P124 Autoantibody subgroups in diffuse cutaneous systemic sclerosis show contrasting myofibroblast differentiation by bulk and single-cell RNA sequencing

Abstract Background/Aims Myofibroblasts are important in the pathogenesis of systemic sclerosis (SSc). TGFβ is a key growth factor driving myofibroblast formation and differentiation; however, the downstream signaling pathways regulated by TGFβ during myofibroblast differentiation are less well understood. Intracellular pathways implicated include the phosphatidylinositol 3-kinase/AKT serine/threonine kinase (PI3K/AKT) signaling pathway. Antinuclear autoantibody subsets are associated with distinct patterns of skin disease and organ manifestations in SSc. Recent studies have defined molecular and cellular interaction differences associated with SSc-specific antibodies, including those with anti-RNA polymerase III (ARA) or anti-topoisomerase-1 (ATA). We examined skin biopsies from well characterized SSc patients to delineate myofibroblast activation by integrating bulk and scRNAsequencing, and quantitative immunostaining for αSMA protein expression. Methods Serial 4mm paired skin biopsies were taken from patients with early dcSSc (n = 21) at baseline, 3 and 12 months, and processed for bulk RNAsequencing. Paraffin embedded biopsies were stained for αSMA and analysed as a proportion of the whole slide. Singe cell (sc)RNAseq was performed on 4mm skin biopsies from a second cohort of patients (n = 12) and healthy controls (HC n = 3). Analysis was carried out using R software (packages ‘Seurat’, ‘gsfisher’ and ‘DESeq2’). Comparisons were made between antibody subsets using ANOVA. Gene set enrichment analysis was performed, with significance at FDR<0.1. Results There were significant differences by IHC at baseline between αSMA+ in the skin in ARA+ patients compared to HC (p < 0.01), but no significant difference in the ATA+ subset and HCs. Over time, the proportion of αSMA+ cells declined in the ATA and other antibody subgroups, whereas the ARA+ subgroup of patients showed no change in αSMA+ cells over 12 months. Bulk RNA sequencing of whole skin defined significantly enriched pathways shared by ARA+ and ATA+ early dcSSc patients including TGFβ signaling pathway (FDR = <0.001 and 0.025 respectively), and inflammatory response pathway (FDR = <0.0001 for both antibody subtypes compared to HC). The PI3K/AKT signaling pathway was only significantly upregulated in ARA+ patients compared to HC (FDR = 0.05), and not in early ATA+ patients (FDR=0.25). scRNAseq analysis of fibroblast clusters confirmed that this difference in PI3K/AKT signaling enrichment is predominantly seen in the early ARA+ patient cohort, whereas later stage disease and ATA+ fibroblasts do not exhibit this upregulation. Conclusion There are significant differences in αSMA positively stained cells between ANA subgroups in early dcSSc patients compared to HC, with more staining in ARA+ patients. Gene expression analysis also confirms key pathways regulating αSMA are upregulated in the ARA+ dcSSc patients and originates from the fibroblast subclusters. Our results suggest that stratification by ANA subset is likely to be important when targeting myofibroblasts in early stage dcSSc. Our data suggest targeting PI3K-AKT is more likely to be beneficial in early-stage ARA+ than ATA+ patients Disclosure K.E.N. Clark: None. C. Campochiaro: None. E. Derrett-Smith: None. V.H. Ong: None. C.D. Buckley: Shareholder/stock ownership; shareholder and founder of Mestag therapeutics. Grants/research support; J&J, UCB Pharma, AbbVie Roche, GSK. C.P. Denton: Consultancies; Janssen, GlaxoSmithKline, Boehringer Ingelheim, Roche, CSL Behring, Corbus, Acceleron, Horizon, Arxx Therapeutics, Lilly, Novartis, Certa Therapeutics, Zurabio. Grants/research support; Abbvie, GlaxoSmithKline, Horizon, Arxx Therapeutics and Servier.