PROJECT SUMMARY The early life environment modifies the risk for childhood allergic diseases, wheezing illnesses, and asthma, and these diseases are less common in children who grow up on farms. Accordingly, in our studies of the Wisconsin Infant Study Cohort ("WISC," funded by the AADCRC program), reductions in atopic dermatitis and respiratory illnesses were related to the intensity of farm exposures during the prenatal and early postnatal periods. Notably, allergies and asthma are rare in children in traditional agrarian (TA) settings (e.g., Amish communities) with intense farm exposures. Early life microbial exposures and colonization are likely to contribute to health benefits by promoting epithelial cell function and immune development. Certain commensal microbes such as Corynebacterium sp. and Dolosigranulum pigrum are inversely associated with bacterial respiratory pathogens and reduce the risks of viral wheezing illnesses and exacerbations of childhood asthma. These findings suggest that airway microbiomes of TA and healthy farm children will be a rich source of microbes that can inhibit viral and bacterial pathogens, increase epithelial integrity, and prime anti- pathogen responses. To test this hypothesis, we have established in vitro models of cultured airway epithelial cells to determine mechanisms for interactions between airway bacteria, viral infections, and epithelial cell composition and function. Our preliminary data indicate that TA children have microbiome community structures enriched for "protective" commensal bacteria with distinct genetic features. Finally, we used single-cell transcriptomics to identify characteristics of airway epithelial cells that are highly susceptible to infection with rhinoviruses (RV), which commonly cause wheezing illnesses during childhood. This project will test for interactions between airway commensal bacteria, airway pathogens, and epithelial cells. First, we will screen commensal bacteria from healthy TA and farm children for metabolites that inhibit viral and bacterial pathogens. Second, we will culture and analyze nasal airway epithelial cells from TA, farm, and non-farm children to determine whether environmental exposures lead to group-related differences in cell composition, antiviral responses, and synthesis of antimicrobial peptides. Finally, we will analyze effects of selected bacterial metabolites from TA and farm children on cultured airway epithelial cells to determine whether they exert beneficial effects on anti-pathogen responses and inhibit T2 inflammatory pathways. The results of these studies could lead to new therapeutic approaches to bring some of the health benefits associated with farm exposures to all children.