Interleukin-24 Induces Inflammatory Chemokine and Cytokine Responses in Fibroblast-Enriched Synovial Cells from Patients with Knee Osteoarthritis

Background: Interleukin-24 (IL-24) is expressed in synovial myofibroblasts and has been associated with pain severity in female patients with knee osteoarthritis (OA). However, the direct effects of IL-24 on inflammatory responses in synovial fibroblasts and the signaling pathways involved remain unclear. This study investigated IL-24-induced transcriptional and inflammatory responses in fibroblast-enriched synovial cells and examined the effects of STAT3 and p38 mitogen-activated protein kinase (MAPK) inhibition. Methods: Fibroblast-enriched synovial cells isolated from synovial tissue obtained from patients with knee OA undergoing total knee arthroplasty were stimulated with IL-24 for 6 and 24 h. RNA sequencing (RNA-seq) was performed using cells from two patients, and overlapping upregulated genes were subjected to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The concentration- and time-course experiment used samples from nine patients; the HJC0152 and SB203580 experiments used ten specimens from eight patients and ten specimens from nine patients, respectively. Selected inflammatory cytokines and chemokines were subsequently evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay in concentration- and time-course experiments using nine specimens from nine patients. To investigate signaling involvement, cells were treated with IL-24 in the presence or absence of the STAT3 inhibitor HJC0152 or the p38 MAPK inhibitor SB203580, each at 5 or 10 μM. Results: RNA-seq identified 27 and 34 overlapping upregulated genes that independently met the DEG criteria in both patients at 6 and 24 h, respectively. These genes were enriched in inflammatory pathways, including TNF, IL-17, NF-κB, chemokine, cytokine–cytokine receptor interaction, Toll-like receptor, and NOD-like receptor signaling pathways. IL-24 significantly increased the mRNA expression of CCL2, CXCL1, CXCL3, IL6, and IL8. HJC0152 and SB203580 at 10 μM both significantly reduced IL-24-associated CCL2, CXCL1, and IL6 expression. At the protein level, both inhibitors attenuated CCL2, CXCL1, and IL-6 concentrations in IL-24-stimulated cells. HJC0152 also reduced CXCL3 expression at the mRNA level, although its protein-level effect was less pronounced. In contrast, the effects of both inhibitors on CXCL3, CXCL10, and IL-8 were limited or inconsistent. Conclusions: IL-24 induces an inflammatory response in fibroblast-enriched synovial cells characterized by increased chemokine and cytokine expression. STAT3 and p38 MAPK inhibition preferentially attenuated IL-24-associated CCL2, CXCL1, and IL-6 responses, suggesting the possible involvement of these pathways in selected IL-24-associated inflammatory responses. These findings provide a basis for further investigation of IL-24-related signaling in OA synovitis.