Alpha2EQ downregulates proinflammatory cytokine and chemokine gene expression in cultured synovial fibroblasts.

Objective: To investigate the ability of the equine orthobiologic Alpha2EQ to control inflammation in cultured synovial fibroblasts. Methods: Equine synovial fibroblasts (n = 16) were cultured in a monolayer, and a targeted transcriptomic analysis (NanoString nCounter) was performed to screen for upregulated inflammatory gene expression. Cells were classified according to their IL-6 expression level. In the first experiment, high IL-6 expression (n = 4) and low IL-6 expression (4) cells were treated with Alpha2EQ, and in the second, cells with basal IL-6 expression were stimulated with 10 ng/mL IL-1β (4) before treatment. Allogeneic Alpha2EQ was pooled from sound healthy horses (n = 6) at a dose of 25% vol/vol of cell culture media. Twenty-four hours later, RNA was isolated for NanoString gene expression analysis. The t tests assessed differences in mean gene expression fold changes between baseline and treatment, while one-way ANOVA or Wilcoxon signed-rank tests were used for multiple comparisons (P < .05). Results: Alpha2EQ downregulated inflammatory genes in 2 cell culture models. Compared to baseline, Alpha2EQ treatment significantly reduced expression of IL-6, IL-15, and CCL2/MCP1 in IL-6HIGH synovial fibroblasts by 1.88- to 4.21-fold, as well as expression of IL-1β, CCL5/RANTES, and PPBP/CXCL7 by 2.14- to 4.07-fold in a 10 ng/mL IL-1β model. In addition, CXCL6/GCP-2 and TNF-α were significantly downregulated by Alpha2EQ in both models (2.64- to 5.38-fold). Conclusions: Alpha2EQ reduces inflammation by modulating the expression of cytokines and chemokines by synovial cells. Clinical Relevance: This study provides early insights into Alpha2EQ's anti-inflammatory mechanisms in vitro and evidence to support its clinical use in the treatment of equine osteoarthritis.