Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis

ABSTRACT Objectives Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/β-catenin signaling, given its emerging importance in arthritis. Methods We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. Results We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/β-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4 hi lining fibroblasts. Trajectory analyses predicted that Prg4 hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes. Conclusions Synovial fibroblasts assume distinct functional identities during PTOA, and Prg4 hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury.