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Introduction: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease in which activated synovial fibroblasts (SF) drive joint erosion. In these cells, integrin expression is increased, enhancing adhesion, migration, and proliferation. Integrin activation is mediated by focal-adhesion proteins such as kindlins and talin-1, yet their precise role in pro-inflammatory RASF activation remains unclear. This study was conducted to identify the significance of kindlins and talin-1 in RASF in vitro under pro-inflammatory stimulation and in vivo in the SCID mouse model.

Methods: RASF were subjected to a single IL-1β dose (10 ng/mL) for 6, 17, 24 or 48 h, or to a repeated 0.05 ng/mL IL-1β pulse every 24 h (16 h stimulation, 8 h recovery) over 3 days, then collected for RNA sequencing. Expression of kindlins-1/2/3 and talin-1 was quantified bei qPCR and immunocytochemistry, while cytokine secretion assessed the inflammatory response by ELISA. In vivo, RASF together with human cartilage were implanted into SCID mice. Three mice at 30, 40, and 45 days after the implantation were randomly selected to assess expression of kindlins and talin-1 as well as cartilage invasion.

Results: Kindlin-2 and talin-1 were highly expressed near vessels and in the synovial lining, sublining, and cartilage-invasion areas of SCID-mouse implants at all time points. After 17 hours, IL-1β stimulation of RASF resulted in a significant short term down-regulation of kindlin-2 (-4.1-fold, p=0.0456) compared to controls. After 48 h, the expression levels returned to baseline. Talin-1 showed the highest mRNA levels (663.1±186.2) followed by kindlin-2 (88.9±23.2), -1 (2.15±2.06) and -3 showing the lowest expression (0.12±0.08). Kindlin-1 expression was significantly increased after 6 h (2.9-fold, p=0.0178) but reduced after 17 h (-1.5-fold, p<0.0001), 24 h (-0.7-fold, p=0.0004) and 48h (-1.2-fold, p<0.0001). These transcriptional changes observed by RNAseq and qPCR were corroborated by immunocytochemistry of RASF and in RA tissue. RNAseq with repeated inflammatory flares showed that kindlin and talin expression remained altered in contrast to reduced inflammatory induction observed after repetitive IL-1β stimulation.

Conclusion: Kindlin-2 and talin-1 are expressed in RASF and RA synovial tissue, including RASF actively invading cartilage in SCID-mouse implants. Inflammatory cues alter the temporal expression of these proteins, activation of RASF likely enhanced their adhesion and migration behaviour.