Proteomics of eosinophil activation

摘要 这项研究的目标是获得关于嗜酸性粒细胞如何影响哮喘和其他嗜酸性粒细胞的新见解。 通过描述非激活和急性和慢性的蛋白质组来描述相关疾病和过程 激活的人类嗜酸性粒细胞。嗜酸性粒细胞在许多方面都是例外的：(1)颗粒含量，(2) 受体和其他控制激活和运输的分子的补体，(3)补体 以及(4)被细胞因子激活进入颗粒室时的极化 核足类，一种特殊的尾足类动物，由嗜酸性粒细胞独特的双叶核占据，顶端有一个 信号体。深入的蛋白质组学分析将有助于加深对细胞范围的过程的理解，例如 极化和颗粒释放，产生关于疾病中改变的路径的假说，并告知 有针对性的研究。这样的研究是可行的。未激活的嗜酸性粒细胞可以从血液中重复纯化， 并被白介素5(IL5)等激动剂激活。从支气管肺泡中提纯嗜酸性粒细胞 节段性抗原攻击后获得的灌洗液提供了一个独特的机会来表征 长期激活的细胞已经进入并持续存在于富含多种介质的组织环境中 嗜酸性炎症。我们将鉴定和定量非激活的和尖锐的和 长期激活的嗜酸性粒细胞，精确定位磷酸化位置，并进行显微免疫- 定位和其他相关研究，以验证蛋白质组学结果并寻找建议的线索 通过数据。根据初步结果提出了倡议，以扩大观察到的 IL-5激活的嗜酸性粒细胞中形成的信号小体与研究得很好的 通过B细胞受体激活的B细胞；表征嗜酸性粒细胞Nance-Horan综合征样蛋白2(NHSL2)， 在初步研究中，它是急性IL5刺激后磷酸化程度最高的蛋白质之一； 并对出人意料地高丰度的嗜酸性粒细胞IL18进行了表征和定位。 了解嗜酸性粒细胞激活过程中蛋白质的动态和翻译后修饰是不必要的 系统地了解人类嗜酸性粒细胞是如何滞留、渗入、 并扰乱哮喘和其他疾病的组织。这项工作将导致识别细胞目标与 它以控制炎症和促进愈合的方式调节嗜酸性粒细胞的激活。展望未来， 我们将建立的实现特定目标的蛋白质组学技术可以用于研究 正常人与嗜酸性粒细胞相关性患者嗜酸性粒细胞蛋白质含量的差异 疾病和嗜酸性粒细胞对激活激动剂和拮抗剂的反应性。

ABSTRACT The goal of this research is to gain new insights in how eosinophils impact asthma and other eosinophil- associated diseases and processes by describing the proteomes of non-activated and acutely and chronically activated human eosinophils. Eosinophils are exceptional in a number of ways: (1) granule content, (2) complement of receptors and other molecules that control activation and trafficking, (3) complement of mediator-generating enzymes, and (4) polarization upon activation by cytokines into a granular compartment and nucleopod, a specialized uropod occupied by the eosinophil's distinctive bilobed nucleus and capped by a signalosome. Deep proteomic analysis will lead to increased understanding of cell-wide processes such as polarization and granule release, generate hypotheses about pathways that are altered in disease, and inform targeted studies. Such studies are feasible. Non-activated eosinophils can be purified reproducibly from blood, and activated acutely with agonists such as interleukin-5 (IL5). Eosinophils purified from broncho-alveolar lavage fluid obtained after segmental antigen challenge provide a unique opportunity to characterize chronically activated cells that have entered and persisted in a tissue milieu enriched in multiple mediators of eosinophilic inflammation. We will identify and quantify the proteins of non-activated and acutely and chronically activated eosinophils, pinpoint sites of phosphorylation, and perform microscopic immuno- localization and other correlative studies to validate the proteomic results and pursue leads that are suggested by the data. Initiatives are proposed based on preliminary results to extend the observation that the signalosome forming in eosinophils activated with IL5 shares components with the well-studied signalosome of B-cells activated via the B-cell receptor; characterize eosinophil Nance-Horan syndrome-like protein 2 (NHSL2), which in preliminary studies was among the proteins most heavily phosphorylated after acute IL5 stimulation; and characterize and localize eosinophil IL18, which was found in unexpectedly high abundance. Understanding dynamics of proteins and post-translational modifications during eosinophil activation is without question necessary to developing a systematic understanding of how human eosinophils arrest in, infiltrate, and perturb tissues in asthma and other diseases. This work will lead to identification of cellular targets with which to modulate eosinophil activation in ways that control inflammation and lead to healing. Going forward, the proteomic techniques that we will establish to accomplish the specific aims can be used to study differences in protein content of eosinophils between normal and individuals with eosinophil-associated diseases and responsiveness of eosinophils to agonists and antagonists of activation.