PROJECT SUMMARY/ABSTRACT - Neutrophil Physiology Core The Neutrophil Physiology Core provides critical services to both Projects 1 and 2. The multi-omics studies provided by the other cores will provide valuable phenotyping information about our patients. The Neutrophil Physiology Core will perform functional assays and provide quantitative data to show how their neutrophils (PMN) are actually performing their roles in the innate immune response. Close communication with the PIs of the two Projects will ensure that the appropriate assays are carried out for each patient sample depending on the clinical context, especially in case of limited samples. PMN will be isolated from peripheral blood and bronchoalveolar lavage (BAL) samples collected simultaneously from the enrolled patients. Isolated PMN will be subjected to a number of assays to determine their ability to adhere to and move across physiologically relevant substrates in response to appropriate inflammatory responses (adhesion to endothelial cells, adhesion to respiratory epithelial cells, transendothelial migration, chemotaxis through extracellular matrix), engage in appropriate effector function responses (phagocytosis of opsonized particles, phagocytosis and killing of bacteria currently infecting the host, ROS generation, secretion of cytokines, release of granule content, generation of regulatory extracellular vesicles (EVs) and production of antibacterial NETs), and to respond to infection and tissue adaptation with relevant changes in metabolism (oxidative reactions, glycolysis, mitochondrial activation). The core will also co-culture patient PMN with patient T cells and endothelial/epithelial cells in physiologic assays to assess whether and how proper intercellular communication is impacted by disease. A parallel arm of the core located at our site at National Jewish Hospital in Denver will carry out similar studies comparing blood, (BAL), and unique sputum samples from their patients. To ensure rigor and reproducibility, endothelial cells are harvested regularly from human umbilical cords and never used beyond passage two. This assures that phenomena are not unique to any particular endothelial cell line. Endothelial cells are treated with the relevant cytokines and checked by flow cytometry and/or immunofluorescence to verify that they are responding appropriately to each stimulus. Similarly, respiratory epithelial cells are harvested from bronchial brushings and cultured in physiological air-liquid interface conditions in early passages. Differentiation will be confirmed by detection of ciliation, mucus production and junctional proteins using imaging approaches. The Core Leads and staff will meet weekly to discuss any issues that might arise regarding assay or reagent reproducibility or workflow. They will meet more frequently should any problems arise. The Core PIs will meet regularly with the PIs of the other cores and projects to review progress toward completion of the ...