IRAK4 Signaling Drives Resistance to Checkpoint Immunotherapy in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: Checkpoint immunotherapy is largely ineffective in pancreatic ductal adenocarcinoma (PDAC). The innate immune nuclear factor (NF)-κB pathway promotes PDAC cell survival and stromal fibrosis, and is driven by Interleukin-1 Receptor Associated Kinase-4 (IRAK4), but its impact on tumor immunity has not been directly investigated. METHODS: We interrogated The Cancer Genome Atlas data to identify the correlation between NF-κB and T cell signature, and a PDAC tissue microarray (TMA) to correlate IRAK4 activity with CD8+ T cell abundance. We performed RNA sequencing (RNA-seq) on IRAK4-deleted PDAC cells, and single-cell RNA-seq on autochthonous KPC (p48-Cre/TP53f/f/LSL-KRASG12D) mice treated with an IRAK4 inhibitor. We generated conditional IRAK4-deleted KPC mice and complementarily used IRAK4 inhibitors to determine the impact of IRAK4 on T cell immunity. RESULTS: We found positive correlation between NF-κB activity, IRAK4 and T cell exhaustion from The Cancer Genome Atlas. We observed inverse correlation between phosphorylated IRAK4 and CD8+ T cell abundance in a PDAC tissue microarray. Loss of IRAK4 abrogates NF-κB activity, several immunosuppressive factors, checkpoint ligands, and hyaluronan synthase 2, all of which drive T cell dysfunction. Accordingly, conditional deletion or pharmacologic inhibition of IRAK4 markedly decreased tumor desmoplasia and increased the abundance and activity of infiltrative CD4+ and CD8+ T cells in KPC tumors. Single-cell RNA-seq showed myeloid and fibroblast reprogramming toward acute inflammatory responses following IRAK4 inhibition. These changes set the stage for successful combination of IRAK4 inhibitors with checkpoint immunotherapy, resulting in excellent tumor control and markedly prolonged survival of KPC mice. CONCLUSION: IRAK4 drives T cell dysfunction in PDAC and is a novel, promising immunotherapeutic target.

Investigation of the effects of the toll-like receptor 4 pathway on immune checkpoint vista in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas. Preclinical studies show that it evades the immune system with immune checkpoints and promotes tumor development. V-domain Ig suppressor of T cell activation (VISTA) is a new immune-check point from the B7 family and is highly expressed in cancer cells. Overexpression of toll like receptor 4 (TLR4) in pancreatic adenocarcinoma is associated with induced tumorigenesis, tumor growth, resistancy to chemotherapy. Naloxone is an opioid and inhibits TLR4-ligand association. In this study, we investigated the relation of TLR4 and downstream pathways with immune-check point VISTA in pancreatic cancer proliferation. We initially collected pancreatic cancer-related datasets using the GEPIA2 and UALCAN databases. Based on this data obtained the effect of various concentrations and incubation times of naloxone were used on PANC-1 cells proliferation. A combination of naloxone and VISTA-siRNA were applied, and the effect of both naloxone and combined treatment on TLR4, Interleukin 1 receptor associated kinase 4 (IRAK4) and VISTA gene expression were analyzed in pancreatic cancer cells. As a result of analysis with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), gene expression levels of TLR4, IRAK4 and VISTA were significantly suppressed and cell proliferation was significantly reduced. We found that administration of naloxone and VISTA-siRNA in combination with PDAC cells suppressed signaling. Therefore, we considered that the relationship between VISTA and TLR4 signaling pathways and the other possible associated signal molecules may be an important marker in determining the response of immune checkpoint inhibitors in cancer treatment.

TRAM-Related TLR4 Pathway Antagonized by IRAK-M Mediates the Expression of Adhesion/Coactivating Molecules on Low-Grade Inflammatory Monocytes.

Low-grade inflammatory monocytes critically contribute to the pathogenesis of chronic inflammatory diseases such as atherosclerosis. The elevated expression of coactivating molecule CD40 as well as key adhesion molecule CD11a is a critical signature of inflammatory monocytes from both human patients with coronary artery diseases as well as in animal models of atherosclerosis. In this study, we report that subclinical superlow-dose LPS, a key risk factor for low-grade inflammation and atherosclerosis, can potently trigger the induction of CD40 and CD11a on low-grade inflammatory monocytes. Subclinical endotoxin-derived monocytes demonstrate immune-enhancing effects and suppress the generation of regulatory CD8+CD122+ T cells, which further exacerbate the inflammatory environment conducive for chronic diseases. Mechanistically, subclinical endotoxemia activates TRAM-mediated signaling processes, leading to the activation of MAPK and STAT5, which is responsible for the expression of CD40 and CD11a. We also demonstrate that TRAM-mediated monocyte polarization can be suppressed by IRAK-M. IRAK-M-deficient monocytes have increased expression of TRAM, elevated induction of CD40 and CD11a by subclinical-dose endotoxin, and are more potent in suppressing the CD8 regulatory T cells. Mice with IRAK-M deficiency generate an increased population of inflammatory monocytes and a reduced population of CD8 T regulatory cells. In contrast, mice with TRAM deficiency exhibit a significantly reduced inflammatory monocyte population and an elevated CD8 T regulatory cell population. Together, our data reveal a competing intracellular circuitry involving TRAM and IRAK-M that modulate the polarization of low-grade inflammatory monocytes with an immune-enhancing function.

The extreme C-terminus of IRAK2 assures full TRAF6 ubiquitination and optimal TLR signaling.

Macrophages are fundamental for initiation, maintenance, and resolution of inflammation. They can be activated by 'Toll-like receptor' (TLR) engagement, which initiates critical pathways to fight infections. 'Interleukin receptor-associated kinase 2' (IRAK2) is part of the membrane-proximal Myddosome formed at IL-1R/TLRs, but utility and regulation of IRAK2 within is not completely understood. In this study, we addressed the importance of the evolutionary conserved extreme C-terminus of IRAK2 in TLR signaling. The last 55 amino acids lack any known functional domain. The C-terminus deletion mutant IRAK2Δ55 was hypofunctional and disabled to conduct TLR4-inducible NF-κB and ERK2 activation. Accordingly, it could neither fully support subsequent CD40 cell surface expression nor IL-6 and nitric oxide release. Interestingly, IRAK2Δ55 was still capable to bind to 'tumor necrosis factor receptor-associated factor 6' (TRAF6), which is requisite to activate TRAF6 as an E3-ubiquitin ligase for further downstream signaling. However, IRAK-dependent auto-ubiquitination of TRAF6 was impaired, when IRAK2Δ55 was bound. Thus, the conserved last 55 amino acids enable IRAK2 to sustain an optimal TLR response. This knowledge might spark ideas how overshooting inflammatory responses could be modified without blocking the entire immune response.

microRNA-1388-5p inhibits NF-κB signaling pathway in miiuy croaker through targeting IRAK1.

Innate immune response is an important response mechanism for the host to achieve self-protection, and it plays an important role in identifying pathogens and resisting pathogen invasion. Growing evidences have shown that microRNA functions as a crucial regulator involved in the host innate immune response. In this study, the regulations of miR-1388-5p to regulate NF-κB signaling pathways via targeting the IRAK1 gene was studied in miiuy croaker. First, through bioinformatics software prediction, we found that IRAK1 is the direct target of miR-1388-5p, and then the prediction results were verified by using dual-luciferase assays. Next, we found that both miR-1388-5p mimics and pre-miR-1388 plasmids inhibit IRAK1 expression by complementing the seed sequence in the 3'-untranslated region (3'-UTR) of IRAK1. Finally, we observed that miR-1388-5p could negatively regulate NF-κB pathways through targeting IRAK1. These results provide new insights into the function of miR-1388-5p in fish innate immunity, meanwhile enriching miRNA-mediated regulatory networks.

Pneumococcal Serotype-specific Opsonophagocytic Activity in Interleukin-1 Receptor-associated Kinase 4-deficient Patients.

BACKGROUND: The antibody response after pneumococcal vaccines and their effectiveness against invasive pneumococcal disease (IPD) in patients with interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency have not been fully evaluated. Here, we evaluated pneumococcal serotype-specific opsonophagocytic activity (OPA) in IRAK4-deficient patients along with their clinical course. METHODS: We investigated 6 IRAK4-deficient patients in Japan, whose attending physicians could be contacted. We performed OPA measurements using stored and more recent serum samples obtained from these patients. RESULTS: All patients had received pneumococcal vaccination. Among the 3 patients who had IPD, 2 had an episode of pneumococcal meningitis and the other developed pneumococcal bacteremia 3 years after the occurrence of pneumococcal meningitis. Only one episode of invasive bacterial infection was caused by a Streptococcus pneumoniae vaccine-type strain. An increased opsonization index was found in the sera after vaccination for all IRAK-deficient patients, including when the 23-valent pneumococcal polysaccharide vaccine was used. CONCLUSIONS: A significant increase in levels of OPA against most of the pneumococcal vaccine antigens was observed for all IRAK4-deficient patients. However, IPD could not be prevented by pneumococcal vaccination alone. Therefore, adequate prophylaxis should be provided with antibiotics at least until 8 years of age, along with regular immunoglobulin therapy, particularly during the infantile period.

Long noncoding RNA IRL regulates NF-κB-mediated immune responses through suppression of miR-27c-3p-dependent IRAK4 downregulation in teleost fish.

Growing pieces of evidence show that the long noncoding RNAs (lncRNAs) as new regulators participate in the regulation of various physiological and pathological processes. The study of lncRNA in lower invertebrates is still unclear compared with that in mammals. Here, we identified a novel lncRNA, termed IRAK4-related lncRNA (IRL), as a key regulator for innate immunity in teleost fish. We find that miR-27c-3p inhibits IRAK4 expression and thus weakens the NF-κB-mediated signaling pathway. Furthermore, the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly upregulated host lncRNA IRL expression. Results indicate that IRL functions as a competing endogenous RNA for miR-27c-3p to regulate protein abundance of IRAK4; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA IRL can competitively adsorb miRNA to regulate the miR-27c-3p/IRAK4 axis that is widespread in teleost fish.

IRAK4 Deficiency Presenting with Anti-NMDAR Encephalitis and HHV6 Reactivation.

IRAK4 deficiency is an inborn error of immunity predisposing patients to invasive pyogenic infections. Currently, there is no established simple assay that enables precise characterization of IRAK4 mutant alleles in isolation. Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is an autoimmune condition that is characterized by psychiatric symptoms, involuntary movement, seizures, autonomic dysfunction, and central hypoventilation. It typically occurs in adult females associated with tumors. Only a few infantile cases with anti-NMDAR encephalitis have been so far reported. We identified a 10-month-old boy with IRAK4 deficiency presenting with anti-NMDAR encephalitis and human herpes virus 6 (HHV6) reactivation. The diagnosis of IRAK4 deficiency was confirmed by the identification of compound heterozygous mutations c.29_30delAT (p.Y10Cfs*9) and c.35G>C (p.R12P) in the IRAK4 gene, low levels of IRAK4 protein expression in peripheral blood, and defective fibroblastic cell responses to TLR and IL-1 (TIR) agonist. We established a novel NF-κB reporter assay using IRAK4-null HEK293T, which enabled the precise evaluation of IRAK4 mutations. Using this system, we confirmed that both novel mutations identified in the patient are deleterious. Our study provides a new simple and reliable method to analyze IRAK4 mutant alleles. It also suggests the possible link between inborn errors of immunity and early onset anti-NMDAR encephalitis.

Grouper (Epinephelus coioides) IRAK-4 regulates activation of NF-κB and expression of inflammatory cytokines in grouper spleen cells.

IRAK-4 is a serine/threonine kinase that can bind to interleukin-1 receptor induced by interleukin-1. It plays a key role in the Toll-like receptor signaling pathway and is involved in innate and adaptive immune responses. In this study, piscine IRAK-4 significantly activated nuclear factor (NF)-κB signaling in grouper spleen cells. Grouper (Epinephelus coioides) IRAK-4 (EcIRAK-4) co-localized with EcMyD88 and did not impair EcMyD88-dependent NF-κB activation. Different doses of EcIRAK-4 caused different degrees of nuclear translocation of the transcription factor NF-κB p65 subunit, and it induced transcription of multiple pro-inflammatory cytokines. Using expression vectors of deletion domains or mutations at important sites of EcIRAK-4, we found that the EcIRAK-4 kinase domain is necessary for its signal transduction function. The conserved amino acid sites performed functions similar to those in mammals, and grouper-specific amino acids such as E339 also played important roles. These findings provide information about the functional characteristics of IRAK-4 in lower vertebrates.

TPL2 enforces RAS-induced inflammatory signaling and is activated by point mutations.

NF-κB transcription factors, driven by the IRAK/IKK cascade, confer treatment resistance in pancreatic ductal adenocarcinoma (PDAC), a cancer characterized by near-universal KRAS mutation. Through reverse-phase protein array and RNA sequencing we discovered that IRAK4 also contributes substantially to MAPK activation in KRAS-mutant PDAC. IRAK4 ablation completely blocked RAS-induced transformation of human and murine cells. Mechanistically, expression of mutant KRAS stimulated an inflammatory, autocrine IL-1ß signaling loop that activated IRAK4 and the MAPK pathway. Downstream of IRAK4, we uncovered TPL2 (also known as MAP3K8 or COT) as the essential kinase that propels both MAPK and NF-κB cascades. Inhibition of TPL2 blocked both MAPK and NF-κB signaling, and suppressed KRAS-mutant cell growth. To counter chemotherapy-induced genotoxic stress, PDAC cells upregulated TLR9, which activated prosurvival IRAK4/TPL2 signaling. Accordingly, a TPL2 inhibitor synergized with chemotherapy to curb PDAC growth in vivo. Finally, from TCGA we characterized 2 MAP3K8 point mutations that hyperactivate MAPK and NF-κB cascades by impeding TPL2 protein degradation. Cancer cell lines naturally harboring these MAP3K8 mutations are strikingly sensitive to TPL2 inhibition, underscoring the need to identify these potentially targetable mutations in patients. Overall, our study establishes TPL2 as a promising therapeutic target in RAS- and MAP3K8-mutant cancers and strongly prompts development of TPL2 inhibitors for preclinical and clinical studies.

Selective inhibition of interleukin-1 receptor-associated kinase 1 ameliorates lipopolysaccharide-induced sepsis in mice.

Interleukin-1 receptor-associated kinases (IRAKs), particularly IRAK1 and IRAK4, are important in transducing signal from Toll-like receptor 4. We interrogated if a selective inhibition of IRAK1 could alleviate lipopolysaccharide (LPS)-induced sepsis. In this study, we tested the impact of a novel selective IRAK1 inhibitor Jh-X-119-01 on LPS-induced sepsis in mice. Survival at day 5 was 13.3% in control group where septic mice were treated by vehicle, while the values were 37.5% (p = 0.046, vs. control) and 56.3% (p = 0.003, vs. control) for 5 mg/kg and 10 mg/kg Jh-X-119-01-treated mice. Jh-X-119-01 alleviated lung injury and reduced production of TNFα and IFNγ in peritoneal macrophages. Jh-X-119-01 decreased phosphorylation of NF-κB and mRNA levels of IL-6 and TNFα in LPS-treated macrophages in vitro. Jh-X-119-01 selectively inhibited IRAK1 phosphorylation comparing with a non-selective IRAK1/4 inhibitor which simultaneously inhibited phosphorylation of IRAK1 and IRAK4. Both Jh-X-119-01 and IRAK1/4 inhibitor increased survival of septic mice, but Jh-X-119-01-treated mice had higher blood CD11b+ cell counts than IRAK1/4 inhibitor-treated ones [24 h: (1.18 ± 0.26) × 106/ml vs. (0.79 ± 0.20) × 106/ml, p = 0.001; 48 h: (1.00 ± 0.30) × 106/ml vs. (0.67 ± 0.23) × 106/ml, p = 0.042]. IRAK1/4 inhibitor induced more apoptosis of macrophages than Jh-X-119-01 did in vitro. IRAK1/4 inhibitor decreased protein levels of anti-apoptotic BCL-2 and MCL-1 in RAW 264.7 and THP-1 cells, an effect not seen in Jh-X-119-01-treated cells. In conclusion, Jh-X-119-01 selectively inhibited activation of IRAK1 and protected mice from LPS-induced sepsis. Jh-X-119-01 showed less toxicity on macrophages comparing with a non-selective IRAK1/4 inhibitor.

Molecular features of interleukin-1 receptor-associated kinase-b and a in Mytilus coruscus, regulating their function by infection of aquatic pathogens and the expression of their serine/threonine protein kinase functional domains.

Interleukin-1 receptor-associated kinases (IRAKs) play important roles in the innate immune system of TLR (Toll-like receptor) signaling pathway. In this paper, interleukin-1 receptor-associated kinase-b (designated as McIRAK-b) and interleukin-1 receptor-associated kinase-a (named as McIRAK-a) were obtained based on the transcriptome data, the full length of McIRAK-b was 1815 bp and McIRAK-a was 3168bp, encoding 532 and 978 amino acids, respectively. BLASTp analysis and phylogenetic relationship strongly suggested that the deduced amino acid sequence of McIRAK-b had high homology with IRAK-4 and McIRAK-a was similar to IRAK-1 of other mollusks, especially at their function domains. The expressions of McIRAK-b and McIRAK-a were detected in six tissues including adductor muscle, hemocyte, gills, gonad and hepatopancreas, and the highest expressions appeared both in gills. The expressions of McIRAK-b and McIRAK-a in gills were observed with time-dependent manners after bacterial infections. After being challenged with Vibrio alginolyticus, McIRAK-b expressed significantly and got the peak at 8 h (9.47 times compared with the control group), but the peak appeared at 4 h by being infected with Vibrio parahaemolyticus (12.02 times compared with the control group). The highest point of McIRAK-a mRNA appeared at 12 h (5.16 times) after being challenged with V.alginolyticus and 8 h (4.21 times) for V.parahaemolyticus challenge. The results suggested that IRAK-b and IRAK-a might be important in immune signaling pathway of mussels. The kinase functional domain sequences (S_TKc) of McIRAK-b and McIRAK-a expressed in BL21(DE3) and purified by Ni-NAT Superflow resin conforming to the expected molecular weight with many active sites for their conferring protein-protein interaction functions. This study may provide some further understandings of the regulatory mechanisms in the bivalve innate immune system for IRAKs family.

Cellular microRNA, miR-1343-5p, modulates IFN-I responses to facilitate feline panleukopenia virus replication by directly targeting IRAK1 gene.

Feline panleukopenia is an acute, highly contagious, and fatal infectious disease caused by feline panleukopenia virus (FPV) and has led to severe consequences on pets, economically important animals, and the wildlife industry. MicroRNAs (miRNAs) play significant roles in the host-pathogen interaction by modulating cellular factors expression which are essential for viral replication or host innate immune response to infection. However, the role of host miRNA response in FPV infection remains to be discovered. In this study, we screened nine host miRNAs associated with FPV infection that were previously implicated in innate immunity or antiviral functions. We found that miR-1343-5p overexpression strongly promoted FPV-BJ04 genomic DNA. Subsequently, the expression of host miR-1343-5p was upregulated by FPV-BJ04 infection in vitro and in vivo. In addition, we demonstrated that miR-1343-5p was a negative regulator of the IFN-I signaling pathway, thereby promoting FPV infection. Bioinformatic analysis combined with molecular biological assay indicated that interleukin-1 receptor-associated kinase 1 (IRAK1) is a putative target of miR-1343-5p. Collectively, our findings emphasize the importance of miR-1343-5p in host defense against FPV, thus, enhancing our understanding of its pathogenic mechanism.

IRAK family in inflammatory autoimmune diseases.

Innate immune signaling plays an important role in inflammation, and dysregulation of signaling components within this pathway has been focused as a critical mediator in initiation, progression of inflammatory autoimmune diseases. Toll-like receptors (TLRs) are the most upstream pattern recognition receptors in the immune cells, detecting pathogen associated molecular patterns, initiating signal transduction, by which interleukin-1 receptor-associated kinase (IRAK) family mediates activating signal from TLRs and interleukin-1 receptor. The family comprises of four members, IRAK1, IRAK2, IRAK-M, IRAK4. The family members have a role in either positive or negative regulation of innate immunity, adaptive immunity and inflammation. Accumulated evidence proves that IRAK performs significantly in the pathogenesis of inflammatory autoimmune disorders. On the one hand, both patients and animal modes reported abnormal expression of the family members. On the other hand, functional study in vivo and in vitro demonstrated that the members are implicated in the development of the diseases. Interestingly, IRAK inhibition has potential therapeutic benefits. In this review, we focus on the family, review the physiological roles in different immune cells, and summarize emerging data for highlighting the importance of them in inflammatory autoimmunity.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

Molecular characterization and expressional modulation of IRAK1 as downstream signaling adaptor molecule of TLR-signaling pathways in Labeo rohita following PAMPs stimulation and bacterial infections.

Interleukin-1 receptor associated kinase (IRAK1) is one of the crucial signal transduction mediators in TLR/IL-1R signaling pathways in host immune system. To investigate about it in rohu (Labeo rohita), one of the economically important freshwater fish species in the Indian subcontinent, we cloned, characterized and analyzed its expression following bacterial infection and pathogens associated molecular patterns (PAMPs) stimulation. The full-length cDNA of rohu IRAK1 (LrIRAK1) consisted of 2765 nucleotide (nt) having an ORF of 2115 nt encoding a polypeptide of 704 amino acids (aa) with a molecular mass of 70.4 kDa. Structurally, LrIRAK1 consisted of twenty-nine helix, twelve strands and forty one coils making one N-terminal death domain (19-94 aa) and a central serine threonine kinase catalytic domain (or kinase domain) (188-489aa). In addition to these two prominent domains, LrIRAK1 also contained highly conserved amino acids viz., lysine 215 and aspartic acid 314 and threonine 185, 361 which were reported to be important for kinase and phosphorylation activity respectively in other animals. Similar to higher vertebrates, LrIRAK1 also consisted of CDK1 (cyclin-dependent kinase1) at 338-352 aa; NEK2 (NIMA-related kinase 2) at 47-61 aa; NEK6 (NIMA-related kinase 6) at 581-595 aa and AMPK (AMP- activated protein kinase) motif at 518-538 aa. Phylogenetically, LrIRAK1 is closely related to cave fish, common carp exhibiting high similarity (~95%) and identity (~90%). In the uninfected fish, the LrIRAK1 expression was highest in liver (~11.5 fold) and lowest in blood. In response to Aeromonas hydrophila, Edwardsiella tarda and Bacillus subtilis infection and various TLR and NLR-ligands stimulation, the expression of LrIRAK1 was markedly enhanced at various time points in almost all the tested tissues. These results together suggest the key role of LrIRAK1 in pattern recognition receptors (PRRs)-mediated host defense against pathogenic insults.

IRAK1 and IRAK4 signaling proteins are dispensable in the response of human neutrophils to Mycobacterium tuberculosis infection.

The involvement of neutrophils in the host response to Mycobacterium tuberculosis (Mtb) infection is not as well recognized as the involvement of macrophages and dendritic cells. Thus, this study gives more insight on the impact of the virulent Mtb H37Rv strain on proapoptotic and proinflammatory functions of human neutrophils in vitro. We found that neutrophils are not able to kill Mtb during the infection process, probably due to the lack of reactive oxygen species and nitric oxide production in response to bacteria. However, infected neutrophils effectively released cytokines, chemoattractant interleukin (IL) 8 and proinflammatory IL-1ß. Moreover, Mtb enhanced the early apoptosis of neutrophils at 2 h postinfection. Additionally, this proapoptotic and proinflammatory response of neutrophils to Mtb infection occurred in an IRAK1- and IRAK4-independent manner. We also found that Mtb did not affect the surface expression of Toll-like receptor (TLR) 2 and slightly enhanced the surface expression of TLR4, but did not influence mRNA levels of both TLRs during the infection process. In conclusion, we show that the inhibition of signaling proteins activated by MyD88-dependent pathway did not participate in the biological activity of neutrophils against Mtb.

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks IRAK4-MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13-dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein-protein interface crucial for initiating innate immune responses.

Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus).

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.

Extracellular vesicles containing miR-146a attenuate experimental colitis by targeting TRAF6 and IRAK1.

Accumulating evidence indicates that microRNA-146a (miR-146a), a well-known anti-inflammatory miRNA, acts as a negative feedback regulator of the innate immune response, but its role in modulation of inflammatory bowel disease (IBD) remains unclear and the issue related to the stability of exogenous miR-146a in blood is up in the air. In this study, extracellular vesicles (EVs) from cultured medium of bone-marrow mesenchymal stem cells (BMSCs) transfected with recombinant lentiviruses can serve as a stable delivery system and overexpress miR-146a, which significantly inhibited TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1) expression in TNBS-induced colitis of rats. Moreover, the increased phosphorylation levels of NF-κB p65 and IκBα were down-regulated by the administration of EVs containing miR-146a. Coupled with the associated influence of over-expressed miR-146a on phosphorylated proteins above, the production of inflammation factors such as tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6) and Interleukin-1ß is apparently suppressed by this non-coding RNA. Collectively, these data elucidated that EVs containing miR-146a ameliorates experimental colitis caused 2,4,6­trinitrobenzenesulfonic acid (TNBS) by targeting TRAF6 and IRAK1.