CD8 T-cells, CD86+ macrophages, and TNF-α signaling pathways are correlated with fetlock osteoarthritis in racehorses

Methods

Fetlock joints from racehorses euthanized on NY racetracks were considered for inclusion. SF and SM were analyzed by flow cytometry using T-cell (CD3, CD4, CD8) and macrophage (CD14, CD86, CD206) surface markers. OA severity was characterized by gross joint evaluation and SM histology.

Bulk RNA-sequencing of SM was performed to identify differentially expressed genes. Flow cytometry data was analyzed with mixed linear modeling, including OA severity as a fixed effect and horse as a random effect. RNA-sequencing was evaluated with mixed linear and quadratic modeling using DREAMSeq method to capture differential gene expression over the range of OA severity.

Results: Twenty-four fetlocks from 12 horses met inclusion criteria. CD8+ T-cells (R2 = 0.25 for SM, 0.35 for SF) and CD86+ macrophages (R2 = 0.14 for SM) were positively correlated with OA severity, while CD4+ T-cells (R2 = 0.17 for SM, 0.31 for SF) were negatively correlated. Macrophages co-expressing CD86 and CD206 correlated with OA severity in SM (R2 = 0.31).

Pathways including those associated with TNF-α signaling, connective tissue development, and cell and neuron projection organization/development were differentially regulated in SM with greater OA scores. Main Limitations: Limited sample size, particularly of horses with severe OA. Conclusions: CD8+ T-cells, CD86+ macrophages, and macrophages co-expressing CD206 and CD86 are enriched in horses with more advanced OA.

Gene set analysis suggests the importance of TNF-α signaling and disinhibition of neuron and cell projection development in OA pathogenesis.