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 ...