OP0092 DECIPHERING THE SYNOVIAL TISSUE AND FIBROBLAST SUBSETS IN SYSTEMIC SCLEROSIS

Abstract

BackgroundArticular involvement is underestimated in systemic sclerosis (SSc), but represents a major cause of disability and is a marker of disease severity. There is no approved effective therapy for arthritis in SSc and immunosuppressive treatments are given to SSc patients by analogy with rheumatoid arthritis (RA), but with limited effect. The last few years have revolutionized the understanding of the pathogenesis of RA by deciphering the heterogeneity of synovium at both the cellular and molecular levels. Nevertheless, the pathogenesis of joint involvement in SSc remains largely unknown.ObjectivesTo characterize the synovium in SSc at tissue and cellular levels and to compare it with RA synovium.MethodsNine consecutive patients fulfilling SSc classification criteria and having joint synovitis were included. Patients with overlap with other autoimmune rheumatic diseases were excluded. Synovial tissues (8 wrists, 1 knee) were obtained by ultrasound-guided biopsy and were compared to those obtained from five RF and CCP positive RA patients (3 wrists, 3 knees). Tolerance of the procedure was assessed after 3-5 days. Histological analysis of the synovium determined the Krenn synovitis score (0-9) and stratified the synovial tissue according to previously published histological features1. One biopsy, without synovial tissue, was not processed further. ScRNA-seq libraries were prepared with 10X Genomics technology and sequenced on NovaSeq 6000. Integrated bioinformatics analysis used Cell Ranger and Seurat software. Overexpressed genes were selected using log2 ratio (>0.25) and false discovery rate adjusted p value (< 0.05). For pathway enrichment analysis, Enrichr software was used.ResultsOf the nine SSc-patients, 6 were women, median age was 65 [IQR: 58-67] years, median disease duration was 2 [0.5-5] years and 2 had diffuse cutaneous subtype. The antibody status was as follows: anti-Scl70 (2), anti-centromere (2), anti-RNA polymerase III (3), anti-Ku (1) and isolated ANA (1). In the RA cohort, 4/5 were women, median age was 56 [54-59] years and median disease duration 6.5 [5-7.5] years. Synovial biopsy was well-tolerated by all the patients. Krenn synovitis score was lower in SSc as compared to RA across the three components of the synovitis score (lining layer, stroma and inflammatory infiltrate). In SSc, 7/8 (87.5%) biopsies were characterized by a pauci-immune pathotype, whereas in RA 3/6 (50%) were pauci-immune. Due to the low inflammatory pauci-immune pathotype in all but one SSc patients, we focused the scRNA sequencing analysis on synovial fibroblasts (SF) (number of SF studied: 4876 in SSc and 5885 in RA). We identified four clusters of SF with respective marker genes: SF1 (CXCL12, APOE, RARRES2, CCL2), SF2 (PRG4, MMP1, MMP3, CD55), SF3 (POSTN, ASPN, COMP, COL1A1) and SF4 (FN1, TIMP1, SERPIN2, PRELP). Comparison between SSc and RA SF subtypes showed differences in the proportion of the clusters between both diseases with higher enrichment of SF2 corresponding to the lining SF in SSc. 741 genes were differentially expressed between SSc and RA SF with 414 genes overexpressed in SSc SF. Pathway enrichment analysis of these 414 genes identified TGF-β (p.adj: 3.708e-18) and interferon (p.adj: 1.071e-8) signaling pathways. TGF-β signaling was enriched across all the clusters of SF, whereas interferon signaling mostly in sublining SF. The most overexpressed genes in SSc included PLCG2 (encoding the signaling enzyme PLCγ2, which plays a regulatory role in various immune pathways), PCOLCE2 (encoding a procollagen C-endopeptidase enhancer) and the transcription factor AHR (negative regulator of TGF-β).ConclusionSynovitis in SSc differs from RA synovitis both at histological and molecular levels. By highlighting the low inflammatory nature of the synovium and the enrichment in TGF-β and interferon signaling pathways in SSc SF, our study questions the use of the same immunosuppressive therapies in RA and SSc. These results are the basis for the development of specific targeted therapies for arthritis in SSc.