OP0192 SYNOVITIS IN SYSTEMIC SCLEROSIS IS CHARACTERISED BY AN INTERFERON SIGNATURE
Authors: C. Geiss, M. Houtman, R. Micheroli, Y. Djeffal, M. Frank Bertoncelj, S. G. Edalat, K. Buerki, C. Pauli, M. Bonelli, T. Karonitsch, O. Distler, C. Ospelt, M. Elhai
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> The pathogenesis of synovitis in systemic sclerosis (SSc) is unknown. Due to the lack of evidence-based therapies, SSc patients with synovitis are treated as rheumatoid arthritis (RA) patients, however with unknown effect. Previously, we showed that SSc synovitis differs from RA synovitis with a prevalent pauci-immune pathotype. <h3>Objectives:</h3> To decipher the histological, cellular, and transcriptional differences between SSc and RA synovitis, specifically in synovial fibroblasts (SF). <h3>Methods:</h3> We collected ultrasound-guided synovial biopsies from 11 SSc and 7 RA patients. All samples were analysed histologically, including Krenn synovitis score (0-9) and pathotype characterisation. Additionally, 7 SSc and 6 RA samples were dissociated and processed for single-cell RNA-sequencing (20,705 cells in SSc and 31,681 in RA). Due to the pauci-immune nature of the synovitis, we focused our analysis on the SF population (4,638 SF in SSc, 6,097 in RA). We performed differential gene expression analysis, over-representation pathway analyses and calculated signature expression scores between SSc and RA SF. Pathway analyses included publicly available datasets and bulk RNA sequencing data from SF stimulated <i>in vitro</i> with TNF-α, IFN-α/b, IFN-γ, and IL-6 from internal experiments and published data<sup>1</sup> (Figure 1A). <h3>Results:</h3> The Krenn synovitis score was lower in SSc than in RA (2.1 ± 1.4 vs. 4.2 ± 1.0, p = 0.01). There was no difference in fibrosis (Elastica van Gieson staining) and vascularisation (CD31 staining) between SSc and RA synovitis. We confirmed a pauci-immune pathotype characteristic of SSc synovitis (91% vs. 43% of RA biopsies, p = 0.04). Consistently, neutrophils were more prevalent in RA as compared to SSc synovitis (p &lt; 0.01). On the single-cell level, we identified seven SF cell states defined by the expression of the marker genes <i>PRG4</i>, <i>CHI3L2</i>, <i>COMP</i>, <i>CXCL12</i>, <i>CXCL14</i>, <i>MFAP5</i>, and <i>POSTN</i> consistent with the current literature in RA<sup>2</sup>. The proportion of SF clusters differed between both diseases with a higher enrichment of <i>MFAP5</i><sup>+</sup> (Fold-change (FC) = 3.81, false discovery rate (FDR) &lt; 0.001) and <i>COMP</i><sup>+</sup> SF in SSc (FC = 2.28, FDR &lt; 0.001), and <i>POSTN</i><sup>+</sup> (FC = 3.12, FDR &lt; 0.001) and <i>CXCL12</i><sup>+</sup> SF in RA (FC = 1.70, FDR &lt; 0.001). We identified 675 significant differentially expressed genes (FDR &lt; 0.01, |log<sub>2</sub>FC| &gt; 0.25) and several differentially activated signalling pathways between SSc and RA SF. In particular, SSc SF were enriched in IFN-α and IFN-γ (both FDR &lt; 0.018) and complement and coagulation pathways (FDR &lt; 0.008), especially in genes of the alternative complement pathway (Figure 1B). RA SF showed a strong enrichment in TNF-α signaling (FDR &lt; 6.3e-12) and inflammatory response genes (FDR &lt; 0.018). Using the transcriptional signature of <i>in vitro</i> stimulated SF as gene sets, we confirmed the IFN response in SSc SF, whereas TNF-α and IL-6 response signatures were more dominant in RA SF (Figure 1C). Interestingly, IFN-α/β and IFN-γ stimulation of SF <i>in vitro</i> led to an activation pattern of alternative complement pathway genes similar to that observed in SSc SF (Figure 1D). IFN and complement signature scores were significantly higher in sublining <i>CXCL12</i><sup>+</sup> SF in SSc compared to RA (both p &lt; 2.22e-16). To identify the drivers of the IFN signature in SSc SF, we assessed the expression of the different IFN genes in synovial cell populations. Only <i>IFNG</i> was expressed, mainly by T cells, which is consistent with the preferential expression of IFN response genes in the sublining synovium. <h3>Conclusion:</h3> This first in-depth characterization of the SSc synovium revealed hallmark tissue, cellular and molecular changes between SSc and RA synovitis. We found a strong enrichment of the IFN signalling pathway in SSc SF, possibly leading to a dysregulation of the alternative complement pathway. Our findings provide the basis for the development of specific targeted therapies for synovitis in SSc. <h3>REFERENCES:</h3> [1] Tsuchiya, Haruka, et al. <i>Annals of the Rheumatic Diseases</i> 80.4 (2021): 440-450. [2] Zhang, Fan, et al. <i>Nature</i> (2023): 1-9. <h3>Acknowledgements:</h3> This project is funded by FOREUM – Foundation for Research in Rheumatology. We thank our patient research partners Joëlle Messmer &amp; Andreas Eisenring. <h3>Disclosure of Interests:</h3> Celina Geiss: None declared, Miranda Houtman: None declared, Raphael Micheroli: None declared, Yannis Djeffal: None declared, Mojca Frank Bertoncelj: None declared, Sam G. Edalat: None declared, Kristina Buerki: None declared, Chantal Pauli: None declared, Michael Bonelli GSK, Galapagos, Thomas Karonitsch: None declared, Oliver Distler 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, Co-founder of CITUS AG, 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant, Amgen, AnaMar, Argenx, Arxx, AstraZeneca, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galderma, Galapagos, Glenmark, Gossamer, Horizon, Janssen, Kymera, Lupin, Medscape, Merck, Miltenyi Biotec, Mitsubishi Tanabe, Novartis, Orion, Prometheus, Redxpharma, Roivant, Topadur and UCB, Caroline Ospelt: None declared, Muriel Elhai: None declared.