Time- and dose-dependent activation of the NLRP3 and MyD88/NF-κB pathways by Trueperella pyogenes membrane vesicles in bovine endometrial epithelial cells.

Trueperella pyogenes is an opportunistic pathogen frequently associated with bovine endometritis, yet the mechanisms by which it induces uterine inflammation remain incompletely understood. In this study, we investigated the effects of T. pyogenes and its membrane vesicles (MVs) on bovine endometrial epithelial cells (BEECs) and explored the underlying inflammatory pathways involved. Bovine endometrial epithelial cells (BEECs) were treated with T. pyogenes (MOI = 100) or MVs at various concentrations (1 × 10⁸, 1 × 10⁷, or 1 × 10⁶ particles/mL) for 6-24 h. Inflammatory cytokines (IL-1β, IL-6, IL-18, or TNF-α) and the activation of the NLRP3 and MyD88/NF-κB signalling pathways were analysed by ELISA, qRT‒PCR, and western blotting. Cell death mechanisms were assessed by flow cytometry and scanning electron microscopy. T. pyogenes significantly upregulated inflammatory cytokine mRNA expression at 6 and 12 h and protein expression at 12 and 24 h. Compared with bacterial stimulation at 12 h, MVs induced earlier activation of the NLRP3 inflammasome at 6 h. High-concentration MVs induced necrosis-like membrane disruption, whereas moderate concentrations promoted apoptosis and pyroptosis. Both T. pyogenes and its MVs activated the MyD88/NF-κB signalling pathway, with significantly increased phosphorylation of P65 at 12 h. Cytokine secretion exhibited time- and dose-dependent trends, aligning with transcriptional changes. Collectively, these findings demonstrate that T. pyogenes MVs contribute to endometrial inflammation through the NLRP3 and MyD88/NF-κB signalling pathways, with distinct forms of cell death determined by MV concentration. These findings highlight MVs as key virulence factors and potential therapeutic targets for bovine endometritis.