Microbial Dysbiosis and Epithelial Dysfunction in Vitamin A-deficient Lungs

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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.