The impact of TLR stimulation on the synovial cells of osteoarthritis patients

Background Osteoarthritis (OA) is a degenerative joint disease affecting 594 million people worldwide (1). Our previous studies have shown that the activation of toll-like receptor 1/2 (TLR1/2) increased the expression of pro-inflammatory cytokines and cartilage–degrading enzymes, and inhibited the mitochondrial respiration in chondrocytes of OA patients (2). This study aimed to assess the impact of TLR stimulation on various types of synovial cells of OA patients. Methods Synovial membrane, synovial fluid, cartilage, and peripheral blood were collected from OA patients who had undergone knee replacement surgeries. The cellular composition of synovial membrane and the expression of TLR1 to 9 in synovial cells were detected using flow cytometric and immunofluorescent analysis. Purified synovial fibroblasts were stimulated with agonists specifically targeting TLR1 to 9 for 3.5 days and then were either directly analyzed or co-cultured with spheroids of chondrocytes from the same patients. The expression of inflammatory factors and cartilage-degrading enzymes was measured using qPCR and Bio-Plex assays. Results Roughly 50% of the synovial membrane cells of OA patients were fibroblasts, 25% were immune cells, and the remaining 25% were a mixture of endothelial cells, mesenchymal stem cells, et. al. For the immune cells, roughly 20% were macrophages, 3% were CD4+ T cells, 1% were CD8+ T cells, and 0.5% were B cells. Synovial fibroblasts could express TLR1 to 9. The expression of inflammation-related genes IL6 and IL8, along with cartilage-degrading enzymes such as MMP1 and MMP3, increased when fibroblasts were activated by TLR1/2, TLR4, TLR5, and TLR2/6 agonists. In addition, stimulation of TLR1/2, TLR4, and TLR2/6 enhanced GCSF, while TLR5 stimulation upregulated IL1B and MMP13 expression in the synovial fibroblasts of OA patients. When TLR1/2-prestimulated fibroblasts were cocultured with chondrocyte spheroids, they suppressed chondrocytes in expressing anabolic factor COL2A1 and enhanced their expression of catabolic factor MMP3 along with inflammatory factor IL6, IL8, and GCSF. For synovial immune cells, such as macrophages and T cells, various types of TLR are also expressed. Conclusions Synovial cells, in particular, synovial fibroblasts, macrophages, and T cells of OA patients, expressed various types of TLR. Stimulation of TLR1/2, TLR4, TLR5, and TLR2/6 resulted in increased secretion of inflammatory factors and cartilage-degrading 2 enzymes in the synovial fibroblasts. Additionally, TLR1/2-pre-stimulated fibroblasts promoted chondrocytes to express catabolic and inflammation-related genes while inhibiting the expression of cartilage-anabolic genes.