Lung Dysbiosis and Increased Host Susceptibility to Respiratory Pathogens

下呼吸道感染是退伍军人的一个重大负担。目前的治疗通常涉及使用 广谱抗生素缺乏病原体特异性，并摧毁人体内的微生物生态。 使用下一代测序发现肺部微生物群确定了多样化的下气道 微生物群落我们的研究小组已经表明，人类口腔中肺部微生物群的富集 链球菌、韦荣氏球菌和普雷沃氏菌等细菌是下呼吸道微生态失调的标志 与炎症和免疫衰竭标记物增加有关。这种生态失调的特征 在健康受试者中，慢性炎性肺病（如慢性 阻塞性肺疾病和肺癌。在退伍军人事务部BLR&D CDA 2重新提交，我寻求 证明慢性肺生态失调如何通过调节 通过厌氧微生物代谢产物如短链脂肪酸（SCFAs）， 临床小鼠模型。在我们的出版物中，下呼吸道生态失调与SCFA增加， T细胞对病原体相关分子模式的应答，以及耗竭标志物表达的增加 和Tibet。我的诱导慢性下呼吸道生态失调的小鼠模型，在我的初步数据显示， 肺中的耗竭途径、PD 1 + T细胞和TcR表达增加，呼吸道感染增加。 病原体易感性表现为鼠死亡率和病原体恢复率。 这项研究将使用我的慢性肺生态失调小鼠模型来确定分子机制 导致宿主对呼吸道病原体易感。在目标1中，我将识别微生物群特征， 使用存在于慢性小鼠下生态失调气道中的元转录组和代谢组方法， 包括测量SCFA水平和影响它们的细菌功能。我还将测试SCFAs是否 足以改变宿主对病原体的易感性在目标2中，我将鉴定和测试宿主功能表型， 在我的慢性下呼吸道生态失调小鼠模型中使用荧光激活细胞分选的免疫耗竭， 单细胞RNA-Seq，以及抗CD 3/CD 28活化后T细胞亚群功能的评估。我会 还通过使用检查点抑制来评估免疫耗竭对病原体易感性的作用， 慢性生态失调，以评估抗PD-1是否会恢复病原体反应。 这些研究将在Leopoldo Segal、Xue-Ru Wu、Jun-Chieh Tsay博士的指导下进行 和杰弗里·威瑟西格尔博士是我的主要导师和退伍军人事务部附属研究员。他是系统专家 利用下一代测序技术评估气道疾病的生物学方法。他的专长在 转化研究的生物信息学方法使他处于下呼吸道多组学研究的最前沿， research.我的共同导师是吴学儒博士，他是尿路上皮疾病的专家， 评估与我的指导工作高度相关的致病机制。虽然他的专长是 专注于尿路上皮，他在实验设计，分子机制评估， 使用鼠模型对我的科学训练至关重要。Jun-Chieh Tsay博士是一名退伍军人事务部医生科学家 研究重点是肺癌的肺部炎症。作为一个共同导师，他将是一个重要的资源， 评估宿主炎症，转录组通路分析，以及我在退伍军人管理局的职业生涯。另外我 聘请Weiser博士作为共同导师，因为他是国际气道粘膜免疫专家 和感染导师和他们的实验室，再加上退伍军人事务部和纽约大学核心的专业知识， 实验室，将为我的研究生涯的发展提供最佳的环境。BLR&D CDA 2将 为我的培训提供必要的方法和技术支持，以完成拟议的 实验这一建议将提供基本的机械理解，以扩展到未来 涉及人类群体的调查。

Lower airway infections are a significant burden for Veterans. Current therapies commonly involve the use of broad-spectrum antibiotics that lack pathogen specificity and decimates microbial ecology in the human body. The discovery of lung microbiota with the use of next-generation sequencing identified a diverse lower airway microbial community. Our group has shown that the enrichment of the lung microbiota with human oral commensals, such as Streptococcus, Veillonella, and Prevotella, is a hallmark of lower airway dysbiosis associated with increased inflammation and immune exhaustion markers. This dysbiotic signature can be seen in healthy subjects and occurs with higher frequency with chronic inflammatory lung disease such as Chronic Obstructive Pulmonary Disease and lung cancer. In this Veterans Affairs BLR&D CDA2 resubmission, I seek to demonstrate how chronic lung dysbiosis is associated with increased pathogen susceptibility by modulation of the lung immune tone through anaerobic microbial metabolites such as short-chain fatty acids (SCFAs) in a pre- clinical murine model. In our publications, lower airway dysbiosis is associated with increased SCFAs, blunted T cell response to pathogen associated molecular patterns, and increased expression of exhaustion markers and Tregs. My mouse model of induced chronic lower airway dysbiosis and in my preliminary data show increased expression of exhaustion pathways, PD1+ T cells and Tregs in the lung, and increased respiratory pathogen susceptibility in the form of murine mortality and pathogen recovery. The proposed study will use my chronic lung dysbiosis murine model to define the molecular mechanisms leading to the host susceptibility to respiratory pathogens. In Aim 1, I will identify microbiota signatures with the use of metatranscriptome and metabolome approaches present in the chronic murine lower dysbiotic airway, including measurement of SCFA levels and bacterial functions affecting them. I will also test whether SCFAs are sufficient to alter host pathogen susceptibility. In Aim 2, I will identify and test host functional phenotypes of immune exhaustion in my chronic lower airway dysbiosis murine model using fluorescent-activated cell sorting, single-cell RNA-Seq, and assessment of T cell subpopulation function following anti-CD3/CD28 activation. I will also evaluate the role of immune exhaustion on pathogen susceptibility by using checkpoint inhibition during chronic dysbiosis to assess if anti-PD-1 will restore pathogen response. These studies will be performed under the mentorship of Drs. Leopoldo Segal, Xue-Ru Wu, Jun-Chieh Tsay and Jeffrey Weiser. Dr. Segal is my primary mentor and a VA affiliated researcher. He is an expert in systems biology approaches utilizing next-generation sequencing to evaluate airway disease. His expertise in bioinformatic approaches to translational research places him at the forefront of lower airway multi-omic research. My co-mentor is Dr. Xue-Ru Wu who is an expert on urothelial disease and built a career in assessing pathogenic mechanisms that will be highly relevant for my mentorship. Although his expertise is focused on the urothelial tract, his training in experimental design, assessment of molecular mechanisms, and use of murine models will be critical for my scientific training. Dr. Jun-Chieh Tsay is a VA physician scientist with a research focus on lung inflammation in lung cancer. As a co-mentor, he will be a significant resource to assess host inflammation, transcriptomic pathway analysis, and for my career with the VA. In addition, I recruited Dr. Weiser as a co-mentor for his expertise as an international expert in airway mucosal immunity and infection. The mentors and their laboratories, together with the expertise of Veterans Affairs and NYU core laboratories, will provide the optimal setting for the development of my research career. The BLR&D CDA2 will provide the support for my training in the methodologies and techniques necessary to complete the proposed experiments. This proposal will provide the fundamental mechanistic understanding to expand to future investigations involving human cohorts.