Cooperativity of Oral Microbes in Evading Neutrophil-Mediated Killing

项目总结/摘要 牙周病是一种复杂的炎症性疾病，是由口腔粘膜的微生态失调引起的。 微生物一起工作来促进疾病。与疾病相关的炎症是由以下成员引发的： 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， 主要负责疾病的进展。牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）是牙周病的重要致病菌， 利用正常的细菌如格氏链球菌（Streptococcus gordonii）（Sg）进行补充和生长辅助， 牙周病的发展需要牙周组织和关键病原体的共同作用。 我们已经发现Pg可以通过改变巨噬细胞活化表型来改变巨噬细胞杀伤Sg的能力 其中Sg在M1巨噬细胞中的存活率高于M2巨噬细胞，这可能是由于 活性氧（ROS）的产生。活性氧的产生是中性粒细胞防御的主要组成部分 针对病原体，其中ROS种类可以在吞噬体内以及外部产生。 中性粒细胞，像巨噬细胞，是有效的吞噬细胞，形成一个强大的防线，对牙周炎 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。中性粒细胞显示出多种表型，包括在口腔环境中， 然而，这些表型并不像它们在巨噬细胞中那样被很好地描述。但若干 中性粒细胞对感染如牙周病的反应的差异已经被表征，包括 ROS的生产。因此，本项目的总体目标是了解口腔细菌， 关键病原体和正常的嗜中性粒细胞，影响和响应于嗜中性粒细胞表型。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得到了很好的理解， 吞噬体发育至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，变化的吞噬体环境对牙周炎相关细菌的影响 在嗜中性粒细胞中疾病尚未被充分探索。我们的中心假设是Sg和Pg的存活 依赖于它们在逃避中性粒细胞防御机制中的协同性。调查这一 假设本项目旨在（1）研究中性粒细胞的激活，包括Pg，如何调节中性粒细胞 吞噬体成熟动力学和（2）研究Pg和Sg如何协同作用以增加它们的存活 和炎症。为了实现这些目标，从经典的细胞生物学和 将使用生物化学技术到先进的显微镜、流式细胞术和图像分析。完成 该项目将是了解肠道细菌合作机制的有价值的一步， 与牙周病原体的接触有助于避免嗜热菌介导的杀伤。它还将为调查人员提供 如提案中进一步概述的那样，通过多样化的指导、科学和专业培训， 成功过渡到独立研究生涯的下一阶段。

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.