Characterization of the single cell landscape in normal and osteoarthritic equine joints

Background: Osteoarthritis (OA) is a major source of pain and disability worldwide. Understanding of disease progression is evolving, but OA is increasingly thought to be a multifactorial disease in which the innate immune system plays a role in regulating and perpetuating low-grade inflammation. The aim of this study was to enhance our understanding of OA immunopathogenesis through characterization of the transcriptomic responses in OA joints, with the goal to facilitate the development of targeted therapies.

Methods: Single-cell RNA sequencing (scRNA-seq) was completed on cells isolated from the synovial fluid of three normal and three OA equine joints. In addition to synovial fluid, scRNA-seq was also performed on synovium from one normal joint and one OA joint.

Results: Characterization of 28,639 cells isolated from normal and OA-affected equine synovial fluid revealed the composition to be entirely immune cells (CD45⁺) with 8 major populations and 26 subpopulations identified. In synovial fluid, we found myeloid cells (macrophage and dendritic cells) to be overrepresented and T cells (CD4 and CD8) to be underrepresented in OA relative to normal joints. Through subcluster and differential abundance analysis of T cells we further identified a relative overrepresentation of IL23R⁺ gamma-delta (γδ) T cells in OA-affected joints (a cell type we report to be enriched in gene signatures associated with T helper 17 mediated immunity). Analysis of an additional 17,690 cells (11 distinct cell type clusters) obtained from synovium of one horse led to the identification of an OA-associated reduction in the relative abundance of synovial macrophages, which contrasts with the increased relative abundance of macrophages in synovial fluid. Completion of cell-cell interaction analysis implicated myeloid cells in disease progression, suggesting that the myeloid-myeloid interactions were increased in OA-affected joints.

Conclusions: Overall, this work provides key insights into the composition of equine synovial fluid and synovium in health and OA. The data generated in this study provides equine-specific cell type gene signatures which can be applied to future investigations. Furthermore, our analysis highlights the potential role of macrophages and IL23R⁺ γδ T cells in OA immunopathogenesis.