ALTERED NONMUSCLE MYOSIN II (NMII) ISOFORM EXPRESSION AND ACTIVATION IN SYSTEMIC SCLEROSIS (SSc) FIBROBLASTS: IMPLICATIONS FOR FIBROTIC DISEASE PROGRESSION

Background: Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by progressive fibrosis and tissue damage. Nonmuscle myosin II (NMII) isoforms play crucial roles in cellular processes, including force generation and cell migration, and have been linked to fibrotic diseases [1]. However, the direct and specific contributions of NMII isoforms to SSc-related fibrosis and how they interact with transforming growth factor beta (TGFβ) signaling, a major fibrogenic mediator in SSc, remain poorly elucidated. Objectives: We investigated the role of NMII isoforms in SSc fibroblasts by examining their expression, cytoskeletal distribution, and activation in response to TGFβ Methods: We utilized immunofluorescence to evaluate the expression and cytoskeletal distribution of NMIIA and NMIIB and their activation through myosin regulatory light chain (MLC) phosphorylation. Primary fibroblasts from eight SSc patients and eight healthy donors were stimulated with recombinant human TGFβ (rhTGFβ). To dissect NMII activation pathways, we employed small molecule inhibitors: Y-27632 (pan-ROCK inhibitor), KD025 (ROCK2 isoform inhibitor), and blebbistatin (NMII motor inhibitor). Additionally, we assessed the production of fibrotic markers (IL-6, type I collagen, and fibronectin) by fibroblasts using ELISA. Results: SSc fibroblasts displayed altered NMII isoform expression and redistribution compared to healthy controls. Specifically, SSc fibroblasts exhibited increased NMIIB filamentation within the actin cytoskeleton, suggesting enhanced NMII activity (n = 8, NMIIB filament/ cytoplasm intensity median [range] HD 1.2 [0.8-1.7] vs SSc 1.8 [1.5-2.2], p = 0.02). HD fibroblasts exhibited a similar redistribution pattern of NMII isoforms upon TGFβ stimulation (n = 8, NMIIB filament/ cytoplasm intensity median, [range] ctrl 1.2 [0.8-1.7] vs TGFβ 1.9 [1.8-2.3], p = 0.02). This dysregulation was accompanied by increased phosphorylation of myosin regulatory light chain (MLC), a critical regulator of NMII motor function (n = 8, ppMLC fluorescence intensity/cell median, [range] ctrl 10000 [8000-1200], TGFβ 15000 [12000-17000], p 0.002). Notably, inhibiting NMII activation with Y-27632 or blebbistatin significantly reduced TGFβ-induced production of IL-6 and type I collagen of HD fibroblasts (n = 10, IL-6 pg/mL median, [range] TGFβ 192.6 [72-572] vs TGFβ+Y 15 [4,6-81] vs TGFβ+blebbistatin 25 [12-200], p = 0.003, p = 0.02 respectively and collagen ng/mL median, [range] TGFβ 248 [109-572] vs TGFβ+Y 109 [73-206]; TGFβ vs TGFβ+blebbistatin 174 [77-244], p = 0.005, p = 0.04, respectively). Inhibition of ROCK2, specifically impaired collagen I production in resting fibroblasts, indicating a distinct role for ROCK2 in collagen synthesis. SSc fibroblasts tended to be less responsive to the inhibition of NMII activation than their healthy counterparts. Conclusion: Our findings indicate dysregulation of NMII isoforms in SSc fibroblasts, emphasizing their critical role in TGFβ-induced fibrogenesis. Specifically, targeting NMII activation pathways may be a promising therapeutic strategy to reverse the progression of SSc-related fibrosis. REFERENCES: [1] Southern BD, Grove LM, Rahaman SO, Abraham S, Scheraga RG, Niese KA, et al. Matrix-driven Myosin II Mediates the Pro-fibrotic Fibroblast Phenotype. J Biol Chem. 2016 Mar 18;291(12):6083–95. Acknowledgements: NIL. Disclosure of Interests: Barbara Russo: None declared, Maria Shutova: None declared, Fanny Noulet: None declared, George Romanescu: None declared, Nicolò Brembilla: None declared, Wolf-Henning Boehncke received honoraria as a speaker or advisor from the following companies: Abbvie, Almirall, BMS, Celgene, Janssen, Leo, Lilly, Novartis, Sun Pharmaceuticals, UCB. LS has no conflict of interest to declare. DOI: 10.1136/annrheumdis-2024-eular.6294 Keywords: Fibroblasts, Skin Citation: , volume 83, supplement 1, year 2024, page 1032Session: Systemic sclerosis (Poster View)

Fibroblasts, Skin