Targeting IRAK4 mitigates osteoarthritis by preserving mitochondrial homeostasis and suppressing MAPK/NF-κB-mediated inflammation

Targeting IRAK4 mitigates osteoarthritis by preserving mitochondrial homeostasis and suppressing MAPK/NF-κB-mediated inflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting IRAK4 mitigates osteoarthritis by preserving mitochondrial homeostasis and suppressing MAPK/NF-κB-mediated inflammation Yong Hu, Xuezhong Wang, Shenglu Cao, Kai Tong, Siliang Ma, Dehong Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7999197/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Osteoarthritis (OA), a prevalent and debilitating condition driven by progressive cartilage degeneration, represents a significant global health challenge due to the absence of effective disease-modifying therapies. Interleukin-1 receptor-associated kinase 4 (IRAK4), a key signaling kinase at the nexus of innate and adaptive immunity, has emerged as a promising therapeutic target for inflammatory diseases. This study elucidates the critical role of IRAK4 in OA pathogenesis. We found IRAK4 expression was significantly upregulated in both osteoarthritic cartilage and IL-1β-stimulated primary chondrocytes. Genetic silencing or pharmacological inhibition with the clinical-stage compound PF-06650833 effectively ameliorated IL-1β-induced inflammatory responses, extracellular matrix degradation, cellular senescence, and mitochondrial dysfunction. Mechanistically, we demonstrated that IRAK4 drives these catabolic processes by activating the MAPK/NF-κB signaling pathway through the TRAF6-TAK1 complex, a process further amplified by METTL3. Crucially, in a rat model of post-traumatic OA induced by DMM, intra-articular injection of an adeno-associated virus carrying shIRAK4 to knock down IRAK4 successfully attenuated overall disease progression, as evidenced by significantly reduced cartilage erosion, osteophyte formation, and aberrant subchondral bone remodeling. Our findings collectively establish IRAK4 as a central driver of OA pathology and highlight the strong translational potential of therapeutic IRAK4 inhibition, exemplified by PF-06650833, as a novel disease-modifying strategy. Health sciences/Medical research/Experimental models of disease Health sciences/Molecular medicine Health sciences/Pathogenesis/Inflammation/Chronic inflammation Health sciences/Medical research/Translational research Health sciences/Medical research/Drug development IRAK4 Mitochondrial dynamics PF-06650833 MAPK/NF-κB signaling METTL3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction For decades, OA has been regarded as the most common musculoskeletal disease affecting millions of elderly people. Its sharply increasing prevalence, along with the enormous economic burden it imposes, drives the need to find effective solutions 1 . OA is an intricate process that involves the entire joint, and the progressive degradation of articular cartilage is regarded as its pivotal feature. Due to its avascularity and lack of nerves, cartilage lacks the ability to self-regenerate 2 . Therefore, clarifying the molecular mechanisms involved in OA cartilage deterioration is urgent and beneficial for developing effective treatments and improving prognosis. Though mainly characterized by biomechanical alterations, increasing evidence indicates that low-grade synovial inflammation (synovitis) exists in OA as well 3 . The release of pro-inflammatory mediators (e.g., IL-1β, TNF-α) into the joint cavity promotes OA cartilage damage 4 . Binding of IL-1β to IL-1 receptor type 1 (IL-1R1) leads to recruitment of the adaptor protein MyD88 and activation of downstream kinases, including the IL-1 receptor-associated kinases (IRAKs), which initiate a signaling cascade that drives inflammatory responses 5 . IRAK4 is a 460-amino acid Ser/Thr protein kinase belonging to the IRAK family, which comprises IRAK-1, IRAK-2, IRAK-M, and IRAK-4. It plays a prominent role in mediating signal transduction by the Toll-like receptor (TLR) and IL-1R families 6,7 . The scaffolding function of IRAK4 is essential for Myddosome assembly and NF-κB activation. Together, the kinase and scaffolding functions of IRAK4 initiate the transcription and expression of pro-inflammatory factors 8 . For example, IRAK4 activation plays a critical role in smoking carcinogen-induced inflammation that promotes lung carcinogenesis 9 . Grant Otto reported that PF-06650833, a small-molecule inhibitor of IRAK4, demonstrates great potential for treating rheumatic diseases by alleviating inflammation in preclinical models of rheumatoid arthritis and lupus, and even reducing basal inflammation in healthy volunteers 10 . Furthermore, the inhibition of IRAK4 holds considerable potential as a therapeutic intervention for various immune-mediated inflammatory diseases 11,12 . Sadiq Umar et al. discovered that IRAK4 inhibition rebalances rheumatoid arthritis metabolic reprogramming by targeting glycolytic pathways and reducing inflammation in macrophages and fibroblasts 13 . A study indicated that adenovirus-mediated knockdown of IRAK4 alleviates OA in a rabbit model by reducing synovitis, while its impact on cartilage and the specific mechanisms involved were not addressed 14 . Therefore, focusing on chondrocytes to study the impact of IRAK4 on cartilage tissue and its potential mechanisms of action could provide new perspectives for the treatment of OA. Genetic factors, modulated at both transcriptional and post-transcriptional levels, have been demonstrated to fundamentally contribute to the development of OA 15 . The RNA modification N6-methyladenosine (m 6 A) is a critical post-transcriptional mechanism that fine-tunes gene expression by influencing RNA stability, translational control, and overall RNA activity, ultimately influencing a wide array of biological functions 16 . The m 6 A methyltransferase complex, which comprises methyltransferase-like 3 (METTL3) as its quintessential methyltransferase, is responsible for writing RNA m 6 A marks and is one of the most critical regulators in the epitranscriptomic machinery 17,18 . Xiang Chen et al. reported that excessive m 6 A modification, primarily mediated by METTL3, suppresses autophagy in OA fibroblast-like synoviocytes (OA-FLS). Silencing METTL3 in OA-FLS enhanced autophagic flux and attenuated cellular senescence propagation within joints, thereby alleviating cartilage damage 19 . Furthermore, mounting evidence supports a mechanism through which METTL3-mediated m 6 A modification of long noncoding RNAs exacerbates OA progression. Mechanistically, METTL3-mediated m 6 A modification of Atg5/Atg7 and BNIP3 was shown to ameliorate OA progression by modulating RNA stability 20 . Nevertheless, the precise mechanisms by which METTL3 contributes to OA progression remain poorly understood, highlighting the necessity for a thorough investigation into how it regulates various genes and signaling pathways. PF-06650833 (zimlovisertib) is a highly potent and selective small-molecule inhibitor of IRAK4, exhibiting an exceptional IC50 value of 0.2 nM—the lowest reported to date 21 . It functions through reversible inhibition of IRAK4, thereby suppressing TLR-mediated signaling and downstream production of pro-inflammatory cytokines such as type I interferons, IL-1, IL-6, IL-12, and TNF-α in human monocytes 22 . These cytokines play a central role in driving autoimmune and inflammatory pathologies. Preclinical studies have demonstrated its efficacy across multiple animal models, including respiratory distress syndrome, psoriasis, and rheumatic diseases 23,24 . Notably, to the best of our knowledge, PF-06650833 is the first IRAK4 inhibitor to enter human clinical trials. It has successfully completed Phase I studies, where it was well-tolerated with no dose-limiting toxicities observed, supporting its further development. A Phase II clinical trial for rheumatoid arthritis is currently underway 25 . Collectively, its strong selectivity, favorable safety profile, and robust anti-inflammatory activity in both experimental and clinical settings validate IRAK4 as a promising therapeutic target for inflammatory and autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, spondyloarthritis, and psoriatic arthritis. However, the efficacy of PF-06650833 in the context of OA has not been established in either preclinical models or clinical trials. Here, an in vitro OA model was generated by treating primary chondrocytes with IL-1β, and an in vivo rat OA model was established via the surgical destabilization of the medial meniscus (DMM). We aimed to examine the contribution of IRAK4 to OA initiation and pathophysiology, along with the involvement of METTL3 in this mechanism. Additionally, PF-06650833 was evaluated in a preclinical study to assess its therapeutic potential for OA. This work intends to offer novel insights into OA treatment strategies. Materials and methods Isolation and culture of rat primary chondrocytes Primary chondrocytes were extracted from the knee articular cartilage of 5-day-old SD rat pups, following established protocols 26 . In brief, articular cartilage was aseptically dissected, cut into 1 mm3 small pieces, and enzymatically digested—first with 0.25% trypsin (Boster, Wuhan, China) for 30 min at 37°C, then with 0.25% type II collagenase (Yeasen, Shanghai, China) for 6 h at 37°C. After the collagenase removal, isolated chondrocytes were resuspended, filtered, and cultured in DMEM/F12 medium (Servicebio, Wuhan, China) supplemented with 10% fetal bovine serum (HyClone, USA), 1% streptomycin and penicillin (Life iLab, Shanghai, China) at 37°C in a humidified 5% CO₂ atmosphere. To maintain chondrocyte phenotypic stability, our study exclusively employed second-generation chondrocytes in the ensuing experiments. Immunofluorescence (IF) staining Chondrocytes were seeded onto sterile glass coverslips placed in 6-well plates. After adhesion and treatment completion, the cells were fixed with 4% paraformaldehyde (PFA, Beyotime Biotechnology, Shanghai, China) at room temperature for 15 min, washed three times with PBS (Boster, Wuhan, China), and permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, Shanghai, China) for 15 min. The cells were then blocked with 5% bovine serum albumin (BSA, Yeasen, Shanghai, China) for 1 h. Following this, chondrocytes were incubated with primary antibodies against alpha-1 type II collagen (Col2, 1:100, Proteintech) and matrix metalloproteinase 13 (MMP13, 1:100, Proteintech) at 4°C overnight. Subsequently, chondrocytes were incubated with fluorescein-conjugated secondary antibodies (Proteintech, Wuhan, China) at 37°C for 1 h. Then, 4',6-diamidino-2-phenylindole (DAPI) (Servicebio, Wuhan, China) was utilized to counterstain the nuclei. Finally, the images were captured using a fluorescence microscope. Detection of intracellular reactive oxygen species (ROS) levels ROS levels in chondrocytes were determined using a commercial kit according to the manufacturer’s protocols. Upon completion of chondrocyte intervention, following addition of DCFH-DA (Beyotime Biotechnology, Shanghai, China) solution to serum-free culture medium at a final concentration of 10 µM, incubation proceeded in the dark for 30 min. The cells were then washed with PBS to remove the fluorescent dye. The images were photographed under a fluorescence microscope. JC-1 staining Employing the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime Biotechnology, Shanghai, China), changes in mitochondrial membrane potential (MMP, ΔΨm) were evaluated. At higher MMP, JC-1 accumulates within the mitochondrial matrix, forming JC-1 aggregates that emit red fluorescence. Conversely, at lower membrane potential, JC-1 remains dispersed in the matrix as monomers, producing green fluorescence. The relative ratio of red-to-green fluorescence intensity serves as a standard indicator for assessing the extent of mitochondrial depolarization. For this experiment, chondrocytes, seeded into 12-well plates and subjected to the designated treatments, were stained with JC-1 working solution at 37°C for 30 min postintervention following the kit instructions. Following aspiration of the supernatant, the cells were washed twice with JC-1 staining buffer and observed under a fluorescence microscope for image acquisition. Cell viability assay Cell viability of chondrocytes was assessed using the CCK-8 assay kit (Solarbio, Beijing, China). In brief, chondrocytes were plated into 96-well plates at a density of 5000 cells per well and incubated at 37°C for 48 hours in the presence of the designated interventions. Subsequently, 10 µL per well of CCK-8 solution diluted in serum-free medium was added and incubation continued for 2 h. Absorbance was measured at 450 nm using a microplate reader. EdU assay Cellular proliferation capacity was evaluated with a commercial 5‑Ethynyl‑2’‑deoxyuridine (EdU) detection kit (Beyotime Biotechnology, Shanghai, China). Briefly, chondrocytes were plated in 12-well plates and maintained in DMEM for 48 hours. After incubation with EdU for 2 h, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized using 0.2% Triton X-100 for 15 min. Subsequently, cells were treated with the click reaction cocktail for 30 min, followed by nuclear staining with DAPI for 10 min. All procedures were performed at room temperature under dark conditions. EdU-positive proliferating cells were counted under a fluorescence microscope. Mito-tracker red staining Biologically active mitochondria in live chondrocytes were labeled with Mito-Tracker Red CMXRos kit (Beyotime Biotechnology, Shanghai, China). The working solution was prepared by diluting the probe in serum-free medium to 200 nM. After 30-min incubation at 37°C, cells were counterstained with Hoechst 33342 (Solarbio, Beijing, China) at 37°C for 10 min. Following three washes with PBS, samples were imaged using a fluorescence microscope. Fluorescence intensity was quantified from captured images using ImageJ. Transmission electron microscopy Mitochondrial ultrastructure in chondrocytes was analyzed via transmission electron microscopy (TEM). Chondrocytes were processed following established interventions. The collected cells were washed twice with PBS, then fixed in 1.5% glutaraldehyde for 4 h, followed by post-fixation in 1% osmium tetroxide for 2 h. Following dehydration through an ethanol gradient and acetone, the samples were embedded in Epon resin. Samples were sectioned at 60 nm thickness using an ultramicrotome, and the resulting ultrathin sections were mounted on copper grids. The sections were sequentially stained with 1% uranyl acetate for 30 min and 0.1% lead citrate for 30 min, and then observed with a TEM. Live/Dead staining Cell viability was qualitatively evaluated using a Calcein-AM/Propidium Iodide (PI) dual-stain kit (Solarbio, Beijing, China). Viable cells were identified by green fluorescence from Calcein-AM staining, whereas dead cells were detected through red fluorescence emitted by PI staining. Briefly, chondrocytes were seeded into 6-well culture plates and subjected to specified interventions. The Calcein-AM/PI working solution was then prepared according to the manufacturer’s instructions. An appropriate volume of the working solution was added to the samples, followed by incubation at 37°C for 30 min in the dark. After incubation, fluorescence microscopy was performed to assess staining: Calcein-AM (green fluorescence; Ex/Em = 494/517 nm) and PI (red fluorescence; Ex/Em = 535/617 nm). Safranin O and toluidine blue staining of chondrocytes Glycosaminoglycan and proteoglycan content, which collectively indicate pathological alterations in the cartilage extracellular matrix, were assessed using Toluidine blue (Servicebio, Wuhan, China) and safranin O (Servicebio, Wuhan, China) staining. After predetermined treatments, the medium was aspirated. Chondrocytes were subsequently washed thrice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. Afterwards, cells were stained by incubation with either toluidine blue or safranin O for 30 minutes at 37°C. Stained chondrocyte samples were imaged with an inverted light microscope. Senescence‑associated β‑galactosidase (SA-β‐gal) staining Using the Senescence β-Galactosidase Staining Kit (Solarbio, Beijing, China), chondrocyte senescence was detected based on the upregulation of SA-β-gal activity during aging. Cells cultured in 6-well plates were processed as follows: the culture medium was aspirated, washed with PBS, and 1 mL of staining fixative solution was added. Cells were fixed at room temperature for 15 minutes. The staining working solution was prepared according to the manufacturer’s instructions. Then, 1 mL of staining working solution was added to each well and incubated at 37°C overnight. Senescent cells were identified by the presence of blue-green staining indicative of SA-β-gal activity observed under light microscopy. Flow cytometry for chondrocyte apoptosis analysis The apoptosis of chondrocytes was assessed by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. Chondrocytes were seeded in 6-well plates and exposed to different designated interventions. Post-treatment, cells were trypsinized, washed twice with PBS, and resuspended in PBS to generate a single-cell suspension. For apoptosis assessment, 500 µL of the suspension was aliquoted into a flow cytometry tube, stained with 5 µL Annexin V-FITC (gentle mixing), followed by addition of 10 µL propidium iodide (PI) solution. After 15-min incubation at room temperature in the dark, samples immediately underwent flow cytometric analysis. Western blotting Upon completion of the designated interventions, chondrocytes were then harvested and lysed in RIPA lysis buffer (Boster, Wuhan, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Boster, Wuhan, China) and phosphatase inhibitor (Solarbio, Beijing, China) for protein preparation. The concentrations of the obtained protein were measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein from each sample were then loaded onto 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature. Subsequently, they were incubated overnight at 4°C with the following primary antibodies against IRAK4 (1:2000, Abclonal), Col2 (1:2000, abcam), MMP13 (1:2500, Abclonal), iNOS (1:5000, Proteintech), COX-2 (1:1000, Servicebio), TNF-α (1:2000, Proteintech), p16 (1:2000, BOSTER), p21 (1:2000, Signalway Antibody, SAB), OPA1 (1:2000, Affinity), Mfn2 (1:5000, Proteintech), p-Drp1 (1:1000, abcam), Drp1 (1:1000, abcam), Fis1 (1:2000, Proteintech), TRAF6 (1:1000, Abclonal), TAK1 (1:2000, Abclonal), p-IKKα (1:1000, Cell Signaling Technology), IKKα (1:2000, Proteintech), p-IκBα (1:1000, abcam), IκBα (1:2000, Abclonal), p-p65 (1:2000, Abclonal), p65 (1:5000, Proteintech), METTL3 (1:2000, Abclonal), p-ERK (1:2000, Servicebio), ERK (1:2000, Proteintech), p-JNK (1:2000, Affinity), JNK (1:10000, Proteintech), p-p38 (1:1000, Abclonal), p38 (1:2000, Abclonal), NOX2 (1:2000, Zen-Bio), GAPDH (1:10000, Proteintech). Following primary antibody incubation, the membranes were washed three times with Tris-buffered saline containing 0.05% Tween-20 (TBST). Finally, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Proteintech, Wuhan, China) for 2 h at room temperature. Protein bands were developed by exposure to the enhanced chemiluminescence (ECL, Abbkine, Wuhan, China) reagent and visualized by a Bio-Rad scanner. Immunoreactive band intensities were quantified using ImageJ software. Band intensities of target proteins were normalized to GAPDH, which served as the internal control. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted from chondrocytes using a Total RNA Extraction Kit (Solarbio, Beijing, China). The extracted RNA was then reverse-transcribed into complementary DNA (cDNA) using a cDNA Synthesis Kit (Servicebio, Wuhan, China). Subsequently, quantitative real-time PCR (qPCR) was performed using the universal SYBR Green fast qPCR mix kit (Servicebio, Wuhan, China) on a Roche LightCycler® 480 instrument. Gene expression levels were normalized to GAPDH (internal reference) and quantified using the 2 −ΔΔCt method. The primer sequences used were as follows: IRAK4 forward: 5’-GCAATCTGAAGTCCCCTCGT-3’ IRAK4 reverse: 5’-GGCTTGCTCATCTTCTACTTCCT-3’ Small-interfering RNA (siRNA) transfection Small interfering RNA (siRNA) oligonucleotides targeting IRAK4, METTL3, and their corresponding negative controls were designed and synthesized by GENE CREATE (Wuhan, China). The sequences were as follows: IRAK4 siRNA-1: 5’-GCAACAGUUUGACCAAGAATT-3’ IRAK4 siRNA-2: 5’-GCGAUGUACUCUGUUGCUATT-3’ IRAK4 siRNA-3: 5’-GGGUGAUGACAGAUACAAUTT-3’ METTL3 siRNA-1: 5’-GGAUUGCGAUGUGAUUGUAGC-3’ METTL3 siRNA-2: 5’-CAGUGGAUCUGUUGUGAUAUC-3’ METTL3 siRNA-3: 5’-GGAGAUCCUAGAGCUAUUAAA-3’ Using Lipofectamine 3000 (Invitrogen, USA), chondrocytes were transfected according to the manufacturer’s instructions. Briefly, siRNA targeting IRAK4 or METTL3 was complexed with Lipofectamine 3000 in Opti-MEM medium for 8 h. After the transfection period, the mixture was removed and replaced with fresh DMEM/F12 medium. Following a further 24 h of culture, cells and supernatant were harvested and processed for further analysis. Establishment of rat OA model All animal procedures were approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (Approval No. 20220603A) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Research Council. Eight-week-old male Sprague-Dawley rats (SD) were group-housed in the Laboratory Animal Center under controlled conditions (23–25°C, 12-h light/dark cycle) with ad libitum access to food and water. Surgically-induced OA models were established in the right knee by transection of the medial meniscotibial ligament and medial meniscectomy (DMM model). Rats were randomly assigned to one of four experimental groups (n = 6 per group): Sham, DMM, DMM + AAV-control, and DMM + AAV-IRAK4 shRNA. Briefly, the DMM surgery was performed as follows: after ensuring adequate anesthesia, the joint capsule was incised. The medial meniscotibial ligament was transected, followed by resection of the medial meniscus. For the sham-operated group, the joint capsule and skin were sutured after incision without any ligament or meniscal damage. In the AAV-IRAK4 shRNA and AAV-control groups, rats received weekly intra-articular (IA) injections of AAV (1 × 10^10 virus particles/30 µL) starting one week after surgery. The Sham and DMM groups received weekly IA injections of an equal volume of phosphate-buffered saline (PBS). At 8 weeks post-surgery, all animals were euthanized under anesthesia, and knee joint tissues were collected for subsequent analysis. Micro-computed tomography (Micro-CT) analysis Micro-computed tomography (Micro-CT) was employed to scan knee joint specimens, enabling 3D reconstruction and visualization for quantitative analysis of subchondral bone structural alterations. In brief, samples underwent high-resolution Micro-CT scanning (SkyScan 1176) at 55 kV, 145 µA, and 300 ms exposure. Image reconstruction (NRecon v1.6) and repositioning (DataViewer v1.5) were performed with alignment of the coronal, sagittal, and axial planes prior to defining a region of interest (ROI) within the medial tibial plateau subchondral bone. Five sequential sagittal-plane ROI images were reconstructed in 3D (CTvol v3.0) for quantification of bone mineral density (BMD), trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) using CTAn v1.15 software. Histological and immunohistochemistry analysis For histological analysis, knee joint samples were fixed in 4% PFA for 48 h at room temperature, followed by decalcification in 10% ethylenediaminetetraacetic acid (EDTA, Beyotime Biotechnology, Shanghai, China) solution for 28 days. Subsequently, tissues were embedded in paraffin and sectioned sagittally into serial sections (5 µm thick). Sections were stained with hematoxylin and eosin (H&E, Servicebio, Wuhan, China) and safranin O/fast green (Servicebio, Wuhan, China) following standard protocols. OA severity assessment employed a modified Osteoarthritis Research Society International (OARSI) scoring system. Scoring was conducted blindly and independently by three trained assessors across at least three joint levels per sample. On this scale, a low score represents minimal damage, and a high score signifies severe damage. For immunohistochemistry analysis, following deparaffinization in xylene and rehydration through a graded alcohol series, cartilage sections were subjected to antigen retrieval using 0.1% trypsin for 30 min. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide for 10 min at room temperature. Nonspecific binding sites were blocked with 10% normal goat serum (Solarbio, Beijing, China). Sections were then incubated overnight at 4°C with primary antibodies targeting IRAK4 (1:100, Abclonal), Col2 (1:100, Proteintech), MMP13 (1:100, Proteintech), p21 (1:50, SAB), and iNOS (1:50, Proteintech). The following day, after thorough PBS washes, sections were incubated for 2 h at room temperature with HRP-conjugated secondary antibodies. Immunoreactivity was visualized using 3,3’-diaminobenzidine (DAB, Beyotime Biotechnology, Shanghai, China) substrate, followed by hematoxylin nuclear counterstaining. Images were captured using a brightfield light microscope. Statistical analysis Statistical analysis was conducted using GraphPad Prism 10 (GraphPad Prism Software, USA). Quantitative data are presented as mean ± standard deviation (SD). Comparisons between two groups were made using Student's t-test, while comparisons among multiple groups used one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. All experiments were independently repeated at least three times. Statistical significance was defined as p < 0.05. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; NS indicates not significant. Results IRAK4 is highly expressed in osteoarthritic articular cartilage in vivo and in chondrocytes induced by IL-1β in vitro To investigate the role of IRAK4 in OA pathogenesis, we first examined its expression levels in osteoarthritic cartilage tissues. Specifically, rat cartilage tissue samples were subjected to hematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemical staining to assess histomorphology and detect IRAK4 expression. Histological analysis with H&E and Safranin O/Fast Green staining revealed a disordered cartilage structure, accompanied by cartilage layer thinning and erosion, alongside a significant decrease in proteoglycan content in the cartilage matrix of the DMM-induced injury group. These pathological changes, together with a higher OARSI score, collectively indicated aggravated cartilage degradation (Fig. 1 A and B). Statistical analysis of the immunohistochemical results further confirmed that the proportion of IRAK4-positive cells was significantly higher in the OA group than in the sham group, suggesting a significant upregulation of IRAK4 expression in OA cartilage tissue (Fig. 1 C and D). Subsequently, to model the OA microenvironment in vitro, primary chondrocytes were treated with IL-1β, followed by an assessment of IRAK4 expression. Western blot analysis confirmed that IL-1β upregulates IRAK4 expression in osteoarthritic chondrocytes in a concentration-dependent manner. IRAK4 levels increased progressively with higher concentrations of IL-1β (0, 10, 20, and 40 ng/mL) (Fig. 1 E and F). Furthermore, a time-course experiment revealed that IL-1β (10 ng/mL) enhanced IRAK4 protein expression in a time-dependent fashion. IRAK4 levels began to increase within 12 hours of stimulation and peaked at 48 hours (Fig. 1 G and H). Quantitative analyses consistently showed significant upregulation of IRAK4 following IL-1β treatment, with both the magnitude and duration of stimulation contributing to enhanced expression. These results indicate that the upregulated IRAK4 is tightly correlated with OA progression both in vivo and in vitro. IRAK4 contributes to chondrocyte phenotype impairments in OA through the promotion of inflammation, cellular senescence, and ECM degradation To elucidate the molecular function of IRAK4, we knocked down its expression in chondrocytes using three distinct siRNAs (siIRAK4-1, -2, and − 3), with a scrambled non-targeting siRNA (siNC) serving as a negative control. As siRNA-2 produced the most robust knockdown, it was chosen for all subsequent IRAK4-silencing experiments (Fig. 2 A and B). Moreover, the upregulation of IRAK4 expression induced by IL-1β (10 ng/mL) was significantly abolished by siIRAK4 knockdown (Fig. 2 C and D). IRAK4 depletion promoted ECM anabolism while inhibiting catabolism, as evidenced by western blot analysis showing the reversal of IL-1β-induced upregulation of MMP13 and downregulation of Col2 in chondrocytes (Fig. 2 E and F). Meanwhile, the immunofluorescence results were consistent with this finding, showing a similar trend (Fig. 2 I-L). Additionally, staining with toluidine blue and safranin O (indicators of proteoglycan content and ECM metabolic status) demonstrated that the reduction in chondrocyte density and staining intensity induced by IL-1β, indicative of proteoglycan loss, was partially rescued by IRAK4 knockdown (Fig. 2 M). Regarding the inflammatory levels, in contrast to the potent upregulation of inflammatory markers (iNOS, COX-2, TNF-α) induced by IL-1β, silencing of IRAK4 potently suppressed their expression in chondrocytes (Fig. 2 E and G). OA is a common degenerative disease of the joints that is strongly associated with advancing age. A key pathological hallmark of OA is the prominent accumulation of senescent chondrocytes. In this study, the induction of cellular senescence by IL-1β, evidenced by upregulated p16 and p21 expression in chondrocytes, was attenuated upon IRAK4 knockdown (Fig. 2 E and H). In line with the WB results, IRAK4 silencing reversed the IL-1β-induced increase in SA-β-gal staining (Fig. 5 A and D). These findings demonstrate that IRAK4 inhibition acts to preserve the chondrocyte phenotype in OA by counteracting key pathological processes: inflammation, cellular senescence, and ECM degradation. The dysregulation of ECM metabolism and inflammation was alleviated by IRAK4 pharmacological inhibition with PF-06650833 We next employed PF-06650833, a potent and selective inhibitor of IRAK4, to further investigate the effects of pharmacological IRAK4 inhibition on the osteoarthritic chondrocyte phenotype in vitro. Figure 3 A depicts the molecular structure of PF-06650833. The results indicated that PF-06650833 did not induce significant cytotoxicity at concentrations ranging from 1 to 10 µM (Fig. 3 B). Both 5 and 10 µM PF-06650833 significantly and dose-dependently attenuated the reduction in cell viability caused by IL-1β (10 ng/mL) (Fig. 3 C). Meanwhile, qPCR showed that PF-06650833 significantly suppressed the IL-1β-induced upregulation of IRAK4 mRNA, confirming its effectiveness as a specific IRAK4 inhibitor (Fig. 3 D). The administration of PF-06650833 rescued matrix homeostasis from IL-1β stimulation, elevating the diminished anabolic structural protein Col2 and reducing the heightened catabolic marker MMP13 (Fig. 3 E and F). In agreement with these results, immunofluorescence indicated that PF-06650833 enhanced Col2 fluorescence intensity and diminished MMP13 level in chondrocytes following IL-1β treatment (Fig. 3 I-L). Furthermore, both toluidine blue and safranin O staining confirmed that PF-06650833 increased the staining intensity relative to the IL-1β-stimulated group, demonstrating that it restored the loss of ECM proteoglycans induced by IL-1β (Fig. 3 M). Western blot analysis further confirmed that IL-1β strongly induced the expression of key inflammatory mediators, including TNF-α, COX-2, and iNOS. PF-06650833 treatment effectively reversed this upregulation, underscoring the central role of IRAK4 in driving inflammatory signaling and suggesting that its inhibition may protect against ECM degradation in OA chondrocytes by dampening pro-inflammatory pathways (Fig. 3 E and G). Moreover, PF-06650833 treatment significantly lowered the increased levels of p16 and p21 induced by IL-1β (Fig. 3 E and H). EdU assays showed that IL-1β reduced chondrocyte proliferation, whereas PF-06650833 treatment significantly restored it (Fig. 3 N and O), suggesting that IRAK4 inhibition promotes proliferation by counteracting inflammation and cellular aging. Taken together, our findings demonstrate that IRAK4 inhibition with PF-06650833 conferred multifaceted protection in vitro, by dampening inflammation, rescuing matrix metabolism, and reducing senescence, ultimately improving chondrocyte function. Notably, for the aforementioned beneficial effects, a high concentration (10 µM) of PF-06650833 exhibited a more pronounced performance compared to the low concentration (5 µM) group. Inhibition of IRAK4 alleviated oxidative stress, promoted mitochondrial fusion, inhibited mitochondrial fission, and restored mitochondrial function. Mitochondria are highly dynamic organelles that continuously undergo fusion and fission to sustain their structural integrity and metabolic flexibility. In OA chondrocytes, preserving such mitochondrial dynamics is essential for energy homeostasis and antioxidant defense. Disruption of mitochondrial fusion/fission balance, together with impaired mitophagy and redox regulation, has been implicated in the pathogenesis of OA 27,28 . We sought to determine whether IRAK4 influences mitochondrial homeostasis by modulating oxidative stress. To this end, we first evidenced reduced oxidative stress—manifested by lower fluorescence intensity of DCFH-DA staining, i.e., ROS levels—upon IRAK4 knockdown in IL-1β-treated chondrocytes (Fig. 4 A and F). Subsequently, we stained mitochondria with Mito-Tracker Red and observed their morphology and structure. IRAK4 silencing attenuated the IL-1β-induced reduction in MitoTracker Red fluorescence intensity, suggesting an increase in mitochondrial number and preservation of mitochondrial biogenesis compared to the IL-1β-stimulated group. Moreover, morphological assessment revealed that chondrocytes under normal conditions contained elongated, tubular mitochondria with highly interconnected networks, typically exhibiting cylindrical or rod-like structures. In contrast, IL-1β stimulation induced severe mitochondrial fragmentation, resulting in shortened, punctate organelles. IRAK4 knockdown was effective in partially rescuing this damaged mitochondrial morphology (Fig. 4 B and G). Densely packed and well-structured cristae drive mitochondrial efficiency. The powerhouse function of mitochondria is powered by its intact, abundant cristae. Subsequently, we further employed transmission electron microscopy (TEM) to observe the mitochondrial ultrastructure. Mitochondria in OA chondrocytes displayed notable ultrastructural aberrations, such as overall swelling, vacuole formation within the matrix, loss and fragmentation of cristae, reduced cristae density, disorganized arrangement, and compromised membrane integrity culminating in outer membrane rupture, which were ameliorated in IRAK4-depleted group (Fig. 4 C). Besides, IRAK4 inhibition restored the IL-1β-induced decrease in mitochondrial membrane potential in chondrocytes, as evidenced by an elevated JC-1 aggregate/monomer ratio (Fig. 4 D and H). Mitochondrial dynamics, which involve the balanced processes of fusion and fission, regulate mitochondrial morphology, quantity, and function, playing a critical role in cellular health and fate. We revealed that IL-1β treatment of chondrocytes promoted mitochondrial fission and reduced fusion, as evidenced by increased expression of fission-related proteins (Fis1, p-Drp1) and decreased expression of fusion-related proteins (OPA1, Mfn2). Conversely, IRAK4 inhibition promotes mitochondrial fusion by downregulating Fis1 and p-Drp1, and upregulating OPA1 and Mfn2 (Fig. 4 E, I, and J). Collectively, these results indicate that IRAK4 inhibition preserves mitochondrial structure and function in OA chondrocytes by alleviating oxidative stress, restoring membrane potential, and rebalancing fusion-fission dynamics against IL-1β-induced damage. IRAK4 inhibition confers protection against IL-1β-induced cell death in chondrocytes Mitochondria function as a critical nexus, where diverse death-inducing pathways converge to orchestrate the execution of programmed cell death, both apoptotic and non-apoptotic 29 . Mitochondrial damage is a hallmark event in the early stages of apoptosis. As a central player in cellular pathways, mitochondrial dysfunction ultimately leads to or exacerbates age-related diseases. In this study, following IL-1β induction, flow cytometry analysis revealed a rise in apoptosis in OA chondrocytes, which was suppressed upon IRAK4 inhibition (Fig. 5 B and E). This result was further corroborated by the Calcein-AM/PI double staining assay. IL-1β treatment resulted in a higher number of dead cells, as evidenced by live/dead staining. In contrast, IRAK4 knockdown significantly enhanced chondrocyte survival (Fig. 5 C and F). We further extended our investigation to explore the role of PF-06650833 in mitigating mitochondrial dysfunction in osteoarthritic chondrocytes. As expected, western blot analysis confirmed that PF-06650833 counteracted the IL-1β-induced increase in the mitochondrial fission markers Fis1 and p-Drp1, as well as the decrease in the fusion markers OPA1 and Mfn2. Moreover, the 10 µM concentration exhibited a more significant ameliorative effect compared to the 5 µM dose (Fig. 5 G-I). In summary, IRAK4 inhibition confers a protective effect against IL-1β-induced chondrocyte death, potentially by restoring mitochondrial dynamics fission/fusion balance. IRAK4 drives the inflammatory response via the MAPK/NF-κB signaling pathway involving METTL3 in chondrocytes Next, to elucidate the mechanism by which IRAK4 activates NF-κB, we examined its effect on key signaling molecules in the NF-κB pathway. First, we observed that in chondrocytes, there was a marked increase in the formation of the TRAF6-TAK1 complex following IL-1β stimulation. IRAK4 depletion significantly reduced the protein levels of both TRAF6 and TAK1 as shown in Fig. 6 A-C. It is well established that the activation of TRAF6-TAK1 during OA acts as a key trigger for major inflammatory signaling pathways, principally NF-κB and MAPK. As expected, with the increase in the level of the TRAF6-TAK1 complex, the IL-1β-induced phosphorylation levels of IKKα, IκBα, and p65 also increased. However, knockdown of IRAK4 significantly reduced the phosphorylation-to-total protein ratios of IKKα, IκBα, and p65 (Fig. 6 A-F). Hence, IRAK4 promotes the inflammatory cascade in IL-1β-treated chondrocytes by engaging the TRAF6-TAK1 complex. We further investigated the involvement of METTL3 in the IRAK4-driven NF-κB inflammatory cascade in chondrocytes. The result indicated that IL-1β treatment had significantly upregulated METTL3 expression, suggesting that METTL3 played an important role in the ensuing inflammatory signaling (Fig. 6 G and H). Interestingly, METTL3 knockdown exerted an additive effect, further reducing the protein levels of TRAF6, p-ERK/ERK, p-JNK/JNK, p-p38/p38, and p-p65/p65 that were already lowered by IRAK4 inhibition (Fig. 6 I-N). NOX2 is one of the major contributors to cellular ROS production. Results indicated that while IRAK4 inhibition attenuated the upregulation of NOX2 caused by IL-1β in chondrocytes, METTL3 knockdown further decreased NOX2 levels beyond this effect (Fig. 6 I and O). Both IRAK4 and METTL3 drive NADPH oxidase to increase ROS production, and inhibiting either of them can exert antioxidant effects. Collectively, our findings identify IRAK4 as a key activator of the TRAF6-TAK1 complex mediated MAPK/NF-κB inflammatory cascade in IL-1β-stimulated chondrocytes, and reveal METTL3 as a critical amplifier of this signaling pathway. IRAK4 inhibition alleviated OA cartilage degeneration in a DMM-induced rat model To investigate the effect of IRAK4 in vivo, we established a post-traumatic OA (PTOA) model in rats by performing DMM surgery—a model that recapitulates human OA pathology. This was followed by an intra-articular injection of an adeno-associated virus carrying shIRAK4 (Fig. 7 A). Given that OA is a whole-joint disease pathologically involving both cartilage and subchondral bone, we extended our analysis to include a general assessment of osteophyte formation and subchondral bone sclerosis. Macroscopically, using micro-CT analysis and 3D reconstruction, we observed that DMM surgery led to aggravated osteophyte formation, uneven bone surfaces of the tibia and femur, and joint space narrowing. Knockdown of IRAK4 partially ameliorated these changes, particularly in reducing the number and volume of osteophytes and improving the smoothness of the bone surfaces (Fig. 7 B). Furthermore, bone remodeling of the subchondral bone in both the medial femoral condyle and the tibial plateau was observed, which is a key pathological feature of OA. In the bone geometry and microstructural parameters of the subchondral bone, micro-CT analysis revealed that, compared with the sham group, DMM rats demonstrated a decrease in the bone volume/tissue volume ratio (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular number (Tb.N), but an increase in trabecular separation (Tb.Sp). IRAK4 knockdown attenuated the aforementioned OA-like pathological changes to a significant degree in DMM rats (Fig. 7 C-G). Microscopically, articular-cartilage integrity and gross morphological changes were evaluated with hematoxylin–eosin and Safranin-O/Fast Green staining. Results showed that DMM-induced rats exhibited significant cartilage degradation, reduced hyaline cartilage thickness, a rough cartilage surface, notable erosion and fissures of the surface articular cartilage, and extensive proteoglycan loss. Our results indicate that intra-articular injection of Ad-shIRAK4 exerted a chondroprotective effect, alleviating the characteristic pathological changes of OA cartilage. Consistent with the observed phenotypes, the OARSI score—a histological measure of OA severity—confirmed that IRAK4 knockdown alleviated OA progression in vivo (Fig. 7 H and I). Not only that, we profiled the expression of key proteins associated with multiple pathological processes (anabolism, catabolism, inflammation, senescence) in OA articular cartilage in vivo using IHC. Firstly, intra-articular injection of Ad-shIRAK4 effectively reduced IRAK4 expression in the articular cartilage of DMM model rats, confirming the efficiency of the knockdown. Then, in terms of anabolism, catabolism, inflammation, and senescence markers, the DMM group exhibited reduced levels of Col2 and elevated levels of MMP13, iNOS, and P21 compared to the sham group. Conversely, the DMM + Ad-shIRAK4 group showed higher expression of Col2 and lower expression of MMP13, iNOS, and P21, which illustrated that IRAK4 inhibition manifested a significant cartilage-protective role in the OA model (Fig. 8 A-F). Overall, the above results provide evidence that IRAK4 knockdown can mitigate articular cartilage damage in an OA model in vivo. Discussion Osteoarthritis (OA) is a prevalent degenerative joint disease whose etiology remains unclear. Despite its high prevalence, effective therapeutic options are still limited, which underscores the significance of this public health problem given its substantial socioeconomic burden. The pathogenesis of OA is primarily characterized by the degradation of cartilaginous tissue, driven by an imbalance between catabolism and anabolism, enhanced oxidative stress, chondrocyte apoptosis, mitochondrial dysfunction, and extracellular matrix (ECM) degradation 30 . Current clinical management strategies are primarily limited to symptom relief, including the use of nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of lubricating supplements, and surgical interventions such as microfracture and mosaicplasty. When these conventional treatments fail, joint arthroplasty remains the final option 31,32 . Although this procedure effectively alleviates pain and restores joint function in most cases, a subset of patients still experiences postoperative complications such as residual pain, functional impairment, and persistent inflammation. Therefore, elucidating the underlying pathological mechanisms of OA and developing novel, effective treatments are top priorities for managing this disease. OA was previously considered merely a sequential degradation of cartilage leading to joint impairment. However, a more dynamic view has now emerged, in which the synovium, cartilage, and subchondral bone all produce inflammatory mediators that ultimately lead to cartilage damage 33 . IL-1β, which is recognized as one of the most significant inflammatory mediators, binds with IL-1R in the presence of TLR to provoke OA phenotype alterations. IRAK4, among the IRAK family, is a critical serine-threonine kinase in TLR/IL-1R signaling pathways 8 . In our study, we found increased IRAK4 expression in rat OA articular cartilage samples, a phenomenon also observed in IL-1β-treated chondrocytes in a dose- and time-dependent manner. The pro-inflammatory role of IRAK4 has been demonstrated in various diseases. Its inhibition alleviates inflammatory skin diseases 11 , ameliorates rheumatoid arthritis and lymphoid malignancy 12,24 , and mitigates oncogenic transformation in lung cancer 9 , among other effects. In our research, IRAK4 inhibition led to significant suppression of MMP13, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). It also reversed the loss of type II collagen and promoted extracellular matrix (ECM) production in IL-1β-treated chondrocytes. Concurrently, the inhibition of cartilage aging-related proteins p16 and p21 helped maintain ECM stability and alleviate inflammatory damage. Therefore, we conclude that IRAK4 promotes ECM degradation via MMP13, accelerates the cellular aging process, stimulates nitric oxide production, and regulates arachidonic acid metabolism to generate inflammatory mediators. This suggests that IRAK4 impairs the OA chondrocyte phenotype by activating inflammatory signaling pathways that primarily drive catabolic processes over anabolic ones. It is well established that aberrant activation of nuclear factor-kappaB (NF-κB) significantly contributes to OA progression 34 . The dynamic balance between NF-κB inhibitors (IκBs) and IκB kinases (IKKs) modulates NF-κB activation. A variety of inflammatory signals, such as TNFα, can activate IKKs. This activation triggers the phosphorylation and degradation of IκBs, leading to the translocation of the NF-κB complex into the nucleus. There, it initiates the transcription of downstream target genes 30 . Our results indicated that IRAK4 inhibition reduced the phosphorylation of IKKα and IκBα, along with the levels of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor beta-activated kinase 1 (TAK1). This suggests that by inhibiting the TRAF6-TAK1 complex, IRAK4 blockade reduces IKKα phosphorylation. This, in turn, diminishes IκBα phosphorylation, thus preventing NF-κB activation and curtailing the inflammatory response in osteoarthritic chondrocytes. Mitochondria are highly dynamic organelles that undergo continuous fission and fusion to maintain quality control 35 . In chondrocytes, mitochondrial functional stability is vital for maintaining cellular energy metabolism and resistance to oxidative stress 36 . Since aging and apoptosis are important mechanisms of OA cartilage degradation, the balance between mitochondrial fission and fusion appears particularly critical 37–39 . Our findings demonstrated that both transfection with IRAK4-targeting siRNA and administration of the IRAK4 inhibitor PF-06650833 reversed the detrimental alterations in the expression of key mitochondrial dynamics proteins induced by IL-1β, specifically restoring the levels of the fusion regulators Mfn2 and Opa1 while reducing the elevated levels of the fission regulators Fis1 and Drp1. Additionally, the increase in ROS levels in IL-1β-treated chondrocytes was attenuated by IRAK4 depletion, suggesting that IRAK4 may contribute to mitochondrial dysfunction by promoting oxidative stress. IRAK4 inhibition also mitigated mitochondrial swelling and structural damage induced by IL-1β, indicating its role in maintaining mitochondrial structural integrity. Given that OA incidence is strongly age-dependent and associated with processes such as the senescence-associated secretory phenotype (SASP) 40 , our results highlight a potential mechanism by which IRAK4 contributes to age-related OA progression. Furthermore, both IRAK4 knockdown and pharmacological inhibition by PF-06650833 alleviated the IL-1β-induced upregulation of the senescence markers p16 and p21 in chondrocytes. Taken together, our study highlights the potential of targeting IRAK4 with PF-06650833 for OA therapy, mechanistically linked to its efficacy in the preservation of mitochondrial integrity and homeostasis. In previous studies, the impact of METTL3 on the pathophysiological process of OA has been shown to be sophisticated and multifaceted, involving regulatory mechanisms across chondrocytes 41,42 , ATDC5 cells 43 , and OA-FLS 19 . Initially, the expression level of METTL3 exhibits a positive correlation with OA severity. METTL3 overexpression mediates an increase in m⁶A methylation modification, which in turn leads to enhanced chondrocyte apoptosis and diminished production of ECM components 42 . A previous study reported that METTL3 mRNA levels were higher in IL-1β-treated ATDC5 cells than in untreated control cells 44 . Intriguingly, seemingly paradoxical findings have been reported. One study revealed through bioinformatic analysis that METTL3 expression was downregulated in OA group, the finding further confirmed in clinical samples. Similarly, in vitro treatment with IL-1β reduced METTL3 expression levels in chondrosarcoma (SW1353) cells. In contrast, overexpression of METTL3 significantly suppressed the IL-1β-induced upregulation of inflammatory cytokines 45 . Furthermore, a recent study demonstrated that the expression of METTL3 was downregulated both in a mouse model of temporomandibular joint OA in vivo and in chondrogenic ATDC5 cells under inflammatory stimulation in vitro. Moreover, METTL3 inhibits inflammation-induced apoptosis and autophagy in ATDC5 cells by enhancing Bcl2 stability through Ythdf1-mediated m⁶A modification 43 . Similarly, METTL3 expression was downregulated in LPS-treated osteoblasts and was found to modulate the downstream MAPK signaling pathway 46 . However, the relationship between IRAK4 and METTL3 in OA pathological process remains unclear. Our results initially demonstrated that METTL3 is overexpressed in IL-1β-treated chondrocytes. Subsequently, we found that depletion of either IRAK4 or METTL3 significantly suppressed the activation of MAPK signaling, and dual knockdown further enhanced this inhibitory effect. Moreover, a similar trend of suppression was observed in the alteration of NADPH oxidase 2 (NOX2), which reflects oxidative pathway activity. These findings suggest that, beyond NF-κB signaling, IRAK4 may facilitate MAPK signaling activation through METTL3, potentially by directly regulating oxidative stress within the inflammatory microenvironment of OA. Conclusion In summary, our findings expand the understanding of mechanisms by which IRAK4 plays a key role in promoting inflammation, ECM degradation, cellular senescence, and mitochondrial dysfunction in OA. Furthermore, IRAK4 primarily activates NF-κB and MAPK signaling through its interaction with METTL3 to mediate these effects. More importantly, this study highlights the immense therapeutic potential of the IRAK4 inhibitor PF-06650833 for treating OA. Declarations Study approval All animal procedures were performed in accordance with the Guidelines for care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (No. 20220603A). Author contributions Xuezhong Wang, Dehong Yang, Lanwei Xu and Yong Hu conceived and designed the project. Xuezhong Wang and Shenglu Cao performed the experiments. Kai Tong and Siliang Ma assisted with the experiments. Xuezhong Wang and Shenglu Cao analyzed the data. Xuezhong Wang, Kai Tong, and Yong Hu performed statistical analyses and prepared figures. Xuezhong Wang and Yong Hu drafted the original manuscript. Xuezhong Wang, Shenglu Cao, and Dehong Yang revised the manuscript. All authors have read and approved the final version of the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was funded by the National Natural Science Foundation of China (No. 82002337). 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BIOCHEM BIOPH RES CO 516 22 (2019) Sang, W., et al.: METTL3 involves the progression of osteoarthritis probably by affecting ECM degradation and regulating the inflammatory response. LIFE SCI 278 119528 (2021) Zhang, Y., Gu, X., Li, D., Cai, L., Xu, Q.: METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling. INT J MOL SCI 21 (2019) Additional Declarations There is NO Competing Interest. Supplementary Files RS1159.pdf Reporting Summary Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":892957,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IRAK4 was elevated in both the DMM-induced rat OA model and in IL-1β-stimulated chondrocytes. (A) Representative images of knee joint sections stained with hematoxylin and eosin (H\u0026amp;E) and Safranin O/Fast Green from the different groups. (B) Statistical analysis of Osteoarthritis Research Society International (OARSI) scores for cartilage histopathology (n=6). (C) Immunohistochemical staining for IRAK4 in articular cartilage from the sham and DMM groups. (D) Quantitative analysis of the percentage of IRAK4-positive cells in immunohistochemical staining (n=6). (E, F) Western blot analysis of IRAK4 expression in chondrocytes treated with increasing concentrations of IL-1β (0, 10, 20, and 40 ng/ml) (n=3). (G, H) Western blot analysis of IRAK4 protein levels in chondrocytes at different time points (0, 12, 24, and 48 h) after stimulation with IL-1β (10 ng/ml) (n=3). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003eIRAK4 contributes to chondrocyte phenotype impairments in OA through the promotion of inflammation, cellular senescence, and ECM degradation\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/4c67d8dca83e1e01041cc189.png"},{"id":96467078,"identity":"21bdc5ce-121b-4504-a0c1-5ab947a14af8","added_by":"auto","created_at":"2025-11-21 11:28:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":831862,"visible":true,"origin":"","legend":"\u003cp\u003eThe function of IRAK4 inhibition in maintaining anabolism, suppressing catabolism, inhibiting inflammatory response, and attenuating cellular senescence upon IL-1β treatment. (A, B) Western blot and quantitative analysis showing the expression levels of IRAK4 in chondrocytes transfected with siIRAK4 (n=3). (C, D) Western blot and quantitative analysis of IRAK4 expression in siIRAK4-transfected chondrocytes following IL-1β treatment (n=3). (E-H) Protein levels and quantitative analysis showing levels of Col2, MMP13, iNOS, COX-2, TNF-α, p16 and p21 (n=3). (I, J) Immunofluorescence images and quantitative analysis of Col2 in siIRAK4-transfected chondrocytes under IL-1β-induced inflammatory conditions (n=5). (K, L) Immunofluorescence images and quantitative analysis of MMP13 in siIRAK4-transfected chondrocytes under IL-1β-induced inflammatory conditions (n=5). (M) Safranin O and toluidine blue staining of chondrocytes following siIRAK4 transfection and IL-1β treatment. The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/e41f636aa666a7cc9d7c2a1d.png"},{"id":96467079,"identity":"2e5bf0b0-c509-407d-8007-f4ebb33f4767","added_by":"auto","created_at":"2025-11-21 11:28:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":726788,"visible":true,"origin":"","legend":"\u003cp\u003eRoles of the IRAK4 inhibitor PF-06650833 in preserving anabolism, blocking catabolism, curbing inflammatory response, and mitigating cellular senescence after IL-1β treatment. (A) Chemical structure of PF-06650833. (B) Cytotoxicity assessment of PF-06650833. Cell viability was measured by CCK-8 assay in chondrocytes treated with increasing concentrations (0, 1, 2, 5, 10 µM) of PF-06650833 (n=5). (C) Cell viability measured by CCK-8 assay in chondrocytes treated with IL-1β in the presence or absence of PF-06650833 (5, 10 µM) (n=5). (D) IRAK4 mRNA expression levels analyzed by qPCR in chondrocytes after treatment with IL-1β and PF-06650833 (5, 10 µM) (n=3). (E-H) Protein expression and quantitative analysis. Representative western blots (E) and quantification (F-H) of Col2, MMP13, iNOS, COX-2, TNF-α, p16, and p21 in chondrocytes treated with IL-1β with or without PF-06650833 (5, 10 µM) (n=3). (I, J) Immunofluorescence images and quantitative analysis of Col2 in chondrocytes under IL-1β-induced inflammatory conditions in the presence or absence of PF-06650833 (5, 10 µM) (n=5). (K, L) Effects of PF-06650833 (5, 10 µM) on MMP13 expression under IL-1β-induced inflammatory conditions, shown by immunofluorescence images and quantitative analysis (n=5). (M) Safranin O and toluidine blue staining of chondrocytes after IL-1β stimulation with or without PF-06650833 (5, 10 µM). (N, O) EdU proliferation assay. Representative images (N) and quantification (O) of EdU-positive cells in chondrocytes following co-treatment with IL-1β and PF-06650833 (5, 10 µM). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/1b6aa037ee6559419e04789c.png"},{"id":96467081,"identity":"3d73a091-d409-4131-a799-87b92b9fe66c","added_by":"auto","created_at":"2025-11-21 11:28:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":602432,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of IRAK4 knockdown on oxidative stress and mitochondrial homeostasis in chondrocytes. (A, F) Assessment of intracellular ROS levels by DCFH-DA staining and quantitative analysis of fluorescence intensity across different groups (n=5). (B, G) Representative images of mitochondrial morphology stained by Mito-Tracker Red and quantitative analysis of fluorescence intensity across experimental groups (n=5). (C) Representative transmission electron microscopy images of mitochondrial ultrastructure in chondrocytes with or without IL-1β stimulation and IRAK4 knockdown (n=3). (D, H) The levels of mitochondrial membrane potential were measured using JC-1 staining, with quantitative analysis of fluorescence intensity across experimental groups (n=5). (E, I, J) Western blot analysis of OPA1, Mfn2, p-Drp1, Drp1, and Fis1 protein expression in chondrocytes under IL-1β stimulation in the presence or absence of IRAK4 knockdown, with quantitative analysis (n=3). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/c678961805e18c696990ffa0.png"},{"id":96603346,"identity":"84ea392a-e72a-428a-90f8-79a3b56f2d9c","added_by":"auto","created_at":"2025-11-24 09:08:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1190998,"visible":true,"origin":"","legend":"\u003cp\u003eUnder IL-1β stimulation, the effect of IRAK4 knockdown on chondrocyte apoptosis, as well as the effect of PF-06650833 on mitochondrial fusion and fission. (A, D) SA-β-gal staining for chondrocyte senescence and quantification of positive cells following IL-1β stimulation and siIRAK4 transfection (n=3). (B, E) Flow cytometric analysis of chondrocyte apoptosis and statistical analysis of the apoptotic cell percentage (n=3). (C, F) Calcein-AM/PI double staining for live/dead cells and statistical analysis of the PI-positive cell percentage (n=3). (G-I) Western blot analysis of OPA1, Mfn2, p-Drp1/Drp1, and Fis1 protein expression in chondrocytes following IL-1β stimulation with or without PF-06650833 (5, 10 µM), with quantification (n=3). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/b32ef0f2bc5ea35acdeeaa46.png"},{"id":96603445,"identity":"e9a61c17-beb9-432f-9992-7ec89c541a2c","added_by":"auto","created_at":"2025-11-24 09:09:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":701383,"visible":true,"origin":"","legend":"\u003cp\u003eUnder IL-1β stimulation, the role of IRAK4 in the MAPK/NF-κB inflammatory cascade in chondrocytes, and the regulatory role of METTL3 in this process. (A-F) Under IL-1β stimulation and IRAK4 knockdown, protein levels and quantitative analysis of TRAF6, TAK1, p-IKKα/IKKα, p-IκBα/IκBα, p-p65/p65 (n=3). (G, H) METTL3 protein expression and quantification in the normal control and IL-1β-induced OA groups (n=3). (I-O) Western blot analysis and quantification of TRAF6, p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-p65/p65, and NOX2 protein levels in IL-1β-stimulated chondrocytes following knockdown of IRAK4 and METTL3. The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/725b0c318098a803ac4444c7.png"},{"id":96467089,"identity":"b7a5fc8d-9734-4112-a900-46d72b3cf42f","added_by":"auto","created_at":"2025-11-21 11:28:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":912255,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of IRAK4 attenuates subchondral bone remodeling and protects against cartilage degradation in DMM-induced rats. (A) Flowchart of animal experiment. (B) Representative three-dimensional micro-CT reconstruction images of rat knee joints, showing the articular surfaces of the femur and tibia and the subchondral bone plate. (C-G) Quantitative analysis of bone microarchitecture parameters, including bone volume fraction (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) (n=6). (H) Representative images of hematoxylin–eosin and Safranin-O/Fast-Green staining in different experimental groups. (I) Osteoarthritis Research Society International (OARSI) scores based on Safranin-O/Fast Green staining of rat knee joints (n=6). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/e60902505c50d146d73cb5cb.png"},{"id":96603168,"identity":"73a6c21f-e728-4253-ab7b-7af9c0de1a68","added_by":"auto","created_at":"2025-11-24 09:07:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":772919,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of IRAK4 alleviates key pathological features of osteoarthritis, thereby slowing disease progression in DMM-induced rats. (A) Immunohistochemical assay of IRAK4, Col2, MMP13, iNOS, and P21 in articular cartilage of the indicated group. (B-F) Quantitative analysis of the percentage of cells positive for IRAK4, Col2, MMP13, iNOS, and P21 based on immunohistochemistry (n=6). The values are presented as the mean ± SD. ns, P \u0026gt; 0.05, not significant, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/f671438c59c30390d5fba14f.png"},{"id":97135360,"identity":"c4644625-abd3-4b42-ab8a-06b6ad52ddb2","added_by":"auto","created_at":"2025-12-01 09:39:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7187007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/486181dd-9ae6-46c4-a34c-e6ae81acd3cc.pdf"},{"id":96467083,"identity":"26797a1f-43ce-459f-b422-a518a629d50d","added_by":"auto","created_at":"2025-11-21 11:28:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2407805,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"RS1159.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7999197/v1/8fa8c25cc9351c257560e987.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Targeting IRAK4 mitigates osteoarthritis by preserving mitochondrial homeostasis and suppressing MAPK/NF-κB-mediated inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor decades, OA has been regarded as the most common musculoskeletal disease affecting millions of elderly people. Its sharply increasing prevalence, along with the enormous economic burden it imposes, drives the need to find effective solutions\u003csup\u003e1\u003c/sup\u003e. OA is an intricate process that involves the entire joint, and the progressive degradation of articular cartilage is regarded as its pivotal feature. Due to its avascularity and lack of nerves, cartilage lacks the ability to self-regenerate\u003csup\u003e2\u003c/sup\u003e. Therefore, clarifying the molecular mechanisms involved in OA cartilage deterioration is urgent and beneficial for developing effective treatments and improving prognosis.\u003c/p\u003e\u003cp\u003eThough mainly characterized by biomechanical alterations, increasing evidence indicates that low-grade synovial inflammation (synovitis) exists in OA as well\u003csup\u003e3\u003c/sup\u003e. The release of pro-inflammatory mediators (e.g., IL-1β, TNF-α) into the joint cavity promotes OA cartilage damage\u003csup\u003e4\u003c/sup\u003e. Binding of IL-1β to IL-1 receptor type 1 (IL-1R1) leads to recruitment of the adaptor protein MyD88 and activation of downstream kinases, including the IL-1 receptor-associated kinases (IRAKs), which initiate a signaling cascade that drives inflammatory responses\u003csup\u003e5\u003c/sup\u003e. IRAK4 is a 460-amino acid Ser/Thr protein kinase belonging to the IRAK family, which comprises IRAK-1, IRAK-2, IRAK-M, and IRAK-4. It plays a prominent role in mediating signal transduction by the Toll-like receptor (TLR) and IL-1R families\u003csup\u003e6,7\u003c/sup\u003e. The scaffolding function of IRAK4 is essential for Myddosome assembly and NF-κB activation. Together, the kinase and scaffolding functions of IRAK4 initiate the transcription and expression of pro-inflammatory factors\u003csup\u003e8\u003c/sup\u003e. For example, IRAK4 activation plays a critical role in smoking carcinogen-induced inflammation that promotes lung carcinogenesis\u003csup\u003e9\u003c/sup\u003e. Grant Otto reported that PF-06650833, a small-molecule inhibitor of IRAK4, demonstrates great potential for treating rheumatic diseases by alleviating inflammation in preclinical models of rheumatoid arthritis and lupus, and even reducing basal inflammation in healthy volunteers\u003csup\u003e10\u003c/sup\u003e. Furthermore, the inhibition of IRAK4 holds considerable potential as a therapeutic intervention for various immune-mediated inflammatory diseases\u003csup\u003e11,12\u003c/sup\u003e. Sadiq Umar et al. discovered that IRAK4 inhibition rebalances rheumatoid arthritis metabolic reprogramming by targeting glycolytic pathways and reducing inflammation in macrophages and fibroblasts\u003csup\u003e13\u003c/sup\u003e. A study indicated that adenovirus-mediated knockdown of IRAK4 alleviates OA in a rabbit model by reducing synovitis, while its impact on cartilage and the specific mechanisms involved were not addressed\u003csup\u003e14\u003c/sup\u003e. Therefore, focusing on chondrocytes to study the impact of IRAK4 on cartilage tissue and its potential mechanisms of action could provide new perspectives for the treatment of OA.\u003c/p\u003e\u003cp\u003eGenetic factors, modulated at both transcriptional and post-transcriptional levels, have been demonstrated to fundamentally contribute to the development of OA\u003csup\u003e15\u003c/sup\u003e. The RNA modification N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) is a critical post-transcriptional mechanism that fine-tunes gene expression by influencing RNA stability, translational control, and overall RNA activity, ultimately influencing a wide array of biological functions\u003csup\u003e16\u003c/sup\u003e. The m\u003csup\u003e6\u003c/sup\u003eA methyltransferase complex, which comprises methyltransferase-like 3 (METTL3) as its quintessential methyltransferase, is responsible for writing RNA m\u003csup\u003e6\u003c/sup\u003eA marks and is one of the most critical regulators in the epitranscriptomic machinery\u003csup\u003e17,18\u003c/sup\u003e. Xiang Chen et al. reported that excessive m\u003csup\u003e6\u003c/sup\u003eA modification, primarily mediated by METTL3, suppresses autophagy in OA fibroblast-like synoviocytes (OA-FLS). Silencing METTL3 in OA-FLS enhanced autophagic flux and attenuated cellular senescence propagation within joints, thereby alleviating cartilage damage\u003csup\u003e19\u003c/sup\u003e. Furthermore, mounting evidence supports a mechanism through which METTL3-mediated m\u003csup\u003e6\u003c/sup\u003eA modification of long noncoding RNAs exacerbates OA progression. Mechanistically, METTL3-mediated m\u003csup\u003e6\u003c/sup\u003eA modification of Atg5/Atg7 and BNIP3 was shown to ameliorate OA progression by modulating RNA stability\u003csup\u003e20\u003c/sup\u003e. Nevertheless, the precise mechanisms by which METTL3 contributes to OA progression remain poorly understood, highlighting the necessity for a thorough investigation into how it regulates various genes and signaling pathways.\u003c/p\u003e\u003cp\u003ePF-06650833 (zimlovisertib) is a highly potent and selective small-molecule inhibitor of IRAK4, exhibiting an exceptional IC50 value of 0.2 nM\u0026mdash;the lowest reported to date\u003csup\u003e21\u003c/sup\u003e. It functions through reversible inhibition of IRAK4, thereby suppressing TLR-mediated signaling and downstream production of pro-inflammatory cytokines such as type I interferons, IL-1, IL-6, IL-12, and TNF-α in human monocytes\u003csup\u003e22\u003c/sup\u003e. These cytokines play a central role in driving autoimmune and inflammatory pathologies. Preclinical studies have demonstrated its efficacy across multiple animal models, including respiratory distress syndrome, psoriasis, and rheumatic diseases\u003csup\u003e23,24\u003c/sup\u003e. Notably, to the best of our knowledge, PF-06650833 is the first IRAK4 inhibitor to enter human clinical trials. It has successfully completed Phase I studies, where it was well-tolerated with no dose-limiting toxicities observed, supporting its further development. A Phase II clinical trial for rheumatoid arthritis is currently underway\u003csup\u003e25\u003c/sup\u003e. Collectively, its strong selectivity, favorable safety profile, and robust anti-inflammatory activity in both experimental and clinical settings validate IRAK4 as a promising therapeutic target for inflammatory and autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus, spondyloarthritis, and psoriatic arthritis. However, the efficacy of PF-06650833 in the context of OA has not been established in either preclinical models or clinical trials.\u003c/p\u003e\u003cp\u003eHere, an in vitro OA model was generated by treating primary chondrocytes with IL-1β, and an in vivo rat OA model was established via the surgical destabilization of the medial meniscus (DMM). We aimed to examine the contribution of IRAK4 to OA initiation and pathophysiology, along with the involvement of METTL3 in this mechanism. Additionally, PF-06650833 was evaluated in a preclinical study to assess its therapeutic potential for OA. This work intends to offer novel insights into OA treatment strategies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIsolation and culture of rat primary chondrocytes\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003ePrimary chondrocytes were extracted from the knee articular cartilage of 5-day-old SD rat pups, following established protocols\u003csup\u003e26\u003c/sup\u003e. In brief, articular cartilage was aseptically dissected, cut into 1 mm3 small pieces, and enzymatically digested\u0026mdash;first with 0.25% trypsin (Boster, Wuhan, China) for 30 min at 37\u0026deg;C, then with 0.25% type II collagenase (Yeasen, Shanghai, China) for 6 h at 37\u0026deg;C. After the collagenase removal, isolated chondrocytes were resuspended, filtered, and cultured in DMEM/F12 medium (Servicebio, Wuhan, China) supplemented with 10% fetal bovine serum (HyClone, USA), 1% streptomycin and penicillin (Life iLab, Shanghai, China) at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. To maintain chondrocyte phenotypic stability, our study exclusively employed second-generation chondrocytes in the ensuing experiments.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eImmunofluorescence (IF) staining\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eChondrocytes were seeded onto sterile glass coverslips placed in 6-well plates. After adhesion and treatment completion, the cells were fixed with 4% paraformaldehyde (PFA, Beyotime Biotechnology, Shanghai, China) at room temperature for 15 min, washed three times with PBS (Boster, Wuhan, China), and permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, Shanghai, China) for 15 min. The cells were then blocked with 5% bovine serum albumin (BSA, Yeasen, Shanghai, China) for 1 h. Following this, chondrocytes were incubated with primary antibodies against alpha-1 type II collagen (Col2, 1:100, Proteintech) and matrix metalloproteinase 13 (MMP13, 1:100, Proteintech) at 4\u0026deg;C overnight. Subsequently, chondrocytes were incubated with fluorescein-conjugated secondary antibodies (Proteintech, Wuhan, China) at 37\u0026deg;C for 1 h. Then, 4',6-diamidino-2-phenylindole (DAPI) (Servicebio, Wuhan, China) was utilized to counterstain the nuclei. Finally, the images were captured using a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eDetection of intracellular reactive oxygen species (ROS) levels\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eROS levels in chondrocytes were determined using a commercial kit according to the manufacturer\u0026rsquo;s protocols. Upon completion of chondrocyte intervention, following addition of DCFH-DA (Beyotime Biotechnology, Shanghai, China) solution to serum-free culture medium at a final concentration of 10 \u0026micro;M, incubation proceeded in the dark for 30 min. The cells were then washed with PBS to remove the fluorescent dye. The images were photographed under a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eJC-1 staining\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eEmploying the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime Biotechnology, Shanghai, China), changes in mitochondrial membrane potential (MMP, ΔΨm) were evaluated. At higher MMP, JC-1 accumulates within the mitochondrial matrix, forming JC-1 aggregates that emit red fluorescence. Conversely, at lower membrane potential, JC-1 remains dispersed in the matrix as monomers, producing green fluorescence. The relative ratio of red-to-green fluorescence intensity serves as a standard indicator for assessing the extent of mitochondrial depolarization. For this experiment, chondrocytes, seeded into 12-well plates and subjected to the designated treatments, were stained with JC-1 working solution at 37\u0026deg;C for 30 min postintervention following the kit instructions. Following aspiration of the supernatant, the cells were washed twice with JC-1 staining buffer and observed under a fluorescence microscope for image acquisition.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eCell viability assay\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eCell viability of chondrocytes was assessed using the CCK-8 assay kit (Solarbio, Beijing, China). In brief, chondrocytes were plated into 96-well plates at a density of 5000 cells per well and incubated at 37\u0026deg;C for 48 hours in the presence of the designated interventions. Subsequently, 10 \u0026micro;L per well of CCK-8 solution diluted in serum-free medium was added and incubation continued for 2 h. Absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eEdU assay\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eCellular proliferation capacity was evaluated with a commercial 5‑Ethynyl‑2\u0026rsquo;‑deoxyuridine (EdU) detection kit (Beyotime Biotechnology, Shanghai, China). Briefly, chondrocytes were plated in 12-well plates and maintained in DMEM for 48 hours. After incubation with EdU for 2 h, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized using 0.2% Triton X-100 for 15 min. Subsequently, cells were treated with the click reaction cocktail for 30 min, followed by nuclear staining with DAPI for 10 min. All procedures were performed at room temperature under dark conditions. EdU-positive proliferating cells were counted under a fluorescence microscope.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eMito-tracker red staining\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eBiologically active mitochondria in live chondrocytes were labeled with Mito-Tracker Red CMXRos kit (Beyotime Biotechnology, Shanghai, China). The working solution was prepared by diluting the probe in serum-free medium to 200 nM. After 30-min incubation at 37\u0026deg;C, cells were counterstained with Hoechst 33342 (Solarbio, Beijing, China) at 37\u0026deg;C for 10 min. Following three washes with PBS, samples were imaged using a fluorescence microscope. Fluorescence intensity was quantified from captured images using ImageJ.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eTransmission electron microscopy\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eMitochondrial ultrastructure in chondrocytes was analyzed via transmission electron microscopy (TEM). Chondrocytes were processed following established interventions. The collected cells were washed twice with PBS, then fixed in 1.5% glutaraldehyde for 4 h, followed by post-fixation in 1% osmium tetroxide for 2 h. Following dehydration through an ethanol gradient and acetone, the samples were embedded in Epon resin. Samples were sectioned at 60 nm thickness using an ultramicrotome, and the resulting ultrathin sections were mounted on copper grids. The sections were sequentially stained with 1% uranyl acetate for 30 min and 0.1% lead citrate for 30 min, and then observed with a TEM.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eLive/Dead staining\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eCell viability was qualitatively evaluated using a Calcein-AM/Propidium Iodide (PI) dual-stain kit (Solarbio, Beijing, China). Viable cells were identified by green fluorescence from Calcein-AM staining, whereas dead cells were detected through red fluorescence emitted by PI staining. Briefly, chondrocytes were seeded into 6-well culture plates and subjected to specified interventions. The Calcein-AM/PI working solution was then prepared according to the manufacturer\u0026rsquo;s instructions. An appropriate volume of the working solution was added to the samples, followed by incubation at 37\u0026deg;C for 30 min in the dark. After incubation, fluorescence microscopy was performed to assess staining: Calcein-AM (green fluorescence; Ex/Em\u0026thinsp;=\u0026thinsp;494/517 nm) and PI (red fluorescence; Ex/Em\u0026thinsp;=\u0026thinsp;535/617 nm).\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eSafranin O and toluidine blue staining of chondrocytes\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eGlycosaminoglycan and proteoglycan content, which collectively indicate pathological alterations in the cartilage extracellular matrix, were assessed using Toluidine blue (Servicebio, Wuhan, China) and safranin O (Servicebio, Wuhan, China) staining. After predetermined treatments, the medium was aspirated. Chondrocytes were subsequently washed thrice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. Afterwards, cells were stained by incubation with either toluidine blue or safranin O for 30 minutes at 37\u0026deg;C. Stained chondrocyte samples were imaged with an inverted light microscope.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eSenescence‑associated β‑galactosidase (SA-β‐gal) staining\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eUsing the Senescence β-Galactosidase Staining Kit (Solarbio, Beijing, China), chondrocyte senescence was detected based on the upregulation of SA-β-gal activity during aging. Cells cultured in 6-well plates were processed as follows: the culture medium was aspirated, washed with PBS, and 1 mL of staining fixative solution was added. Cells were fixed at room temperature for 15 minutes. The staining working solution was prepared according to the manufacturer\u0026rsquo;s instructions. Then, 1 mL of staining working solution was added to each well and incubated at 37\u0026deg;C overnight. Senescent cells were identified by the presence of blue-green staining indicative of SA-β-gal activity observed under light microscopy.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eFlow cytometry for chondrocyte apoptosis analysis\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eThe apoptosis of chondrocytes was assessed by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (Solarbio, Beijing, China) according to the manufacturer\u0026rsquo;s protocol. Chondrocytes were seeded in 6-well plates and exposed to different designated interventions. Post-treatment, cells were trypsinized, washed twice with PBS, and resuspended in PBS to generate a single-cell suspension. For apoptosis assessment, 500 \u0026micro;L of the suspension was aliquoted into a flow cytometry tube, stained with 5 \u0026micro;L Annexin V-FITC (gentle mixing), followed by addition of 10 \u0026micro;L propidium iodide (PI) solution. After 15-min incubation at room temperature in the dark, samples immediately underwent flow cytometric analysis.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eWestern blotting\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eUpon completion of the designated interventions, chondrocytes were then harvested and lysed in RIPA lysis buffer (Boster, Wuhan, China) supplemented with protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Boster, Wuhan, China) and phosphatase inhibitor (Solarbio, Beijing, China) for protein preparation. The concentrations of the obtained protein were measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein from each sample were then loaded onto 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for separation, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 2 h at room temperature. Subsequently, they were incubated overnight at 4\u0026deg;C with the following primary antibodies against IRAK4 (1:2000, Abclonal), Col2 (1:2000, abcam), MMP13 (1:2500, Abclonal), iNOS (1:5000, Proteintech), COX-2 (1:1000, Servicebio), TNF-α (1:2000, Proteintech), p16 (1:2000, BOSTER), p21 (1:2000, Signalway Antibody, SAB), OPA1 (1:2000, Affinity), Mfn2 (1:5000, Proteintech), p-Drp1 (1:1000, abcam), Drp1 (1:1000, abcam), Fis1 (1:2000, Proteintech), TRAF6 (1:1000, Abclonal), TAK1 (1:2000, Abclonal), p-IKKα (1:1000, Cell Signaling Technology), IKKα (1:2000, Proteintech), p-IκBα (1:1000, abcam), IκBα (1:2000, Abclonal), p-p65 (1:2000, Abclonal), p65 (1:5000, Proteintech), METTL3 (1:2000, Abclonal), p-ERK (1:2000, Servicebio), ERK (1:2000, Proteintech), p-JNK (1:2000, Affinity), JNK (1:10000, Proteintech), p-p38 (1:1000, Abclonal), p38 (1:2000, Abclonal), NOX2 (1:2000, Zen-Bio), GAPDH (1:10000, Proteintech). Following primary antibody incubation, the membranes were washed three times with Tris-buffered saline containing 0.05% Tween-20 (TBST). Finally, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Proteintech, Wuhan, China) for 2 h at room temperature. Protein bands were developed by exposure to the enhanced chemiluminescence (ECL, Abbkine, Wuhan, China) reagent and visualized by a Bio-Rad scanner. Immunoreactive band intensities were quantified using ImageJ software. Band intensities of target proteins were normalized to GAPDH, which served as the internal control.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eReverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from chondrocytes using a Total RNA Extraction Kit (Solarbio, Beijing, China). The extracted RNA was then reverse-transcribed into complementary DNA (cDNA) using a cDNA Synthesis Kit (Servicebio, Wuhan, China). Subsequently, quantitative real-time PCR (qPCR) was performed using the universal SYBR Green fast qPCR mix kit (Servicebio, Wuhan, China) on a Roche LightCycler\u0026reg; 480 instrument. Gene expression levels were normalized to GAPDH (internal reference) and quantified using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primer sequences used were as follows:\u003c/p\u003e\u003cp\u003eIRAK4 forward: 5\u0026rsquo;-GCAATCTGAAGTCCCCTCGT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eIRAK4 reverse: 5\u0026rsquo;-GGCTTGCTCATCTTCTACTTCCT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eSmall-interfering RNA (siRNA) transfection\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eSmall interfering RNA (siRNA) oligonucleotides targeting IRAK4, METTL3, and their corresponding negative controls were designed and synthesized by GENE CREATE (Wuhan, China). The sequences were as follows:\u003c/p\u003e\u003cp\u003eIRAK4 siRNA-1: 5\u0026rsquo;-GCAACAGUUUGACCAAGAATT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eIRAK4 siRNA-2: 5\u0026rsquo;-GCGAUGUACUCUGUUGCUATT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eIRAK4 siRNA-3: 5\u0026rsquo;-GGGUGAUGACAGAUACAAUTT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eMETTL3 siRNA-1: 5\u0026rsquo;-GGAUUGCGAUGUGAUUGUAGC-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eMETTL3 siRNA-2: 5\u0026rsquo;-CAGUGGAUCUGUUGUGAUAUC-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eMETTL3 siRNA-3: 5\u0026rsquo;-GGAGAUCCUAGAGCUAUUAAA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003eUsing Lipofectamine 3000 (Invitrogen, USA), chondrocytes were transfected according to the manufacturer\u0026rsquo;s instructions. Briefly, siRNA targeting IRAK4 or METTL3 was complexed with Lipofectamine 3000 in Opti-MEM medium for 8 h. After the transfection period, the mixture was removed and replaced with fresh DMEM/F12 medium. Following a further 24 h of culture, cells and supernatant were harvested and processed for further analysis.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eEstablishment of rat OA model\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eAll animal procedures were approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (Approval No. 20220603A) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Research Council. Eight-week-old male Sprague-Dawley rats (SD) were group-housed in the Laboratory Animal Center under controlled conditions (23\u0026ndash;25\u0026deg;C, 12-h light/dark cycle) with ad libitum access to food and water. Surgically-induced OA models were established in the right knee by transection of the medial meniscotibial ligament and medial meniscectomy (DMM model). Rats were randomly assigned to one of four experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group): Sham, DMM, DMM\u0026thinsp;+\u0026thinsp;AAV-control, and DMM\u0026thinsp;+\u0026thinsp;AAV-IRAK4 shRNA. Briefly, the DMM surgery was performed as follows: after ensuring adequate anesthesia, the joint capsule was incised. The medial meniscotibial ligament was transected, followed by resection of the medial meniscus. For the sham-operated group, the joint capsule and skin were sutured after incision without any ligament or meniscal damage. In the AAV-IRAK4 shRNA and AAV-control groups, rats received weekly intra-articular (IA) injections of AAV (1 \u0026times; 10^10 virus particles/30 \u0026micro;L) starting one week after surgery. The Sham and DMM groups received weekly IA injections of an equal volume of phosphate-buffered saline (PBS). At 8 weeks post-surgery, all animals were euthanized under anesthesia, and knee joint tissues were collected for subsequent analysis.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eMicro-computed tomography (Micro-CT) analysis\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eMicro-computed tomography (Micro-CT) was employed to scan knee joint specimens, enabling 3D reconstruction and visualization for quantitative analysis of subchondral bone structural alterations. In brief, samples underwent high-resolution Micro-CT scanning (SkyScan 1176) at 55 kV, 145 \u0026micro;A, and 300 ms exposure. Image reconstruction (NRecon v1.6) and repositioning (DataViewer v1.5) were performed with alignment of the coronal, sagittal, and axial planes prior to defining a region of interest (ROI) within the medial tibial plateau subchondral bone. Five sequential sagittal-plane ROI images were reconstructed in 3D (CTvol v3.0) for quantification of bone mineral density (BMD), trabecular bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) using CTAn v1.15 software.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eHistological and immunohistochemistry analysis\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eFor histological analysis, knee joint samples were fixed in 4% PFA for 48 h at room temperature, followed by decalcification in 10% ethylenediaminetetraacetic acid (EDTA, Beyotime Biotechnology, Shanghai, China) solution for 28 days. Subsequently, tissues were embedded in paraffin and sectioned sagittally into serial sections (5 \u0026micro;m thick). Sections were stained with hematoxylin and eosin (H\u0026amp;E, Servicebio, Wuhan, China) and safranin O/fast green (Servicebio, Wuhan, China) following standard protocols. OA severity assessment employed a modified Osteoarthritis Research Society International (OARSI) scoring system. Scoring was conducted blindly and independently by three trained assessors across at least three joint levels per sample. On this scale, a low score represents minimal damage, and a high score signifies severe damage.\u003c/p\u003e\u003cp\u003eFor immunohistochemistry analysis, following deparaffinization in xylene and rehydration through a graded alcohol series, cartilage sections were subjected to antigen retrieval using 0.1% trypsin for 30 min. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide for 10 min at room temperature. Nonspecific binding sites were blocked with 10% normal goat serum (Solarbio, Beijing, China). Sections were then incubated overnight at 4\u0026deg;C with primary antibodies targeting IRAK4 (1:100, Abclonal), Col2 (1:100, Proteintech), MMP13 (1:100, Proteintech), p21 (1:50, SAB), and iNOS (1:50, Proteintech). The following day, after thorough PBS washes, sections were incubated for 2 h at room temperature with HRP-conjugated secondary antibodies. Immunoreactivity was visualized using 3,3\u0026rsquo;-diaminobenzidine (DAB, Beyotime Biotechnology, Shanghai, China) substrate, followed by hematoxylin nuclear counterstaining. Images were captured using a brightfield light microscope.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was conducted using GraphPad Prism 10 (GraphPad Prism Software, USA). Quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons between two groups were made using Student's t-test, while comparisons among multiple groups used one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. All experiments were independently repeated at least three times. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Significance levels are denoted as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; NS indicates not significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIRAK4 is highly expressed in osteoarthritic articular cartilage in vivo and in chondrocytes induced by IL-1β in vitro\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of IRAK4 in OA pathogenesis, we first examined its expression levels in osteoarthritic cartilage tissues. Specifically, rat cartilage tissue samples were subjected to hematoxylin and eosin (H\u0026amp;E) staining, Safranin O/Fast Green staining, and immunohistochemical staining to assess histomorphology and detect IRAK4 expression. Histological analysis with H\u0026amp;E and Safranin O/Fast Green staining revealed a disordered cartilage structure, accompanied by cartilage layer thinning and erosion, alongside a significant decrease in proteoglycan content in the cartilage matrix of the DMM-induced injury group. These pathological changes, together with a higher OARSI score, collectively indicated aggravated cartilage degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Statistical analysis of the immunohistochemical results further confirmed that the proportion of IRAK4-positive cells was significantly higher in the OA group than in the sham group, suggesting a significant upregulation of IRAK4 expression in OA cartilage tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D).\u003c/p\u003e\u003cp\u003eSubsequently, to model the OA microenvironment in vitro, primary chondrocytes were treated with IL-1β, followed by an assessment of IRAK4 expression. Western blot analysis confirmed that IL-1β upregulates IRAK4 expression in osteoarthritic chondrocytes in a concentration-dependent manner. IRAK4 levels increased progressively with higher concentrations of IL-1β (0, 10, 20, and 40 ng/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F). Furthermore, a time-course experiment revealed that IL-1β (10 ng/mL) enhanced IRAK4 protein expression in a time-dependent fashion. IRAK4 levels began to increase within 12 hours of stimulation and peaked at 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and H). Quantitative analyses consistently showed significant upregulation of IRAK4 following IL-1β treatment, with both the magnitude and duration of stimulation contributing to enhanced expression. These results indicate that the upregulated IRAK4 is tightly correlated with OA progression both in vivo and in vitro.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIRAK4 contributes to chondrocyte phenotype impairments in OA through the promotion of inflammation, cellular senescence, and ECM degradation\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the molecular function of IRAK4, we knocked down its expression in chondrocytes using three distinct siRNAs (siIRAK4-1, -2, and \u0026minus;\u0026thinsp;3), with a scrambled non-targeting siRNA (siNC) serving as a negative control. As siRNA-2 produced the most robust knockdown, it was chosen for all subsequent IRAK4-silencing experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). Moreover, the upregulation of IRAK4 expression induced by IL-1β (10 ng/mL) was significantly abolished by siIRAK4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). IRAK4 depletion promoted ECM anabolism while inhibiting catabolism, as evidenced by western blot analysis showing the reversal of IL-1β-induced upregulation of MMP13 and downregulation of Col2 in chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). Meanwhile, the immunofluorescence results were consistent with this finding, showing a similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-L). Additionally, staining with toluidine blue and safranin O (indicators of proteoglycan content and ECM metabolic status) demonstrated that the reduction in chondrocyte density and staining intensity induced by IL-1β, indicative of proteoglycan loss, was partially rescued by IRAK4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Regarding the inflammatory levels, in contrast to the potent upregulation of inflammatory markers (iNOS, COX-2, TNF-α) induced by IL-1β, silencing of IRAK4 potently suppressed their expression in chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and G). OA is a common degenerative disease of the joints that is strongly associated with advancing age. A key pathological hallmark of OA is the prominent accumulation of senescent chondrocytes. In this study, the induction of cellular senescence by IL-1β, evidenced by upregulated p16 and p21 expression in chondrocytes, was attenuated upon IRAK4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and H). In line with the WB results, IRAK4 silencing reversed the IL-1β-induced increase in SA-β-gal staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and D). These findings demonstrate that IRAK4 inhibition acts to preserve the chondrocyte phenotype in OA by counteracting key pathological processes: inflammation, cellular senescence, and ECM degradation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe dysregulation of ECM metabolism and inflammation was alleviated by IRAK4 pharmacological inhibition with PF-06650833\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eWe next employed PF-06650833, a potent and selective inhibitor of IRAK4, to further investigate the effects of pharmacological IRAK4 inhibition on the osteoarthritic chondrocyte phenotype in vitro. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA depicts the molecular structure of PF-06650833. The results indicated that PF-06650833 did not induce significant cytotoxicity at concentrations ranging from 1 to 10 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Both 5 and 10 \u0026micro;M PF-06650833 significantly and dose-dependently attenuated the reduction in cell viability caused by IL-1β (10 ng/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, qPCR showed that PF-06650833 significantly suppressed the IL-1β-induced upregulation of IRAK4 mRNA, confirming its effectiveness as a specific IRAK4 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The administration of PF-06650833 rescued matrix homeostasis from IL-1β stimulation, elevating the diminished anabolic structural protein Col2 and reducing the heightened catabolic marker MMP13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). In agreement with these results, immunofluorescence indicated that PF-06650833 enhanced Col2 fluorescence intensity and diminished MMP13 level in chondrocytes following IL-1β treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-L). Furthermore, both toluidine blue and safranin O staining confirmed that PF-06650833 increased the staining intensity relative to the IL-1β-stimulated group, demonstrating that it restored the loss of ECM proteoglycans induced by IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Western blot analysis further confirmed that IL-1β strongly induced the expression of key inflammatory mediators, including TNF-α, COX-2, and iNOS. PF-06650833 treatment effectively reversed this upregulation, underscoring the central role of IRAK4 in driving inflammatory signaling and suggesting that its inhibition may protect against ECM degradation in OA chondrocytes by dampening pro-inflammatory pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and G). Moreover, PF-06650833 treatment significantly lowered the increased levels of p16 and p21 induced by IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and H). EdU assays showed that IL-1β reduced chondrocyte proliferation, whereas PF-06650833 treatment significantly restored it (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN and O), suggesting that IRAK4 inhibition promotes proliferation by counteracting inflammation and cellular aging. Taken together, our findings demonstrate that IRAK4 inhibition with PF-06650833 conferred multifaceted protection in vitro, by dampening inflammation, rescuing matrix metabolism, and reducing senescence, ultimately improving chondrocyte function. Notably, for the aforementioned beneficial effects, a high concentration (10 \u0026micro;M) of PF-06650833 exhibited a more pronounced performance compared to the low concentration (5 \u0026micro;M) group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eInhibition of IRAK4 alleviated oxidative stress, promoted mitochondrial fusion, inhibited mitochondrial fission, and restored mitochondrial function.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eMitochondria are highly dynamic organelles that continuously undergo fusion and fission to sustain their structural integrity and metabolic flexibility. In OA chondrocytes, preserving such mitochondrial dynamics is essential for energy homeostasis and antioxidant defense. Disruption of mitochondrial fusion/fission balance, together with impaired mitophagy and redox regulation, has been implicated in the pathogenesis of OA\u003csup\u003e27,28\u003c/sup\u003e. We sought to determine whether IRAK4 influences mitochondrial homeostasis by modulating oxidative stress. To this end, we first evidenced reduced oxidative stress\u0026mdash;manifested by lower fluorescence intensity of DCFH-DA staining, i.e., ROS levels\u0026mdash;upon IRAK4 knockdown in IL-1β-treated chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and F). Subsequently, we stained mitochondria with Mito-Tracker Red and observed their morphology and structure. IRAK4 silencing attenuated the IL-1β-induced reduction in MitoTracker Red fluorescence intensity, suggesting an increase in mitochondrial number and preservation of mitochondrial biogenesis compared to the IL-1β-stimulated group. Moreover, morphological assessment revealed that chondrocytes under normal conditions contained elongated, tubular mitochondria with highly interconnected networks, typically exhibiting cylindrical or rod-like structures. In contrast, IL-1β stimulation induced severe mitochondrial fragmentation, resulting in shortened, punctate organelles. IRAK4 knockdown was effective in partially rescuing this damaged mitochondrial morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and G). Densely packed and well-structured cristae drive mitochondrial efficiency. The powerhouse function of mitochondria is powered by its intact, abundant cristae. Subsequently, we further employed transmission electron microscopy (TEM) to observe the mitochondrial ultrastructure. Mitochondria in OA chondrocytes displayed notable ultrastructural aberrations, such as overall swelling, vacuole formation within the matrix, loss and fragmentation of cristae, reduced cristae density, disorganized arrangement, and compromised membrane integrity culminating in outer membrane rupture, which were ameliorated in IRAK4-depleted group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Besides, IRAK4 inhibition restored the IL-1β-induced decrease in mitochondrial membrane potential in chondrocytes, as evidenced by an elevated JC-1 aggregate/monomer ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and H). Mitochondrial dynamics, which involve the balanced processes of fusion and fission, regulate mitochondrial morphology, quantity, and function, playing a critical role in cellular health and fate. We revealed that IL-1β treatment of chondrocytes promoted mitochondrial fission and reduced fusion, as evidenced by increased expression of fission-related proteins (Fis1, p-Drp1) and decreased expression of fusion-related proteins (OPA1, Mfn2). Conversely, IRAK4 inhibition promotes mitochondrial fusion by downregulating Fis1 and p-Drp1, and upregulating OPA1 and Mfn2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, I, and J). Collectively, these results indicate that IRAK4 inhibition preserves mitochondrial structure and function in OA chondrocytes by alleviating oxidative stress, restoring membrane potential, and rebalancing fusion-fission dynamics against IL-1β-induced damage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIRAK4 inhibition confers protection against IL-1β-induced cell death in chondrocytes\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eMitochondria function as a critical nexus, where diverse death-inducing pathways converge to orchestrate the execution of programmed cell death, both apoptotic and non-apoptotic\u003csup\u003e29\u003c/sup\u003e. Mitochondrial damage is a hallmark event in the early stages of apoptosis. As a central player in cellular pathways, mitochondrial dysfunction ultimately leads to or exacerbates age-related diseases. In this study, following IL-1β induction, flow cytometry analysis revealed a rise in apoptosis in OA chondrocytes, which was suppressed upon IRAK4 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and E). This result was further corroborated by the Calcein-AM/PI double staining assay. IL-1β treatment resulted in a higher number of dead cells, as evidenced by live/dead staining. In contrast, IRAK4 knockdown significantly enhanced chondrocyte survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and F). We further extended our investigation to explore the role of PF-06650833 in mitigating mitochondrial dysfunction in osteoarthritic chondrocytes. As expected, western blot analysis confirmed that PF-06650833 counteracted the IL-1β-induced increase in the mitochondrial fission markers Fis1 and p-Drp1, as well as the decrease in the fusion markers OPA1 and Mfn2. Moreover, the 10 \u0026micro;M concentration exhibited a more significant ameliorative effect compared to the 5 \u0026micro;M dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I). In summary, IRAK4 inhibition confers a protective effect against IL-1β-induced chondrocyte death, potentially by restoring mitochondrial dynamics fission/fusion balance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIRAK4 drives the inflammatory response via the MAPK/NF-κB signaling pathway involving METTL3 in chondrocytes\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eNext, to elucidate the mechanism by which IRAK4 activates NF-κB, we examined its effect on key signaling molecules in the NF-κB pathway. First, we observed that in chondrocytes, there was a marked increase in the formation of the TRAF6-TAK1 complex following IL-1β stimulation. IRAK4 depletion significantly reduced the protein levels of both TRAF6 and TAK1 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C. It is well established that the activation of TRAF6-TAK1 during OA acts as a key trigger for major inflammatory signaling pathways, principally NF-κB and MAPK. As expected, with the increase in the level of the TRAF6-TAK1 complex, the IL-1β-induced phosphorylation levels of IKKα, IκBα, and p65 also increased. However, knockdown of IRAK4 significantly reduced the phosphorylation-to-total protein ratios of IKKα, IκBα, and p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). Hence, IRAK4 promotes the inflammatory cascade in IL-1β-treated chondrocytes by engaging the TRAF6-TAK1 complex. We further investigated the involvement of METTL3 in the IRAK4-driven NF-κB inflammatory cascade in chondrocytes. The result indicated that IL-1β treatment had significantly upregulated METTL3 expression, suggesting that METTL3 played an important role in the ensuing inflammatory signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and H). Interestingly, METTL3 knockdown exerted an additive effect, further reducing the protein levels of TRAF6, p-ERK/ERK, p-JNK/JNK, p-p38/p38, and p-p65/p65 that were already lowered by IRAK4 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-N). NOX2 is one of the major contributors to cellular ROS production. Results indicated that while IRAK4 inhibition attenuated the upregulation of NOX2 caused by IL-1β in chondrocytes, METTL3 knockdown further decreased NOX2 levels beyond this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and O). Both IRAK4 and METTL3 drive NADPH oxidase to increase ROS production, and inhibiting either of them can exert antioxidant effects. Collectively, our findings identify IRAK4 as a key activator of the TRAF6-TAK1 complex mediated MAPK/NF-κB inflammatory cascade in IL-1β-stimulated chondrocytes, and reveal METTL3 as a critical amplifier of this signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eIRAK4 inhibition alleviated OA cartilage degeneration in a DMM-induced rat model\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effect of IRAK4 in vivo, we established a post-traumatic OA (PTOA) model in rats by performing DMM surgery\u0026mdash;a model that recapitulates human OA pathology. This was followed by an intra-articular injection of an adeno-associated virus carrying shIRAK4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Given that OA is a whole-joint disease pathologically involving both cartilage and subchondral bone, we extended our analysis to include a general assessment of osteophyte formation and subchondral bone sclerosis. Macroscopically, using micro-CT analysis and 3D reconstruction, we observed that DMM surgery led to aggravated osteophyte formation, uneven bone surfaces of the tibia and femur, and joint space narrowing. Knockdown of IRAK4 partially ameliorated these changes, particularly in reducing the number and volume of osteophytes and improving the smoothness of the bone surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Furthermore, bone remodeling of the subchondral bone in both the medial femoral condyle and the tibial plateau was observed, which is a key pathological feature of OA. In the bone geometry and microstructural parameters of the subchondral bone, micro-CT analysis revealed that, compared with the sham group, DMM rats demonstrated a decrease in the bone volume/tissue volume ratio (BV/TV), bone mineral density (BMD), trabecular thickness (Tb.Th), and trabecular number (Tb.N), but an increase in trabecular separation (Tb.Sp). IRAK4 knockdown attenuated the aforementioned OA-like pathological changes to a significant degree in DMM rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-G). Microscopically, articular-cartilage integrity and gross morphological changes were evaluated with hematoxylin\u0026ndash;eosin and Safranin-O/Fast Green staining. Results showed that DMM-induced rats exhibited significant cartilage degradation, reduced hyaline cartilage thickness, a rough cartilage surface, notable erosion and fissures of the surface articular cartilage, and extensive proteoglycan loss. Our results indicate that intra-articular injection of Ad-shIRAK4 exerted a chondroprotective effect, alleviating the characteristic pathological changes of OA cartilage. Consistent with the observed phenotypes, the OARSI score\u0026mdash;a histological measure of OA severity\u0026mdash;confirmed that IRAK4 knockdown alleviated OA progression in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and I). Not only that, we profiled the expression of key proteins associated with multiple pathological processes (anabolism, catabolism, inflammation, senescence) in OA articular cartilage in vivo using IHC. Firstly, intra-articular injection of Ad-shIRAK4 effectively reduced IRAK4 expression in the articular cartilage of DMM model rats, confirming the efficiency of the knockdown. Then, in terms of anabolism, catabolism, inflammation, and senescence markers, the DMM group exhibited reduced levels of Col2 and elevated levels of MMP13, iNOS, and P21 compared to the sham group. Conversely, the DMM\u0026thinsp;+\u0026thinsp;Ad-shIRAK4 group showed higher expression of Col2 and lower expression of MMP13, iNOS, and P21, which illustrated that IRAK4 inhibition manifested a significant cartilage-protective role in the OA model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-F). Overall, the above results provide evidence that IRAK4 knockdown can mitigate articular cartilage damage in an OA model in vivo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOsteoarthritis (OA) is a prevalent degenerative joint disease whose etiology remains unclear. Despite its high prevalence, effective therapeutic options are still limited, which underscores the significance of this public health problem given its substantial socioeconomic burden. The pathogenesis of OA is primarily characterized by the degradation of cartilaginous tissue, driven by an imbalance between catabolism and anabolism, enhanced oxidative stress, chondrocyte apoptosis, mitochondrial dysfunction, and extracellular matrix (ECM) degradation\u003csup\u003e30\u003c/sup\u003e. Current clinical management strategies are primarily limited to symptom relief, including the use of nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of lubricating supplements, and surgical interventions such as microfracture and mosaicplasty. When these conventional treatments fail, joint arthroplasty remains the final option\u003csup\u003e31,32\u003c/sup\u003e. Although this procedure effectively alleviates pain and restores joint function in most cases, a subset of patients still experiences postoperative complications such as residual pain, functional impairment, and persistent inflammation. Therefore, elucidating the underlying pathological mechanisms of OA and developing novel, effective treatments are top priorities for managing this disease.\u003c/p\u003e\u003cp\u003eOA was previously considered merely a sequential degradation of cartilage leading to joint impairment. However, a more dynamic view has now emerged, in which the synovium, cartilage, and subchondral bone all produce inflammatory mediators that ultimately lead to cartilage damage\u003csup\u003e33\u003c/sup\u003e. IL-1β, which is recognized as one of the most significant inflammatory mediators, binds with IL-1R in the presence of TLR to provoke OA phenotype alterations. IRAK4, among the IRAK family, is a critical serine-threonine kinase in TLR/IL-1R signaling pathways\u003csup\u003e8\u003c/sup\u003e. In our study, we found increased IRAK4 expression in rat OA articular cartilage samples, a phenomenon also observed in IL-1β-treated chondrocytes in a dose- and time-dependent manner.\u003c/p\u003e\u003cp\u003eThe pro-inflammatory role of IRAK4 has been demonstrated in various diseases. Its inhibition alleviates inflammatory skin diseases\u003csup\u003e11\u003c/sup\u003e, ameliorates rheumatoid arthritis and lymphoid malignancy\u003csup\u003e12,24\u003c/sup\u003e, and mitigates oncogenic transformation in lung cancer\u003csup\u003e9\u003c/sup\u003e, among other effects. In our research, IRAK4 inhibition led to significant suppression of MMP13, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). It also reversed the loss of type II collagen and promoted extracellular matrix (ECM) production in IL-1β-treated chondrocytes. Concurrently, the inhibition of cartilage aging-related proteins p16 and p21 helped maintain ECM stability and alleviate inflammatory damage. Therefore, we conclude that IRAK4 promotes ECM degradation via MMP13, accelerates the cellular aging process, stimulates nitric oxide production, and regulates arachidonic acid metabolism to generate inflammatory mediators. This suggests that IRAK4 impairs the OA chondrocyte phenotype by activating inflammatory signaling pathways that primarily drive catabolic processes over anabolic ones.\u003c/p\u003e\u003cp\u003eIt is well established that aberrant activation of nuclear factor-kappaB (NF-κB) significantly contributes to OA progression\u003csup\u003e34\u003c/sup\u003e. The dynamic balance between NF-κB inhibitors (IκBs) and IκB kinases (IKKs) modulates NF-κB activation. A variety of inflammatory signals, such as TNFα, can activate IKKs. This activation triggers the phosphorylation and degradation of IκBs, leading to the translocation of the NF-κB complex into the nucleus. There, it initiates the transcription of downstream target genes\u003csup\u003e30\u003c/sup\u003e. Our results indicated that IRAK4 inhibition reduced the phosphorylation of IKKα and IκBα, along with the levels of tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor beta-activated kinase 1 (TAK1). This suggests that by inhibiting the TRAF6-TAK1 complex, IRAK4 blockade reduces IKKα phosphorylation. This, in turn, diminishes IκBα phosphorylation, thus preventing NF-κB activation and curtailing the inflammatory response in osteoarthritic chondrocytes.\u003c/p\u003e\u003cp\u003eMitochondria are highly dynamic organelles that undergo continuous fission and fusion to maintain quality control\u003csup\u003e35\u003c/sup\u003e. In chondrocytes, mitochondrial functional stability is vital for maintaining cellular energy metabolism and resistance to oxidative stress\u003csup\u003e36\u003c/sup\u003e. Since aging and apoptosis are important mechanisms of OA cartilage degradation, the balance between mitochondrial fission and fusion appears particularly critical\u003csup\u003e37\u0026ndash;39\u003c/sup\u003e. Our findings demonstrated that both transfection with IRAK4-targeting siRNA and administration of the IRAK4 inhibitor PF-06650833 reversed the detrimental alterations in the expression of key mitochondrial dynamics proteins induced by IL-1β, specifically restoring the levels of the fusion regulators Mfn2 and Opa1 while reducing the elevated levels of the fission regulators Fis1 and Drp1. Additionally, the increase in ROS levels in IL-1β-treated chondrocytes was attenuated by IRAK4 depletion, suggesting that IRAK4 may contribute to mitochondrial dysfunction by promoting oxidative stress. IRAK4 inhibition also mitigated mitochondrial swelling and structural damage induced by IL-1β, indicating its role in maintaining mitochondrial structural integrity. Given that OA incidence is strongly age-dependent and associated with processes such as the senescence-associated secretory phenotype (SASP)\u003csup\u003e40\u003c/sup\u003e, our results highlight a potential mechanism by which IRAK4 contributes to age-related OA progression. Furthermore, both IRAK4 knockdown and pharmacological inhibition by PF-06650833 alleviated the IL-1β-induced upregulation of the senescence markers p16 and p21 in chondrocytes. Taken together, our study highlights the potential of targeting IRAK4 with PF-06650833 for OA therapy, mechanistically linked to its efficacy in the preservation of mitochondrial integrity and homeostasis.\u003c/p\u003e\u003cp\u003eIn previous studies, the impact of METTL3 on the pathophysiological process of OA has been shown to be sophisticated and multifaceted, involving regulatory mechanisms across chondrocytes\u003csup\u003e41,42\u003c/sup\u003e, ATDC5 cells\u003csup\u003e43\u003c/sup\u003e, and OA-FLS\u003csup\u003e19\u003c/sup\u003e. Initially, the expression level of METTL3 exhibits a positive correlation with OA severity. METTL3 overexpression mediates an increase in m⁶A methylation modification, which in turn leads to enhanced chondrocyte apoptosis and diminished production of ECM components\u003csup\u003e42\u003c/sup\u003e. A previous study reported that METTL3 mRNA levels were higher in IL-1β-treated ATDC5 cells than in untreated control cells\u003csup\u003e44\u003c/sup\u003e. Intriguingly, seemingly paradoxical findings have been reported. One study revealed through bioinformatic analysis that METTL3 expression was downregulated in OA group, the finding further confirmed in clinical samples. Similarly, in vitro treatment with IL-1β reduced METTL3 expression levels in chondrosarcoma (SW1353) cells. In contrast, overexpression of METTL3 significantly suppressed the IL-1β-induced upregulation of inflammatory cytokines\u003csup\u003e45\u003c/sup\u003e. Furthermore, a recent study demonstrated that the expression of METTL3 was downregulated both in a mouse model of temporomandibular joint OA in vivo and in chondrogenic ATDC5 cells under inflammatory stimulation in vitro. Moreover, METTL3 inhibits inflammation-induced apoptosis and autophagy in ATDC5 cells by enhancing Bcl2 stability through Ythdf1-mediated m⁶A modification\u003csup\u003e43\u003c/sup\u003e. Similarly, METTL3 expression was downregulated in LPS-treated osteoblasts and was found to modulate the downstream MAPK signaling pathway\u003csup\u003e46\u003c/sup\u003e. However, the relationship between IRAK4 and METTL3 in OA pathological process remains unclear. Our results initially demonstrated that METTL3 is overexpressed in IL-1β-treated chondrocytes. Subsequently, we found that depletion of either IRAK4 or METTL3 significantly suppressed the activation of MAPK signaling, and dual knockdown further enhanced this inhibitory effect. Moreover, a similar trend of suppression was observed in the alteration of NADPH oxidase 2 (NOX2), which reflects oxidative pathway activity. These findings suggest that, beyond NF-κB signaling, IRAK4 may facilitate MAPK signaling activation through METTL3, potentially by directly regulating oxidative stress within the inflammatory microenvironment of OA.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our findings expand the understanding of mechanisms by which IRAK4 plays a key role in promoting inflammation, ECM degradation, cellular senescence, and mitochondrial dysfunction in OA. Furthermore, IRAK4 primarily activates NF-κB and MAPK signaling through its interaction with METTL3 to mediate these effects. More importantly, this study highlights the immense therapeutic potential of the IRAK4 inhibitor PF-06650833 for treating OA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eStudy approval\u003c/p\u003e\n\u003cp\u003eAll animal procedures were performed in accordance with the Guidelines for care and Use of Laboratory Animals of the National Institutes of Health and approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University (No. 20220603A).\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXuezhong Wang, Dehong Yang, Lanwei Xu and Yong Hu conceived and designed the project. Xuezhong Wang and Shenglu Cao performed the experiments. Kai Tong and Siliang Ma assisted with the experiments. Xuezhong Wang and Shenglu Cao analyzed the data. Xuezhong Wang, Kai Tong, and Yong Hu performed statistical analyses and prepared figures. Xuezhong Wang and Yong Hu drafted the original manuscript. Xuezhong Wang, Shenglu Cao, and Dehong Yang revised the manuscript. All authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDeclaration of competing interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No. 82002337).\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors wish to express their sincere gratitude to all the staff members at the Central Laboratory of Renmin Hospital of Wuhan University and to our colleagues at the School of Basic Medical Sciences of Southern Medical University for their invaluable assistance and support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCourties, A., Kouki, I., Soliman, N., Mathieu, S., Sellam, J.: 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DRP1 regulates mitochondrial dynamics and apoptosis in chondrocytes. \u003cem\u003eOSTEOARTHR CARTILAGE\u003c/em\u003e 30 315 (2022)\u003c/li\u003e\n\u003cli\u003eYang, J., et al.: Progress in Understanding Oxidative Stress, Aging, and Aging-Related Diseases. \u003cem\u003eANTIOXIDANTS-BASEL\u003c/em\u003e 13 (2024)\u003c/li\u003e\n\u003cli\u003eLoeser, R.F., Collins, J.A., Diekman, B.O.: Ageing and the pathogenesis of osteoarthritis. \u003cem\u003eNAT REV RHEUMATOL\u003c/em\u003e 12 412 (2016)\u003c/li\u003e\n\u003cli\u003eDiekman, B.O., Loeser, R.F.: Aging and the emerging role of cellular senescence in osteoarthritis. \u003cem\u003eOSTEOARTHR CARTILAGE\u003c/em\u003e 32 365 (2024)\u003c/li\u003e\n\u003cli\u003eRen, J., Li, Y., Wuermanbieke, S., Hu, S., Huang, G.: N(6)-methyladenosine (m(6)A) methyltransferase METTL3-mediated LINC00680 accelerates osteoarthritis through m(6)A/SIRT1 manner. \u003cem\u003eCELL DEATH DISCOV\u003c/em\u003e 8 240 (2022)\u003c/li\u003e\n\u003cli\u003eTang, Y., et al.: METTL3-mediated m(6)A modification of IGFBP7-OT promotes osteoarthritis progression by regulating the DNMT1/DNMT3a-IGFBP7 axis. \u003cem\u003eCELL REP\u003c/em\u003e 42 112589 (2023)\u003c/li\u003e\n\u003cli\u003eHe, Y., et al.: Mettl3 inhibits the apoptosis and autophagy of chondrocytes in inflammation through mediating Bcl2 stability via Ythdf1-mediated m(6)A modification. \u003cem\u003eBONE\u003c/em\u003e 154 116182 (2022)\u003c/li\u003e\n\u003cli\u003eLiu, Q., Li, M., Jiang, L., Jiang, R., Fu, B.: METTL3 promotes experimental osteoarthritis development by regulating inflammatory response and apoptosis in chondrocyte. \u003cem\u003eBIOCHEM BIOPH RES CO\u003c/em\u003e 516 22 (2019)\u003c/li\u003e\n\u003cli\u003eSang, W., et al.: METTL3 involves the progression of osteoarthritis probably by affecting ECM degradation and regulating the inflammatory response. \u003cem\u003eLIFE SCI\u003c/em\u003e 278 119528 (2021)\u003c/li\u003e\n\u003cli\u003eZhang, Y., Gu, X., Li, D., Cai, L., Xu, Q.: METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling. \u003cem\u003eINT J MOL SCI\u003c/em\u003e 21 (2019)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"IRAK4, Mitochondrial dynamics, PF-06650833, MAPK/NF-κB signaling, METTL3","lastPublishedDoi":"10.21203/rs.3.rs-7999197/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7999197/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoarthritis (OA), a prevalent and debilitating condition driven by progressive cartilage degeneration, represents a significant global health challenge due to the absence of effective disease-modifying therapies. Interleukin-1 receptor-associated kinase 4 (IRAK4), a key signaling kinase at the nexus of innate and adaptive immunity, has emerged as a promising therapeutic target for inflammatory diseases. This study elucidates the critical role of IRAK4 in OA pathogenesis. We found IRAK4 expression was significantly upregulated in both osteoarthritic cartilage and IL-1β-stimulated primary chondrocytes. Genetic silencing or pharmacological inhibition with the clinical-stage compound PF-06650833 effectively ameliorated IL-1β-induced inflammatory responses, extracellular matrix degradation, cellular senescence, and mitochondrial dysfunction. Mechanistically, we demonstrated that IRAK4 drives these catabolic processes by activating the MAPK/NF-κB signaling pathway through the TRAF6-TAK1 complex, a process further amplified by METTL3. Crucially, in a rat model of post-traumatic OA induced by DMM, intra-articular injection of an adeno-associated virus carrying shIRAK4 to knock down IRAK4 successfully attenuated overall disease progression, as evidenced by significantly reduced cartilage erosion, osteophyte formation, and aberrant subchondral bone remodeling. Our findings collectively establish IRAK4 as a central driver of OA pathology and highlight the strong translational potential of therapeutic IRAK4 inhibition, exemplified by PF-06650833, as a novel disease-modifying strategy.\u003c/p\u003e","manuscriptTitle":"Targeting IRAK4 mitigates osteoarthritis by preserving mitochondrial homeostasis and suppressing MAPK/NF-κB-mediated inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 11:28:50","doi":"10.21203/rs.3.rs-7999197/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"aa1ccbe0-b433-4084-b4e2-5d2f488708f9","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57799771,"name":"Health sciences/Medical research/Experimental models of disease"},{"id":57799772,"name":"Health sciences/Molecular medicine"},{"id":57799773,"name":"Health sciences/Pathogenesis/Inflammation/Chronic inflammation"},{"id":57799774,"name":"Health sciences/Medical research/Translational research"},{"id":57799775,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2025-12-19T11:10:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 11:28:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7999197","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7999197","identity":"rs-7999197","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}