Activation of the S100A8/A9 Alarmin Amplifies Inflammatory Pathways in Equine Ascending Placentitis

Ascending placentitis is a significant cause of equine pregnancy loss, yet the upstream inflammatory triggers are poorly defined. Recently, we identified S100A8/S100A9 (S100A8/A9) alarmins as potential upstream regulators in a chronic equine placentitis model. The current study aimed to determine whether this upregulation is sustained in the acute model and in clinical cases, and to elucidate the expression of their downstream inflammatory mediators. Using an experimental model, we quantified <i>S100A8/A9</i> mRNA expression in acute (<i>n</i> = 5) and chronic (<i>n</i> = 6) placentitis induced by <i>Streptococcus equi</i> ssp. <i>zooepidemicus</i>. We found mRNA expression of <i>S100A8</i> and <i>S100A9</i> was significantly upregulated in chorioallantois during both acute (<i>p</i> < 0.001) and chronic (<i>p</i> < 0.0001) disease compared to controls (<i>n</i> = 5), demonstrating their role is not limited to chronic pathology. A strong positive correlation (<i>r</i> = 0.945) underscored their coordinated expression. Immunohistochemistry revealed minimal staining in controls but dense infiltrations of S100A8/A9-positive neutrophils and macrophages in placentitis tissues. To define the clinical relevance of the downstream pathway, we analyzed RNA sequencing data from clinical placentitis cases (placentitis, <i>n</i> = 4) compared to normal postpartum placenta (control, <i>n</i> = 4). This confirmed upregulation of <i>S100A8/A9</i> and revealed a concurrent increase in their receptors (<i>TLR4</i>, <i>RAGE</i>) and a spectrum of NF-κB-driven effectors, including pro-inflammatory cytokines (<i>IL1β</i>, <i>IL6</i>, <i>TNF</i>), chemokines (<i>CXCL8</i>, <i>CCL2</i>, <i>CXCL10</i>), and the apoptotic mediator <i>CASP3</i>. Our findings establish that S100A8/A9 upregulation is a sustained feature of equine placentitis and delineates a coherent S100A8/A9-TLR4/RAGE-NF-κB signaling axis that drives inflammation and tissue damage in clinical disease. These findings highlight the diagnostic potential of S100A8/A9 and position this alarmin system as a promising therapeutic target for mitigating infection-induced pregnancy loss.