DNA methylation modifications induced by corticosteroid treatment of human cartilage explants in vitro

Purpose: Osteoarthritis (OA) is both the most common form of arthritis and a leading cause of chronic disability in the US. One of the most commonly used therapeutic strategies to treat knee OA is injection of the joint with corticosteroids. Although intraarticular steroid concentrations drop within hours following injection, patients experience pain relief for weeks or months. We hypothesized that the long acting benefits of steroid injection were associated with epigenetic changes within human chondrocytes. Methods: Matched samples of macroscopically eroded (n=8) and intact cartilage (n=8) explants from patients undergoing total knee arthroplasty for primary knee osteoarthritis (OA) were obtained and cultured. Samples were divided into two portions. To one, corticosteroid was added to the culture media (triamcinolone, final concentration equivalent to 40mg injection into a human knee = 8mg/mL) for 24 hours, then media were changed, and samples were cultured for an additional 72 hours. The second portion of explants were treated with vehicle. Samples were then cryogenically ground and DNA isolated, treated with sodium bisulfite (Zymo EZ DNA methylation kit) and loaded onto Illumina Infinium MethylationEPIC chips, which quantify genome-wide DNA methylation at >850,000 CpG sites across the genome. DNA methylation levels (beta values) were extracted from raw chip imaging and processed in Genome Studio (Illumina). CpG sites within 5bp of known SNPs and those on sex chromosomes were excluded from analysis CpG sites were considered significantly differentially methylated if false data rate-corrected (Benjamini-Hochberg) group p≤0.01 with absolute DNA methylation difference between groups (delta-beta) of ≥10% criteria were met. Genes associated with differentially methylated CpG sites were evaluated ontologically using the Ingenuity Pathway Analysis (IPA) software package. Results: Corticosteroid treatment induced substantial changes in DNA methylation levels. In eroded cartilage, steroid treatment resulted in differential methylation of 4326 CpG sites associated with 1925 distinct genes. These genes were clustered in inflammatory pathways, including Fcy receptor-mediated phagocytosis in macrophages and monocytes, IL8 signaling, B and T lymphocyte signaling etc., but also included several OA-related pathways, including apelin endothelial signaling, gap junction signaling, axonal guidance signaling, nitric oxide signaling, etc. Upstream regulator analysis identified TGFB1 as most overrepresented among differentially methylated genes. In intact cartilage, steroid treatment induced methylation changes in 502 CpG sites corresponding to 328 distinct genes. Pathways among these genes included platelet-derived growth factor signaling, PPAR alpha signaling, B and T cell receptor signaling, and others. Interestingly, 77 genes overlapped between the two sets, pathways included pyrimidine salvage, cAMP-mediated signaling, beta-adrenergic signaling, PDGF signaling, and Th17 activation. Conclusions: Herein, we identify significant changes in DNA methylation patterns in human cartilage explants following exposure to corticosteroids. These epigenetic alterations offer a novel candidate mechanism to potentially partially explain the durable improvements patients experience following short-acting steroid injection. Future work should focus on confirming gene expression alterations associated with these epigenetic changes, and on examining the specific contributions of epigenetic changes within steroid-associated pathways to OA.