Genetic and Pharmacologic Evaluation of the Role of HIF-1a in asthma

描述(由申请人提供)： 哮喘是一种极其常见的疾病，6.4%的美国成年人在一生中都会受到影响。确定哮喘发生和发展背后的分子机制将导致新的治疗目标、更具体的治疗选择和更好的疾病控制。哮喘是一种炎症性疾病，以平滑肌肥大、呼吸道阻塞和粘液生成增加为特征。已知树突状细胞、嗜酸性粒细胞和肥大细胞在哮喘的发生和发展中起作用，但其背后的分子机制尚未阐明。缺氧诱导因子-11(HIF-11)是一种转录因子，最近被确认为髓系细胞炎症能力的主要调节因子4.HIF-11在炎症中介导巨噬细胞和中性粒细胞的许多关键功能4.由于它存在于所有哺乳动物细胞中，调节超过100个功能基因，并调节已知在过敏性炎症过程中上调的NF-B通路7；8，我假设HIF-11调节哮喘的过敏性炎症。哮喘的发病机制可以分为两个部分。首先，启动对特定过敏原的过敏反应。其次，呼吸道炎症是对过敏原再次暴露的反应。本研究的目的是探索转录因子缺氧诱导因子-11(HIF-11)如何调控哮喘的发病机制，并从机制上探讨HIF-11发挥这一作用的分子途径。我假设：(A)低氧诱导因子-11是参与过敏性炎症的分子通路的关键调节因子；(B)树突状细胞中的HIF-11在启动过敏性呼吸道反应中起重要作用；以及(C)嗜酸性粒细胞中的HIF-11在气道炎症的持续和发展中发挥作用。我将探索对白细胞中HIF-11水平的遗传操作如何影响哮喘易感性，并研究从药物上抑制HIF-11的治疗策略是否能够有效地减少呼吸道炎症和反应性。我们将利用卵蛋白(OVA)在小鼠中建立过敏性炎性呼吸道疾病模型。将通过IgE、组胺、IL-4和IL-13水平、呼吸道阻力、炎性肺浸润和气道重塑来评估小鼠的过敏反应程度。对嗜酸性粒细胞和树突状细胞的详细机制研究将在体外进行，细胞来自小鼠骨髓。这些研究将利用迁移分析、FACS分析、ELISA、Western blotts和实时定量PCR。在这项提案中，我将确定HIF-11是否启动或维持小鼠过敏性炎症性呼吸道疾病的发展。具体地说，这些研究将严格测试HIF-11是否调节树突状细胞和嗜酸性粒细胞在导致哮喘的过敏性炎症中的功能。独特的小鼠遗传试剂，其中HIF-11水平在白细胞谱系中显著降低或结构性增加，使我可以在体内和体外以详细的、机械的方式明确地测试这些假说。除了这些强大的遗传方法，我还提出了一系列平行的药理学实验，利用小分子抑制剂在全球范围内抑制HIF-11。我假设，由于HIF-11是在蛋白质水平上调节的，半衰期为9秒，抗HIF-11免疫抑制剂将导致炎症迅速减少，这种方式在停药后迅速可逆。因此，如果重度哮喘患者长期使用HIF-11抑制剂治疗并发生感染，可以暂时停止使用HIF-11拮抗剂，以提供正常白细胞功能的快速恢复。以及其他慢性炎症性疾病(类风湿性关节炎、狼疮等)，我推测抑制HIF-11可能为哮喘的治疗提供了一种令人兴奋的策略。 公共卫生相关性： 哮喘影响6.4%的美国成年人，包括退伍军人，对退伍军人医疗系统造成重大负担1。特别是在军事人员中对哮喘的发病率进行了研究，发现哮喘不仅非常普遍，而且随着时间的推移还会增加。1980年至1999年，3900名海军人员首次因哮喘住院，在此期间，一些哮喘患者的住院率翻了一番。在过去的几年里，哮喘的治疗没有重大的改进，因此需要研究哮喘的具体机制，以找到更好的治疗和预防方法。这里提出的研究与一种在退伍军人中常见的疾病相关，并将极大地提高我们对哮喘发生和发展的分子机制的了解，并导致开发更有效的靶向疗法，以提高我们美国退伍军人的医疗质量。

DESCRIPTION (provided by applicant): Asthma is an extremely common disease, affecting 6.4% of American adults over the course of their lives1. Determining the molecular mechanisms behind the development and progression of asthma will lead to new treatment targets, more specific treatment options, and better control of the disease. Asthma is an inflammatory disease characterized by hypertrophy of smooth muscle, airway obstruction, and increased mucous production3. Dendritic cells, eosinophils, and mast cells are known to play a role in the development and progression of asthma, however the molecular mechanisms behind the disease have not been elucidated. Hypoxia inducible factor-11 (HIF-11) is a transcription factor recently identified as a master regulator of the inflammatory capacity of myeloid cells4; 5. HIF-11 mediates many critical functions of macrophages and neutrophils in inflammation4; 6. Because it is found in all mammalian cells, regulates over 100 functional genes, and modulates NF-:B pathways that are known to be up regulated during allergic inflammation7; 8, I hypothesize that HIF-11 regulates allergic inflammation in asthma. The pathogenesis underlying the development of asthma can be broken down into two components. First, an allergic response to a specific allergen is initiated. Second, airway inflammation develops in response to allergen re-exposure. The goal of the work proposed here is to explore how the transcription factor Hypoxia Inducible Factor-11 (HIF-11) modulates the pathogenesis of asthma, and to probe mechanistically the molecular pathways through which HIF-11 acts in this capacity. I hypothesize that (a) Hypoxia Inducible Factor-11 is a critical regulator of molecular pathways involved in allergic inflammation; (b) HIF-11 in dendritic cells plays a significant role in the initiation of the allergic airway response; and (c) HIF-11 in eosinophils plays a role in the perpetuation and progression of airway inflammation. I will explore how genetic manipulation of HIF-11 levels in leukocytes influences asthma susceptibility, and examine whether a therapeutic strategy to pharmacologically inhibit HIF-11 can be effective in reducing airway inflammation and reactivity. We will utilize an ovalbumin (OVA) model of allergic inflammatory airways disease in mice. Mice will be evaluated for the magnitude of allergic response via IgE, histamine, IL-4, and IL-13 levels, airway resistance, inflammatory pulmonary infiltrates, and airway remodeling. Detailed, mechanistic studies of eosinophils and dendritic cells will be done in vitro, with cells derived from mouse bone marrow. These studies will utilize migration assays, FACS analysis, ELISAs, Western blots, and real-time quantitative PCR. In this proposal, I will determine whether HIF-11 initiates or sustains allergic inflammatory airway disease development in mice. Specifically, these studies will rigorously test whether HIF-11 regulates dendritic cells and eosinophil function in allergic inflammation leading to asthma. Unique mouse genetic reagents, in which HIF-11 levels have been markedly reduced or constitutively increased in leukocyte lineages, are available for me to definitively test these hypotheses in a detailed, mechanistic fashion in vivo and ex vivo. Beyond these powerful genetic approaches, I propose a parallel series of pharmacologic experiments utilizing small molecule inhibitors to globally suppress HIF-11. I hypothesize that because HIF-11 is regulated at the protein level with a half-life of seconds9, anti-HIF-11 immunosuppressant agents will lead to a rapid decrease in inflammation in a manner that is rapidly reversible upon discontinuation of the agent. Therefore, if severe asthmatic patients were treated chronically with a HIF-11 inhibitor and developed an infection, the HIF-11 antagonist could be temporarily stopped to provide a rapid return of normal leukocyte function. As well as other chronic inflammatory diseases (rheumatoid arthritis, lupus, etc.), I hypothesize that HIF-11 inhibition may offer an exciting strategy for the management of asthma. PUBLIC HEALTH RELEVANCE: Asthma affects 6.4% of American adults, including Veterans, causing a major burden on the VA healthcare system1. The incidence of asthma has been studied in military personnel in particular, and has been found to not only be highly prevalent, but also increasing over time. Between 1980-In 1999, 3,900 Navy personnel were first hospitalized for asthma, and during this time the incidence of hospitalization doubled for some asthmatic populations10. There have been no major improvements in asthmatic treatments over the past several years, therefore research into the specific mechanisms underlying asthma is needed, to find better ways of treating and preventing it. The research proposed here is relevant to a disease commonly found in the Veteran population, and will greatly enhance our understanding of the molecular mechanisms underlying the development and progression of asthma, and lead towards the development of more effective targeted therapies to increase the quality of healthcare for our U.S. Veterans.