Pathogens-derived TLR2/4 ligands disrupt the uterine immune response to bovine sperm in vitro

Abstract

Subclinical endometritis (SE) is known as one of the major problems in reproduction of dairy cattle where the risk of the disease is often not noticed due to its silent mode. Bovine endometrium senses and responds to bacteria (their pathogen-associated molecules; PAMs) by the secretion of inflammatory chemokines and cytokines. Our recent results showed that Toll-like receptor 2/4 (TLR2/4) pathway is involved in mediating sperm-bovine endometrial epithelial cells (BEECs) inflammatory process. This study aimed to (1) elucidate the role of peptidoglycan (PGN-SA) and lipopolysaccharide (LPS O55:B5) at extremely low concentrations in BEECs monolayer model, (2) determine how these molecules interact with the sperm-induced response of BEECs, thus possibly interfere their inflammatory response. BEECs monolayers were incubated with each ligand for 24 h independently, prior to their co-culture with/without sperm for 6 h, and cells were harvested for real-time PCR analysis for pro-inflammatory genes TNFA, IL-1B, IL-8, PGES, TLR2, and TLR4. First, the pathologically high doses of PGN (100 ng and 1 mg/ml) and LPS (100 ng and 1 mg/ml) stimulated the mRNA expression of TNFA, IL-1B, IL-8 and PGES, indicating a strong pro-inflammatory response. Sperm alone upregulated TNFA, IL-1B, IL-8 and PGES expressions, while suppressed the TLR2 expression in BEECs. Whereas low doses of (PGN 10 and 100 pg/ml) or (LPS 10 pg/ml) did not induce a significant pro-inflammatory response in BEECs, they suppressed the stimulatory effect of sperm to induce the pro-inflammatory genes in BEECs. The results suggest that low concentrations at 10 pg/ml of PGN (TLR2-ligand) and LPS (TLR4-ligand) can potentially compete with sperm for activating its specific receptors of BEECs, thereby disrupting the intact immune response to sperm. This may underline the possible mechanisms for SE partly mediated by TLR2/4 system in the cow.