RA and PsA synovial tissue single-cell analysis demonstrates differential fibroblast populations with distinct phenotypes and functional capacities

Objectives This study determines the phenotypic and functional characteristics that define distinct fibroblast-like synoviocyte (FLS) populations in rheumatoid arthritis (RA) vs psoriatic arthritis (PsA). Methods Single-cell RNA sequencing and multiparametric flow cytometry analysis (21 markers) were performed on RA and PsA synovial cell suspensions to determine FLS phenotype/function. Podoplanin (PDPN) + FLS and sorted THY1 + FLS vs THY1⁻ FLS function in RA vs PsA was assessed by flow cytometry, enzyme-linked immunosorbent assay (ELISA), quantitative real -time quantitative polymerase chain reaction (qPCR), and metabolic analysis ex vivo, in vitro, and in the presence of verteporfin (Hippo signalling blockade). Results Flow analysis of PDPN + FLS demonstrated significant increases in human leukocyte antigen (HLA)-DR + , yes-associated protein (YAP) + , cadherin 11 (Cad11) + , and phosphorylated protein S6 + FLS in RA (all P < .05), while CD55 was increased in PsA-FLS ( P < .001). Polyfunctionality demonstrated enhanced coexpression of Cad11 + CD44 + HLA-DR + ICAM-1 + FLS ( P < .01) and CD44 + HLA-DR + ICAM-1 + VCAM-1 + FLS ( P < .05) in RA, whereas PsA-FLS exhibited increased coexpression of pAKT + pmTOR + ( P < .01). THY1 + FLS populations were dominant in RA ( P < .05), while THY1⁻ FLS were dominant in PsA ( P < .05), with differential angiogenic, chemokine, and matrix metalloproteinase expression observed. THY1 + FAP + FLS correlated with disease activity score (DAS28) ( r = 0.55, P < .05) and synovitis ( r = 0.54, P < .05). Further flow analysis identified 6 main distinct FLS populations, with enrichment of THY1 + CD34⁻CD55⁻FAP + FLS and THY1 + CD34 + CD55⁻FAP + FLS in RA ( P < .05), while PsA displayed enrichment of THY1 + CD34 - CD55 + FAP + FLS ( P < .05) and THY1⁻CD34⁻CD55 + FAP + FLS ( P < .001). Immune/adhesive markers were significantly higher in RA subpopulations, whereas metabolic/osteogenic markers were higher in PsA subpopulations. ScRNA-seq identified 16 distinct FLS clusters, with 3 sublining clusters enriched in RA compared with PsA, including THY1 hi POSTN + , THY1 hi CXCL12 + CHI3L2 + , and THY1 hi CXCL12 + APOE + . Pathway enrichment analysis identified heightened Hippo signalling in RA THY1 hi POSTN + FLS, and blockade of this signalling inhibited FLS pathogenic function. Conclusions RA and PsA possess distinct FLS populations with unique functional and metabolic properties, which may facilitate improved understanding of disease pathogenesis and therapeutic response.