Enterococcus faecalis is an important contributor to the persistence of chronic apical periodontitis. However, the mechanism by which E. faecalis infection in the root canals and dentinal tubules affects periapical tissue remains unclear. Bacterial extracellular vesicles (EVs) act as natural carriers of microbe-associated molecular patterns (MAMPs) and have recently attracted considerable attention. In this study, we investigated the role of EVs derived from E. faecalis in the pathogenesis of apical periodontitis. We observed that E. faecalis EVs can induce inflammatory bone destruction in the periapical areas of mice. Double-labeling immunofluorescence indicated that M1 macrophage infiltration was increased by E. faecalis EVs in apical lesions. Moreover, in vitro experiments demonstrated the internalization of E. faecalis EVs into macrophages. Macrophages tended to polarize toward the M1 profile after treatment with E. faecalis EVs. Pattern recognition receptors (PRRs) can recognize MAMPs of bacterial EVs and, in turn, trigger inflammatory responses. Thus, we performed further mechanistic exploration, which showed that E. faecalis EVs considerably increased the expression of NOD2, a cytoplasmic PRR, and that inhibition of NOD2 markedly reduced macrophage M1 polarization induced by E. faecalis EVs. RIPK2 ubiquitination is a major downstream of NOD2. We also observed increased RIPK2 ubiquitination in macrophages treated with E. faecalis EVs, and E. faecalis EV-induced macrophage M1 polarization was notably alleviated by the RIPK2 ubiquitination inhibitor. Our study revealed the potential for EVs to be considered a virulence factor of E. faecalis and found that E. faecalis EVs can promote macrophage M1 polarization via NOD2/RIPK2 signaling. To our knowledge, this is the first report to investigate apical periodontitis development from the perspective of bacterial vesicles and demonstrate the role and mechanism of E. faecalis EVs in macrophage polarization. This study expands our understanding of the pathogenic mechanism of E. faecalis and provides novel insights into the pathogenesis of apical periodontitis.

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粪肠球菌细胞外囊泡促进根尖周炎

粪肠球菌是慢性根尖周炎持续存在的重要因素。然而，根管和牙本质小管中粪肠球菌感染影响根尖周组织的机制仍不清楚。细菌细胞外囊泡（EV）作为微生物相关分子模式（MAMP）的天然载体，最近引起了相当大的关注。在这项研究中，我们研究了源自粪肠球菌的EV在根尖周炎发病机制中的作用。我们观察到粪肠球菌 EVs 可以诱导小鼠根尖周区域的炎症性骨破坏。双标记免疫荧光表明顶端病变中粪肠球菌 EV 增加了 M1 巨噬细胞浸润。此外，体外实验证明粪肠球菌EV可内化至巨噬细胞中。用粪肠球菌 EV 处理后，巨噬细胞倾向于向 M1 方向极化。模式识别受体（PRR）可以识别细菌 EV 的 MAMP，进而引发炎症反应。因此，我们进行了进一步的机制探索，结果表明粪肠球菌 EV 显着增加了细胞质 PRR NOD2 的表达，并且抑制 NOD2 显着降低了粪肠球菌 EV 诱导的巨噬细胞 M1 极化。 RIPK2 泛素化是 NOD2 的主要下游。我们还观察到用粪肠球菌 EV 处理的巨噬细胞中 RIPK2 泛素化增加，并且 RIPK2 泛素化抑制剂显着减轻了粪肠球菌 EV 诱导的巨噬细胞 M1 极化。我们的研究揭示了 EV 被视为粪肠球菌毒力因子的潜力，并发现粪肠球菌 EV 可以通过 NOD2/RIPK2 信号传导促进巨噬细胞 M1 极化。据我们所知，这是第一篇从细菌囊泡角度研究根尖周炎发展并论证粪肠球菌EV在巨噬细胞极化中的作用和机制的报告。这项研究拓展了我们对粪肠球菌致病机制的理解，并为根尖周炎的发病机制提供了新的见解。