Antimicrobial and inflammatory epithelial responses modulated by P. gingivalis-induced PLA2-IIA

描述(申请人提供)：虽然特定的口腔致病细菌种类[即牙龈假单胞菌(PG)]已与已建立的牙周病变相关，并可在动物模型中重现疾病，但在复杂的微生物生态中，与上皮-口腔病原体相互作用相关的早期事件仍不清楚，该微生物生态创造了使牙周炎(即牙周生物失调)发生的微环境。我们实验室目前对暴露于口腔细菌物种的口腔上皮细胞(OECs)免疫炎症基因表达谱的研究表明，PG能够诱导分泌型磷脂酶A2组IIA(Pla2g2a)的转录显著增加，其表达水平明显超过经典免疫炎症介质(如细胞因子/趋化因子和抗菌肽)的20-30倍。蛋白质水平测定表明，PLA2-IIA的表达似乎是PG特异性的反应，因为其他口腔G+和G-细菌物种未能诱导类似的PLA2-IIA反应。磷脂酶A2-IIA是一种由多个细胞产生的高度阳离子脂解酶，在感染和炎症中起关键作用。 通过其强大的抗微生物特性(特别是对革兰氏阳性细菌)，以及 其活化炎性细胞和产生脂质介质的作用。此外，有证据表明，Notch-1是黏膜免疫反应的中枢调节因子，并参与肠上皮细胞表达PLA2-IIA。我们一致地观察到PG诱导了OECs中NOTCH1基因的表达和激活。因此，调节上皮细胞PLA2-IIA水平，特别是粘膜表面的PLA2-IIA水平，可能是致病菌打破宿主-微生物区系共生作用导致炎症性疾病的一种可行的策略。因此，我们假设牙龈假单胞菌通过参与Notch-1受体的机制，通过PLA2-IIA的差异激活来调节嗅鞘细胞的抗微生物和免疫炎症反应。为了验证这一假说，我们提出了以下两个具体目标：(I)确定参与牙龈假单胞菌诱导的PLA2-IIA的OEC信号通路；(Ii)确定OEC产生的PLA2-IIA响应Pggivalis的细胞位置和功能。为了解决这些知识空白，我们将使用口腔上皮细胞培养模型以及不同的PG菌株，结合使用单抗、化学抑制剂或siRNA方法的中和实验：(I)识别PG诱导的PLA2-IIA所涉及的OEC受体/途径，以及(Ii)确定PG攻击后OEC产生的PLA2-IIA的细胞位置和功能，以更全面地了解这种有趣的分子在口腔黏膜感染和炎症初始状态中的潜在作用。这一贡献的意义将使我们能够更好地理解PG诱导的PLA2-IIA所涉及的细胞和分子机制，作为PG诱导的生物失调的潜在机制。这些结果有望为未来的研究奠定坚实的循证基础，这些研究旨在确定与PG诱导的上皮细胞PLA2-IIA在体外(使用原代OECs和器官型培养)和体内(使用非人灵长类动物模型)产生相关的分子靶点(S)，这可能会增加口腔粘膜生态失调的风险。对这一失调过程的更有针对性的控制最终将为开发预防/治疗牙周炎的创新方法提供新的机会。

DESCRIPTION (provided by applicant): Although specific oral pathogenic bacterial species [i.e., P. gingivalis (Pg)] have been related to established periodontal lesions and can recapitulate disease in animal models, the early events associated with epithelial-oral pathogen interactions in a complex microbial ecology that creates a microenvironment enabling the initiation of periodontitis (i.e., periodontal dysbiosis) remain unclear. Current studies in our laboratory identifying global gene expression profiles of immunoinflammatory genes in oral epithelial cells (OECs) exposed to oral bacteria species, indicated that Pg is able to induce a remarkable increase in the transcription of secreted phospholipase A2-group IIA (PLA2G2A), whose expression levels clearly exceeded 20-30 times those of classical immunoinflammatory mediators (e.g., cytokines/chemokines and antimicrobial peptides). Protein levels determination indicated that PLA2-IIA expression appears to be Pg-specific response since other oral G+ and G- bacterial species failed to induce similar PLA2-IIA responses. PLA2-IIA is a highly cationic lipolytic enzyme produced by multiple cells and plays a critical role in infection and inflammation through its potent antimicrobial properties (especially against Gram-positive bacteria), as well as its effects on activating inflammatory cells and lipid mediator production. Moreover, evidence indicates that Notch-1 is a central regulator of mucosal immune responses and is involved in PLA2-IIA expression by intestinal epithelial cells. Consistently, we observed that Pg induced the expression of the NOTCH1 gene and its activation in OECs. Thus, modulation of epithelial PLA2-IIA levels particularly at mucosal surfaces could be a plausible strategy for pathogenic bacterial species to break the host-microbiota symbiotic interactions leading to inflammatory disease. Therefore, we hypothesize that antimicrobial and immunoinflammatory responses of OECs are modulated by P. gingivalis through differential activation of PLA2-IIA in a mechanism that involves the Notch-1 receptor. To test this hypothesis we propose the following two specific aims: (i) to identify the OEC signaling pathways involved in P. gingivalis-induced PLA2-IIA, and (ii) to determine the cellular location and function of OEC- produced PLA2-IIA in response to P. gingivalis. To address these knowledge gaps, we will use oral epithelial cell culture models, as well as different Pg strains, combined with neutralization experiments using monoclonal antibodies, chemical inhibitors or siRNA approaches to: (i) identify OEC receptors/pathways involved in Pg-induced PLA2-IIA, and (ii) determine the cellular location and function of OEC-produced PLA2-IIA after Pg challenge to more fully understand the potential role of this interesting molecule in the initial states of infection and inflammation of the oral mucosa. This contribution's significance will enable a better understanding of the cellular and molecular mechanisms involved in Pg-induced PLA2-IIA as a potential mechanism involved in Pg-induced dysbiosis. The results are expected to contribute to a strong evidence-based foundation for future studies designed to identify molecular target(s) associated with Pg-induced epithelial PLA2-IIA production in vitro (using primary OECs and organotypic cultures) and in vivo (using a nonhuman primate model) that could increase the risk for oral mucosa dysbiosis. More focused control of this dysbiosis process should ultimately provide new opportunities for the development of innovative approaches to prevent/treat periodontitis.