PROJECT SUMMARY Accumulating evidence indicates that infections with a newly-discovered species of rhinovirus, RV-C, are asso- ciated with severe respiratory tract infections and asthma exacerbations often requiring hospitalization. In addi- tion, recent data suggest a possible role for early-life RV-C infections in asthma development. Despite increasing recognition of RV-C as a cause of asthmatic disease, virtually nothing is known about the pathogenesis of RV-C infections. To accomplish this, we infected mice with RV-C15 (the RV-C15 infec- tious clone and HeLa-E8 cells overexpressing a variant of the human RV-C receptor, cadherin related family member 3, were obtained from James Gern, University of Wisconsin). Our pilot studies show that RV-C15- infected mice show increased type 2 cytokine and mucin gene expression, BAL eosinophils and lineage-nega- tive, CD25+, CD127+ type 2 innate lymphoid cells (ILC2s) compared to RV-A1B-infected mice. In addition, pi- lot studies from children with natural RV-C infections show increased type 2 cytokine production. In this application, we will test the general hypothesis that, after RV-C infection, airway innate cytokine ex- pression drives ILC2 expansion and development of eosinophilic inflammation and mucous metaplasia. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1. Determine the contribution of epithelial-derived innate cytokines to RV-C15-induced eosinophilic airway inflammation and hyperresponsiveness (AHR). We hypothesize that: 1) compared to RV-A, RV-C infection of mature mice induces greater lung expression of innate cytokines (IL-25, IL-33, TSLP); 2) IL-25 is produced by doublecortin-like kinase (DCLK)-1-positive airway tuft cells; 3) innate cytokines are re- quired for eosinophilic inflammation; 4) RV-C engagement of CDHR3 activates distinct signaling pathways leading to innate cytokine expression. Specific Aim 2. Determine the contribution of lung ILC2s and macrophages to RV-C-induced airway inflammation and AHR. We hypothesize that: 1) RV-C infection of mature mice induces innate cytokine-de- pendent expansion of ILC2s; 2) ILC2s promote eosinophilic inflammation, macrophage polarization and AHR; 3) house dust mite (HDM) and RV-C have additive effects on eosinophilic inflammation and AHR; 4) ILC2s convey corticosteroid resistance; and 5) nasal aspirates from human subjects infected with RV-C show in- creased expression of type 2 cytokines and ILC2s compared to samples from RV-A-infected subjects. Specific Aim 3. Determine the effects of early-life RV-C infection on the established asthma pheno- type. We have found that RV-A1B infection of six day-old mice, but not mature mice, induces long-lasting mu- cous metaplasia and AHR which is dependent on IL-13-producing ILC2s. We hypothesize that: 1) RV-C infec- tion of 6 day-old mice induces greater and more long-lasting mucous metaplasia than RV-A; 2) early-life RV-C infection increases the number of IL-25-producin...