Interleukin-1 Receptor-associated Kinase Research Articles

Abstract 5020: IRAK1 is a critical mediator of hyaluronic acid induced stemness

Abstract Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due in part to late-stage diagnoses. While the overall response rate to first line therapy is encouraging (~80%), the majority of women develop recurrent disease, characterized by resistance to standard chemotherapy. There is convincing evidence that cancer stem cells (CSCs) have implications in resistance and recurrence of OvCa. To understand the pathways involved in enhancing this stem-like phenotype, we performed RNA sequencing and identified TLR-ILR1 (TIR) pathways as highly activated in cisplatin resistant OvCa and stem-cell populations. Subsequently, using a tumor microarray, we observed that interleukin receptor-associated kinase 1 (IRAK1), a critical mediator of TIR signaling, is upregulated in OvCa patient tissues compared to normal. This expression further correlated with younger diagnosis age and shorter overall survival, suggesting a role in OvCa tumorigenesis. Knockdown of IRAK1 by specific shRNA in OvCa cells, significantly impaired CSC enriched spheroid growth and orthotopic tumor growth in mouse models replicating advanced peritoneal OvCa. RNA sequencing of IRAK1 knocked down cells identified downregulation of CSC related genes including STAT3, MYC, NOTCH1, and NOTCH3. In addition, there was a reduction in the cisplatin efflux transporter ABCC1 and stemness marker gene CD44. Advanced OvCa is often associated (~90% in stage III and IV) with the development of malignant ascites. We demonstrate for the first time that significant amounts of low molecular weight hyaluronic acid fragments (LMW HA) are present in ascites. LMW HA induced IRAK1 dependent activation of non-canonical signaling through PKC-β leading to induction of STAT3 phosphorylation and MYC. This was further accompanied by increased spheroid formation, demonstrating a critical role for IRAK1 in HA-induced stemness. We have further identified a selective IRAK1 inhibitor, TCS2210 (1,2-Dihydro-N-hydroxy-2-oxo-3-(3-phenylpropyl)-6-quinoxalinecarboxamide). TCS2210 abrogated LMW HA induced activation of IRAK1 and expression of stemness genes MYC, NOTCH1, and NOTCH3. Lastly, we found that TCS2210 effectively suppresses OvCa cell growth in vitro and in vivo, synergizing with standard-of-care cisplatin. Our data suggests a role of IRAK1 in OvCa, by enhancing cancer cell growth and stemness. It further suggests that IRAK1 is a putative target for advanced OvCa. Citation Format: David Standing, Prasad Dandawate, Jaimie Johnson, Sumedha Gunewardena, Michele T. Pritchard, Harsh Pathak, Dineo Khabele, Katherine Roby, Andrew Godwin, Roy Jensen, Scott Weir, Shrikant Anant. IRAK1 is a critical mediator of hyaluronic acid induced stemness. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5020.

Abstract 525: Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells

Abstract Therapies for acute myeloid leukemia (AML) patients are suboptimal due mainly to relapse of disease after treatment. Leukemia stem cell (LSC) populations maintain the disease and serve as a reservoir for relapse. Therefore, treatments that can eradicate LSCs will improve the outcomes of patients with AML. Our preliminary data have shown that the interleukin-1 (IL-1) pathway is upregulated following treatment and disease progression in AML and plays an important role in LSCs in vitro and in vivo. To target IL-1 signaling, we disclose the structure-activity relationship guided development of UR241-2, a novel small molecule compound of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4), which are critical downstream factors in IL-1/Toll-Like Receptor (TLR) signaling. UR241-2, at single dose (5 nM) screening exhibited high target selectivity; other than IRAK1/4 only 9 kinases in a panel of 682 kinases were inhibited by >25% activity. The NanoBRET assay using HEK293 cells transfected with targeted kinase vectors showed notably selective target engagement of UR241-2 against IRAK4 (IC50 = 6.07 nM). By using the in vitro NF-kB reporter assay and immunoblotting analysis, we identified that UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB and the phosphorylation of P65 and P38, following IL-1 stimulation. Next, we treated murine MLL-AF9 AML cells and human MDSL cells with UR241-2 and two IRAK4 inhibitors (PF-06650833 and CA-4948) that are currently in clinical or preclinical studies. We found that UR241-2 significantly inhibits colony-forming ability in AML and myelodysplastic syndromes, with similar or better efficacy compared to PF-06650833 or CA-4948. We also determined that UR241-2 suppresses LSC clonogenicity in primary human AML cells at diagnosis and in relapse while minimally impacting normal hematopoietic stem and progenitor cell function. Last, we investigated the impacts of UR241-2 on LSC engrafted ability in vivo. We treated murine MLL-AF9 cells with UR241-2 and PF-06650833 ex vivo for three days and transplanted them into recipient mice. After four weeks, we observed that UR241-2 significantly suppresses the engraftment levels of AML cells in bone marrow. It also decreases the WBC in peripheral blood and increases the RBC, HGB and PLT counts compared to the vehicle control group. Taken together, our findings demonstrated that targeting IRAK1/4 using the novel compound UR241-2 is able to impair LSC clonogenicity and engraftment capability in AML following treatment and disease progression. This provides a potential therapeutic strategy to eradicate the LSC population and improve the outcome of AML patients. Citation Format: Tzu-Chieh Ho, Rakesh K. Singh, Hiroki Kawano, Mark W. LaMere, Jian Wang, Paula M. Vertino, Nikolay V. Dokholyan, Laura M. Calvi, Craig T. Jordan, Michael W. Becker. Targeting interleukin-1 receptor-associated kinase 1/4 in acute myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 525.

Interactions between dietary protein level and water temperature on the growth performance, innate immunity and disease resistance of juvenile abalone Haliotis discus hannai Ino

A 100-day feeding trial was performed to examine the effects of dietary protein level, water temperature and their interactions on the growth performance, innate immunity and disease resistance of abalone Haliotis discus hannai with an initial body weight of 12.20 ± 0.1 g and an initial shell length of 42.64 ± 0.29 mm. With graded levels of dietary protein (9.33%, 27.53% and 40.42%, respectively), three isolipidic and isoenergetic diets were made and used in two water temperatures (17 °C and 23 °C) in a form of a 3 × 2 factorial design. The results indicated that the weight gain rate (WGR) and daily increment in shell length of abalones (DISL) were significantly higher in 27.53% of dietary protein level at both 17 °C and 23 °C water temperatures. At 17 °C and 23 °C of water temperatures, 27.53% of dietary protein significantly increased the trypsin and α- amylase as well as decreased the malondialdehyde content and glutamate dehydrogenase activities of abalone. Dietary protein and water temperature interact significantly with WGR and α-amylase. The histological analysis of abalone’s digestive gland showed that 27.53% of dietary protein caused normal development at both water temperatures. The mRNA levels of tumor necrosis factor alpha, toll-like receptor 2 and 4, inhibitor κBα, iκb kinase α, myeloid differentiation primary response gene 88, interleukin1 receptor-associated kinase 4, caspase 3 and B-cell lymphoma protein-2-associated X protein were significantly down-regulated by 27.53% of dietary protein at 17 °C of water temperature. The total hemocyte counts (THC), respiratory burst (RB) and B-cell lymphoma 2 (Bcl-2) were significantly increased by 27.53% of dietary protein at 17 °C water temperature. Dietary protein and water temperature interact significantly with THC, RB and Bcl-2. The use of 27.53% of dietary protein at 17 °C of water temperature showed no mortality when challenged with V. parahaemolyticus. In conclusion, 27.53% of dietary protein level at 17 °C of water temperature activated the innate immunity by reducing inflammation and apoptosis response, hence increasing disease resistance in abalones after the Vibrio challenge test.

The Expression of Plasmacytoid Dendritic Cells and TLR7/9-MyD88-IRAKs Pathway in Chronic Eczema Lesions.

To investigate the expression of Plasmacytoid Dendritic Cells (pDCs) and TLR7/9-MyD88-IRAKs pathway in chronic eczema lesions. Lesional tissues and the surrounding healthy tissues were collected from 25 individuals with chronic eczema, and immunohistochemistry was used to detect and comparatively analyze the expression profile of CD123, CD2AP, toll-like receptor 7 (TLR7), toll-like receptor 9 (TLR9) along with interleukin-1 receptor-associated kinase 1 (IRAK1) and interferon regulatory factor 7 (IRF7) in signaling pathways. The positive rates of CD2AP + pDC and CD123 + pDC in lesional tissues were significantly elevated compared to the surrounding healthy tissues (P < 0.05). They were distributed in both the epidermal and dermal layers of the lesional tissue, but the majority were in the dermal layer. The TLR7, TLR9, IRAK1 and IRF7 were more expressed in dermal layers of the lesional tissue with higher positive rates of expression compared to the surrounding healthy tissues (P < 0.05). IRAK1 and IRF7 were expressed in a small amount in the epidermal layer with higher positive rates of expression than in the surrounding healthy tissues (P < 0.05), while the positive rates of TLR7 and TLR9 expression in the epidermal layer were not statistically different from those in the surrounding healthy tissues (P > 0.05). PDC and TLR7/9-MyD88-IRAKs pathways are actively expressed in chronic eczema lesions and may be involved in pathogenesis and disease progression.

LMP2 deficiency causes abnormal metabolism, oxidative stress, neuroinflammation, myelin loss and neurobehavioral dysfunctions

BackgroundSubstantial evidence suggests that immunoproteasome is implicated in the various neurological diseases such as stroke, multiple sclerosis and neurodegenerative diseases. However, whether the immunoproteasome itself deficiency causes brain disease is still unclear. Therefore, the aim of this study was to explore the contribution of the immunoproteasome subunit low molecular weight protein 2 (LMP2) in neurobehavioral functions.MethodsMale LMP2 gene completed knockout (LMP2-KO) and littermate wild type (WT) Sprague–Dawley (SD) rats aged 12-month-old were used for neurobehavioral testing and detection of proteins expression by western blotting and immunofluorescence. A battery of neurobehavioral test tools including Morris water maze (MWM), open field maze, elevated plus maze were used to evaluate the neurobehavioral changes in rats. Evans blue (EB) assay, Luxol fast blue (LFB) and Dihydroethidium (DHE) staining were applied to explore the blood–brain barrier (BBB) integrity, brain myelin damage and brain intracellular reactive oxygen species (ROS) levels, respectively.ResultsWe firstly found that LMP2 gene deletion did not cause significantly difference in rats’ daily feeding activity, growth and development as well as blood routine, but it led to metabolic abnormalities including higher levels of low-density lipoprotein cholesterol, uric acid and blood glucose in the LMP2-KO rats. Compared with the WT rats, LMP2-KO rats displayed obviously cognitive impairment and decreased exploratory activities, increased anxiety-like behavior and without strong effects on gross locomotor abilities. Furthermore, multiple myelin loss, increased BBB leakage, downregulation of tight junction proteins ZO-1, claudin-5 and occluding, and enhanced amyloid-β protein deposition were observed in brain regions of LMP2-KO rats. In addition, LMP2 deficiency significantly enhanced oxidative stress with elevated levels of ROS, caused the reactivation of astrocytes and microglials and markedly upregulated protein expression levels of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1), IL-6 and tumor necrosis factor-α (TNF-α) compared to the WT rats, respectively.ConclusionThese findings highlight LMP2 gene global deletion causes significant neurobehavioral dysfunctions. All these factors including metabolic abnormalities, multiple myelin loss, elevated levels of ROS, increased BBB leakage and enhanced amyloid-β protein deposition maybe work together and eventually led to chronic oxidative stress and neuroinflammation response in the brain regions of LMP2-KO rats, which contributed to the initial and progress of cognitive impairment.

A novel IRAK4/PIM1 inhibitor ameliorates rheumatoid arthritis and lymphoid malignancy by blocking the TLR/MYD88-mediated NF-κB pathway.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal enzyme in the Toll-like receptor (TLR)/MYD88 dependent signaling pathway, which is highly activated in rheumatoid arthritis tissues and activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL). Inflammatory responses followed by IRAK4 activation promote B-cell proliferation and aggressiveness of lymphoma. Moreover, proviral integration site for Moloney murine leukemia virus 1 (PIM1) functions as an anti-apoptotic kinase in propagation of ABC-DLBCL with ibrutinib resistance. We developed a dual IRAK4/PIM1 inhibitor KIC-0101 that potently suppresses the NF-κB pathway and proinflammatory cytokine induction invitro and invivo. In rheumatoid arthritis mouse models, treatment with KIC-0101 significantly ameliorated cartilage damage and inflammation. KIC-0101 inhibited the nuclear translocation of NF-κB and activation of JAK/STAT pathway in ABC-DLBCLs. In addition, KIC-0101 exhibited an anti-tumor effect on ibrutinib-resistant cells by synergistic dual suppression of TLR/MYD88-mediated NF-κB pathway and PIM1 kinase. Our results suggest that KIC-0101 is a promising drug candidate for autoimmune diseases and ibrutinib-resistant B-cell lymphomas.

A small molecule potent IRAK4 inhibitor abrogates lipopolysaccharide-induced macrophage inflammation in-vitro and in-vivo

Increasing evidence supports vanillin and its analogs as potent toll-like receptor signaling inhibitors that strongly attenuate inflammation, though, the underlying molecular mechanism remains elusive. Here, we report that vanillin inhibits lipopolysaccharide (LPS)-induced toll-like receptor 4 activation in macrophages by targeting the myeloid differentiation primary-response gene 88 (MyD88)-dependent pathway through direct interaction and suppression of interleukin-1 receptor-associated kinase 4 (IRAK4) activity. Moreover, incubation of vanillin in cells expressing constitutively active forms of different toll-like receptor 4 signaling molecules revealed that vanillin could only able to block the ligand-independent constitutively activated IRAK4/1 or its upstream molecules-associated NF-κB activation and NF-κB transactivation along with the expression of various proinflammatory cytokines. A significant inhibition of LPS-induced IRAK4/MyD88, IRAK4/IRAK1, and IRAK1/TRAF6 association was evinced in response to vanillin treatment. Furthermore, mutations at Tyr262 and Asp329 residues in IRAK4 or modifications of 3-OMe and 4-OH side groups in vanillin, significantly reduced IRAK4 activity and vanillin function, respectively. Mice pretreated with vanillin followed by LPS challenge markedly impaired LPS-induced IRAK4 activation and inflammation in peritoneal macrophages. Thus, the present study posits vanillin as a novel and potent IRAK4 inhibitor and thus providing an opportunity for its therapeutic application in managing various inflammatory diseases.

IRAK-M suppresses the activation of microglial NLRP3 inflammasome and GSDMD-mediated pyroptosis through inhibiting IRAK1 phosphorylation during experimental autoimmune encephalomyelitis

The activation of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome triggers pyroptosis proinflammatory cell death in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanisms of the inflammatory processes of microglia in EAE remain unclear. Our previous studies suggested that interleukin-1 receptor-associated kinase (IRAK)-M down-regulates the toll-like receptor 4/interleukin-1 receptor signaling pathway. Here, we used IRAK-M knockout (IRAK-M−/−) mice and their microglia to dissect the role of IRAK-M in EAE. We found that deletion of IRAK-M increased the incidence rate and exacerbated the clinical symptoms in EAE mice. We then found that IRAK-M deficiency promoted the activation of microglia, activated NLRP3 inflammasomes, and enhanced GSDMD-mediated pyroptosis in the microglia of EAE. In contrast, over-expression of IRAK-M exerted inhibitory effects on neuroinflammation, NLRP3 activation, and pyroptosis. Moreover, IRAK-M deficiency enhanced the phosphorylation of IRAK1, while IRAK-M over-expression downregulated the level of phosphorylated IRAK1. Finally, we found upregulated binding of IRAK1 and TNF receptor-associated factor 6 (TRAF6) in IRAK-M−/− EAE mice compared to WT mice, which was blocked in AAVIRAK-M EAE mice. Our study reveals a complex signaling network of IRAK-M, which negatively regulates microglial NLRP3 inflammasomes and pyroptosis by inhibiting IRAK1 phosphorylation during EAE. These findings suggest a potential target for the novel therapeutic approaches of multiple sclerosis (MS)/EAE and NLRP3-related inflammatory diseases.

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.

Hyperfunction variant rs708035 of interleukin 1 receptor-associated kinases 2 gene involved in the predisposition of leprosy infection.

Mycobacterium leprae (slow-growing bacteria) is the etiological agent for leprosy infection, which is a chronic granulomatous disease. Symptoms initiate with the loss of sensation in the affected areas, which can lead to severe injuries, cuts and burns. IRAK2 (interleukin-1 receptor-associated kinases 2) is reported to function in the regulation of the NFκB pathway. The frequency of the IRAK2 polymorphism (rs708035) was unknown in the Pakistani population. Therefore, the study was designed to identify the role of the rs708035 SNP (single nucleotide polymorphism) in susceptibility to leprosy. The case-control study was designed, and participants were selected by Ridley-Jopling Classification. Blood samples from healthy individuals and patients were collected after ethical approval. Genomic DNA was extracted for the amplification of selected polymorphisms by tetra-primer amplification refractory mutation system polymerase chain reaction. The desired products were observed via agarose gel (2.5%) electrophoresis followed by data analysis using bioinformatics tools (SNP Stats and SHEsis) and statistical tests (odds ratio, OR, and chi square). The study revealed that the mutant genotype (TT) was found to be frequent among cases (22.80%) in comparison with the controls (1.66%). The SNP rs708035 was significantly associated with the progression of leprosy (χ2 = 17.62, p < 0.0001). The targeted SNP significantly increases the risk of leprosy 2.3 times (OR =2.3119, 95% CI1.2729-4.1989, p< 0.01). The genetic model also confirms the significant association of the A/T genotype with leprosy in the over-dominant model (OR =2.83, 95% CI1.16-6.89, p< 0.001). The study revealed a significant association of the targeted SNP with leprosy and provided baseline data regarding the association of rs708035. The current research could be utilized for the preparation of biomarkers by considering a larger sample size. The patients suffering from leprosy faced various comorbidities, including hypertension and diabetes. The study reports for the first time a significant association of interleukin 1 receptor associated kinases 2 (IRAK2) single nucleotide polymorphism (SNP) rs708035 among the Pakistani population (Karachi). The current study provides baseline data to develop diagnostic biomarkers for early detection of leprosy.

Jianpiyiqi decoction inhibits proliferation and invasion by suppressing the Caspase-1/IRAKs/NF-KB signalling pathway in hepatoma Huh-7 cells

IntroductionAlthough the inflammatory response and inflammatory factors mediated by Caspase-1/Interleukin-1 Receptor Associated Kinases (IRAKs)/Nuclear Transcription Factor-κB (NF-κB) signaling pathway play an important role in hepatocellular carcinoma, the relationship between Caspase-1/IRAKs/NF-κB signaling pathway and proliferation, invasion, and apoptosis of hepatoma cells remains elusive. This study investigated whether a Chinese herbal formula named the traditional Chinese medicine formula Jianpiyiqi Decoction (JPYQD) could inhibit hepatoma cell proliferation and invasion through the Caspase-1/IRAKs/NF-κB signaling pathway. MethodsThis study investigated the inflammatory response, proliferation, invasion, and apoptosis of the traditional Chinese medicine JPYQD in human hepatoma Huh-7 cells in an inflammatory microenvironment. Huh-7 human hepatoma cells were treated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to induce inflammation and mimic the inflammatory microenvironment of hepatoma cells. Consequently, three different doses of JPYQD and positive control (VX-765) were used for intervention in hepatoma cells for 12 consecutive hours. The present study evaluated the proliferation and invasion of hepatoma cells, cycle and apoptosis levels, inflammatory factor secretion, oxidative stress homeostasis, Caspase-1/IRAKs/NF-κB signaling-related proteins, and mRNA expression levels to observe the effect and mechanism of JPYQD on hepatoma cells in the inflammatory microenvironment. ResultsThis study found that JPYQD inhibited the proliferation and invasion of hepatoma cells in an inflammatory microenvironment by reducing the production of interleukin-1β (IL-1β) and interleukin-18 (IL-18) and inhibiting NF-κB inflammatory factors, which could be associated with inhibition of Caspase-1/IRAKs/NF-κB signaling. Furthermore, JPYQD promoted increased ROS levels in hepatoma cells, balancing oxidative stress damage and inflammatory factor release with an inflammatory microenvironment in hepatoma cells. ConclusionJianpiyiqi decoction ameliorates Caspase-1/IRAKs/NF-κB signaling and oxidative stress homeostasis in hepatoma cells in an inflammatory microenvironment. This study provides a theoretical basis for the clinical development of therapeutic drugs focused on treating hepatocellular carcinoma in traditional Chinese medicine.

Ancestry-dependent genetic structure of the Xq28 risk haplotype in the Mexican population and its association with childhood-onset systemic lupus erythematosus.

Here we aimed to investigate the association of the Xq28 risk haplotype (H1) with susceptibility to childhood-onset systemic lupus erythematosus (SLE), and to compare its frequency and genetic structure in the Mexican population with those in other continental populations. We genotyped 15 single-nucleotide variants (SNVs) that form the H1 haplotype, using TaqMan real-time PCR. The association analysis [case-control and transmission disequilibrium test (TDT)] included 376 cases and 400 adult controls, all of whom were mestizos (MEZ). To identify risk alleles in Mexican Indigenous individuals, SNVs were imputed from whole-exome sequencing data of 1,074 individuals. The allelic frequencies determined in MEZ and Indigenous individuals were compared with those of the continental populations from the 1,000 Genomes database phase 3. Linkage disequilibrium (LD) analysis of risk alleles was performed on all populations. Interleukin-1 receptor associated kinase 1 (IRAK1) and methyl CpG binding protein 2 (MECP2) mRNA levels were determined using real-time PCR. Case-control analysis revealed genetic association with childhood-onset SLE for all 15 SNVs (OR = 1.49-1.75; p = 0.0095 to 1.81 × 10-4) and for the Xq28 risk haplotype (OR = 1.97, p = 4 × 10-6). Comparing with individuals of European ancestry (0.14-0.16), the frequencies of the risk alleles were significantly higher in the MEZ individuals (0.55-0.68) and even higher in Indigenous individuals (0.57-0.83). LD analysis indicated a differential haplotype structure within the Indigenous groups, which was inherited to the MEZ population as a result of genetic admixture. Individuals homozygous for the Xq28 risk haplotype exhibited decreased levels of both MECP2A and B transcripts. We found that the H1 risk haplotype differs in its conformation in the Mexican population. This difference could be attributed to positive selection within the Indigenous population, with its inheritance now having an autoimmune health impact in both the Mexican Indigenous and MEZ populations.

Interleukin-1 receptor-associated kinase 2 promotes inflammatory reactions by activating the nuclear factor kappa-B signaling pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is a major complication of diabetes. Interleukin-1 receptor-associated kinase 2 (IRAK2) has been implicated in various diseases. This study aimed to investigate the role of IRAK2 in DN progression and its association with inflammation and the nuclear factor-kappa B (NF-κB) signaling pathway. DN model mice were generated by intraperitoneal injection of streptozotocin. IRAK2 expression was upregulated in the DN model mice. IRAK2 knockdown increased weight and reduced blood glucose levels in DN model mice. In addition, IRAK2 downregulation improved glomerular morphology in DN mice. IRAK2 knockdown reduced the levels of kidney damage biomarkers (24-h urinary protein, urine albumin-creatinine ratio, and plasma creatinine) and inflammatory cytokines (IL-6, tumor necrosis factor [TNF]-α, TNF-1R, and TNF-2R). Moreover, IRAK2 activated the NF-κB signaling pathway in DN model mice. Overexpression of NF-κB exacerbated DN progression, and IRAK2 knockdown reversed these effects. IRAK2 promoted DN progression and inflammation by activating the NF-κB signaling pathway. These findings suggest that IRAK2 is a potential therapeutic target for DN treatment.

IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways.

Lung cancer is a globally prevailing malignancy, and the predominant histological subtype is lung adenocarcinoma (LUAD). IL-1 receptor-associated kinase 3 (IRAK3) has been identified in connection with innate immune and inflammatory response. The aim of this study is to investigate the impact of IRAK3 on prognosis and immunotherapy efficacy in LUAD, which remains incompletely elucidated. Our study delved into multiple online databases to find out expression, methylation and prognostic potentials of IRAK3 in LUAD and other malignancies. We employed tissue microarrays to assess IRAK3 protein levels in our LUAD cohort [National Cancer Center (NCC), China] and explore prognostic values. The correlations between IRAK3 and immune infiltration based on The Cancer Genome Atlas (TCGA) data were analyzed by corresponding algorithms. The contribution of IRAK3 to immunotherapy response was explored through the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm. Both LinkedOmics database and gene set enrichment analysis (GSEA) were applied to investigate how IRAK3 influences the tumor immune microenvironment and regulates immunotherapy response. We applied single-cell RNA sequencing datasets for the investigation of IRAK3 expression across diverse immune cells. Moreover, we employed genomics of drug sensitivity in cancer (GDSC) databases to examine how IRKA3 expression correlates with different drug responses. Compared with normal tissues, various tumor tissues had lower IRAK3 expression which could be regulated by its high methylation level. Reduced IRAK3 protein level was observed to correlate with advanced tumor stages and unfavorable prognosis among patients with LUAD, especially individuals with lymph node metastasis. Gene set enrichment analysis (GSEA) and tumor infiltration analysis proved that IRAK3 provoked immune infiltration. Macrophages/monocytes, CD4+ T cells, CD8+ T cells and neutrophils correlated significantly with IRAK3 expression. With TIDE algorithm, IRAK3 was verified to be related to poor immune checkpoint blockade (ICB) response. IRAK3 demonstrated positive associations with T-cell dysfunction score and immune checkpoint markers. Conversely, it exhibited negative correlations with microsatellite instability (MSI) and tumor mutation burden (TMB). High IRAK3 expression exacerbated cytotoxic T lymphocyte (CTL) dysfunction and predicted immunotherapy resistance by involvement of multiple inflammation-related pathways including IL-6/JAK/STAT3 signaling, inflammatory response and interferon-gamma (IFN-γ) response pathways. Additionally, elevated IRAK3 expression was predicted to be related with better responses to chemotherapeutic and molecular targeted drugs. Our findings indicated that IRAK3 could function as an independent prognostic predictor and an immunotherapeutic indicator in LUAD through involvement of multiple inflammation-related pathways.

Lipid Nanoparticle-Mediated Delivery of miRNA Mimics to Myeloid Cells.

MicroRNA (miRNA) dysregulation is known to be associated with a variety of human diseases, including cancers and immune disorders. MiR146a represents one of the best characterized regulators of the immune response, as well as cellular survival through the negative feedback inhibition of nuclear factor-kappa B (NF-ĸB) signaling in myeloid cells. Restoration of miR146a levels would be an attractive therapeutic strategy for reducing exaggerated immune responses or to prevent certain types of blood cancers. However, delivery of synthetic miRNA mimics to target myeloid cells remains challenging. Here, we describe an optimized lipid nanoparticle (LNP) strategy for the delivery of miRNA mimics to myeloid immune cells and provide detailed protocols for characterization of LNP complexes and their biological activity. The encapsulation of miR146a within a lipid complex protects the nucleic acid from nuclease degradation, while allowing for rapid uptake by target myeloid immune cells. The strategy results in an efficient inhibition of target interleukin (IL) 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor receptor associated factor 6 (TRAF6) protein expression, thereby resulting in reduced NF-ĸB activity in mouse macrophages in vitro. The LNP-encapsulated miR146a effectively inhibits expression of IL-6, a major proinflammatory mediator downstream from NF-ĸB. This LNP-based strategy is suitable for testing of other miRNAs or RNA therapeutics targeting myeloid immune cells.

Inhibitory Mechanism of IL-6 Production by Orento in Oral Squamous Cell Carcinoma Cell Line CAL27 Stimulated by Pathogen-Associated Molecular Patterns from Periodontopathogenic Porphyromonas gingivalis.

Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.

IRAK-M Regulates Proliferative and Invasive Phenotypes of Lung Fibroblasts.

Lung fibroblasts play an important role in subepithelial fibrosis, one feature for airway remodeling. IL-1 receptor-associated kinase (IRAK)-M was shown to involve fibrosis formation in airways and lung through regulation of inflammatory responses. IRAK-M is expressed by lung fibroblasts, whether IRAK-M has direct impact on lung fibroblasts remains unclear. In this investigation, we evaluated in vitro effect of IRAK-M on phenotypes of lung fibroblasts by silencing or overexpressing IRAK-M. Murine lung fibroblasts (MLg) were stimulated with house dust mite (HDM), IL-33, and transforming growth factor (TGF) β1. Techniques of small interfering RNA or expression plasmid were employed to silence or overexpress IRAK-M in MLg fibroblast cells. Proliferation, migration, invasiveness, and fibrosis-related events were evaluated. Significant upregulation of IRAK-M expression in MLg cells was caused by these stimuli. Silencing IRAK-M significantly increased proliferation, migration, and invasiveness of lung fibroblasts regardless of stimulating conditions. By contrast, IRAK-M overexpression significantly inhibited proliferation and motility of MLg lung fibroblasts. IRAK-M overexpression also significantly decreased the expression of fibronectin, collagen I, and α-SMA in MLg cells. Under stimulation with TGFβ1 or IL-33, IRAK-M silencing reduced MMP9 production, while IRAK-M overexpression increased MMP9 production. Modulation of IRAK-M expression affected cytokines production, either decreased or increased expression of TNFα and CXCL10 by the cells regardless of stimulation. Our in vitro data reveal that IRAK-M directly impacts on lung fibroblasts through modulation of cellular motility, release of inflammatory, and fibrotic cytokines of lung fibroblasts. These might suggest a new target by regulation of IRAK-M in slowing airway remodeling.

Suppression of TLR signaling by IRAK-1 and -4 dual inhibitor decreases TPF-resistance-induced pro-oncogenic effects in HNSCC.

Combination of docetaxel, cisplatin and 5-FU, known as TPF, is an FDA-approved treatment for head and neck squamous cell carcinoma (HNSCC). Acquired chemo-resistance to TPF, a primary reason for non-responsiveness to the treatment and relapse of tumor is a major concern for treatment failure, especially in elder patients. In this study, we investigated the role of Interleukin-1 receptor-associated kinases (IRAK) mediated Toll-like receptor (TLR)-signaling in chemo-resistance using a cell line-based in-vitro TPF-resistant HNSCC model of laryngeal origin. TPF chemo-resistant state showed over-expression and phosphorylation of the active downstream kinases IRAK-1 and IRAK-4 along with enhanced proliferative potential, survival, stemness and metastatic capability as compared to the parent cell line. Pharmacological inhibition of IRAK-1 and -4 had a cytostatic effect on chemo-resistant cells and re-sensitized them to chemotherapy. The treatment also decreased the pro-oncogenic effects of the chemo-resistant cells. Our study provides insights into the pro-oncogenic role of amplified IRAK-1 and-4 mediated TLR signaling in TPF-resistant HNSCC. Pharmacological inhibition of IRAK-1 and-4 signaling is a promising therapeutic strategy for TPF-resistant HNSCC. It can also be used as a combination therapy or a chemo-drug sparing regimen in HNSCC.

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.

Tumor cell-derived exosomal microRNA-146a promotes non-small cell lung cancer cell invasion and proliferation by inhibiting M1 macrophage polarization.

Tumor-associated macrophages (TAMs) affects the outcomes of non-small cell lung cancer (NSCLC). NSCLC cells released exosomes to suppress the antitumor activity of TAMs. MiR-146a is a critical regulator in TAM polarization. We hypothesized that NSCLC cells released exosomal miR-146a to regulate TAM polarization and thus affected its antitumor activity. We used H1299 cells-derived exosomes to stimulate THP-1 cells that was pretreated with phorbol 12-myristate 13-acetate (M0 macrophage). Flow cytometry and reverse transcription-quantitative polymerase chain reaction (PCR) were used to determine the polarization of macrophages. The conditioned medium of exosome-treated M0 cells was used to culture H1299 cells, and the Cell Counting Kit-8, Ki67, transwell and scratch wound assays were used to determine the biological behavior of H1299 cells. To investigate whether exosomal miR-146a regulates TAM macrophages through targeting tumor necrosis factor receptor-associated factor 6 (TRAF-6) and interleukin-1 receptor-associated kinase 1 (IRAK-1), we used small interfering RNA to knockdown the expressions of them. Upregulation of miR-146a inhibited M1 polarization and thus impaired the antitumor activity of TAMs. Exosomes released by H1299 cells can be taken by M0 macrophage, and they upregulated the expression of miR-146a in M0 macrophage. The exosome suppresses M1 polarization by exosomal miR-146a. TRAF-6 and IRAK-1 mediated the inhibitive effects of exosomal miR-146a on M1 polarization. NSCLC cells released exosomal miR-146a to inhibit the expressions of TRAF-6 and IRAK-1 in TAMs, resulting in the impaired antitumor activity of TAMs. NSCLC cell-derived exosomal miR-146a represents a novel therapeutic target for NSCLC treatment.