Endometritis is a common and costly disorder in horses that results in poor reproductive performance. Current therapies are supportive, and the industry suffers from a paucity of immunomodulatory treatment modalities for this disease that is characterized by inflammatory dysregulation. Recently, mesenchymal stromal cell conditioned media (MSC-CM) is being explored as a treatment option for various inflammatory conditions due to its immunoregulatory, antimicrobial, biofilm disruption, and tissue repair properties. Our objective was to evaluate changes in the endometrial inflammatory response after treatment with MSC-CM in vitro. We hypothesized that endometrial cells in culture would have altered cytokine expression and production. Five estrous mares were biopsied to establish primary endometrial cell cultures. After cell dissociation, endometrial cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution (Sigma Aldrich) at 37°C with 5% CO2. At 80% confluence, cultures were hormonally supplemented with 1nM Estradiol-17β, 100 nM Progesterone, or no supplementation; and 24 hours later, treated with MSC-CM or DMSO as control. Supernatants from the cell cultures were analyzed for cytokine expression using an equine cytokine Multiplex assay (Luminex Corp. Austin, TX) quantifying inflammatory biomarkers including IFN-α, IFN-γ, IL-4, IL-17, IL-10, IL1-β, CCL2, CCL3, CCL5, CCL11, TNF-α, and sCD14. In parallel, cells were lysed and RNA extracted for Real-time PCR using the ΔCT method with ACTB as the reference gene for geneexpression of IL-6, IL-8, PTGS2, PTGES, PTGIS, TNFα, TGFB, and NFKb. Data analysis was performed with JMP Pro 16 with level of significant p<0.05. A restricted maximum likelihood model with treatment, supplementation and their interaction and mare as a random factor was used. There was no effect of hormone supplementation on supernatant cytokine concentrations, but treatment with MSC-CM altered their concentration. MSC-CM treatment increased the concentration of CCL2 pg/ml [median (interquartile)] [1862554 (32482, 240080) (p≤0.02); and decreased CCL3 [66 (0.1, 99)] (p≤ 0.01) and IL-10 [6.5 (0.1, 9.5)] (p≤0.007) compared to DMSO control [3114 (2398.5, 7483); 109 (0.1, 154); and 13 (0.1, 17)], respectively. Gene expression analysis showed that MSC-CM-treated cells had increased expression of IL-6 (p≤0.001), IL-8 (p≤0.001), PTGS2 (p≤0.002), PTGES (p≤0.0012), and NFKb (p≤0.01). Additionally, gene expression analysis revealed an effect of hormone supplementation, with increased expression of IL-8 (p≤0.027) and PTGS2 (p≤0.0443) in estrogen or progesterone supplemented cultures compared to no supplementation. Taken together, treatment with MSC-CM resulted in increased inflammatory marker production and greater gene expression of inflammatory cytokines and prostaglandin-related genes. These results are supportive of the potential role of MSC-CM in equine endometrial immunomodulation. Further studies in vivo are warranted to investigate the clinical outcome of this immunomodulatory effect after intrauterine infusion.
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Nov 1, 2023
Ragavan Varadharajan Suresh + 5
Open Access
