Cooperativity of Oral Microbes in Evading Neutrophil-Mediated Killing

项目摘要/摘要 牙周病是一种复杂的炎症性疾病，由口腔微生物群落引起。 微生物一起工作，促进疾病的发生。与疾病相关的炎症是由 口腔生物膜，但宿主对微生物的反应，包括中性粒细胞的炎症反应，是 主要负责疾病的进展。牙龈卟啉单胞菌(Pg)是牙周的重要致病菌， 利用通常共生的细菌，如戈登链球菌(Sg)进行招募和生长辅助， 与共生菌和基石病原体，两者都需要促进牙周病的发展。 我们发现PG可以通过改变巨噬细胞的激活表型来改变巨噬细胞杀伤Sg的能力 Sg在M1巨噬细胞中的存活率高于在M2巨噬细胞中的存活率，这可能是由于 活性氧(ROS)的产生。ROS的产生是中性粒细胞防御的主要组成部分 抗病原体，其中ROS物种既可以在吞噬小体内产生，也可以在外部产生。 中性粒细胞和巨噬细胞一样，是一种有效的吞噬细胞，可以形成一道强大的防线来抵御牙周疾病。 具有一系列防御机制的病原体，包括吞噬作用、中性粒细胞胞外陷阱(NET) 生产和脱粒。中性粒细胞表现出多种表型，包括在口腔环境中， 然而，这些表型没有在巨噬细胞中描述得那么好。然而，一些人 中性粒细胞对牙周病等感染反应的差异已经被表征，包括 在ROS的制作中。因此，这个项目的总体目标是了解口腔细菌是如何 Keystone病原体和通常共生，影响和响应中性粒细胞表型。中性粒细胞的作用 在先天免疫中的作用在整个科学研究界都是众所周知的，正如 吞噬小体发展为中性粒细胞介导的杀伤。尽管这些细胞是主要的吞噬细胞 在牙周组织中，不断变化的吞噬小体环境对牙周细菌的影响 中性粒细胞中的疾病尚未得到充分研究。我们的中心假设是Sg和Pg的生存 依赖于它们在逃避中性粒细胞防御机制方面的协同性。来调查这件事 假设本项目的目的是(1)研究中性粒细胞的激活，包括PG对中性粒细胞的调节 吞噬小体成熟动力学和(2)研究Pg和Sg如何协同作用以提高其存活率 和炎症。为了实现这些目标，从经典的细胞生物学和 生物化学技术向先进的显微镜、流式细胞仪和图像分析方向发展。完成 该项目将是理解共生细菌的合作机制的有价值的一步 牙周病原体有助于逃避中性粒细胞介导的杀伤。它还将为调查员提供 通过不同的指导、科学和专业培训，如提案中进一步概述的那样，使 成功过渡到独立研究事业的下一阶段。

Project Summary/Abstract Periodontal disease is a complex inflammatory disease resulting from a dysbiotic community of oral microbes working together to promote disease. Inflammation associated with disease is initiated by members of the oral biofilm, but the host response to the microorganisms, including inflammatory reactions by neutrophils, is primarily responsible for disease progression. Porphyromonas gingivalis (Pg), a keystone periodontal pathogen, utilizes normally commensal bacteria such as Streptococcus gordonii (Sg) for recruitment and growth assistance, with commensals, as well as keystone-pathogens, both required to promote periodontal disease development. We have found Pg can alter the ability of macrophages to kill Sg by modifying macrophage activation phenotypes where Sg survives in M1 macrophages at a higher rate than in M2 macrophages, likely due to differences in reactive oxygen species (ROS) production. ROS production is a major component of neutrophils’ defense against pathogens, where ROS species can be produced both within the phagosome as well as externally. Neutrophils, like macrophages, are efficient phagocytes that form a powerful line of defense against periodontal pathogens with an array of defense mechanisms including phagocytosis, neutrophil extracellular trap (NET) production and degranulation. Neutrophils display a variety of phenotypes, including in the oral environment, however, these phenotypes are not as well described as they are in macrophages. However, a number of differences in neutrophil response to infections such as periodontal disease have been characterized, including in ROS production. Therefore, the overall objective of this project is to understand how oral bacteria, both keystone pathogens and normally commensal, affect and respond to neutrophil phenotypes. The role neutrophils play in innate immunity is well understood throughout the scientific research community as is the importance of phagosome development to neutrophil mediated killing. Despite these cells being the predominant phagocytes in periodontal tissue, the influence of a changing phagosome environment on bacteria involved in periodontal disease has yet to be fully explored in neutrophils. Our central hypothesis is that the survival of Sg and Pg is dependent on their cooperativity in evading neutrophil defense mechanisms. To investigate this hypothesis this project aims to (1) investigate how activation of neutrophils, including by Pg, modulates neutrophil phagosomal maturation dynamics and (2) investigate how Pg and Sg act collaboratively to increase their survival and inflammation. To achieve these aims a wide variety of techniques, from classical cell biology and biochemical techniques to advanced microscopy, flow cytometry and image analysis will be used. Completion of this project will be a valuable step in understanding how the cooperative mechanisms of commensal bacteria with periodontal pathogens contribute to evading neutrophil-mediated killing. It will also provide the investigator with diverse mentoring, scientific and professional training, as further outlined in the proposal, to allow a successful transition to the next stage of an independent research career.