Background/Purpose: Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA) encompass two of the most common forms of Inflammatory Arthritis. While common pathogenic mechanisms are involved in driving inflammation in both arthropathies there are significant differences, including the presence/absence of autoantibodies, immune-cell infiltrates, molecular signaling, enthesitis and response to therapy. Therefore, the aim is to identify unique mediators that drive differential synovial fibroblast (FLS) populations and function in RA vs PsA, with implication for the inflammatory responses.

Methods: Single cell (Sc) RNAseq was performed on RA and PsA synovial cell suspensions and FLS populations were then defined by advanced bioinformatic analysis. Subsequently, primary RA FLS were cultured with IL-1β (1ng/ml), TGF-β (10ng/ml), or a combination of both. Analysis of key FLS activation and functional markers was performed by flow cytometry, and secreted pro-inflammatory cytokines and matrix metalloproteinases were quantified by ELISA/MSD multiplex assays. Metabolic capacity was assessed using the Seahorse-XFe-technology and fluorescence-lifetime imaging (FLIM) and glycolytic genes were quantified by real-time PCR. mRNA expression of mitochondrial fission/fusion proteins (DRP1/MFN1/MFN2) and endoplasmic reticulum (ER) stress proteins (XBP1S/BIP/ATF6) were quantified by real-time PCR and immunofluorescent staining was utilized to examine ER stress.

Results: ScRNAseq analysis demonstrated 11 distinct FLS populations in RA and PsA, with FAPα+THY1+ invasive FLS enriched in RA patients. ScRNAseq receptor-ligand interactions demonstrated that T cell-derived TGF-β and macrophage derived IL-1β synergistically drive the transcriptional profile of these FAPα+THY1+ invasive FLS in RA. We further demonstrated at a functional level that IL-1β and TGF- β synergistically interact to promote secretion of pro-inflammatory cytokines IL-6, IL-8, and MCP-1, the growth factor VEGF (all p< 0.05), cell surface expression of the adhesion molecule ICAM-1 (p< 0.01), in addition to secretion of MMP3 (p< 0.05), with no synergistic effects observed for MMP1, CXCR3, CXCR4 and CXCR5. Furthermore, this synergistic interaction also altered the metabolic profile of the RA FLS, towards a more glycolytic phenotype, with synergistic induction of basal glycolysis (p< 0.05), proton efflux rate (p< 0.05), % PER from glycolysis (p< 0.05) and compensatory glycolysis (p< 0.001), which supports their invasive phenotype. Significant increases were also observed in key glycolytic markers GLUT-1, HIF1A and PKM2 (all p< 0.05) and ER stress genes XBP1S, BIP and ATF6. In parallel, a reduction in mitochondrial size (p< 0.05) which was associated with an increase mitochondrial fission protein DRP-1 (p< 0.05) with no effect observed for fusion proteins MFN1/MFN2 was demonstrated.

Conclusion: Distinct FLS subtypes were identified in RA and PsA, the invasive phenotype of which were driven by synergistic interactions between T-cell and macrophage derived cytokines. Blocking specific immune-stromal cell interactions may offer new therapeutic intervention in RA and PsA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-cell-derived-cytokines-synergistically-interact-to-drive-synovial-fibroblast-invasive-function-and-metabolic-capacity/