PROJECT SUMMARY/ABSTRACT – PROJECT 1 Lower respiratory tract infection (pneumonia) causes almost 80% of deaths due to infection. For many otherwise fatal lung diseases, lung transplantation is the only curative option yet carries a dismal 5-year survival of 50%. Primary graft dysfunction following lung transplantation is responsible for the bulk of the early mortality following lung transplantation and is the dominant risk factor for chronic lung allograft dysfunction, the major barrier to long term survival in lung transplant recipients. We and others determined that a profound neutrophilic alveolitis is central to both pneumonia and PGD, yet mechanisms underlying the pathobiology of neutrophilic lung injury in these important diseases remain unclear. At Northwestern, we have created robust research programs engaged in high-volume, serial bronchoalveolar lavage fluid, lung tissue, and blood sample collection from patients with pneumonia as part of routine clinical care as well as from donor lungs throughout the course of lung transplantation. Hence, we are well-positioned to test mechanistic questions about the activation, migration, and function of lung neutrophils in well-phenotyped patients with neutrophilic alveolitis. Historically, neutrophils have been viewed as a homogenous, single-function population; however, our preliminary data point to substantial heterogeneity in lung and blood neutrophils. For example, we identified heterogeneity in expression of the integrin CD11b, which we found serves as a negative regulator of inflammatory signaling. We also observed in preliminary data that type I and II interferons (IFNs) have different expression levels in the circulation versus in the alveolar space and that lung epithelial and endothelial cells are sources of the key neutrophil cytokine CSF3. Hence, we hypothesize that CD11b/TLR/MYD88/NFκB signaling and systemic and tissue-derived signals (CSF3 and type I and II IFN) regulate neutrophil activation, migration, and function in patients with severe pneumonia and PGD. To test our hypothesis in humans, we will perform systematic sampling and multidimensional assessment using single-cell sequencing and spatial profiling of neutrophils in the circulation and alveolar compartments of hundreds of patients with severe pneumonia and PGD. We will integrate these molecular datasets with ex vivo functional assays and deep clinical phenotypes. In Aim 1, we will determine whether CD11b/TLR/MYD88/NFκB signaling and type I and II IFN control neutrophil activation, migration, and function in patients with severe pneumonia and PGD. In Aim 2, we will determine whether CSF3 generated within the alveolus determines neutrophil maturation, activation, migration, and function and whether arginase- 1 and BAFF link persistent neutrophilia to adaptive immunity in patients with pneumonia and PGD. In both Aims, we will use cutting-edge computational strategies to test whether these pathways are associated with disease e...