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

描述（由申请人提供）：虽然特定的口腔致病菌种[即，牙龈卟啉单胞菌（Pg）]与已建立的牙周病变有关，并且可以在动物模型中重现疾病，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，所述微生物生态产生能够引发牙周炎的微环境（即，牙周生态失调）仍不清楚。我们实验室目前的研究鉴定了暴露于口腔细菌物种的口腔上皮细胞（OEC）中免疫炎症基因的全局基因表达谱，表明Pg能够诱导分泌型磷脂酶A2-IIA组（PLA 2G 2A）的转录显著增加，其表达水平明显超过经典免疫炎症介质（例如，细胞因子/趋化因子和抗微生物肽）。蛋白质水平测定表明，PLA 2-IIA表达似乎是PG特异性反应，因为其他口服G+和G-细菌物种未能诱导类似的PLA 2-IIA反应。PLA 2-IIA是一种由多种细胞产生的高度阳离子脂肪分解酶，在感染和炎症中起关键作用 通过其有效的抗菌性能（特别是对革兰氏阳性菌），以及 其对活化炎性细胞和脂质介质产生的作用。此外，有证据表明Notch-1是粘膜免疫应答的中心调节剂，并参与肠上皮细胞的PLA 2-IIA表达。因此，我们观察到Pg诱导OECs中NOTCH 1基因的表达和激活。因此，调节上皮PLA 2-IIA水平，特别是在粘膜表面，可能是致病细菌物种打破宿主-微生物群共生相互作用导致炎性疾病的合理策略。因此，我们假设OECs的抗菌和免疫炎症反应是由牙龈卟啉单胞菌通过涉及Notch-1受体的机制中的PLA 2-IIA的差异活化来调节的。为了检验这一假设，我们提出了以下两个具体目标：（i）鉴定参与牙龈卟啉单胞菌诱导的PLA 2-IIA的OEC信号传导途径，和（ii）确定响应牙龈卟啉单胞菌的OEC产生的PLA 2-IIA的细胞定位和功能。为了解决这些知识差距，我们将使用口腔上皮细胞培养模型，以及不同的Pg菌株，结合使用单克隆抗体，化学抑制剂或siRNA方法的中和实验，以：（i）鉴定参与Pg诱导的PLA 2-IIA的OEC受体/途径，和（ii）确定OEC产生的PLA 2-的细胞定位和功能。在Pg激发后进行IIA，以更充分地了解这种有趣的分子在口腔粘膜感染和炎症的初始状态中的潜在作用。这一贡献的意义，将使一个更好地理解的细胞和分子机制参与Pg诱导的PLA 2-IIA作为一个潜在的机制参与Pg诱导的生态失调。这些结果有望为未来的研究提供强有力的循证基础，这些研究旨在确定与体外（使用原代OEC和器官型培养物）和体内（使用非人灵长类动物模型）Pg诱导的上皮PLA 2-IIA产生相关的分子靶标，这些靶标可能会增加口腔粘膜生态失调的风险。更集中地控制这种微生态失调的过程，最终应该提供新的机会，创新的方法来预防/治疗牙周炎的发展。

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.