IL8 and GATA3-mediated Pathways in Asthma Exacerbations

描述(由申请人提供)： 在这一应用中，我们提出了一种新的方法来理解儿童哮喘加重(AE)的发病机制。我们推测有两条途径参与了AE的触发：一条以IL-8和中性粒细胞为中心，另一条由GATA3和嗜酸性粒细胞介导。我们基于我们的初步数据，表明IL8和GATA3的单核苷酸多态(SNPs)与亚利桑那州图森市一项哮喘纵向研究中登记的儿童的AEs相关。IL8-251T与6岁以前哮喘儿童继发AE的风险显著正相关。GATA3-7018C，在6岁以后哮喘儿童中显著增加，但仅与过敏有关。功能研究将澄清这种关联是否真的是由于GATA3/-7018或与其连锁不平衡的另一个SNP所致。 我们打算招募一组具有良好特征的哮喘儿童，并对他们进行跟踪，直到他们患上第一例急性呼吸窘迫综合征。我们将比较这些SNPs不同基因携带者首次发生AE的时间。我们还将评估这些AE的急性免疫表型与我们假设参与调节AE的两个SNP的关系。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，即有过敏背景的儿童中AE的一个重要决定因素。具体地说，我们建议：1.确定IL8-251T-&GT；A是否通过影响哮喘儿童病毒感染过程中IL-8的表达和中性粒细胞炎症来调节AE的临床表型。我们将研究IL8启动子中的SNP IL8-251T-&gt；A在调节IL-8依赖的途径中的作用，该途径决定了上皮细胞和炎性细胞产生IL-8，以及随后对病毒感染的中性粒细胞反应。2.确定GATA3-7018C-&GT；G是否通过影响呼吸道病毒感染时嗜酸性粒细胞GAT-A3和IL-13的表达来调节AE的临床表现。我们将评估AEs期间和术后诱导痰中嗜酸粒细胞GATA3和IL13mRNA和/或蛋白的表达，并将其与GATA3-7018基因和AEs表型进行相关性分析。3.确定GATA3-7018C-&gt；G是否以及如何影响嗜酸性粒细胞中GATA3转录的调节。我们将利用GATA3报告结构的等位基因变体进行功能研究，以确定嗜酸性粒细胞中GATA3 5‘沉默因子的功能是否受到GATA3-7018C-&GT；G的影响，从而导致GATA3转录的差异。将分析野生型和多态沉默基因上的蛋白质/DNA相互作用，以确定导致GATA3差异表达的机制。 本申请中提出的体外分析和机制研究的新组合将使我们能够确定拟议的IL-8/中性粒细胞和GATA3/嗜酸性粒细胞相关通路在AE中所起的作用，并可能导致发现预防和治疗AEs的新的治疗靶点。(摘要结束)

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)