Single-cell sequencing reveals synovial fluid γδ T-cell expansion in equine experimental osteoarthritis.

OBJECTIVE: Define temporal cellular changes following joint injury using single-cell RNA sequencing in experimental equine posttraumatic osteoarthritis (PTOA). METHODS: PTOA was induced in 4 Quarter Horses (3 to 5 years) via carpal osteochondral fragmentation and high-speed treadmill exercise. Synovial fluid (SF) cells and synovium were sampled over 18 weeks (November 2023 to April 2024). Single-cell suspensions were processed (10x Genomics Chromium iX), then aligned to the equine genome (Cell Ranger). Downstream analysis was completed in the R Seurat package. Differential gene expression (log2[fold change] > 1; P < .05) and differential abundance analyses were performed (P < .1). RESULTS: Cartilage injury had a modest impact on gene expression changes and cell abundance shifts in SF. Integrated analysis of 90,323 SF cells across 4 time points revealed 9 distinct cell types, primarily T cells (73 ± 19%) followed by myeloid cells (20 ± 13%). Subcluster analysis of T cells revealed 9 transcriptomically distinct subtypes (3 CD8, 2 CD4, 3 γδ, and 1 cycling). Differential abundance analyses of temporal changes identified increased γδ T and decreased CD4+ T-cell subsets in joints over time. Expanded populations of IL-23 receptor-positive γδ T cells exhibited increased T-helper 17 signatures. CONCLUSIONS: IL-23 receptor-positive γδ T-cell expansion, associated with joint inflammation, occurred in PTOA. Limitations include small sample size and individual heterogeneity; further investigation over extended timeframe is necessary to confirm whether later stages of the experimental model reflect natural chronic OA. CLINICAL RELEVANCE: Cellular immunotherapy targeting γδ T cells and IL-23/IL-17 blockade may warrant investigation to mitigate equine OA progression.