IL8 and GATA3-mediated Pathways in Asthma Exacerbations

描述（由申请人提供）： 在本申请中，我们提出了一种新的方法来了解儿童哮喘急性发作（AE）的发病机制。我们假设两种途径参与触发AE：一种以IL-8和中性粒细胞为中心，另一种由GAT A3和嗜酸性粒细胞介导。我们的初步数据表明，IL 8和GATA 3的单核苷酸多态性（SNP）与亚利桑那州图森市哮喘纵向研究中招募的儿童的AE相关。IL 8 - 251 T与6岁之前患有哮喘的儿童随后发生AE的风险显著正相关。GATA 3 - 7018 C是GATA 3 5'沉默子中的SNP，与6岁以后患有哮喘的儿童的AE频率增加显著相关，但仅在过敏症中。功能研究将阐明这种关联是否真的是由于GATA 3/-7018或与其连锁不平衡的不同SNP。 我们打算招募一组特征明确的哮喘儿童，并对其进行随访，直至发生首次AE。我们将比较这些SNP的不同基因型携带者至首次AE的时间。我们还将评估这些AE与我们假设参与调节AE的两个SNP相关的急性免疫表型。我们将特别讨论GAT A3中一个SNP的功能基因组学，我们认为这是严重AE风险最高的儿童中AE的重要决定因素，即，有过敏背景的人。具体而言，我们建议：1。确定IL 8 - 251 T->A是否通过影响哮喘儿童病毒感染期间IL-8的表达和嗜酸性炎症来调节AE期间的临床表型。我们将研究IL-8 - 251 T->A（IL-8启动子中的SNP）在IL-8依赖性途径调节中的作用，该途径决定上皮细胞和炎症细胞产生IL-8以及随后对病毒感染的嗜酸性反应。2.确定GATA 3 - 7018 C->G是否通过影响病毒性呼吸道感染期间气道嗜酸性粒细胞中GATA 3和IL 13的表达来调节AE的临床表现。我们将评估AE期间和之后从诱导痰中分离的气道嗜酸性粒细胞中GATA 3和IL 13 mRNA和/或蛋白的表达，并将其与GATA 3 -7018基因型和AE表型相关联。 3.确定GATA 3 - 7018 C->G是否以及如何影响嗜酸性粒细胞中GATA 3转录的调节。我们将使用GATA 3报告基因构建体的等位基因变体进行功能研究，以确定嗜酸性粒细胞中GATA 3 5'沉默子的功能是否受GATA 3 - 7018 C->G的影响，从而导致差异GATA 3转录。将分析野生型和多态性沉默子的蛋白质/DNA相互作用，以鉴定负责差异GATA 3表达的机制。 本申请中提出的离体分析和机制研究的新组合将使我们能够确定所提出的IL-8/中性粒细胞和GATA 3/嗜酸性粒细胞相关途径在AE中发挥的作用，并可能导致识别用于预防和治疗AE的新治疗靶点。 (End摘要）

DESCRIPTION (provided by applicant): 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)