Proteomics of eosinophil activation

嗜酸性粒细胞活化的蛋白质组学

• 批准号: 9274150
• 负责人: JOSHUA J COON
• 金额: $ 37.61万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-16 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9274150
• 关键词: Acute; Agonist; Asthma; B-Lymphocytes; Benign; Blood; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Catalogs; Cell Nucleus; Cells; Chronic; Complement; Complement Receptor; Correlative Study; Coupled; Custom; Cytokine Activation; Cytoplasmic Granules; Data; Disease; Enzymes; Eosinophilic Esophagitis; Event; Feasibility Studies; G-Protein-Coupled Receptors; Goals; Helper-Inducer T-Lymphocyte; Hour; Human; IL18 gene; Immune system; Immunoblotting; Immunofluorescence Immunologic; Individual; Indolent; Inflammation; Inflammatory Response; Interleukin-5; Label; Lead; Learning; Leukocytes; Liquid Chromatography; Lymphocyte; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Methodology; Microscopic; Nance-Horan syndrome; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Population; Post-Translational Protein Processing; Process; Protein Dynamics; Proteins; Proteome; Proteomics; Receptors, Antigen, B-Cell; Research; Signal Pathway; Techniques; Technology; Tissues; Work; antigen challenge; base; burden of illness; cellular targeting; cytokine; eosinophil; eosinophilic inflammation; experimental study; healing; insight; irritation; mass spectrometer; response; trafficking

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.

抽象的 这项研究的目的是获得有关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，急性和慢性的蛋白质组来通过描述相关的疾病和过程 活化的人嗜酸性粒细胞。嗜酸性粒细胞在多种方面是例外：（1）颗粒含量，（2） 控制激活和运输的受体和其他分子的补充，（3）补充 介体生成酶，（4）细胞因子激活成颗粒室后的极化 和核足类是嗜酸性粒细胞独特的双核核占据的一种专业uropod，并由a盖住 信号体。深度蛋白质组学分析将导致人们对细胞范围的过程的了解增加 极化和颗粒释放，产生关于疾病改变的途径的假设，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纯化， 并用白介素5（IL5）等激动剂急性激活。从支气管 - 肺泡纯化的嗜酸性粒细胞 节段抗原挑战后获得的灌洗液提供了一个独特的机会来表征 长期活化的细胞已进入并持续在组织环境中，富含多个介体 嗜酸性炎症。我们将识别并量化非激活和急性的蛋白质，以及 长期激活的嗜酸性粒细胞，磷酸化的精确位点，并进行微观免疫 - 本地化和其他相关研究以验证蛋白质组学结果和追求潜在客户 通过数据。提出了基于初步结果的倡议，以扩大观察 用IL5共享的嗜酸性粒细胞中的信号体形成，具有良好的信号体 通过B细胞受体激活的B细胞；表征了嗜酸性粒细胞nance-horan综合征样蛋白2（NHSL2）， 在初步研究中，这是急性IL5刺激后最严重磷酸化的蛋白质之一。 并表征和定位嗜酸性粒细胞IL18，这是出乎意料的高丰度。 了解嗜酸性粒细胞激活期间蛋白质和翻译后修饰的动态 对人类嗜酸性粒细胞如何浸入浸润，浸润， 和哮喘和其他疾病中的扰动组织。这项工作将导致识别具有 可以通过控制炎症并导致愈合的方式调节嗜酸性粒细胞的激活。展望， 我们将建立实现特定目标的蛋白质组学技术可用于研究 嗜酸性粒细胞相关的正常和个体之间嗜酸性粒细胞蛋白质含量的差异 嗜酸性粒细胞对激动剂和激活的拮抗剂的疾病和反应性。