Peptidoglycan derived from Lacticaseibacillus rhamnosus and Lactobacillus acidophilus suppress TLR2/1-mediated inflammation in bovine endometrial epithelial cells

Elham Waehama^1,2Kenji Fukuda¹Alireza Mansouri¹Malinda Hulugalla¹Ihshan Akthar¹Mohamed Samy Yousef^1,3Akio Miyamoto^1*

• ¹Global Agromedicine Research Center (GAMRC), Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan
• ²Faculty of Agriculture, Princess of Naradhiwas University, Narathiwat, Thailand
• ³Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt

Bacteria and associated products are factors in the pathogenesis of bovine endometrial inflammation, contributing to reproductive dysfunction. While peptidoglycan derived from Staphylococcus aureus (PGN-Sa) has been demonstrated to induce pro-inflammatory responses and disrupt sperm-immune interactions in bovine endometrial epithelial cells (BEECs) via Toll-like receptor 2/1 (TLR2/1), the immunomodulatory potential of peptidoglycan from lactic acid bacteria (LAB) within the female reproductive tract remains unexplored. This study investigated the in vitro immunomodulatory effects of LAB-derived peptidoglycan (PGN-L) on TLR2/1-mediated inflammation in BEECs, the specific TLR2/1 agonist PAM3CSK4 (PAM3) as an inflammatory stimulus. PGN-L was extracted and characterized from Lacticaseibacillus rhamnosus (PGN-Lr) and Lactobacillus acidophilus (PGN-La), and its structural composition was compared to that of commercial PGN-Sa. Subsequently, BEECs were pre-incubated with PGN-L (Lr, La) or PGN-Sa (1 ng/mL) for 24 hours before stimulation with PAM3 (100 ng/mL) for 3 hours. The expression of inflammatory genes (TNF, CXCL8, IL1B, and PTGES) and TLRs (TLR1, TLR2, TLR4, and TLR6) was quantified by RT-qPCR. Protein expression of TNF, PTGES, and TLR2 was detected using immunofluorescence, while PGE2 concentrations in the culture media were measured by ELISA. PGN-Lr and PGN-La shared the GlcNAc-MurNAc backbone with PGN-Sa, while PGN-L had a unique modification. PGN-L and PGN-Sa contained lysine at the cross-bridge stem, composed of glycine in PGN-Sa and likely modified D-aspartate in PGN-L. While PGN-Sa and PAM3 significantly upregulated the expression of inflammatory mediators, neither PGN-Lr nor PGN-La alone induced a pro-inflammatory response in BEECs. Importantly, pretreatment with both PGN-Lr and PGN-La significantly reduced PAM3-induced inflammatory gene expression and reduced PGE2 secretion. In silico molecular findings suggested a potential mechanism whereby PGN-L may act as a TLR2/1 antagonist, contrasting with the agonistic effects of PGN-Sa and PAM3, which promoted TLR2/1 heterodimerization. These findings suggest that PGN-Lr and PGN-La can suppress TLR2/1-mediated uterine inflammation in vitro, potentially by modulating TLR2/1 signaling in BEECs. Further investigation of PGN-L holds promise for the development of therapeutic strategies to enhance bovine reproductive efficiency.