? ? In this application, we propose a novel approach to the understanding of the pathogenesis of asthma exacerbations (AE) in children. We postulate two pathways involved in triggering AE: one centered on IL-8 and neutrophils, the other mediated by GAT A3 and eosinophils. We base this hypothesis on our preliminary data showing that single nucleotide polymorphisms (SNPs) in IL8 and GATA3 are associated with AEs in children enrolled in a longitudinal study of asthma in Tucson, AZ. IL8-251T was significantly and positively associated with the risk of having subsequent AE in children who had asthma by age 6. GATA3-7018C, a SNP in the GATA3 5' silencer, was significantly associated with increased frequency of AE after age 6 in children who had asthma by that age, but only among allergies. Functional studies will clarify if this association is truly due to GATA3/-7018 or to a different SNP in linkage disequilibrium with it. ? ? We intend to enroll a group of children with well characterized asthma and follow them until they develop the first AE. We will compare the time to the first AE in carriers of different genotypes for these SNPs. We will also assess the acute immunophenotype of these AE in relation to the two SNPs we postulate to be involved in modulating AE. We will particularly address the functional genomics of one SNP in GAT A3, which we believe is an important determinant of AE in the children at highest risk for severe AE, i.e., those with an allergic background. Specifically we propose to: 1. Determine whether IL8-251T->A modulates the clinical phenotype during AE by affecting the expression of IL-8 and neutrophilic inflammation during viral infections in children with asthma. We will study the role ofIL8-251T->A, a SNP in the IL8 promoter, in the regulation of an IL-8-dependent pathway that determines the production of IL-8 by epithelial and inflammatory cells and the consequent neutrophilic response to viral infections. 2. Determine whether GATA3-7018C->G modulates the clinical manifestations of AE by affecting the expression of GAT A3 and IL13 in airway eosinophils during viral respiratory infections. We will assess the expression of GAT A3 and IL13 mRNA and/or protein in airway eosinophils isolated from induced sputum during and after AEs, and correlate it with GATA3-7018 genotype and AE phenotypes. 3. Determine whether and how GATA3-7018C->G affects the regulation of GATA3 transcription in eosinophils. We will perform functional studies using allelic variants of GATA3 reporter constructs to determine whether the function of the GATA3 5' silencer in eosinophils is affected by GATA3-7018C->G, resulting in differential GATA3 transcription. Protein/DNA interactions at the wild-type and polymorphic silencer will be analyzed to identify the mechanisms responsible for differential GATA3 expression. ? ? The novel combination of ex vivo analysis and mechanistic studies proposed in this application will allow us to define the role played by the proposed IL-8/neutrophil- and GATA3/eosinophil-associated pathways in AE and may lead to the identification of new therapeutic targets for the prevention and treatment of AEs. (End of Abstract) ? ? ? ?
