PROJECT SUMMARY OVERALL The main objective of our U19 proposal is to define the contribution of airway epithelial cells to age-related dysfunction of the tissue-resident immune system, particularly in the context of lung responses to viral infections. Indeed, age is a major risk factor for increased susceptibility to infectious diseases, including severe seasonal influenza virus infection. Recently, this risk was brought into even greater focus by the COVID-19 pandemic, with poor outcomes disproportionately affecting individuals over 65 years of age. While it is well documented that aging impacts both innate and adaptive immunity, the mechanisms underlying these effects, especially in lung tissue, are not well understood. In addition, little is known about how the aged airway epithelium contributes to functional immune alterations during respiratory virus infections. Based on our preliminary studies, we hypothesize that age-related epithelial changes contribute to chronic inflammation and altered tissue- resident lung immunity. This hypothesis directly builds upon our current U19 lung immunity research program that is advancing the concept that the lung epithelium, as the first site of respiratory virus infection and replication, acts as a critical pathogen barrier both as a modulator and an effector of the immune system. The workhorses of our program are primary human ex vivo air-liquid-interface (ALI) cultures, derived from airway epithelial progenitors, which closely resemble in vivo airway epithelial cell composition and responses to viruses. ALI cultures allow for 1) modelling, in time and space, of interactions between airway epithelium, pathogenic viruses (influenza and SARS-CoV-2), and immune cells; 2) genetically altering ALI genomes to study specific genes/pathways; 3) measuring the magnitude and kinetics of transcriptional responses to inflammatory insults that are difficult to measure in vivo; and 4) studying epigenetic regulation, RNA splicing, and impact of the microbiome in immune responses. Our findings will be validated using precision tissue slice assays from uninvolved lung tissue. We will also continue increasing the complexity of our 3D lung models, by incorporating alveolar space, integrating immune cells, and leveraging 3D bioprinting. To achieve our objective, we structured our Center around: two integrated research Projects focused on epigenetic, transcriptional, and alternative splicing mechanisms that we propose lead to a skewed isoform repertoire in aging lung epithelial cells and dysfunctional tissue-resident immunity; a Technology Development Project that will create sophisticated cellular models and gene editing tools to support research Project objectives; a Sample Core for storage and distribution of human tissues; and a Data Science Core for integrative analysis and data dissemination. The Center brings together clinicians and experts in lung immunology, bioengineering, genomics, and computational biology to maxi...