¹Justus Liebig University Gießen, Campus Kerckhoff, Department of Rheumatology and Clinical Immunology, Bad Nauheim
²Kerckhoff-Klinik, Rheumatologie, Bad Nauheim
³Agaplesion Markus Hospital, Dept. of Orthopaedics and Trauma Surgery, Frankfurt am Main

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Introduction: Rheumatoid arthritis (RA) synovial fibroblasts (RASF) contribute to inflammation and cartilage degradation in RA. Our research indicates that RASF from patients in remission exhibit reduced levels of interleukin-36β (IL-36β). IL-36β plays a crucial role in immune regulation and was evaluated regarding its effects on RASF.

Methods: RA patients were classified as in remission or active RA according to clinical and laboratory parameters. RNA sequencing was performed on RASF from both patients groups with/without stimulation with 0.05ng/ml IL-1β followed by pathway analysis and top 50 dysregulated genes. Adhesion assays for 6h with 1ng/ml IL-1β and/or IL-36β pre-treated RASF were performed for 2 h and the number of adhesive RASF quantified. Pre-stimulated RASF were compared after migration through 8µm membranes towards an FBS gradient. The surface of crystal violet stained RASF was quantified. Proliferation was measured using RASF stimulated with IL-1β or/and IL-36β. After 19–24 h, the BrdU-assay was performed. To evaluate differences of the IL36 receptor (IL-36R) expression, immunofluorescence of synovial tissues from RA (active/remission) and osteoarthritis patients were performed and signal intensity and area of signal/visual field measured.

Results: RNA sequencing revealed that RASF from patients in remission showed significantly lower levels of IL-36β and IL-36Ra compared to patients with active disease (n=4). RA-36R was increased in RA tissue from patients with active disease. IL36R was primarily located in the lining layer in all synovial tissues. Patients with active RA had stronger IL36R signals. IL-36β-stimulated RASF showed significantly lower adhesion compared to unstimulated cells and IL-1β-stimulated cells (p<0.0075, n=14). IL-36β-stimulated RASF showed significantly increased migration compared to unstimulated controls (p<0.002, n=14). IL-36β-stimulated RASF from patients in remission showed significantly increased migration compared to unstimulated controls (p<0.026, n=7). Proliferation was increased in IL36β-stimulated cells compared to controls with no difference between active disease and remission.

Conclusion: The altered migration and adhesion of IL-36β-stimulated cells suggests a potential for increased migration towards sites of tissue destruction in RA. The increased proliferation of RASF by IL36β, and the impaired IL36R synovial signal in patients with active disease highlights the pathogenicity of elevated IL36β levels observed in active disease.