Epigenetic Regulation of Acute Lung Injury Resolution

急性肺损伤消退的表观遗传调控

• 批准号: 8590629
• 负责人: Benjamin David Singer
• 金额: $ 5.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8590629
• 关键词: Acetylation; Acute; Acute Lung Injury; Adoptive Transfer; Adult Respiratory Distress Syndrome; Affect; Alveolar; Animals; Biological Assay; CD4 Positive T Lymphocytes; Cell physiology; Cells; Chromatin; Clinical; Code; DNA; DNA Methylation; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNA methyltransferase inhibition; Data; Defect; Deoxycytidine; Development; Diffuse; Disease; Enzymes; Epigenetic Process; Exhibits; Family; Fellowship; Flow Cytometry; Foundations; Genes; Genetic Transcription; Goals; Histone Acetylation; Histones; Homeodomain Proteins; Human; IL2RA gene; Immune response; Immune system; Individual; Inflammation; Injury; Kinetics; Lead; Lipopolysaccharides; Lung; Lung diseases; Lymphocyte; Lymphocyte Subset; Maintenance; Measures; Mediating; Mentors; Messenger RNA; Modification; Morbidity - disease rate; Mus; National Research Service Awards; Outcome; Patients; Pharmaceutical Preparations; Phenotype; Physicians; Proteins; Regulation; Regulatory T-Lymphocyte; Research; Resolution; Respiratory Failure; Role; Scientist; Spleen; Time; Tissues; Training; Transcription Factor 3; Transforming Growth Factor beta; United States; Western Blotting; Wild Type Mouse; Work; career; chromatin immunoprecipitation; design; enzyme activity; improved; injured; insight; lung injury; molecular phenotype; mortality; mouse model; novel; novel therapeutics; promoter; protein expression; public health relevance; repaired; research study; response; skills; therapeutic target

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.

描述（由申请人提供）：该 NRSA 个人奖学金的主要目标有两个：1）促进基本技能的发展，使候选人成为一名有效的医师科学家；2）研究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具体的培训计划。该计划包括严格的研究部分，为成功的职业生涯奠定基础。该提案涉及急性肺损伤（ALI）及其临床对应的急性呼吸窘迫综合征（ARDS）。 ALI 和 ARDS 是弥漫性肺部疾病，在美国造成巨大的发病率和死亡率。尽管对导致 ALI 的炎症进行了数十年的研究，但尚未找到可以加速其消退并挽救患者生命的特定疗法。关于 ALI 如何解决的信息很少。然而，调节性 T 细胞 (Treg)——减少炎症并促进受损组织修复的免疫系统细胞——可以在小鼠模型中解决 ALI。 ARDS 患者肺部的 Tregs 也会增加，这表明它们可能在人类对 ALI 的免疫反应中发挥重要作用。 尽管与体内所有细胞具有相同的 DNA 代码，但在任何给定时间，细胞内只有某些基因（包括 Tregs）会“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如何通过染色质（DNA 和附着在 DNA 上的组蛋白）的变化而“开启”或“关闭”的学科。该候选者的初步数据表明，染色质修饰药物（5-aza-2'-脱氧胞苷 [AZA]）可增强小鼠 ALI 的缓解并增加肺部 Treg 的数量。 AZA 阻断 DNA 甲基转移酶的活性，这种酶已知可以“关闭”诱导 Treg 发育和功能的基因。该提案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 Tregs。该提案的长期希望是确定可能为 ARDS 患者提供治疗选择的新机制。 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 Tregs 的影响。多色流式细胞术和 AZA 处理的 Treg 过继转移到受伤的淋巴细胞缺陷小鼠中将用于确定 ALI 消退过程中 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。在具体目标 2 中，将使用染色质免疫沉淀和实时 PCR 确定肺损伤后 DNA 甲基转移酶抑制对关键 Treg 基因 (Foxp3) 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致命疾病的患者预后。