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导致将类乳清蛋白1样3（ORMDL 3）鉴定为哮喘易感性的候选基因。除了最近的研究表明ORMDL 3作为酵母中鞘脂稳态的介导剂和内质网介导的Ca 2+信号传导和细胞应激的调节剂起作用外，关于这种蛋白在哮喘发病机制中所起的作用知之甚少。包括哮喘在内的过敏性气道炎症的特征在于大量炎性细胞浸润气道、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，有助于疾病的发病机制和恶化。我们首次证明Eos（鼠和人）在mRNA和蛋白水平表达ORMDL 3。此外，响应于细胞内Ca 2+水平的变化以及暴露于过敏性炎症介质如IL-3（~10倍）和嗜酸细胞活化趋化因子，诱导鼠Eos中的ORMDL 3表达。基于这一观察结果，我们假设ORMDL 3调节Eos活化，并可能调节其粘附功能，导致Eos在血管中的运输增加，并募集到发炎的肺部。因此，在本申请中，在目的1中，我们提出在肺微血管中的血流条件下评估ORMDL 3在细胞运输事件中的作用，所述细胞运输事件例如涉及Ca 2+动员的细胞滚动、粘附和迁移，作为募集到哮喘气道的前奏。使用2-彩色活体视频显微镜技术，我们将研究正常EOS与EOS的粘附相互作用，这些EOS通过基因沉默或EOS过度表达小鼠肺血管内的ORMDL 3而被赋予ORMDL 3缺陷。此外，还将评价过敏性（蟑螂抗原诱导的）哮喘模型中这些Eos群体向肺部的募集。由于发现气道炎症介质（例如IL-3和嗜酸性粒细胞趋化因子）诱导Eos中的ORMDL 3表达，因此在目的2中，我们将进一步详细评价除了IL-3和嗜酸性粒细胞趋化因子之外，存在于炎症部位的细胞因子和趋化因子如IL-5、GMCSF、IL-13、IL-17、IL-23和C5 α对ORMDL 3表达的调节。还将研究通过CD 48介导的途径对IL-3诱导的ORMDL 3表达的潜在调节。此外，我们将评估ORMDL 3表达是否与Eos存活和Ca 2+动员相关，以及ORMDL 3是否与其他细胞内Eos蛋白相互作用。最后，我们将使用过敏性炎症的小鼠模型来确定招募到气道的Eos是否表达ORMDL 3。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，对此知之甚少。我们预计这些研究将阐明EOS表达的ORMDL 3的功能作用，对哮喘的发病机制具有重要意义，并满足NIAID/NHLBI了解，治疗或预防过敏和肺部疾病的目标。 公共卫生相关性：仅在美国，包括哮喘在内的过敏性气道炎症就影响着约2000万人。本申请将阐明ORMDL 3，一种由最近被鉴定为哮喘的潜在危险因素的基因编码并由嗜酸性粒细胞表达的蛋白质（本研究的新观察结果），是否促进血管中嗜酸性粒细胞的粘附相互作用，以使其在过敏性炎症期间能够招募到肺部。了解ORMDL 3的表达如何在嗜酸性粒细胞中响应特定信号如过敏性炎症介质（细胞因子和趋化因子）或细胞内钙水平进行调节，可能会提供新的方法来阻断这种蛋白质的功能，从而减少嗜酸性粒细胞向肺部的募集，从而可能减轻与过敏性炎症（包括哮喘）相关的痛苦。

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.