Microbial Dysbiosis and Epithelial Dysfunction in Vitamin A-deficient Lungs

摘要 曾经被认为是无菌的，最近的研究表明，居住在健康肺部的微生物在 患有慢性阻塞性肺疾病(COPD)、哮喘、结核病(TB)和SARS-CoV-2的患者 感染。营养不良的患者患呼吸道感染和发病的风险增加，根据 最近的研究表明，代谢状态和肺内稳态之间存在潜在联系。这些和其他 研究表明，肺中存在宿主-微生物-维生素A相互作用并影响 呼吸道免疫反应，但详细说明肺部微生物群的变化是原因还是 肺病理的后果，或者维生素A的生物活性代谢物维甲酸(RA)是否可以 以一种不依赖宿主的方式直接影响肺微生物群在很大程度上是未知的。我们假设 膳食VAD诱导的呼吸道上皮重建促进了肺内微生物的失调，进一步 通过宿主-微生物相互作用使呼吸道上皮功能障碍永久化。目标1将决定是否 膳食VAD直接改变下层上调的机会性微生物的代谢途径 成年VAD小鼠肺的呼吸道(LRT)。我们的显微镜核心设施提供最先进的 旨在捕捉高质量荧光图像的显微镜，使我们能够识别微生物 组成、蛋白质表达和在呼吸道内的定位(1.1)。自由使用qRT-PCR机 我在肺中心可以接触到，我们将测量机会主义者相对丰富的变化 在饮食调整后3周和8周，VAS和VAD肺中的微生物上调(1.2)。目标2将 确定宿主局部来源的代谢变化与远端来源的代谢变化是否会影响呼吸道 下呼吸道的上皮重塑。我们将测量VAS气管外植体的纤毛运动 和VAD肺使用我们实验室已经完成的纤毛运动方案(2.1)。我们还将调查 微生物是否具有独立于宿主影响上皮重塑和纤毛功能的能力 代谢状态。我们的目标是在体外将VAS和VAD气管外植体暴露于来自相反饮食的微生物 (2.2)并重复目标1中提出的纤毛运动技术。我们实验室的后翻译工作与 肺科在气管内病毒注射和细胞培养技术方面的成熟专业知识使 这一分目标很容易在拟议的时间框架内实现。目标3将确定与以下各项相关的机制 在缺乏维生素A的情况下微生物代谢功能失调。我们将测量RNA转录本 通过qRT-PCR从目标2(3.1)中提到的解释组织中收集我们上调的微生物。我们 还将使用基于硅的社区研究微生物-微生物和微生物-环境的相互作用 建模程序名为Comets(3.2)，这是我们在BU微生物组倡议的合作者创建的一个平台。 这项工作的结果旨在揭示VAD肺中特定的宿主-微生物和微生物-微生物的相互作用 对健康的肺功能是必不可少的。

Abstract Once believed to be sterile, recent studies now show microbes inhabiting healthy lungs that are dysregulated in patients with chronic obstructive pulmonary disease (COPD), asthma, tuberculosis (TB), and SARS-CoV-2 infection. Malnourished patients have an increased risk of respiratory infections and pathogenesis according to recent studies, indicating a potential link between metabolic status and lung homeostasis. These and other studies indicate the presence of host-microbe-vitamin A interactions that are present in the lung and influence the respiratory immune response, however details of whether changes to the lung microbiome is a cause or consequence of lung pathology, or whether retinoic acid (RA) – the bioactive metabolite of Vitamin A – can directly influence the lung microbiome in a host-independent manner are largely unknown. We hypothesize that dietary VAD-induced airway epithelial remodeling promotes microbial dysbiosis in the lung, further perpetuating respiratory epithelial dysfunction via host-microbe interactions. Aim 1 will determine whether dietary VAD directly alters metabolic pathways of opportunistic microbes found upregulated in the lower respiratory tract (LRT) of adult VAD mouse lungs. Our Microscopy core facilities offer state-of-the-art microscopes designed to capture high-quality fluorescent images that will enable us to identify microbial composition, protein expression, and localization within the airway (1.1). Using qRT-PCR machines freely accessible to me in the Pulmonary Center, we will measure changes in relative abundance of the opportunistic microbes upregulated in VAS and VAD lungs at 3 weeks and 8 weeks post-dietary modulation (1.2). Aim 2 will determine whether locally-derived vs. distally-derived metabolic changes in the host could influence airway epithelial remodeling in the lower respiratory tract. We will measure ciliary motility in tracheal explants of VAS and VAD lungs using a ciliary motility protocol already completed by our laboratory (2.1). We will also investigate whether microbes have the ability to influence epithelial remodeling and ciliary function independent of host metabolic status. We aim to expose VAS and VAD tracheal explants to microbes from opposing diets in-vitro (2.2) and repeat the ciliary motility technique proposed in Aim 1. Our lab’s metatranscriptomic work paired with the Pulmonary department’s proven expertise in intratracheal viral injections and cell culture techniques makes this sub-aim easily achievable within the proposed time frame. Aim 3 will identify mechanisms associated with microbial metabolic functions that are dysregulated in the absence of vitamin A. We will measure RNA transcripts of our upregulated microbes via qRT-PCR gathered from our explanted tissues mentioned in Aim 2 (3.1). We will also investigate microbe-microbe and microbe-environment interactions using an in-silico community-based modeling program called COMETS (3.2), a platform created by our collaborators at the BU Microbiome Initiative. The results of this work aims to uncover specific host-microbe and microbe-microbe interactions in VAD lungs that are essential for healthy lung function.