Epigenetic Regulation of Acute Lung Injury Resolution

该NRSA个人奖学金的广泛目标有两个方面：1）促进基本技能的发展，使候选人成为一名有效的医生-科学家，2）调查指导免疫系统解决严重肺部疾病的机制。候选人和他的导师设计了一个具体的培训计划。该计划包括一个严格的研究组成部分，奠定了成功的职业生涯的基础。该提案涉及急性肺损伤（ALI）及其临床对应物急性呼吸窘迫综合征（ARDS）。急性肺损伤和急性呼吸窘迫综合征是弥漫性肺部疾病，在美国造成巨大的发病率和死亡率。尽管对驱动ALI的炎症进行了数十年的研究，但尚未发现任何特定的治疗方法来加速其解决并挽救患者的生命。关于ALI如何解决的信息很少。然而，调节性T细胞（Tcells）-免疫系统细胞，减少炎症和促进受损组织的修复-解决了该疾病的小鼠模型中的ALI。在ARDS患者的肺中，凝血酶也会增加，这表明它们可能在人类对ALI的免疫反应中很重要。 尽管与身体中的所有细胞具有相同的DNA代码，但在任何给定的时间内，细胞内只有某些基因（包括Tregs）被“打开”。“开启”的特定基因调节Treg的发育和功能。表观遗传学是研究基因是如何通过染色质的变化而“打开”或“关闭”的：DNA和附着在DNA上的蛋白质，称为组蛋白。该候选人的初步数据表明，染色质修饰药物（5-氮杂-2 '-脱氧胞苷[AZA]）可增强小鼠ALI的消退，并增加其肺部的TcR数量。AZA阻断DNA甲基转移酶的活性，这种酶已知会关闭诱导Treg发育和功能的基因。该建议的重点是进一步研究通过DNA甲基转移酶介导的表观遗传现象如何影响ALI后的TdR。该提案的长期希望是确定可能导致ARDS患者治疗选择的新机制。 在具体目标1中，候选人将检查DNA甲基转移酶抑制对小鼠肺损伤后TcR的影响。将使用多色流式细胞术和AZA处理的Treg过继转移至损伤的淋巴细胞缺陷型小鼠，以确定在ALI消退期间DNA甲基转移酶抑制对Treg功能和表型的影响。抑制测定将测量离体Treg功能。在特定目标2中，将在肺损伤后使用染色质免疫沉淀和实时PCR确定DNA甲基转移酶抑制对关键Treg基因（Foxp 3）的影响。这些实验将探索ALI后控制Treg功能的机制，最终目标是改善这种经常致命的疾病的患者结局。

DESCRIPTION (provided by applicant): The broad objectives of this NRSA Individual Fellowship are two-fold: 1) to facilitate development of essential skills that will allow the candidate to become an effective physician-scientist, and 2) to investigate the mechanisms that direct the immune system to resolve a severe lung disease. The candidate and his mentors have designed a specific training plan. The plan includes a rigorous research component that lays the foundation for a successful career. The proposal concerns acute lung injury (ALI) and its clinical counterpart, the acute respiratory distress syndrome (ARDS). ALI and ARDS are diffuse lung diseases that cause tremendous morbidity and mortality in the United States. Despite decades of research into the inflammation that drives ALI, no specific therapies have been found to accelerate its resolution and save the lives of afflicted patients. There is a paucit of information about how ALI resolves. However, regulatory T cells (Tregs)- immune system cells that decrease inflammation and promote repair of damaged tissues-resolve ALI in a mouse model of the disease. Tregs also increase in the lungs of patients with ARDS, suggesting that they may be important in the human immune response to ALI. Despite having the same DNA code as all cells in the body, only certain genes are turned "on" within cells-including Tregs-at any given time. The specific genes that are "on" regulate Treg development and function. Epigenetics is the study of how genes are turned "on" or "off" by changes to chromatin: DNA and the proteins attached to DNA known as histones. The candidate has preliminary data demonstrating that a chromatin-modifying drug (5-aza-2'-deoxycytidine [AZA]) enhances resolution of ALI in mice and increases the number of Tregs in their lungs. AZA blocks activity of DNA methyltransferase, an enzyme known to turn "off" genes that induce Treg development and function. The focus of this proposal is to further examine how epigenetic phenomena mediated via DNA methyltransferase affect Tregs following ALI. The long-term hope of the proposal is to identify novel mechanisms that may lead to treatment options for patients with ARDS. In Specific Aim 1, the candidate will examine the effect of DNA methyltransferase inhibition on Tregs following lung injury in mice. Multicolor flow cytometry and adoptive transfer of AZA-treated Tregs to injured lymphocyte-deficient mice will be used to define the effect of DNA methyltransferase inhibition on Treg function and phenotype during ALI resolution. A suppression assay will measure ex vivo Treg function. In Specific Aim 2, the effect of DNA methyltransferase inhibition on a critical Treg gene (Foxp3) will be determined following lung injury using chromatin immunoprecipitation and real-time PCR. These experiments will explore the mechanisms controlling Treg function following ALI with the ultimate goal of improving patient outcomes in this oftentimes fatal disease.