Project Summary/Abstract Pneumonia remains a significant cause of morbidity and mortality, which has come into sharp focus recently during the COVID-19 pandemic. The lung is immunologically unique, and neutrophils traversing the lungs must be ready to respond to potential pathogen challenges without causing injury to surrounding tissues. Disruptions of this balance lead to many severe complications of pneumonia, but we lack therapeutics directed toward neutrophil-mediated immune dysfunction. Our preliminary data demonstrate that neutrophils undergo transcriptional remodeling as they travel from the circulation to the alveolar space during pneumonia. A subset of pro-inflammatory genes is upregulated as neutrophils move from the vasculature to the interstitium, while genes related to metabolism and degranulation are upregulated as cells move from interstitium to the alveolar space. Maintenance of appropriate neutrophil function and development requires tight regulation of gene changes, yet the specific pathways underlying such changes constitute a major knowledge gap. The goal of this proposal is to delineate the transcriptional and functional changes neutrophils undergo as they migrate from the circulation to the site of infection during pneumonia. Our central hypothesis is that during pneumonia, appropriate neutrophil function is achieved by proscribed responses to specific environmental stimuli, resulting in a heterogenous population defined by their interaction history. Aim 1: Test the hypothesis that location-specific neutrophil responses in pneumonia are achieved by the tuning of neutrophil transcriptome and function by environmental stimuli. Using complementary in vitro and in vivo models we will determine the contributions of migration, pathogen interaction and cytokine signaling to neutrophil gene programs and function. Outcomes will include transcriptional and functional changes predicted by our preliminary studies. These findings will then be confirmed in an in vivo model of pneumonia, employing exogenously added labeled neutrophils. Aim 2: Test the hypothesis that during transit to the alveolar space, neutrophils differentiate into subpopulations unique to the lung, with transcriptional and functional heterogeneity. Using a combination of in vivo cell labeling, flow cytometry assisted cell sorting, and single cell RNA sequencing, we will track modulations in the neutrophil transcriptome from circulation to alveolar space at a single cell level. Aim 3: NFκB-RelA is a central transcription factor in neutrophils, driving the upregulation of a pro-inflammatory transcriptional program during migration to the alveolar space. Using complementary in vitro and in vivo loss-of function models we will determine the effect of neutrophil NFκB-RelA on pneumonia outcomes neutrophil transcriptome and neutrophil function. The results of these studies will have a positive impact the field of pneumonia biology furthering our knowledge of neutrophil functi...