IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

Cutting edge: molecular structure of the IL-1R-associated kinase-4 death domain and its implications for TLR signaling

IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 A resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.     

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.     

TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway

Polymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient's functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient's PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient's PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency.     

Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp, Penaeus monodon

The black tiger shrimp (Penaeus monodon) is economically important in many parts of the world, including Thailand. Shrimp immunity is similar to that of other invertebrate organisms; it consists of an innate immunity system. Toll or Toll-like receptors (TLRs) play an essential role in recognizing the cleaved form of the cytokine Sp?tzle, which is processed by a series of proteolytic cascades activated by secreted recognition molecules. We isolated a full-length Toll receptor from P. monodon. The cloned full-length sequence of the PmToll cDNA consists of 4144 nucleotides, containing a 5'-UTR with 366 nucleotides, a 3'-terminal UTR with 985 nucleotides, with a classical polyadenylation signal sequence AATAAA, a poly A-tail with 27 nucleotides, and an open reading frame coding for 931 amino acids. The deduced amino acid sequence of PmToll is a typical type I membrane domain protein, characteristic of TLR functional domains. It includes a putative signal peptide, an extracellular domain consisting of leucine-rich repeats, flanked by cysteine-rich motifs, a single-pass transmembrane portion, and a cytoplasmic TLR domain. PmToll was expressed in all tissues tested, including gill, hemocytes, heart, hepatopancreas, lymphoid organs, muscle, nerve, pleopod, stomach, testis, and ovary. The deduced amino acid of PmToll is closely related to that of other shrimp Tolls, especially FcToll. Further studies elucidating the mechanism of action of Tolls will be of benefit for understanding the defense mechanisms of this economically important aquatic species.     

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.     

Association pattern of interleukin-1 receptor-associated kinase-4 gene polymorphisms with allergic rhinitis in a Han Chinese population

Objective

Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population.

Methods

A population of 379 patients with AR and 333 healthy controls was studied. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 11 single nucleotide polymorphisms (SNPs) in IRAK-4 were selected and individually genotyped.

Results

Significant allelic differences between cases and controls were obtained for the SNP of rs3794262 in the IRAK-4 gene. In the stratified analysis for gender, two SNPs (rs4251431 and rs6582484) in males appeared as significant associations. Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262, rs4251481). None of the selected SNPs in IRAK-4 was associated with total IgE level. The haplotype analyisis indicated GCCTGCGA was significantly associated with AR. The SNP-SNP interaction information analysis indicated that the selected sets of polymorphisms had no synergistic effect.

Conclusions

Our findings did not support the potential contribution of the IRAK-4 gene to serum IgE levels. However, the results demonstrated a gender- and allergen-dependant association pattern between polymorphisms in IRAK-4 and AR in Chinese population.     

Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus

Toll receptors are cell-surface receptors acting as pattern recognition receptors (PRRs) that are involved in the signaling pathway for innate immunity activation and are genetically conserved from insects to mammals. Tolls from penaeid shrimp are found in white leg shrimp Litopenaeus vannamei (lToll) and black tiger shrimp Penaeus monodon (PmToll). However, the molecular ligand-recognition patterns and identification of these penaeid Toll classes remain unknown. Here, we report cDNA cloning of a new type of Toll receptor gene (MjToll) from kuruma shrimp, Marsupenaeus japonicus, and the modulation of expression by immunostimulation. The full length cDNA of MjToll gene has 3095 nucleotides coding for a putative protein of 1009 amino acids. The MjToll gene is constitutively expressed in the gill, gut, lymphoid organ, heart, hematopoietic organ, hemocyte, ventral abdominal nerve cord, eyestalk neural ganglia and brain tissues. The MjToll gene expression was significantly increased (76-fold) as compared to a control in lymphoid organ stimulated with peptidoglycan at 12h, in vitro. lToll gene showed high similarity to PmToll gene with 96.9% identity; however, MjToll gene exhibited a percentage identity of 59% with that of penaeid Toll homologues. Therefore, this suggests that the identified MjToll gene belongs to the other class of Toll receptors in shrimp.     

IRAK-4 in macrophages contributes to inflammatory osteolysis of wear particles around loosened hip implants

TLRs recognizing PAMPS play a role in local immunity and participate in implant-associated loosening. TLR-mediated signaling is primarily regulated by IL-1 receptor associated kinase-M (IRAK-M) negatively and IRAK-4 positively. Our previous studies have proved that wear particles promote endotoxin tolerance in macrophages by inducing IRAK-M. However, whether IRAK-4 is involved in inflammatory osteolysis of wear particles basically, and the specific mechanism of IRAK-4 around loosened hip implants, is still unclear. IRAK-4 was studied in the interface membranes from patients in vivo and in particle-stimulated macrophages to clarify its role. Also, IL-1β and TNF-α levels were measured after particle and LPS stimulation in macrophages with or without IRAK-4 silenced by siRNA. Our results showed that the interface membranes around aseptic and septic loosened prosthesis expressed more IRAK-4 compared with membranes from osteoarthritic patients. IRAK-4 in macrophages increased upon particle and LPS stimulation. In the former, IL-1β and TNF-α levels were lower compared with those of LPS stimulation, and IRAK-4 siRNA could suppress production of pro-inflammatory cytokines. These findings suggest that besides IRAK-M, IRAK-4 also plays an important role in the local inflammatory reaction and contributes to prosthesis loosening.     

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.     

Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.     

Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.     

Metal ion-mediated reduction in surface entropy improves diffraction quality of crystals of the IRAK-4 death domain.

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is an essential component of innate immunity in mice and humans. IRAK-4 is a bipartite protein composed of a death domain (DD) that mediates molecular recognition, and a catalytic kinase domain. Structure determination of the proteolytically stable, soluble IRAK-4 DD was hampered by poor diffraction quality. Addition of manganese (II) chloride to the crystallization solution produced significant improvements in diffraction, and the structure has been determined to 1.7-Angstrom resolution. Examination of the IRAK-4 DD crystal structure reveals a single manganese ion coordinated to surface residues lysine-21 and aspartate-24. Coordination of the manganese ion resulted in a reduction in the surface entropy at this region of the molecule, by generating a contact-forming and conformationally homogenous surface patch. Prior studies have shown that surface entropy reduction by mutation of surface residues with large flexible side chains (i.e., Lys and Glu) to smaller side chains results in the production of diffraction-quality crystals. The intrinsic high surface entropy of Lys residues can also be decreased by reductive methylation. Our results suggest that screening of manganese ions as a crystallization additive may also facilitate ordered crystallization by reduction of surface entropy. Given the quick and inexpensive nature of screening, this technique is likely to be amenable to high-throughput methods such as those employed by Protein Structure Initiatives.     

Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis

As a crucial component in TLR/IL-1R signaling pathways, IRAK-4 plays a central role in innate and adaptive immunity. In the present study, the cDNA of IRAK-4 was cloned for the first time from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of csIRAK-4 was 2149 bp and contained a 168 bp 5′ UTR, a 580 bp 3′ UTR and a 1401 bp CDS. The predicted protein sequence of csIRAK-4 had two typical domains, a death domain (DD) at the N terminus and a serine/threonine/tyrosine protein kinase domain (STYKc) at the C terminus. RT-PCR showed that csIRAK-4 mRNA was detected in all tested tissues, especially in immune-related organs, gonads and brain. After injected with inactivated Vibrio anguillarum, the expressions of csIRAK-4 were up-regulated significantly (P < 0.05) in spleen and head kidney. During development, csIRAK-4 was expressed at all selected stages and low-level expression was detected at metamorphosis. Taken together, the present study indicated that csIRAK-4 played a crucial role in immune responses and might be involved in the process of development.     

Regulation of IRAK-4 kinase activity via autophosphorylation within its activation loop

Interleukin-1 stimulation leads to the recruitment of MyD88, interleukin-1 receptor-associated kinase 1 (IRAK-1) and interleukin-1 receptor-associated kinase 4 (IRAK-4) to the IL-1 receptor. The formation of the IL-1 receptor complex triggers a series of IRAK-1 autophosphorylations, which result in activation. IRAK-4 is upstream of IRAK-1 and may act as IRAK-1 kinase to transmit the signal. To date, there is no upstream kinase reported for IRAK-4; the activation mechanism of IRAK-4 remains poorly understood. Here, for the first time, we report three autophosphorylation sites that are responsible for IRAK-4 kinase activity. LC-MS/MS analysis has identified phosphorylations at T342, T345, and S346, which reside within the activation loop. Site-directed mutants at these positions exhibit significant reductions in the catalytic activity of IRAK-4 (T342A: 57%; T345A: 66%; S346A: 50%). The absence of phosphorylation in kinase-dead IRAK-4 indicates that phosphorylations in the activation loop result from autophosphorylation rather than from phosphorylation by an upstream kinase. Finally, we demonstrate that autophosphorylation is an intramolecular event as wild-type IRAK-4 failed to transphosphorylate kinase-inactive IRAK-4. The present data indicate that the kinase activity of IRAK-4 is dependent on the autophosphorylations at T342, T345, and S346 in the activation loop.     

Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases

Members of the Toll-like receptor (TLR) and IL-1 receptor (IL-1R) family initiate signalling pathways that shape innate immunity. Pellino proteins have recently been implicated as evolutionary conserved scaffold proteins in TLR/IL-1R signalling leading to nuclear factor-kappaB and mitogen activated protein kinase-dependent gene expression. We found that Pellino proteins contain a new RING-like motif. Because RING motifs are a feature of a subclass of E3-ubiquitin-ligases that target specific proteins for ubiquitination, we suggest that Pellino proteins are involved in TLR/IL-1R signalling not only as scaffold proteins but also as RING E3-ubiquitin-ligases. In support of this hypothesis we show that Pellino proteins induce IRAK-1 polyubiquitination in a RING-dependent manner. We further propose a model in which Pellino-mediated IRAK-1 polyubiquitination regulates TLR/IL-1R signalling.