PROJECT SUMMARY Asthma is an immune-mediated disorder, and early life allergic sensitization (T2 inflammation) and respiratory illnesses are the most common precursors to the development of lifelong asthma. With over 300 million patients affected worldwide, asthma is a significant health care burden and a lifelong illness for most individuals. New information is needed to design preventive treatments, and answers may come from studying children who grow up in farming or traditional agrarian (TA) environments, who have intense and unique microbial exposures and are much less likely to develop allergic diseases and asthma. We hypothesize that TA microbial exposures and colonization promote immune development and airway epithelial cell functions to reduce the risks of respiratory illnesses, allergies and asthma. To test this hypothesis, we started the Wisconsin Infant Study Cohort (WISC) in 2013 and added a cohort of TA families in 2018 to determine mechanisms for protection against allergic diseases and asthma. We find that Wisconsin farm children have distinct gastrointestinal and nasopharyngeal microbiome features and unique gene expression patterns in nasal airway epithelial cells at age 2 years. These findings correspond with lower rates of atopic dermatitis, often a precursor to asthma and respiratory illnesses. The differences in immune and microbial signatures are even more pronounced in TA children. The microbiota in TA children's stool and nasal secretions are enriched for commensals that may promote immune development and reduce rates of allergic diseases and respiratory illnesses. Notably, the bacterial species identified in the TA samples contain genes distinct from those found in samples from the farm and non-farm children. We therefore propose that TA bacteria of the GI tract and airways and their metabolites are mediators of protection from allergic diseases and will be a rich source to determine the mechanistic pathways that promote health. To test our overall hypothesis, we propose three highly interactive projects. Project I will determine how TA children differ in immune development, nasal airway epithelial cell profiles, and clinical outcomes related to respiratory illnesses and allergic diseases. Project II will evaluate how TA children's distinct microbiota and their metabolites relate to their unique patterns of immune development and the resulting clinical outcomes. Project III will use in vitro models of airway epithelial cells to determine the mechanisms of TA microbial metabolites that inhibit viral and bacterial pathogens and modify the immune and inflammatory responses of airway epithelial cells.