSF) were analysed quantitatively by Proximity Extension Assay (PEA). RESULTS: Initial results revealed that normalised protein expression differed in 35 proteins. For the purposes of this report five proteins that are involved in immune system were selected for analysis: IFNLR1 (p = 0.003), IL10 (p = 0.007), IRAK4 (p < 0.001) and IL6 (p = 0.035); all had lower protein concentrations in SIDS cases compared to controls except for CD28 (p = 0.024) which had higher protein concentrations in SIDS cases. CONCLUSION: The results confirm previous studies indicating that a dysregulation of the immune system may be a predisposing factor for SIDS. The results may indicate that these aberrant protein concentrations could lead to an inadequate response to immune triggers and uncontrolled defence mechanisms towards the common cold or other non-fatal infections.

The recent advance of Interleukin-1 receptor associated kinase 4 inhibitors for the treatment of inflammation and related diseases.

The interleukin-1 receptor associated kinase 4 (IRAK-4) is a member of serine-threonine kinase family, which plays an important role in the regulation of interleukin-1 receptors (IL-1R) and Toll-like receptors (TLRs) related signaling pathways. At present, the IRAK-4 mediated inflammation and related signaling pathways contribute to inflammation, which are also responsible for other autoimmune diseases and drug resistance in cancers. Therefore, targeting IRAK-4 to develop single-target, multi-target inhibitors and proteolysis-targeting chimera (PROTAC) degraders is an important direction for the treatment of inflammation and related diseases. Moreover, insight into the mechanism of action and structural optimization of the reported IRAK-4 inhibitors will provide the new direction to enrich the clinical therapies for inflammation and related diseases. In this comprehensive review, we introduced the recent advance of IRAK-4 inhibitors and degraders with regards to structural optimization, mechanism of action and clinical application that would be helpful for the development of more potent chemical entities against IRAK-4.

Design, synthesis, and pharmacological evaluation of N-(3-carbamoyl-1H-pyrazol-4-yl)-1,3-oxazole-4-carboxamide derivatives as interleukin-1 receptor-associated kinase 4 inhibitors with reduced potential for cytochrome P450 1A2 induction.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical molecule in Toll-like receptor/interleukin-1 receptor signaling and an attractive therapeutic target for a wide range of inflammatory and autoimmune diseases as well as cancers. In our search for novel IRAK4 inhibitors, we conducted structural modification of a thiazolecarboxamide derivative 1, a lead compound derived from high-throughput screening hits, to elucidate structure-activity relationship and improve drug metabolism and pharmacokinetic (DMPK) properties. First, conversion of the thiazole ring of 1 to an oxazole ring along with introduction of a methyl group at the 2-position of the pyridine ring aimed at reducing cytochrome P450 (CYP) inhibition were conducted to afford 16. Next, modification of the alkyl substituent at the 1-position of the pyrazole ring of 16 aimed at improving CYP1A2 induction properties revealed that branched alkyl and analogous substituents such as isobutyl (18) and (oxolan-3-yl)methyl (21), as well as six-membered saturated heterocyclic groups such as oxan-4-yl (2), piperidin-4-yl (24, 25), and dioxothian-4-y (26), are effective for reducing induction potential. Representative compound AS2444697 (2) exhibited potent IRAK4 inhibitory activity with an IC50 value of 20 nM and favorable DMPK properties such as low risk of drug-drug interactions mediated by CYPs as well as excellent metabolic stability and oral bioavailability.

Design, synthesis and pharmacological evaluation of 2,3-dihydrobenzofuran IRAK4 inhibitors for the treatment of diffuse large B-cell lymphoma.

Interleukin-1 receptor associated kinase 4 (IRAK4) is a critical mediator of MYD88 L265P-induced NF-κB activation, indicating it is a promising therapeutic target for diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of 2,3-dihydrobenzofuran IRAK4 inhibitors through structure-based drug design. The representative compound 22 exhibited strong IRAK4 inhibitory potency (IRAK4 IC50 = 8.7 nM), favorable kinase selectivity and high antiproliferative activity against the MYD88 L265P DLBCL cell line (OCI-LY10 IC50 = 0.248 µM). Compound 22 also exhibited the ability to inhibit the activation of IRAK4 signaling pathway and induce apoptosis in MYD88 L265P DLBCL cell line. In combination with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, 22 showed enhanced apoptosis-inducing effect and antiproliferative potency. The most advanced compound 22 in this inhibitor series holds promise for further development into efficacious and selective IRAK4 inhibitors for the treatment of DLBCL.

Deciphering the intrinsic dynamics of unphosphorylated IRAK4 kinase bound to type I and type II inhibitors.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a vital protein involved in Toll-like and interleukin-1 receptor signal transduction. Several studies have reported regarding the crystal structure, dynamic properties, and interactions with inhibitors of the phosphorylated form of IRAK4. However, no dynamic properties of inhibitor-bound unphosphorylated IRAK4 have been previously studied. Herein, we report the intrinsic dynamics of unphosphorylated IRAK4 (uIRAK4) bound to type I and type II inhibitors. The corresponding apo and inhibitor-bound forms of uIRAK4 were subjected to three independent simulations of 500 ns (total 1.5 µs) each, and their trajectories were analyzed. The results indicated that all three systems were relatively stable, except for the type II inhibitor-bound form of uIRAK4, which exhibited less compact folding and higher solvent surface area. The intra-hydrogen bonds corroborated the structural deformation of the type-II inhibitor-bound complex, which could be attributed to the long molecular structure of the type-II inhibitor. Moreover, the type II inhibitor bound to uIRAK4 showed higher binding free energy with uIRAK4 than the type I inhibitor. The free energy landscape analysis showed a reorientation of Phe330 side chain from the DFG motif at different metastable states for all the systems. The intra-residual distance between residues Lys213, Glu233, Tyr262, and Phe330 suggests a functional interplay when the inhibitors are bound to uIRAK4, thereby hinting at their crucial role in the inhibition mechanism. Ultimately, the intrinsic dynamics study observed between type I/II inhibitor-bound forms of uIRAK4 may assist in better understanding the enzyme and designing therapeutic compounds.

Recent advances in small molecule inhibitors of interleukin‑1 receptor-associated kinase / 药学学报

Interleukin-1 receptor associated kinase 4 (IRAK-4), acting as a serine threonine kinase, is considered as a key signal node for the transduction of IL-1R family and TLRs signal pathway. Studies have found that IRAK-4 has a hand in many signal pathways, involving the inflammatory response of human joints, intestines, liver and nervous system, as well as other autoimmune diseases. It is also one of the causes of drug resistance of some cancer cells. Therefore, IRAK-4 tends to be an effective therapeutic target for inflammatory diseases and cancer. The prospects for the development of drugs in this pathway is to develop novel IRAK-4 small molecule inhibitors and investigate their safety and effectiveness, enrich the clinical treatment of inflammatory and cancer diseases finally. This paper classified and summarized the latest research progress on small molecule inhibitors of IRAK-4 signaling pathway according to structures of the compounds, in order to provide assistances and references for the research and development of related drugs.

Effects of adenovirus-mediated knockdown of IRAK4 on synovitis in the osteoarthritis rabbit model.

BACKGROUND: The use of interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor as a treatment for the inflammatory joint disease is a promising method. However, its underlying mechanism in osteoarthritis (OA) remains unclear. The purpose of this study is to look into the effects of adenovirus-mediated knockdown of IRAK4 on synovitis in the OA rabbit model. METHODS: Ad-shIRAK4 was injected two weeks after anterior cruciate ligament resection. Six weeks later, the rabbits were killed. The expression of IRAK4, TNFR-associated factor 6(TRAF6), TGF-activated kinase 1(TAK1), p-IKB kinase (p-IKK), p-nuclear factor kappa-B (p-NFκB), p38, and p-p38 in the synovial membrane was detected by western blot, qRT-PCR, and immunohistochemistry analysis. Immunohistochemistry was to detect the expression of IRAK4 proteins in articular cartilage. H&E staining was to assess the pathological changes of synovium and cartilage. The levels of interleukin (IL)-1ß, tumor necrosis factor-α(TNF-α), and MMP-13 in the synovial fluid were measured by ELISA. X-ray and micro-computerized tomography (µCT) scans were used to assess knee joint conditions and microstructure of subchondral bone. RESULTS: IRAK4 expression levels in synovial tissues of the OA model group exhibited a significant upward trend. Ad-shIRAK4 significantly reduced IRAK4 mRNA expression in synovium tissues. Notably, Ad-shIRAK4 suppressed the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling. In addition, in the Ad-shIRAK4 treatment group, we can see less inflammatory cell infiltration and reduced hyperplasia and angiogenesis. The levels of IL-1ß, TNF-α, and MMP-13 in the synovial fluid in the OA model group were significantly higher than that in the control group, which were reduced by Ad-shIRAK4 treatment. Finally, Results of HE stains, immunohistochemistry, and µCT showed that Ad-shIRAK4 treatment has a protective effect on cartilage damage. CONCLUSIONS: IRAK4 is significantly upregulated in the synovium from the osteoarthritis rabbit model. In addition, Ad-shIRAK4 reduced the expression of IRAK4 and suppressed TLR/IL-1R signaling in the synovium from the osteoarthritis rabbit model. Ad-shIRAK4 could alleviate synovitis and cartilage degradation in the osteoarthritis rabbit model, and thus alleviate the symptoms of OA and prevent the progression of OA.

Creating Awareness for Primary Immunodeficiencies in Japan.

Primary immunodeficiency (PID) is primarily characterized by susceptibility to infectious diseases. In addition, patients with some type of PID are prone to develop autoimmune, autoinflammatory, or malignant diseases. Therefore, the term, inborn errors of immunity (IEI), has been more used rather than PID. In recent years, the number of diseases which belong to PID has been increasing. There were approximately 110 diseases in the report of International Union of Immunological Societies in 1999. Since then, the number increased to 430 diseases in the latest IUIS report in 2019. We conducted PID nationwide survey in Japan for 3 times in the last 15 years. These studies were focused on incidence and complications of PID, the clinical course of viral infection, and methods to prevent infectious diseases in PID patients. For the awareness of PID, it is essential to know the general and fundamental information of PID patients. Needless to say, we need it to offer appropriate medical services for PID patients. Moreover, chances to provide answers to the questionnaires and seeing the results of the analysis should contribute to the awareness of PID among doctors. In this review, I am going to summarize the results of 3 nationwide survey in Japan, and pick up interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency as an example for creating awareness for its appropriate management.

Case Report: Pseudomonas can take a toll on a patient.

Pseudomonas aeruginosa ( P. aeruginosa) is an aerobic Gram-negative bacterium that is implicated in the development of severe systemic infections among pediatric patients.  It is identified in hospitalized chronically ill pediatric patients in association with genitourinary, respiratory tract, and skin or soft tissue infections as well as severe and life-threating infection including sepsis.  A variety of immunologic mechanisms play a vital role in the host defense mechanisms against invasive infections with P. aeruginosa. Rarely, specific inborn errors of immune function are implicated in deficiencies that predispose to invasive infections with P. aeruginosa.  Innate immune function including germ-line encoded pattern recognition receptors such as toll-like receptors (TLRs) and their downstream signaling is vital in the host defense against P. aeruginosa through the generation of antimicrobial peptides, cytokines/chemokines, and shaping of adaptive immune responses. Herein, we describe a previously healthy two-year-old female with an invasive skin, soft tissue, and central nervous system infection secondary to P. aeruginosa.  The invasive nature of this infection prompted a careful evaluation for an inborn error of immunity. Decreased cytokine response to agonists of TLRs was documented. Targeted sequencing of interleukin-1 receptor-associated kinase (IRAK)-4 documented a homozygous deletion of exons 8-13 consistent with IRAK-4 deficiency.  This report provides a vital educative message in the existing scientific literature by underscoring the importance of considering inborn errors of immunity in all patients with severe P. aeruginosa infections.  Functional assessments of immune function often in combination with sequencing can accurately assign a diagnosis in a timely fashion allowing for definitive treatment and the use of necessary supportive care.

Interleukin-1 receptor-associated kinase 4 as a potential biomarker: Overexpression predicts poor prognosis in patients with glioma.

The undetectable onset of glioma and the difficulty of surgery lead to a poor prognosis. Appropriate biomarkers for diagnosis and treatment need to be identified. Interleukin-1 receptor-associated kinase 4 (IRAK4) is involved in the initiation and progression of cancer. However, up until now, no report has revealed the relationship between IRAK4 and glioma. The present study aimed to examine the expression of IRAK4 in glioma, and to determine if there was a relationship between IRAK4 expression and clinical outcomes or survival prognosis. Thousands of glioma tissue samples and corresponding clinical information were obtained from various databases. Then a series of bioinformatics methods were used to reveal the role of IRAK4 in glioma. Finally, reverse transcription-quantitative PCR technology was used to verify the bioinformatics results. The study found that the expression of IRAK4 was significantly increased in glioma compared with the control brain tissue samples, and IRAK4, as an independent prognostic factor, shortened the overall survival time of patients with glioma. Gene Set Enrichment Analysis showed that IRAK4 promoted the activation of cell signalling pathways, such as NOD-like and Toll-like receptor signalling pathways. Co-expression analysis showed that the expression of IRAK4 was correlated with CMTM6, MOB1A and other genes. The present study demonstrated the role of IRAK4 as an oncogene in the pathological process of glioma for the first time, and highlights the potential of IRAK4 as a biomarker for prognostic evaluation and treatment of glioma.

Interleukin-1 receptor-associated kinase-4 gene variation may increase post-bronchiolitis asthma risk.

AIM: Evidence based on studies of the encoding genes suggests that interleukin-1 receptor-associated kinase-4 (IRAK4) plays a role in childhood asthma and allergy. Our aim was to evaluate the associations of six IRAK4 gene polymorphisms with presence of asthma and allergic rhinitis and use of inhaled corticosteroids (ICSs) for asthma at 5-7 and 11-13 years of ages after hospitalisation for bronchiolitis at younger than 6 months of age. METHODS: IRAK4 rs4251513, rs4251520, rs4251522, rs4251578, rs79154645 and rs13852554 polymorphisms were determined in 141 former bronchiolitis patients prospectively followed up until 5-7 and in 125 children until 11-13 years of age. RESULTS: The homozygous variant IRAK4 rs4251513 genotype was associated with the presence of asthma and allergic rhinitis and use of ICSs at 5-7 and 11-13 years of ages in univariate analyses. Statistical significance remained for the presence of asthma and use of ICSs but was lost in the case of allergic rhinitis in multivariate analyses. The adjusted odds ratios were 3.48 and 4.16 for asthma and 5.22 and 14.00 for ICS use at these two ages. CONCLUSION: The homozygous variant IRAK4 rs4251513 genotype was constantly associated with post-bronchiolitis asthma and asthma medication in school-aged children.

Case Report: Extensive Phosphorylation of Interleukin-1 Receptor-Associated Kinase 4 in a Patient With Schnitzler Syndrome.

Schnitzler syndrome (SchS) is a rare autoinflammatory disease, characterized by urticarial rash, recurrent fever, osteo-articular pain/arthritis with bone condensation, and monoclonal gammopathy. Diagnosis may be difficult due to overlapping signs with other diseases. Here, we describe the case of a 62-year-old man with SchS, who was initially misdiagnosed with multicentric Castleman disease (MCD). As excessive release of IL-6 is characteristic of MCD, in contrast to IL-1 in SchS, we measured the phosphorylation of intracellular signaling proteins of the respective pathways by flow cytometry. We found a distinct increase of phosphorylated IRAK-4 in our patient's B cells and monocytes while phosphorylation of STAT-3 was low, suggesting predominant IL-1 signaling. In accordance with these results and the classification criteria, we established the diagnosis of SchS instead of MCD and commenced therapy with the IL-1 receptor antagonist anakinra. We observed a rapid remission of signs accompanied by a reduction of phosphorylated IRAK-4 to normal levels. In conclusion, we propose phosphorylated IRAK-4 in B cells and monocytes as a potential marker for diagnosis of SchS and for treatment response to IL-1 blockade.

Investigational IRAK-4 inhibitors for the treatment of rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease that can lead to permanent disability and deformity. Despite current treatment modalities, many patients are still unable to reach remission. Interleukin-1 receptor-associated kinase 4 (IRAK-4) inhibitors are novel agents designed to suppress immune signaling pathways involved in inflammation and joint destruction in RA. Four IRAK-4 inhibitors have entered clinical trials. AREAS COVERED: This review summarizes the current stage of development of IRAK-4 inhibitors in clinical trials, detailing their chemistry, pharmacokinetics, and therapeutic potential in the treatment of RA. PubMed, Embase and restricted Google searches were conducted using the term 'IRAK-4', and publicly accessible clinical trial databases were reviewed. EXPERT OPINION: IRAK-4 inhibitors are an exciting therapeutic option in RA management because unlike other targeted disease-modifying agents, they target the innate immune system. The role of IRAK-4 as a key component of Toll/Interleukin-1 receptor signaling and its potential for a low rate of infectious complications is particularly exciting and this may facilitate their use in combination treatment. A key aspect of upcoming clinical trials will be the identification of biomarkers predictive of treatment efficacy, which will help to define if and how they will be used in the clinic.

Interleukin-1 receptor-associated kinase 4 inhibitor interrupts toll-like receptor signalling and sensitizes chronic lymphocytic leukaemia cells to apoptosis.

Chronic lymphocytic leukaemia (CLL) cells are strongly influenced by microenvironmental signals through the activation of distinct membrane receptors including the B-cell receptor and toll-like receptors (TLR). Recapitulating TLR stimulation in vitro by treating CLL cells with the TLR9 ligand CpG can induce metabolic activation and protection from apoptosis. We hypothesized that interfering with TLR signalling may be beneficial for treating CLL, and we tested in preclinical studies the effect of a specific interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitory small molecule on primary leukaemic cells isolated from the peripheral blood of patients. We observed that IRAK4, an upstream kinase of the TLR pathway, is expressed in patients with CLL, and lower IRAK4 mRNA levels associate with a better outcome. The specific IRAK4 inhibitor disrupted TLR signalling as assessed by reduction of the specific biomarkers NFKBIZ and interleukin-6 mRNAs, and restrained the protective effect of in vitro TLR stimulation on cell viability. To note, IRAK4 inhibitor induced p53 and triggered apoptosis. Co-treatment of CLL cells with increasing concentrations of IRAK4i and the Bruton tyrosine kinase inhibitor ibrutinib demonstrated a synergistic effect. Our results suggest that targetting IRAK4 may represent a novel approach in CLL and may be combined with other signalling inhibitors.

Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the treatment of mutant MYD88 L265P diffuse large B-cell lymphoma.

Harboring MYD88 L265P mutation triggers tumors growth through the activation of NF-κB by interleukin-1 receptor associated kinase 4 (IRAK4) in diffuse large B-cell lymphoma (DLBCL), highlighting IRAK4 as a therapeutic target for tumors driven by aberrant MYD88 signaling. Herein, we report the design, synthesis, and structure-activity relationships of imidazo[1,2-b]pyridazines as potent IRAK4 inhibitors. The representative compound 5 exhibited excellent IRAK4 potency (IRAK4 IC50 = 1.3 nM) and favorable kinase selectivity profile. It demonstrated cellular selectivity for activated B cell-like (ABC) subtype DLBCL with MYD88 L265P mutation in cytotoxicity assay. The kinase inhibitory efficiency of compound 5 was further validated by Western blot analysis of phosphorylation of IRAK4 and downstream signaling in OCI-LY10 and TMD8 cells. Besides, combination of compound 5 and BTK inhibitor ibrutinib synergistically reduced the viability of TMD8 cells. These results indicated that compound 5 could be a promising IRAK4 inhibitor for the treatment of mutant MYD88 DLBCL.

A novel interleukin-1 receptor-associated kinase-4 from thick shell mussel Mytilus coruscus is involved in inflammatory response.

Interleukin-1 receptor-associated kinase-4 (IRAK4) is considered as the most upstream kinase of IRAKs and plays a vital role in Toll-like receptor/Interleukin-1 receptor (TLR/IL-1R) signal transduction. In the present study, IRAK4 from thick shell mussel Mytilus coruscus (McIRAK4) was identified and characterized. McIRAK4 showed the most similarity to its counterparts in bivalves. The conserved death domain (DD) and catalytic domain of serine/threonine kinases (STKc) were predicted in all examined IRAK4s. McIRAK4 transcripts were constitutively expressed in all examined tissues with the higher expression level in immune related tissues, and were significantly induced in haemocytes upon lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further, the expression of McIRAK4 was obviously repressed by dsRNA mediated RNA interference (RNAi), meanwhile the proinflammatory cytokines TNF-alpha and IL17 were down-regulated while the antiinflammatory cytokine TGF-ß was up-regulated. Additionally, McIRAK4 showed a global cytoplasmic localization in HEK293T cells through fluorescence microscopy. These results collectively indicated that McIRAK4 is one member of IRAK4 subfamily and might play the potential signal transducer role in inflammatory response. The present study provides supplement for TLR-mediated signaling pathway triggered by pathogenic invasions in thick shell mussel, and contributes to the clarification of the innate immune response in molluscs.

Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Cloning, expression analysis and functional characterization of an interleukin-1 receptor-associated kinase 4 from Apostichopus japonicus.

Interleukin 1 receptor-associated kinase 4 (IRAK4) is a key factor in TLR-mediated host immune function. In this study, an IRAK4 homologue molecule was identified from Apostichopus japonicus (designated as AjIRAK4) by RACE approach. The full-length cDNA of AjIRAK4 was of 2024 bp containing an open reading frame of 1311 bp encoding a 436-amino-acid (aa) residue polyprotein with the typical death domain (10-113aa) and the kinase domain (160-426aa). The mRNA transcripts of AjIRAK4 displayed constitutively expressed in all examined tissues with highest expression in the muscles (7.20-fold increase compared to the coelomocytes). The pathogen Vibrio splendidus challenge and LPS exposure could both significantly up-regulate the mRNA expression of AjIRAK4. Silencing AjIRAK4 in vitro and in vivo could inhibit the expression of TLR members at mRNA and protein levels, suggesting AjIRAK4 was an important component of TLR cascade in sea cucumber. More importantly, knockdown of AjIRAK4 by specific siRNA resulted in the significant promotion of coelomocyte apoptosis by 1.82-fold increase in vitro and 1.95-fold in vivo. Taken together, all our results suggested that AjIRAK4 might be served as coelomocyte apoptosis regulator via TLR cascade.