Background/Purpose: Rheumatoid arthritis synovial fibroblasts (RASF) play a central role in driving inflammation and promoting cartilage degradation in Rheumatoid arthritis (RA). In our study, we observed that RASF from patients in clinical remission exhibit reduced expression levels of Interleukin-36β (IL-36β). As IL-36β is a pro-inflammatory cytokine involved in immune regulation, we investigated its role in modulating RASF behavior.

Methods: Patients with RA were stratified into remission or active disease groups based on clinical assessments and laboratory markers. RNA sequencing was conducted on RASF isolated from both groups, with and without stimulation using 0.05ng/ml IL-1β. Subsequent analyses included pathway enrichment and identification of the top 50 dysregulated genes. To assess cell adhesion, RASF pre-treated with 1ng/ml IL-1β and/or IL-36β were subjected to a 6-hour assay, followed by quantification of adherent cells after 2 hours. Cell migration was evaluated using transwell assays with 8μm pores and an FBS gradient, comparing pre-stimulated RASF populations based on the stained surface area after crystal violet labeling. Proliferation was determined via BrdU assay 19–24 hours after stimulation with IL-1β, IL-36β, or both. Immunofluorescence staining was employed to analyze IL-36 receptor (IL-36R) expression in synovial tissues from RA patients (active/remission) and individuals with osteoarthritis, with quantification of signal intensity per visual field.

Results: RNA sequencing revealed a marked reduction in IL-36β (-7,14 fold) and its antagonist IL-36Ra (-11,49 fold) in RASF derived from patients in remission compared to those with active rheumatoid arthritis (n=4). Consistent with these findings, elevated IL-36β expression was detected in synovial tissue samples from patients with active disease. Immunofluorescence analysis demonstrated that IL-36 receptor (IL-36R) was predominantly localized to the synovial lining layer across all RA samples, with a more pronounced signal intensity observed in tissues from patients with active RA. Functional assays showed that IL-36β stimulation significantly reduced the adhesive capacity of RASF compared to both unstimulated and IL-1β-stimulated cells (p < 0.0075,n = 14). In contrast, IL-36β stimulation led to a significant increase in RASF migration relative to unstimulated controls (p< 0.002,n=14), an effect also observed specifically in remission-derived RASF (p< 0.026,n = 7). IL-36β treatment increased RASF proliferation compared to IL-1β stimulated cells (p< 0.009,n=5), with no significant difference between cells derived from patients with active disease versus remission.

Conclusion: The modulation of adhesion and enhanced migratory capacity of IL-36β-stimulated RASF indicates a potential mechanism by which these cells may more effectively localize to sites of tissue damage in RA. Moreover, the IL-36β-induced increase in RASF proliferation, combined with the diminished IL-36R signal in synovial tissues from patients with active disease, highlights the pathogenic relevance of elevated IL-36β levels in the context of RA progression.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-36%ce%b2-modulates-the-progression-of-rheumatoid-arthritis-by-altering-the-behavior-of-rheumatoid-arthritis-synovial-fibroblasts/