Regulation of inflammatory cell migration in asthma

描述（由申请人提供）：控制细胞表面的氧化还原环境调节关键的细胞功能，包括细胞-细胞识别、细胞粘附和细胞迁移。我们发现半乳糖凝集素-9通过细胞外基质调节特定类型的白细胞、Th2细胞和嗜酸性粒细胞的迁移。半乳糖凝集素-9与T细胞表面的硫还原酶蛋白二硫异构酶（PDI）结合，并将酶保留在细胞表面，在那里酶修饰细胞表面蛋白质以增加这些蛋白质上的还原硫醇的数量。细胞表面PDI已被提出通过修饰整合素来调节其他类型的白细胞（如血小板）的迁移，这意味着半凝集素-9结合是调节整合素介导的T细胞通过细胞外基质迁移的新机制。本研究的目的是：1)阐明半乳糖凝集素-9通过细胞外基质调控Th2细胞和嗜酸性粒细胞迁移的机制；2)利用小鼠哮喘模型，确定半乳糖凝集素-9在体内Th2细胞和嗜酸性粒细胞向炎症组织浸润中的作用。由于半乳糖凝集素-9在肥大细胞、树突状细胞和嗜酸性粒细胞等炎症细胞以及内皮细胞和支气管上皮细胞中表达，并且Th2细胞和嗜酸性粒细胞是哮喘的主要效应细胞，我们将确定半乳糖凝集素-9在哮喘模型中的作用。因此，在Aim1中，我们将通过亲和色谱和质谱法鉴定所有结合半凝集素-9的T细胞和嗜酸性粒细胞糖蛋白受体。我们将确定负责半乳糖凝集素-9结合的聚糖配体。我们将研究半乳糖凝集素-9结合受体增强迁移的机制，检查受体在细胞表面的流动性、受体构象和下游基因表达。在Aim 2中，我们将比较半乳糖凝集素-9和半乳糖凝集素-1缺失小鼠对气道炎症诱导剂的反应性以及产生的一系列效应细胞和细胞因子。我们将确定外源性半乳糖凝集素-9对气道炎症程度和各种效应细胞募集的影响。我们将通过检查半乳糖凝集素-9嵌合小鼠的气道炎症来确定白细胞来源的半乳糖凝集素-9与组织来源的半乳糖凝集素-9的功能。对哮喘中白细胞迁移新机制的研究将为改善哮喘肺部炎症和组织损伤提供新的潜在治疗途径。关于半乳糖凝集素-9通过调节细胞表面氧化还原环境来调节白细胞功能的假说是非常新颖的，哮喘模型对我们小组来说是一个全新的研究领域。这个项目是高风险的，但潜在的非常高的收益，因此适合R21应用程序。

DESCRIPTION (provided by applicant): Control of the redox environment at the cell surface regulates critical cellular functions, including cell-cell recognition, cell adhesion and cell migration. We have found that galectin-9 regulates migration of specific types of leukocytes, Th2 cells and eosinophils, through extracellular matrix. Galectin-9 binds to the thioreductase Protein Disulfide Isomerase (PDI) on surface of T cells and retains the enzyme on the cell surface, where the enzyme modifies cell surface proteins to increase the number of reduced thiols on these proteins. Cell surface PDI has been proposed to regulate migration of other types of leukocytes, such as platelets, through modification of integrins, implicating galectin-9 binding as a novel mechanism for regulating integrin-mediated T cell migration through extracellular matrix. The goals of this proposal are 1) to elucidate the mechanism by which galectin-9 regulates Th2 cell and eosinophil migration through extracellular matrix, and 2) to determine the role of galectin-9 in infiltration of Th2 cells and eosinophils into inflamed tissue in vivo, using a murine model of asthma. As galectin-9 is expressed by inflammatory cells, such as mast cells, dendritic cells, and eosinophils, as well as by endothelial cells and bronchial epithelial cells, and as Th2 cells and eosinophils are primary effector cells in asthma, we will determine the role of galectin-9 in an asthma model in vivo. Thus, in Aim1, we will identify all T cell and eosinophil glycoprotein receptors that bind galectin-9 by affinity chromatography and mass spectrometry. We will identify glycan ligands responsible for galectin-9 binding. We will investigate mechanisms by which galectin-9 binding to receptors enhances migration, examining receptor mobility on the cell surface, receptor conformation, and downstream gene expression. In Aim 2, we will compare galectin-9 and galectin-1 null mice for responsiveness to inducers of airway inflammation and the array of effector cells and cytokines produced. We will determine the effect of exogenously administered galectin-9 on the extent of airway inflammation and recruitment of various effector cells. We will determine the function of leukocyte derived vs. tissue derived galectin-9 by examining airway inflammation in galectin-9 chimeric mice. Investigation of this new mechanism of leukocyte migration in asthma will identify new potential therapeutic approaches to ameliorating the lung inflammation and tissue damage in asthma. The hypothesis that galectin-9 regulates leukocyte function by regulating the cell surface redox environment is very novel, and asthma models are an entirely new area of research for our group. This project is high- risk but potentially very high yield, and thus appropriate for an R21 application. PUBLIC HEALTH RELEVANCE: Relevance to human health: Novel therapies are needed for asthma, a disease that affects approximately 10% of American children and adults. Asthma patients develop permanent tissue damage in their lungs, due to the chronic inflammation that occurs in this devastating disease. Preventing the inflammation in asthma, by blocking the ability of inflammatory cells to enter into and migrate through tissue, is a critical goal in the design of new therapeutic approaches. Our proposal investigates a previously unknown effect of a molecule produced in asthmatic lung that we have found promotes the migration of inflammatory cells. Understanding how this molecule triggers cell migration will allow development of new approaches to prevent the inflammation and subsequent tissue damage seen in the lungs of asthma patients.