Uterine proteomic and cytokine profiling show that Streptococcus equi subsp. zooepidemicus induces immune-metabolic dysregulation and may predispose mares to endometrosis.

Abstract: This study aims to characterize and compare the uterine fluid proteome and cytokine profile of reproductively healthy mares (HM; n = 15) and mares with Streptococcus equi subsp. zooepidemicus (S. zooepidemicus)-associated endometritis (SAE; n = 9). Uterine flush samples collected during the follicular phase were analyzed by shotgun proteomics and multiplex cytokine assays. Equine proteins were mapped to human orthologs, and enrichment and network analyses were performed using g:Profiler, Metascape, ToppGene, and STRING/MCODE. SAE mares showed higher levels of IL-1α, IL-1β, GRO, IL12p70, and Eotaxin, and exclusive detection of IL-6, IL-8, IP10, IL-10 and TNFα, consistent with a proinflammatory and profibrotic environment. Proteomic analysis identified 67 differentially expressed proteins, indicating activation of innate immunity, metabolic reprogramming, and tissue remodeling. Upregulated proteins included neutrophil effectors linked to NET formation, acute-phase proteins, glycolytic enzymes, and chromatin-associated histones, whereas anti-inflammatory, antioxidant, or contractility-related proteins were reduced (SPI2, SOD3, ALDH1A1, CALM1). Enrichment and network analyses highlighted neutrophil degranulation/NETosis, glycolysis-gluconeogénesis, extracellular vesicle activity, and actin-cytoskeleton regulation, indicating immune-metabolic reprogramming of the uterine environment. Age-related decline in immune regulation may further exacerbate inflammatory and fibrotic responses in infected mares. The findings suggest that S. zooepidemicus infection induces a persistent neutrophil-driven inflammatory state and oxidative stress that compromise uterine clearance and may predispose mares to fibrosis. Sixteen candidate biomarkers related to chromatin remodeling, antimicrobial defense, and metabolic regulation were identified, providing potential diagnostic value. Therapeutic strategies targeting dysregulated inflammation and NETosis, alongside antimicrobial therapy, may improve fertility outcomes in mares affected by infectious endometritis.