Project Summary Birth cohort studies have found significant associations between early-life wheezing-associated respiratory tract infections and the development of asthma in children up to 13 years of age. These studies suggest that early life respiratory tract infections have a direct effect on lung and/or immune cell development and the risk of asthma. To determine possible mechanisms, we established a mouse model of early-life RV infection. Infection of 6 day-old mice, but not mature mice, induces long-lasting mucous metaplasia, eosinophilic inflammation and airways hyperresponsiveness (AHR) which is associated with type 2 innate lymphoid cell (ILC2) expansion and dependent on IL-13, IL-25 and IL-33. For this renewal application, we have developed preliminary data showing that early-life RV infection increases the number of airway IL-25+ DCLK1+ brush cells, providing a mechanism for a persistent ILC2-dependent asthma-like phenotype. In addition, we have found that, in immature mice, activation of the NLRP3 inflammasome and IL-1β maturation inhibits type 2 cytokine expression and mucous metaplasia. In this proposal, we will test the general hypothesis that, following early- life RV infection, development of ILC2-dependent type 2 airway inflammation and mucous metaplasia represents a balance between tuft cell RV-induced IL-25 signaling (promotes the phenotype) and NLRP3- dependent IL-1β signaling (suppresses the phenotype). To test this, we propose the following Aims: Specific Aim 1. Determine the contribution of airway brush (tuft) cells to viral-induced IL-25 production. We hypothesize that: 1) early-life RV infection stimulates a persistent increase in the number of IL-25-producing airway tuft cells; 2) tuft cells are required for RV-induced ILC2 expansion, mucous metaplasia and AHR; 3) RV-induced IL-25 and IL-13 production (by ILC2s and M2 polarized macrophages) constitute a feed-forward mechanism for tuft cell development. Specific Aim 2. Determine the role of IL-1β on the development of RV-induced mucous metaplasia and AHR. We hypothesize that: 1) in immature mice, RV-induced, NLRP3 inflammasome-dependent IL-1β production suppresses the asthma-like phenotype; 2) IL-1β inhibits epithelial cell innate cytokine expression; and 3) LPS and dog-associated house dust each attenuate development of the mucous metaplasia phenotype by stimulating inflammasome priming and activation. Specific Aim 3. Determine the effects of early-life RV-C infection. We hypothesize that: 1) compared to RV-A, RV-C infection of 6 day-old mice induces more type 2 inflammation, mucous metaplasia and AHR; 2) RV-C induces greater expansion of tuft cells; 3) RV-C elicits inflammasome priming but not activation, thereby permitting greater and more long-lasting type 2 cytokine expression and mucous metaplasia. Immature mice and infants with respiratory viral infections will be studied. Completion of the proposed work will provide new insight into the pathogenesis of asthma...