Background/Purpose: Rheumatoid arthritis (RA) is pathologically characterized by autoimmunity against citrullinated proteins, proliferative synovitis, and ultimately joint destruction. Synovial fibroblasts (SFs) are, when activated, capable of hyperproliferating and producing large amounts of proinflammatory mediators including IL-6, thus considered to be the key effector cells in RA pathogenesis. SFs obtained from RA patients have high autophagy activity, probably contributing their active phenotype [1]. Besides, our recent study suggests SFs’ autoimmune potential by demonstrating the increase of citrullinated vimentin and its interaction with MHC class II when treated with IFN-γ and autophagy inducers [2]. JAK1 is an emerging therapeutic target in RA, but its roles in the active phenotype of SFs remain to be elucidated. To clarify the role of JAK1 in the regulation of autophagy and in the inflammatory and autoimmune potentials in SFs.

Methods: SFs were derived from synovial tissue specimens obtained from RA patients during joint replacement surgery and used between passages 4-8 for all experiments. To inhibit JAK1, SFs were treated with its selective inhibitor upadacitinib for 6-24 h with the optimal concentration determined by BrdU assay. To induce autophagy, SFs were starved using serum-free medium for 2h or treated with 10μM of the proteasome inhibitor MG132 for 24h in the presence or absence of 5mM of the autophagy inhibitor 3-methyladenin. The expression of autophagy-related proteins/genes, including LC3-II, BECN1, ATG5 and ATG7, were analyzed by Western blotting or real-time PCR. The expression of IL-6 was measured in cell culture supernatants by ELISA. The interaction between citrullinated vimentin and MHC class II (HLA-DR) were analyzed by in situ proximity ligation assays. P values were calculated by ratio paired t-test and considered significant when less than 0.05.

Results: JAK1 inhibition with upadacitinib resulted in the significantly decreased expression of LC-3II (p =0.016 triplicated, n =3), BECN1 (p = 0.01, n =5), ATG5 (p = 0.03, n =5) and ATG7 (p = 0.02, n =5) in starved SFs. Similar results were obtained in SFs treated with IFN-γ. The treatment of SFs with 100 ng/mL of IFN-γ for 24 h increased the expression of IL-6 in cell culture supernatants (p =0.017, n =3), which was inhibited by upadacitinib (p = 0.02, n = 5). The interaction between citrullinated vimentin and MHC class II was increased in SFs following treatment with starvation and IFN-γ (100ng/mL, 24h) (p = 0.003, n = 6). The effect was cancelled by the addition of upadacitinib (p = 0.009, n = 6) or 3-methyladenin (p = 0.02, n = 6).

Conclusion: Our current results indicate that JAK1 positively regulates autophagy and reinforces the inflammatory and autoimmune potentials in SFs. The mode of action of JAK inhibitors would include the mitigation of SFs’ active phenotype.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/jak1-regulates-autophagy-and-reinforces-the-inflammatory-and-autoimmune-potentials-in-rheumatoid-arthritis-synovial-fibroblasts/