Expression Of Interleukin-1 Receptor-associated Kinase Research Articles

MiR-146a Is Mutually Regulated by High Glucose-Induced Oxidative Stress in Human Periodontal Ligament Cells.

The high-glucose conditions caused by diabetes mellitus (DM) exert several effects on cells, including inflammation. miR-146a, a kind of miRNA, is involved in inflammation and may be regulated mutually with reactive oxygen species (ROS), which are produced under high-glucose conditions. In the present study, we used human periodontal ligament cells (hPDLCs) to determine the effects of the high-glucose conditions of miR-146a and their involvement in the regulation of oxidative stress and inflammatory cytokines using Western blotting, PCR, ELISA and other methods. When hPDLCs were subjected to high glucose (24 mM), cell proliferation was not affected; inflammatory cytokine expression, ROS induction, interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) expression increased, but miR-146a expression decreased. Inhibition of ROS induction with the antioxidant N-acetyl-L-cysteine restored miR-146a expression and decreased inflammatory cytokine expression compared to those under high-glucose conditions. In addition, overexpression of miR-146a significantly suppressed the expression of the inflammatory cytokines IRAK1 and TRAF6, regardless of the glucose condition. Our findings suggest that oxidative stress and miR-146a expression are mutually regulated in hPDLCs under high-glucose conditions.

Abstract 1464: Smoking carcinogen-induced inflammation promotes lung carcinogenesis via IRAK4 activation

Abstract Lung cancer is globally the most common cancer, and most cases are associated with smoking. Smoking exposes respiratory epithelial tissues to various carcinogens. Evaluating the effects of these carcinogens administered intratracheally in mouse models would allow us to study lung carcinogenesis in a relevant manner and determine therapeutically targetable pathways. Cigarette smoking exposure was mimicked in vivo using a smoke machine and the condensate was subjected to chemical analysis. In addition to nicotine, cigarette smoking condensate included significant levels of the carcinogens, Nicotine-derived nitrosamine ketone (NNK) and Benzo(α)pyrene (BP). Next, a mouse model of cigarette smoking-induced carcinogenesis was developed by exposing ICR mice to intratracheal exposure to NNK and BP three times a week for 18 months. Mice chronically exposed to NNK and BP developed epithelial dysplasia at 4 months of exposure and lung cancers at 8-12 mo. of exposure at significantly increased rates relative to controls. Histology revealed myeloid inflammation in murine lung tissues. Exposure of lung epithelial cells to cigarette smoking condensate led to increased production of pro-inflammatory IL-1β. Downstream mediator of IL-1β signaling, Interleukin 1 receptor associated kinase-4 (IRAK4), was overexpressed in murine lung tissues exposed to carcinogens in vivo. Two-thirds of human tissue sections obtained from archived lung cancers also exhibited overactivated IRAK4 expression. In lung cancer cell lines, IRAK4 immunofluorescence revealed a microtubule like pattern of expression in lung cancer cells. Immunoblotting confirmed IRAK4 colocalization within purified microtubules. Using mass spectrometry on isolated microtubules, we observed that phosphorylation decreased in various tubular proteins after IRAK4 inhibition. Inhibition of IRAK4 using small molecule specific inhibitors resulted in decreased invasion in lung cancer cell lines. These data show that chronic intratracheal exposure of smoking associated carcinogens leads to dysplasia and malignancies in mouse models. This exposure also leads to myeloid inflammation and activation of IRAK4 in lung tissues. Therapeutic targeting of IRAK4 can have potentially beneficial effects in lung cancer models. Citation Format: Ritesh K. Aggarwal, Simone Sidoli, Srabani Sahu, Srinivas Aluri, Charan Vegivinti, Divij Verma, Shanisha Gordon-Mitchell, Beamon Agarwal, Tanya Verma, Daniel T. Starczynowski, Ulrich G. Steidl, Balazs Halmos, Lindsay M. LaFave, Haiying Cheng, Amit Verma, Yiyu Zou. Smoking carcinogen-induced inflammation promotes lung carcinogenesis via IRAK4 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1464.

Saikosaponin-b2 Inhibits Primary Liver Cancer by Regulating the STK4/IRAK1/NF-κB Pathway.

The development of primary liver cancer (PLC) is associated with chronic liver inflammation and the loss of associated tumor suppressor genes, which characterizes inflammation-related tumors. In this study, we aimed to explore the effect of saikosaponin-b2 (SS-b2) on the development of PLC and its effect of the STK4 expression and IRAK1/NF-κB signaling axis. In vitro and in vivo experiments showed that SS-b2 exerted potent anti-inflammatory and antitumor effects. A PLC model was induced in vivo by treating male BALB/c mice with diethylnitrosamine, while an inflammatory model was induced in vitro by exposing RAW 264.7 macrophages to lipopolysaccharides (LPS). After treating cancer mice with SS-b2, the serum levels of alpha-fetoprotein, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly reduced. Ki67 expression also decreased. The carcinomatous lesions of the liver were attenuated. Similar results were observed in liver tissue and RAW 264.7 macrophages, where SS-b2 significantly elevated serine/threonine protein kinase 4 (STK4) expression and decreased the expression of interleukin-1 receptor-associated kinase 1 (IRAK1), nuclear factor-kappaB (NF-κB), and downstream inflammatory cytokines, thus exerting anti-cancer and anti-inflammatory effects. Moreover, we employed siRNA to silence the STK4 expression in HepG2 to investigate the anti-tumor effect of SS-b2 in vitro. The STK4 knockdown would upregulate IRAK1 and thus the activation of NF-κB activity revealed by the increase in the levels of proinflammatory cytokines, consequently impairing SS-b2-induced inhibition of liver cancer development. Consequently, SS-b2 effectively inhibited PLC by upregulating STK4 to suppress the IRAK1/NF-κB signaling axis and is a promising agent for treating this disease.

Ketogenic diet and β-Hydroxybutyrate alleviate ischemic brain injury in mice via an IRAKM-dependent pathway

Ketogenic diet (KD) is a classical nonpharmacological therapy that has recently been shown to benefit cerebral ischemia, but the mechanism remains unclear. This study investigated the neuroprotective effects of KD pretreatment and β-hydroxybutyrate (BHB, bioactive product of KD) post-treatment in a mouse model of temporary middle cerebral artery occlusion (tMCAO). Neurological function, infarct volume, as well as inflammatory reactions are evaluated 24 h after ischemia. Results showed that both KD pretreatment or BHB post-treatment improved the Bederson score and Grip test score, reduced infarct volume and the extravasation of IgG, suppressed the over-activation of microglia, and modulated the expression of cytokines. Mechanically, we found that both KD pretreatment or BHB post-treatment significantly stimulated the expression of interleukin-1 receptor-associated kinase M (IRAKM) and then inhibited the nuclear translocation of NF-κB. IRAKM deletion (Irakm−/−) exacerbated tMCAO-induced neurovascular injuries, and aggravated neuroinflammatory response. Moreover, KD pretreatment or BHB post-treatment lost their neuroprotection in the tMCAO-treated Irakm−/− mice. Our results support that KD pretreatment and BHB post-treatment alleviate ischemic brain injury in mice, possibly via an IRAKM-dependent way.

MicroRNA-146a negatively regulates inflammation via the IRAK1/TRAF6/NF-κB signaling pathway in dry eye

Inflammation is a key factor in the pathogenesis of dry eye disease (DED). We aimed to investigate the role of microRNA-146a (miR-146a) in regulating corneal inflammation in a mouse model of benzalkonium chloride (BAC)-induced dry eye and the TNF-α-induced NF-κB signaling pathway in human corneal epithelial cells (HCECs). A mouse model of dry eye was established by administering with BAC to BALB/c mice, and the expression of TNF-α, IL-1β, IL-6, IL-8, cyclooxygenase 2 (COX2), interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) in the corneas of dry eye model mice was significantly increased; this was accompanied by the upregulation of miR-146a and activation of the NF-κB pathway. In vitro, TNF-α induced miR-146a expression in HCECs, while the NF-κB inhibitor SC-514 reduced the expression of miR-146a. Overexpression of miR-146a decreased the expression of IRAK1 and TRAF6, which have been identified as targets of miR-146a. Furthermore, overexpression of miR-146a suppressed NF-κB p65 translocation from the cytoplasm to the nucleus. Moreover, overexpression of miR-146a attenuated the TNF-α-induced expression of IL-6, IL-8, COX2 and intercellular adhesion molecule 1 (ICAM1), while inhibition of miR-146a exerted the opposite effect. Our results suggest that miR-146a mediates the inflammatory response in DED. MiR-146a negatively regulates inflammation in HCECs through the IRAK1/TRAF6/NF-κB pathway, and this may serve as a potential therapeutic approach for the treatment of DED.

Radiation induces IRAK1 expression to promote radioresistance by suppressing autophagic cell death via decreasing the ubiquitination of PRDX1 in glioma cells

Radiotherapy is the standard adjuvant treatment for glioma patients; however, the efficacy is limited by radioresistance. The function of Interleukin-1 receptor associated kinase 1 (IRAK1) in tumorigenesis and radioresistance remains to be elucidated. IRAK1 expression and its correlation with prognosis were analyzed in glioma tissues. We found that glioma patients with overexpressed IRAK1 show a poor prognosis. Notably, ionizing radiation (IR) remarkably induces IRAK1 expression, which was decreased by STING antagonist H-151 treatment. JASPAR prediction, ChIP assays, and dual luciferase reporter assays indicated that transcription factor FOXA2, suppressed by STING inhibition, directly binds to the IRAK1 promoter region and activates its transcription. IRAK1 knockdown inhibits malignancy and enhances the radiosensitivity of glioma in vitro and in vivo. To explore the potential IRAK1 interacting targets mediating the radioresistance of glioma cells, IP/Co-IP, LC-MS/MS, GST pull-down, and ubiquitination analyses were conducted. Mechanistically, IRAK1 bound to PRDX1, a major member of antioxidant enzymes, and further prevents ubiquitination and degradation of PRDX1 mediated by E3 ubiquitin ligase HECTD3; Both the DOC and HECT domains of HECTD3 directly interacted with PRDX1 protein. Overexpression of PRDX1 reverses the radiotherapy sensitization effect of IRAK1 depletion by diminishing autophagic cell death. These results suggest the IRAK1-PRDX1 axis provides a potential therapeutic target for glioma patients.

Oral IRAK-4 Inhibitor CA-4948 Is Blood-Brain Barrier Penetrant and Has Single-Agent Activity against CNS Lymphoma and Melanoma Brain Metastases.

An ongoing challenge in cancer is the management of primary and metastatic brain malignancies. This is partly due to restrictions of the blood-brain barrier and their unique microenvironment. These challenges are most evident in cancers such as lymphoma and melanoma, which are typically responsive to treatment in systemic locations but resistant when established in the brain. We propose interleukin-1 receptor-associated kinase-4 (IRAK-4) as a potential target across these diseases and describe the activity and mechanism of oral IRAK-4 inhibitor CA-4948. Human primary central nervous system lymphoma (PCNSL) and melanoma brain metastases (MBM) samples were analyzed for expression of IRAK-4 and downstream transcription pathways. We next determined the central nervous system (CNS) applicability of CA-4948 in naïve and tumor-bearing mice using models of PCNSL and MBM. The mechanistic effect on tumors and the tumor microenvironment was then analyzed. Human PCNSL and MBM have high expression of IRAK-4, IRAK-1, and nuclear factor kappa B (NF-κB). This increase in inflammation results in reflexive inhibitory signaling. Similar profiles are observed in immunocompetent murine models. Treatment of tumor-bearing animals with CA-4948 results in the downregulation of mitogen-activated protein kinase (MAPK) signaling in addition to decreased NF-κB. These intracellular changes are associated with a survival advantage. IRAK-4 is an attractive target in PCNSL and MBM. The inhibition of IRAK-4 with CA-4948 downregulates the expression of important transcription factors involved in tumor growth and proliferation. CA-4948 is currently being investigated in clinical trials for relapsed and refractory lymphoma and warrants further translation into PCNSL and MBM.

Expression of Steroid Receptor RNA Activator 1 (SRA1) in the Adipose Tissue Is Associated with TLRs and IRFs in Diabesity.

Steroid receptor RNA activator gene (SRA1) emerges as a player in pathophysiological responses of adipose tissue (AT) in metabolic disorders such as obesity and type 2 diabetes (T2D). We previously showed association of the AT SRA1 expression with inflammatory cytokines/chemokines involved in metabolic derangement. However, the relationship between altered adipose expression of SRA1 and the innate immune Toll-like receptors (TLRs) as players in nutrient sensing and metabolic inflammation as well as their downstream signaling partners, including interferon regulatory factors (IRFs), remains elusive. Herein, we investigated the association of AT SRA1 expression with TLRs, IRFs, and other TLR-downstream signaling mediators in a cohort of 108 individuals, classified based on their body mass index (BMI) as persons with normal-weight (N = 12), overweight (N = 32), and obesity (N = 64), including 55 with and 53 without T2D. The gene expression of SRA1, TLRs-2,3,4,7,8,9,10 and their downstream signaling mediators including IRFs-3,4,5, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), and nuclear factor-κB (NF-κB) were determined using qRT-PCR and SRA1 protein expression was determined by immunohistochemistry. AT SRA1 transcripts' expression was significantly correlated with TLRs-3,4,7, MyD88, NF-κB, and IRF5 expression in individuals with T2D, while it associated with TLR9 and TRAF6 expression in all individuals, with/without T2D. SRA1 expression associated with TLR2, IRAK1, and IRF3 expression only in individuals with obesity, regardless of diabetes status. Furthermore, TLR3/TLR7/IRAK1 and TLR3/TLR9 were identified as independent predictors of AT SRA1 expression in individuals with obesity and T2D, respectively. Overall, our data demonstrate a direct association between the AT SRA1 expression and the TLRs together with their downstream signaling partners and IRFs in individuals with obesity and/or T2D.

Expression of IRAK1 in Hepatocellular Carcinoma, Its Clinical Significance, and Docking Characteristics with Selected Natural Compounds.

This study aimed to explore clinical significance of interleukin-1 receptor-associated kinase 1 (IRAK1) in the diagnosis, prognosis, and targeted therapy of hepatocellular carcinoma. A systematic analysis based on the cancer genome atlas (TCGA) indicated that IRAK1 was highly expressed in 18 cancer types (p < 0.01) and may be a pan-cancer biomarker. In hepatocellular carcinoma, the alteration rate of IRAK1 was rather high (62.4%), in which mRNA high relative to normal predominated (58.9%). Higher expression was associated with shorter overall survival (p < 0.01). IRAK1 expression correlated positively with pathology stage and tumor grade (for the latter there was only a slight trend). Interestingly, it correlated positively with TP53 mutation (p < 0.001), suggesting a possible strategy for targeting TP53 via IRAK1. Immunohistochemistry experiments confirmed a higher positive rate of IRAK1 in carcinoma than in para-carcinoma tissues (χ2 = 18.006, p < 0.001). Higher tumor grade correlated with more strongly positive staining. Molecular docking revealed cryptotanshinone, matrine, and harmine as the best hit compounds with inhibition potential for IRAK1. Our findings suggest that IRAK1 may play biologically predictive roles in hepatocellular carcinoma. The suppression of IRAK1/NF-κB signaling via inhibition of IRAK1 by the hit compounds can be a potential strategy for the targeted therapy.

IL-1 receptor-associated kinase 1 participates in the modulation of the NLRP3 inflammasome by tumor-associated macrophages in hepatocellular carcinoma

Hepatocellular carcinomas (HCCs) occur frequently in the digestive system and are associated with high mortality. This current study examined the regulatory relationship between interleukin (IL)-1 receptor-associated kinase 1 (IRAK1), NLR family pyrin domain-containing 3 (NLRP3) inflammasomes, and tumor-associated macrophages (TAMs) in the growth and metastasis of HCC. The expression of IRAK1 and NLRP3 was assessed in tissues and cells via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Immunohistology was performed to detect the macrophage markers CD68, CD163, and CD168 in tumor tissues. Small interfering (si)RNA targeting IRAK1 (si-IRAK1) was designed to silence IRAK1 expression. Following si-IRAK1 transfection and/or co-culture with TAMs, HCC cell viability, proliferation, migration, and invasion, as well as the expression of NLRP3 and pro-inflammatory cytokines IL-1 β, IL-18, and monocyte chemotactic protein 1 (MCP-1) were assessed. HCC tissues showed elevated expression of IRAK1 and NLRP3, as well as increased expression of the macrophage markers CD68, CD163, and CD168, compared to adjacent healthy tissues. Silencing of IRAK1 expression in HepG2 and Huh7 cells resulted in suppression of cell proliferation, migration, and invasion, and also reduced expression of NLRP3 and the pro-inflammatory cytokines IL-1β, IL-18, and MCP-1. Moreover, TAMs promoted HepG2 and Huh7 cell proliferation, migration, and invasion, and elevated the expression of NLRP3, IL-1β, IL-18, and MCP-1. Furthermore, IRAK1 silencing reversed the effects of TAMs on HepG2 and Huh7 cells. The expression of IRAK1 was associated with HCC growth and metastasis, as well as NLRP3 inflammasome activation. The ability of TAMs to promote HCC growth and metastasis may be activated by NLRP3 inflammasomes and regulated by IRAK1.

CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

ObjectiveIgG4‐related disease (IgG4‐RD) is a fibro‐inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7‐transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll‐like receptor 7 (TLR‐7)–positive CD163+ M2 macrophage infiltration in SGs from IgG4‐RD patients. We undertook this study to examine the fibrotic mechanism via the TLR‐7 pathway.MethodsGene expression in SGs from human TLR7‐transgenic mice and IgG4‐RD patients was analyzed using DNA microarrays. We extracted the common up‐regulated TLR‐7–related genes in SGs from TLR7‐transgenic mice and IgG4‐RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR‐7 agonist loxoribine.ResultsIn TLR7‐transgenic mice and IgG4‐RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real‐time polymerase chain reaction validated the up‐regulation of only IRAK4 in IgG4‐RD patients compared with the other groups (P < 0.05). Interleukin‐1 receptor–associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4‐related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4‐positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF‐κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05).ConclusionCD163+ M2 macrophages promote fibrosis in IgG4‐RD by increasing the production of fibrotic cytokines via TLR‐7/IRAK4/NF‐κB signaling.

Circ-TFF1 Promotes Breast Cancer Progression Through the miR-129-2-3p/IRAK1 Axis.

Breast cancer (BC) is a common malignant tumor, and circular RNA-trefoil factor 1 (circ-TFF1; hsa_circ_0061825) has been found to be highly expressed in BC tissues and cells and is associated with the poor prognosis of BC patients. However, the interaction between circ-TFF1 and microRNA in BC has not been studied. Quantitative real-time PCR was used to detect the expression of circ-TFF1, miR-129-2-3p, and interleukin (IL)-1 receptor-associated kinase 1 (IRAK1). Through the detection of cell proliferation, migration, invasion, tube formation, and apoptosis, cell function was assessed. The expression levels of angiogenesis-related proteins were detected by western blot. The interaction between miR-129-2-3p and circ-TFF1 or IRAK1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Xenotransplantation experiments were used to confirm the function of circ-TFF1 in vivo. Circ-TFF1 and IRAK1 were significantly high expressed in BC tissues and cells. Silencing of circ-TFF1 reduced the proliferation, migration, invasion and tube formation, while increased the apoptosis of MDA-MB-361 and SK-Br-3 cells. MiR-129-2-3p was a target of circ-TFF1. Silencing of circ-TFF1 inhibited the malignant behavior of BC cells by releasing miR-129-2-3p. In addition, IRAK1 was a target of miR-129-2-3p. Overexpression of IRAK1 partially restored the inhibitory effect of miR-129-2-3p on cell progression. Animal experiments confirmed the anti-tumor effect of circ-TFF1 knockdown in vivo. Circ-TFF1 regulated the expression of IRAK1 by sponging miR-129-2-3p, thereby, promoting the development of BC. These data provided a novel targeted therapy for BC.

C-Myc participates in high glucose-mediated endothelial inflammation via upregulation of IRAK1 expression in diabetic nephropathy

Diabetic nephropathy (DN) is a common vascular complication of diabetes. Endothelial adhesion molecules are involved in physiopathology of DN. Interleukin-1 receptor-associated kinase 1 (IRAK1) and c-Myc participate in inflammation in DN. We hypothesized c-Myc modulates IRAK1 expression, contributing to hyperglycemia-mediated endothelial inflammation. The expression of endothelial adhesion molecules and IRAK1 were increased in glomerular endothelium of DN patients and rats. Our cellular experiments indicated high glucose-induced endothelial cell inflammation was inhibited by si-IRAK1. Additionally, high glucose increased c-Myc expression. si-c-Myc inhibited high glucose-mediated increase of IRAK1 levels and endothelial cell inflammation. c-Myc overexpression-mediated endothelial cell inflammation was counteracted by si-IRAK1. c-Myc also interacted with lysine methyltransferase 5A (KMT5A). Furthermore, high glucose decreased KMT5A expression and histone H4 lysine 20 methylation (H4K20me1). KMT5A upregulation decreased high glucose-mediated increase of IRAK1 levels as well as endothelial inflammation. KMT5A silencing-mediated endothelial inflammation was reversed by si-IRAK1. Mechanistic research indicated that c-Myc and H4K20me1 occupied IRAK1 promoter region. KMT5A silencing augmented the active action of c-Myc on IRAK1 levels. Our in vivo experiments represented KMT5A is downregulated and c-Myc is upregulated in DN patients and rats. KMT5A interacts with c-Myc to modulate IRAK1 expression, thus contributing to hyperglycemia-mediated endothelial inflammation in DN.

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

BackgroundThe 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.MethodsAd-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)-1β, 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.ResultsIRAK4 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-1β, 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.ConclusionsIRAK4 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.Graphical abstract

Mitofusin 2 Deficiency Causes Pro-Inflammatory Effects in Human Primary Macrophages.

Mitofusin 2 (MFN2) is a mitochondrial outer membrane GTPase, which modulates mitochondrial fusion and affects the interaction between endoplasmic reticulum and mitochondria. Here, we explored how MFN2 influences mitochondrial functions and inflammatory responses towards zymosan in primary human macrophages. A knockdown of MFN2 by small interfering RNA decreased mitochondrial respiration without attenuating mitochondrial membrane potential and reduced interactions between endoplasmic reticulum and mitochondria. A MFN2 deficiency potentiated zymosan-elicited inflammatory responses of human primary macrophages, such as expression and secretion of pro-inflammatory cytokines interleukin-1β, -6, -8 and tumor necrosis factor α, as well as induction of cyclooxygenase 2 and prostaglandin E2 synthesis. MFN2 silencing also increased zymosan-induced nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases inflammatory signal transduction, without affecting mitochondrial reactive oxygen species production. Mechanistic studies revealed that MFN2 deficiency enhanced the toll-like receptor 2-dependent branch of zymosan-triggered responses upstream of inhibitor of κB kinase. This was associated with elevated, cytosolic expression of interleukin-1 receptor-associated kinase 4 in MFN2-deficient cells. Our data suggest pro-inflammatory effects of MFN2 deficiency in human macrophages.

Targeting Interleukin‐1 Receptor‐Associated Kinase 1 (IRAK1) In Ovarian Cancer

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due largely to late-stage diagnoses and the development of chemoresistant disease. Nearly 70% of affected women develop recurrent disease, characterized by resistance to standard chemotherapy despite initial response. Cancer stem cells (CSCs) are a subset of cells that have an innate ability to self-renew and enter a dormant state and have been further implicated in resistance to chemotherapy due to high expression of the ATP-binding Cassette (ABC) transporters. To understand the pathways that may be implicated in enhancing this stem-like phenotype, RNA sequencing was performed on cisplatin sensitive and isogenic cisplatin-resistant OvCa cell lines, as well as cells grown in 2- dimensional (2-D) or 3-dimensional (3-D) cultures. OvCa cells grown as spheroids, a 3-D growth model, are enriched with CSCs, and therefore represent a surrogate for stemness. Interestingly, the Toll-like (TLR) or interleukin-1 (IL-1) receptor pathways were highly enriched in resistant cancer cells and spheroid cultures. A critical factor in these signaling pathways is the interleukin receptor associated kinase 1 (IRAK1). IRAK1 is also highly expressed in chemoresistant OvCa and is responsible for activating numerous downstream pathways involved in tumor progression, such as NF-kB, P38/MAPK, and C-Jun. High expression of IRAK1 is associated with poor overall survival based on analysis of the Cancer Genome Atlas (TCGA) publicly available datasets. Due to the important role IRAK1 has in OvCa growth signaling and patient outcomes, IRAK1 represents an attractive target for CSC-targeted drug therapy. The present study is aimed at identifying small molecule inhibitors of IRAK1 by using in-silico molecular docking-based screening. FDA-approved compound libraries were screened using Autodock Vina software. Compounds were assessed for binding within the c-terminal kinase domain. The compounds are ranked-based on their binding energies (BE) and hydrogen bonding patterns. Our screen identified multiple compounds, one of which included mebendazole, an anthelminthic with a BE of -10.3 kCal/mol. This suggests potent interaction with the kinase domain of IRAK1. To confirm the binding in vitro, we performed cellular thermal shift stability assays (CETSA). We observed that mebendazole treatment protected IRAK1 protein from thermal denaturation, confirming the in silico docking data. Lastly, we performed hexosaminidase proliferation assays to assess the effectiveness of mebendazole on inhibiting cancer cell growth. Cisplatin sensitive OvCar3, A1847, A2780 OvCa and cisplatin resistant C30 OvCa cell lines were treated with increasing doses of mebendazole. The inhibitory concentration 50 (IC50) was determined to be approximately 300 nM at 72h for all cell lines. This suggests that mebendazole has potent anti-cancer activity that may be, in part, mediated through the inhibition of IRAK1.

Interferon-α activates interleukin-1 receptor-associated kinase 1 to induce regulatory T-cell apoptosis in patients with systemic lupus erythematosus.

Impaired regulatory T-cell (Treg) responses and upregulated interleukin-1 receptor-associated kinase 1 (IRAK1) expression are associated with the development of human systemic lupus erythematosus (SLE). Here, we show that the levels of upregulated IRAK1 expression in circulating Tregs are correlated with the percentages of apoptotic Tregs, Systemic Lupus Erythematosus Disease Activity Index scores, and serum complement C3 levels in SLE patients. High levels of plasma interferon (IFN)-α in SLE patients induced IRAK1 phosphorylation to trigger Treg apoptosis, which was mitigated by IRAK1 inhibitor (IRAK-Inh) treatment. Bioinformatics indicated that IRAK1 activation was related to the IFN-α/β and mitogen-activated protein kinase (MAPK) signaling in Tregs and IFN-α treatment induced the p38 and MAPK/ERK kinase 3/6 phosphorylation, which was attenuated by IRAK-Inh in Tregs. Treatment with IRAK-Inh effectively alleviated renal injury and promoted the survival of lupus-prone B6.MRL-Faslpr /Nju mice. Therefore, IFN-α induced IRAK1 activation to promote Treg apoptosis, contributing to the pathogenesis of SLE and IFN-α/IRAK1 may be therapeutic targets for SLE.

MiR‑146a reduces depressive behavior byinhibiting microglial activation.

Depression is one of the major psychiatric diseases affecting the quality of life for individuals worldwide. Numerous reports have investigated depression, although its etiology remains to be elucidated. microRNA (miR)-146a is suggested to regulate innate immune and inflammatory responses. However, it is unclear whether miR-146a is involved in depression. Depression model mice were established using lipopolysaccharide-induced depression and chronic unpredictable mild stress, separately. miR-146a mimic and short interfering RNA were used to treat depressed mice. Depression-like behaviors and levels of pro-inflammatory cytokines were measured, while ionized calcium binding adapter molecule 1 (Iba-1) expression in hippocampus was quantified by immunohistochemistry. Neuroinflammatory factor levels in hippocampus were measured by western blotting. BV-2 cells were used to confirm that miR-146a suppressed microglia activation. Compared with control mice, the two depressed mouse models showed clearly decreased sucrose preference and significantly increased immobility time in the forced swimming test and tail suspension test (P<0.05). miR-146a overexpression significantly increased sucrose preference and reduced immobility time in depressed mice (P<0.05). However, total distance traveled in the locomotor activity test did not differ among groups. Compared with controls, expression levels of Iba-1, inducible nitric oxide, IL-1β, TNF-α, interleukin 1 receptor associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6) and phosphorylated NF-κB p65 were significantly increased in depressed mice (P<0.05). miR-146a overexpression effectively inhibited expression of these neuroinflammatory proteins, while miR-146a silencing significantly upregulated their expression (P<0.05). Consistent with these in vivo results, miR-146a mimic treatment inhibited TNF-α, IL-1β, IRAK1 and TRAF6 expression in BV-2 cells. miR-146a improved depressive behaviors in depressed model mice by inhibiting microglial activation and neuroinflammatory factor expression.

Resveratrol suppresses the reprogramming of macrophages into an endotoxin-tolerant state through the activation of AMP-activated protein kinase

Resveratrol has been reported to have beneficial effects on sepsis by regulating the inflammatory response. However, it remains unclear if resveratrol plays a role in the development of endotoxin tolerance. Treatment with resveratrol in macrophages stimulated with primary lipopolysaccharide (LPS) resulted in the increased production of TNF-α and IL-6 induced by a 2nd dose of LPS (by 74.5 ± 12.9% and 63.4 ± 12%, respectively, compared to untreated cells, P < 0.05). This effect was inhibited by compound C, an AMPK inhibitor, and STO609, a calcium/calmodulin-dependent protein kinase-kinase (CaMKK) inhibitor. Resveratrol diminished the expression of interleukin-1 receptor-associated kinase M (IRAK-M) and Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) by prolonging the exposure of cells to LPS (by 60.8 ± 16.3% and 70.3 ± 18.1%, respectively, compared to LPS only). The effect of resveratrol on the LPS-induced expression of IRAK-M and SHIP1 was inhibited by compound C or STO609. After a 2nd dose of LPS, resveratrol increased phosphorylation of ERK1/2, p38, and JNK in endotoxin tolerant macrophages. In vivo systemic administration of resveratrol prevented a significant increase in mortality rate by cecal ligation and puncture in LPS-induced endotoxin-tolerant mice. These results indicate that resveratrol induces AMPK activation through the Ca2+/CaMKKβ pathway and suppresses the development of endotoxin tolerance by inhibiting LPS-induced expression of IRAK-M and SHIP1.

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.