Function of secreted PLA2 group X in eosinophil biology

分泌型 PLA2 X 组在嗜酸性粒细胞生物学中的功能

基本信息

批准号：
8401088
负责人：
TEAL S HALLSTRAND
金额：
$ 23.18万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-15 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8401088
关键词：
Abbreviations Antibodies Arachidonate 5-Lipoxygenase Arachidonic Acids Asthma Attenuated Biology Cell Line Cell membrane Cells Confocal Microscopy Cytolysis Cytoplasmic Granules Data Eicosanoids Enzymes Figs - dietary G Protein-Coupled Receptor Genes G-Protein-Coupled Receptors Generations HL60 Human Inflammation Inflammation Mediators Inflammatory Interleukin-3 Interleukin-5 JUN gene Label Life Location MAPK14 gene Mediating Mediator of activation protein Modeling Organelles Pathogenesis Patients Phospholipase Phospholipase A2 Phospholipids Phosphotransferases Play Production Public Health Role Signal Transduction Site Source Stimulus Subcellular structure TNF gene TSLP gene Tissues United States airway inflammation cysteinyl-leukotriene cytokine eosinophil extracellular human TSLP protein in vivo inhibitor/antagonist lentiviral-mediated lipid mediator novel overexpression peripheral blood small hairpin RNA therapeutic target translational study young adult

项目摘要

DESCRIPTION (provided by applicant): The synthesis of cysteinyl leukotrienes (CysLT)s by eosinophils plays an important role in the pathogenesis of asthma. We have discovered that secreted PLA2 group X (sPLA2-X) is involved in eosinophil CysLT synthesis, contradicting the long-held view that cytosolic PLA2¿ (cPLA2¿) is the only major PLA2 involved in CysLT formation in eosinophils. Our overall hypothesis is that sPLA2-X is upregulated in eosinophils during inflammation of the airways and plays an important role in CysLT formation by eosinophils. We have recently demonstrated that exogenous sPLA2-X initiates CysLT synthesis by human eosinophils through a mechanism that is dependent upon the enzymatic function of sPLA2-X, but also involves the activation of cPLA2¿, through p38 and c-Jun-terminal kinase (JNK). In preliminary data, we demonstrate that sPLA2-X is endogenously expressed in human eosinophils and that a highly selective inhibitor of sPLA2-X attenuates CysLT formation by fMLP-stimulated eosinophils. In the first specific aim, we will examine the intracellular location of sPLA2-X in human eosinophils using confocal microscopy. We will determine if the expression of sPLA2-X in eosinophils is increased by IL-3, IL-5, GMCSF and other cytokines that "prime" eosinophils for activation as they enter the airways. In a translational portion of this study, we will determine if sPLA2-X expression is increased in eosinophils from patients with asthma. In the second specific aim, we will examine the function of sPLA2-X in eosinophils first through selective inhibitors of sPLA2-X and cPLA2¿, and then through lentiviral-mediated shRNA knockdown of sPLA2-X in the eosinophil-like cell line HL-60 clone-15. Since sPLA2-X can act within cells and after secretion, we will determine if sPLA2-X co-localizes with 5-lipoxygenase (5-LO) during activation, and if it is released during piecemeal degranulation, allowing the enzyme to act on the outer cell membrane. Finally, we will examine the potential function of sPLA2-X in cell-free eosinophil granules that act as secretion competent organelles infiltrating the airway tissues of patients with asthma. Establishing the important function of sPLA2-X in eosinophil CysLT formation will provide a strong rationale for this enzyme as a therapeutic target in asthma. PUBLIC HEALTH RELEVANCE: Asthma is one of the most important public health problems in young adults in the United States. This study examines the function of a novel enzyme that may serve as a key regulator of inflammatory lipid mediator formation in asthma. The results will provide a strong rationale for this enzyme as a therapeutic target in asthma.

描述（由申请人提供）：嗜酸性粒细胞合成半胱氨酰白三烯（CysLT）在哮喘的发病机制中起着重要作用。我们发现分泌的PLA2基团X（sPLA2-X）参与嗜酸性粒细胞CysLT的合成，这与之前的研究相矛盾。长期以来的观点是，细胞质 PLA2¿ (cPLA2¿) 是参与嗜酸性粒细胞中 CysLT 形成的唯一主要 PLA2，我们的总体假设是，气道炎症期间 sPLA2-X 在嗜酸性粒细胞中上调，并且在嗜酸性粒细胞形成 CysLT 中发挥重要作用。 sPLA2-X 通过依赖于酶功能的机制启动人嗜酸性粒细胞合成 CysLT sPLA2-X，但也涉及 cPLA2 的激活¿ ，通过 p38 和 c-Jun 末端激酶 (JNK) 在初步数据中，我们证明 sPLA2-X 在人嗜酸性粒细胞中内源性表达，并且 sPLA2-X 的高选择性抑制剂可减弱 fMLP 刺激的嗜酸性粒细胞的 CysLT 形成。第一个具体目标是，我们将使用共聚焦技术检查人嗜酸性粒细胞中 sPLA2-X 的细胞内位置在本研究的翻译部分中，我们将确定嗜酸性粒细胞中 sPLA2-X 的表达是否因 IL-3、IL-5、GMCSF 和其他细胞因子的增加而增加，这些细胞因子在嗜酸性粒细胞进入气道时“启动”它们。我们将确定哮喘患者嗜酸性粒细胞中 sPLA2-X 的表达是否增加。在第二个具体目标中，我们将检查 sPLA2-X 在哮喘患者中的功能。首先通过 sPLA2-X 和 cPLA2 的选择性抑制剂抑制嗜酸性粒细胞，然后通过慢病毒介导的 shRNA 在嗜酸性粒细胞样细胞系 HL-60 克隆-15 中敲低 sPLA2-X，因为 sPLA2-X 可以在细胞内发挥作用并在分泌后，我们将确定 sPLA2-X 是否与 sPLA2-X 共定位。 5-脂氧合酶 (5-LO) 在激活过程中，如果它在逐步脱粒过程中释放，则允许酶作用于外细胞膜。最后，我们将检查 sPLA2-X 在无细胞嗜酸性粒细胞颗粒中的潜在功能，这些颗粒作为浸润哮喘患者气道组织的分泌细胞器，建立 sPLA2-X 在嗜酸性粒细胞 CysLT 形成中的重要功能将提供强有力的理论依据。将该酶作为哮喘的治疗靶点。公共健康相关性：哮喘是美国年轻人最重要的公共健康问题之一。这项研究检查了一种新型酶的功能，该酶可能作为哮喘炎症脂质介质形成的关键调节剂。这种酶作为哮喘治疗靶点的有力理由。