Eosinophil ORMDL3 and allergic inflammation

描述（由申请人提供）：涉及全基因组关联和微阵列方法的研究已经鉴定出orosomucoid 1-like 3 （ORMDL3）作为哮喘易感性的候选基因。除了最近的研究表明ORMDL3在酵母中作为鞘脂稳态的介质和内质网介导的Ca2+信号和细胞应激的调节剂，关于该蛋白在哮喘发病机制中所起的作用知之甚少。包括哮喘在内的变应性气道炎症的特点是气道被大量炎症细胞浸润，气道高反应性和重塑。嗜酸性粒细胞（Eos）是最促炎的粒细胞之一，有助于疾病的发病和恶化。我们首次证明Eos（小鼠和人类）在mRNA和蛋白水平上表达ORMDL3。此外，ORMDL3在小鼠Eos中的表达被诱导响应于细胞内Ca2+水平的变化以及暴露于变应性炎症介质，如IL-3（~10倍）和eotaxin。基于这一观察，我们假设ORMDL3调节Eos的激活，并可能调节其粘附功能，导致Eos在血管中的运输增加，并向发炎的肺部募集。因此，在当前的应用中，在Aim 1中，我们建议评估ORMDL3在细胞运输事件中的作用，如涉及Ca2+动员的细胞滚动、粘附和迁移，在肺微血管血流条件下，作为向哮喘气道招募的前奏。使用双色活体视频显微镜技术，我们将检查正常Eos与通过基因沉默导致ORMDL3缺失的Eos或在小鼠肺血管中过度表达ORMDL3的Eos之间的粘附相互作用。此外，将评估这些Eos种群在过敏性哮喘（蟑螂抗原诱导）模型中的肺部招募情况。由于发现气道炎症介质（如IL-3和eotaxin）可诱导Eos中ORMDL3的表达，在Aim 2中，我们将进一步详细评估炎症部位存在的细胞因子和趋化因子（如IL-5、GMCSF、IL-13、IL-17、IL-23和C5a）对ORMDL3表达的调节。通过CD48介导的途径，il -3诱导ORMDL3表达的潜在调控也将被研究。此外，我们将评估ORMDL3表达是否与Eos存活和Ca2+动员相关，以及ORMDL3是否与其他细胞内Eos蛋白相互作用。最后，我们将通过小鼠变应性炎症模型确定气道募集的Eos是否表达ORMDL3。重要的是，我们正试图探索这种相对较新的蛋白质在Eos中的潜在作用，目前对其知之甚少。我们期望这些研究将揭示eos表达的ORMDL3的功能作用，对哮喘发病机制具有重要意义，并实现NIAID/NHLBI了解、治疗或预防过敏和肺部疾病的目标。

DESCRIPTION (provided by applicant): Studies involving genome-wide association and microarray approaches have led to the identification of orosomucoid 1-like 3 (ORMDL3) as a candidate gene for susceptibility to asthma. Apart from very recent studies demonstrating that ORMDL3 functions as a mediator of sphingolipid homeostasis in yeast and a regulator of endoplasmic reticulum-mediated Ca2+ signaling and cellular stress, very little is known regarding the role played by this protein in the pathogenesis of asthma. Allergic airway inflammation including asthma is characterized by infiltration of the airways by a large number of inflammatory cells, airway hyperresponsiveness and remodeling. Eosinophils (Eos) are one of the most proinflammatory granulocytes that contribute to the pathogenesis and exacerbation of the disease. We have, for the first time, demonstrated that Eos (murine and human) express ORMDL3 at the mRNA and protein level. Further, ORMDL3 expression in murine Eos was induced in response to changes in intracellular Ca2+ levels as well as exposure to mediators of allergic inflammation such as IL-3 (~10 fold) and eotaxin. Based on this observation, we hypothesize that ORMDL3 regulates Eos activation and potentially its adhesive function leading to increased trafficking of Eos in blood vessels and recruitment to inflamed lungs. Accordingly, in the current application, in Aim 1, we propose to evaluate the role of ORMDL3 in cellular trafficking events, such as cell rolling, adhesion and migration that involve Ca2+ mobilization, under conditions of blood flow in lung microvessels as a prelude to recruitment to the asthmatic airways. Using the technique of 2-color intravital video microscopy, we will examine the adhesive interaction of normal Eos versus Eos that have been rendered ORMDL3 deficient by gene silencing or Eos that over-express ORMDL3 within blood vessels of the mouse lung. In addition, recruitment of these Eos populations to the lungs in a model of allergic (cockroach antigen-induced) asthma will be evaluated. Since mediators of airway inflammation (e.g. IL-3 and eotaxin) were found to induce ORMDL3 expression in Eos, in Aim 2 we will evaluate regulation of ORMDL3 expression by cytokines and chemokines present at sites of inflammation such as IL-5, GMCSF, IL-13, IL-17, IL-23 and C5a in addition to IL-3 and eotaxin in further detail. The potential regulation of IL-3-induced ORMDL3 expression via a CD48- mediated pathway will also be investigated. Additionally, we will evaluate whether ORMDL3 expression is associated with Eos survival and Ca2+ mobilization and if ORMDL3 interacts with other intracellular Eos proteins. Lastly, we will ascertain whether Eos recruited to the airways express ORMDL3 using a mouse model of allergic inflammation. Importantly, we are attempting to explore the potential role of this relatively new protein in Eos, regarding which little is known. We anticipate that these studies will shed light on the functional role of Eos-expressed ORMDL3 with important implications on pathogenesis of asthma as well as meet the objectives of NIAID/NHLBI to understand, treat or prevent allergy and lung diseases. PUBLIC HEALTH RELEVANCE: Allergic airway inflammation including asthma affects an estimated 20 million people in the US alone. The current application will elucidate whether ORMDL3, a protein which is encoded by a gene recently identified as a potential risk factor for asthma and expressed by eosinophhils (a novel observation of this study), promotes adhesive interactions of eosinophils in blood vessels to enable their recruitment to the lungs during allergic inflammation. An understanding of how expression of ORMDL3 is regulated in eosinophils in response to specific signals such as mediators of allergic inflammation (cytokines and chemokines) or intracellular calcium levels might provide new approaches to block the function of this protein and thus reduce recruitment of eosinophils to the lungs leading to the likely alleviation of suffering associated with allergic inflammation including asthma.