Proteomics of eosinophil activation

摘要 这项研究的目的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。 通过描述非激活的、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很特殊：(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) 介体生成酶，以及（4）细胞因子激活后极化进入颗粒区室 和核足类，一种特殊的尾足类动物，被嗜酸性粒细胞独特的双叶核占据，并被一个 信号体。深入的蛋白质组分析将加深对细胞范围内过程的理解，例如 极化和颗粒释放，产生有关疾病中改变的途径的假设，并提供信息 有针对性的研究。这样的研究是可行的。可以从血液中重复纯化未活化的嗜酸性粒细胞， 并用白细胞介素 5 (IL5) 等激动剂急性激活。从支气管肺泡中纯化的嗜酸性粒细胞 分段抗原攻击后获得的灌洗液提供了一个独特的机会来表征 长期激活的细胞已进入并持续存在于富含多种介质的组织环境中 嗜酸性粒细胞炎症。我们将快速识别和量化非活化的蛋白质 长期激活的嗜酸性粒细胞，精确定位磷酸化位点，并进行显微免疫 定位和其他相关研究，以验证蛋白质组结果并寻找建议的线索 通过数据。根据初步结果提出了一些举措，以扩展观察结果： IL5 激活的嗜酸性粒细胞中形成的信号小体与经过充分研究的信号小体共享成分 通过 B 细胞受体激活 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 刺激后磷酸化最严重的蛋白质之一； 并对嗜酸性粒细胞 IL18 进行表征和定位，发现其丰度出乎意料地高。 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没有必要的 系统地了解人类嗜酸性粒细胞如何滞留、渗透、 并扰乱哮喘和其他疾病的组织。这项工作将导致细胞目标的识别 它以控制炎症和导致愈合的方式调节嗜酸性粒细胞活化。展望未来， 我们为实现特定目标而建立的蛋白质组学技术可用于研究 正常人和嗜酸性粒细胞相关个体之间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。

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.