Yang Yanling, Liu Runze, Sun Qing, Chen Zhuo, Fan Wei
The State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan, 430079, China.
Odontology. 2025 Sep 13. doi: 10.1007/s10266-025-01202-5.
Programmed death-ligand 1 (PD-L1) is a critical immune checkpoint molecule that negatively regulates T-cell activation and serves as a characteristic marker of exhausted T cells in bacterial and viral infections. In this study, we found that Enterococcus faecalis (E. faecalis) infection modulated macrophage immune function under inflammatory conditions by upregulating PD-L1 expression. The aim of this study was to investigate the effect and potential regulatory mechanisms of E. faecalis and its virulence factor lipoteichoic acid (LTA) on the expression of PD-L1 in macrophages. RAW264.7 cells were treated with E. faecalis or LTA, respectively. Cellular immunofluorescence staining, flow cytometry, quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were employed to assess the expression of PD-L1 and endoplasmic reticulum (ER) stress-related proteins, including inositol-requiring enzyme 1 α (IRE1 α) and X-box binding protein 1 (XBP1), in macrophages. Following inhibition of the IRE1 α/XBP1 pathway and treatment with E. faecalis, qRT-PCR, flow cytometry, and WB were performed to detect the expression of PD-L1 and XBP1. Macrophage apoptosis was quantified by flow cytometry. Toll-like receptor 2 (TLR2) was knocked down using small interfering RNA (siRNA), and the expression of PD-L1, IRE1 α, and XBP1 in TLR2-silenced macrophages stimulated by E. faecalis was evaluated by qRT-PCR and WB. Statistical significance was analyzed using the Mann-Whitney test and Kruskal-Wallis test. The results demonstrated that E. faecalis and LTA significantly enhanced the expression of PD-L1, IRE1 α, and XBP1s in macrophages. Inhibition of the IRE1 α/XBP1 pathway reduced XBP1s and PD-L1 expression as well as apoptosis in E. faecalis-stimulated macrophages. TLR2 silencing decreased PD-L1, IRE1 α, and XBP1s expression levels in E. faecalis-stimulated macrophages. These findings reveal a novel mechanism by which E. faecalis induces persistent apical periodontitis and provide a foundation for further exploration of immune checkpoint molecules in the pathogenesis and treatment of this disease.
程序性死亡配体1（PD-L1）是一种关键的免疫检查点分子，它对T细胞活化起负调节作用，并且是细菌和病毒感染中耗竭T细胞的特征性标志物。在本研究中，我们发现粪肠球菌感染通过上调PD-L1表达在炎症条件下调节巨噬细胞免疫功能。本研究的目的是探讨粪肠球菌及其毒力因子脂磷壁酸（LTA）对巨噬细胞中PD-L1表达的影响及潜在调控机制。分别用粪肠球菌或LTA处理RAW264.7细胞。采用细胞免疫荧光染色、流式细胞术、定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法（WB）评估巨噬细胞中PD-L1以及内质网（ER）应激相关蛋白（包括肌醇需求酶1α（IRE1α）和X盒结合蛋白1（XBP1））的表达。在抑制IRE1α/XBP1途径并用粪肠球菌处理后，进行qRT-PCR、流式细胞术和WB检测PD-L1和XBP1的表达。通过流式细胞术对巨噬细胞凋亡进行定量分析。使用小干扰RNA（siRNA）敲低Toll样受体2（TLR2），并用qRT-PCR和WB评估在粪肠球菌刺激的TLR2沉默巨噬细胞中PD-L1、IRE1α和XBP1的表达。采用Mann-Whitney检验和Kruskal-Wallis检验分析统计学意义。结果表明，粪肠球菌和LTA显著增强巨噬细胞中PD-L1、IRE1α和XBP1s的表达。抑制IRE1α/XBP1途径降低了粪肠球菌刺激的巨噬细胞中XBP1s和PD-L1的表达以及细胞凋亡。TLR2沉默降低了粪肠球菌刺激的巨噬细胞中PD-L1、IRE1α和XBP1s的表达水平。这些发现揭示了粪肠球菌诱导持续性根尖周炎的新机制，并为进一步探索免疫检查点分子在该疾病发病机制和治疗中的作用提供了基础。
