Waehama Elham, Fukuda Kenji, Mansouri Alireza, Hulugalla Malinda, Akthar Ihshan, Yousef Mohamed Samy, Miyamoto Akio
Global Agromedicine Research Center (GAMRC), Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan.
Faculty of Agriculture, Princess of Naradhiwas University, Narathiwat, Thailand.
Front Immunol. 2025 Jun 18;16:1622307. doi: 10.3389/fimmu.2025.1622307. eCollection 2025.
Bacteria and associated products are factors in the pathogenesis of bovine endometrial inflammation, contributing to reproductive dysfunction. While peptidoglycan derived from (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 immunomodulatory effects of LAB-derived peptidoglycan (PGN-L) on TLR2/1-mediated inflammation in BEECs, with the specific TLR2/1 agonist PAM3CSK4 (PAM3) as an inflammatory stimulus. PGN-L was extracted and characterized from (PGN-Lr) and (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 h before stimulation with PAM3 (100 ng/mL) for 3 h. The expression of inflammatory genes (, , , and ) and TLRs (, , , and ) was quantified by RT-qPCR. The protein expression of TNF, PTGES, and TLR2 was detected using immunofluorescence, while PGE 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 PGE secretion. 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 , by potentially modulating TLR2/1 signaling in BEECs. Further investigation of PGN-L holds promise for the development of therapeutic strategies to enhance bovine reproductive efficiency.
细菌及其相关产物是牛子宫内膜炎发病机制中的因素，会导致生殖功能障碍。虽然已证明源自金黄色葡萄球菌的肽聚糖（PGN-Sa）可通过Toll样受体2/1（TLR2/1）诱导牛子宫内膜上皮细胞（BEECs）产生促炎反应并破坏精子-免疫相互作用，但女性生殖道中乳酸菌（LAB）肽聚糖的免疫调节潜力仍未得到探索。本研究调查了LAB衍生的肽聚糖（PGN-L）对BEECs中TLR2/1介导的炎症的免疫调节作用，以特定的TLR2/1激动剂PAM3CSK4（PAM3）作为炎症刺激物。从鼠李糖乳杆菌（PGN-Lr）和嗜酸乳杆菌（PGN-La）中提取并鉴定了PGN-L，并将其结构组成与市售PGN-Sa进行了比较。随后，在用PAM3（100 ng/mL）刺激3小时之前，将BEECs与PGN-L（Lr、La）或PGN-Sa（1 ng/mL）预孵育24小时。通过RT-qPCR定量炎症基因（IL-1β、IL-6、TNF-α和PTGS2）和TLRs（TLR2、TLR4、TLR7和TLR9）的表达。使用免疫荧光检测TNF、PTGES和TLR2的蛋白表达，同时通过ELISA测量培养基中PGE的浓度。PGN-Lr和PGN-La与PGN-Sa共享GlcNAc-MurNAc主链，而PGN-L具有独特的修饰。PGN-L和PGN-Sa在交联茎处含有赖氨酸，PGN-Sa由甘氨酸组成，而PGN-L中可能是修饰的D-天冬氨酸。虽然PGN-Sa和PAM3显著上调了炎症介质的表达，但单独的PGN-Lr和PGN-La均未在BEECs中诱导促炎反应。重要的是，用PGN-Lr和PGN-La预处理均显著降低了PAM3诱导的炎症基因表达并减少了PGE分泌。分子研究结果提示了一种潜在机制，即PGN-L可能作为TLR2/1拮抗剂发挥作用，这与促进TLR2/1异二聚化的PGN-Sa和PAM3的激动作用形成对比。这些发现表明，PGN-Lr和PGN-La可以通过潜在地调节BEECs中的TLR2/1信号传导来抑制TLR2/1介导的子宫炎症。对PGN-L的进一步研究有望开发出提高牛繁殖效率的治疗策略。
