Regulatory T cell Uhrf1 and DNA methylation in repair of acute lung injury

调节性T细胞Uhrf1和DNA甲基化在急性肺损伤修复中的作用

• 批准号: 9118337
• 负责人: Benjamin David Singer
• 金额: $ 16.22万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9118337
• 关键词: Acute; Acute Lung Injury; Adult Respiratory Distress Syndrome; Affect; Alveolar; Boxing; Breeding; CD4 Positive T Lymphocytes; Cell Therapy; Cells; Communication; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Data; Data Analyses; Development; Epigenetic Process; Experimental Designs; Flow Cytometry; Fostering; Genetic Transcription; Goals; Growth; Guanine; Healed; Immune system; In Vitro; Inflammation; Inflammatory; Injury; Knowledge; Laboratories; Laboratory Procedures; Lead; Lipopolysaccharides; Logic; Lung; Lung Inflammation; Lung diseases; Maintenance; Mediating; Mentors; Messenger RNA; Methods; Methylation; Modification; Morbidity - disease rate; Mus; Patients; Pattern; Peer Review; Pharmacotherapy; Phenotype; Physicians; Play; Population; Process; Proteins; Public Health Schools; Pulmonary Pathology; RNA Interference; Recruitment Activity; Regulatory T-Lymphocyte; Research; Resolution; Role; Scientist; System; Techniques; Testing; Therapeutic; Tissues; Training; Training Programs; Transcription Factor 3; Transferase; Translating; Undifferentiated; United States; Universities; Untranslated RNA; adapter protein; career; demethylation; design; healing; improved; in vivo; inflammatory lung disease; insight; knock-down; laboratory experience; lung injury; lung repair; molecular marker; mortality; mouse model; novel; novel therapeutics; outcome forecast; programs; protein expression; public health relevance; repaired; research study; skills; targeted treatment; therapeutic target; tool

 DESCRIPTION (provided by applicant): The broad objectives of this K08 proposal are two-fold: 1) to foster the development of essential scientific and professional skills that will allow he candidate, Dr. Benjamin Singer, to achieve his long-term goal of becoming an independent physician-scientist concentrating on epigenetic modifications as therapeutic targets in lung pathology, and 2) to investigate mechanisms that direct the immune system to resolve a severe lung disease. Through laboratory experience, coursework in the Bloomberg School of Public Health, and the peer review process, Dr. Singer will gain expertise in experimental design, laboratory procedures, data analysis, and scientific communication. Dr. Singer and his mentors at Johns Hopkins University, Drs. Srinivasan Yegnasubramanian and Franco D'Alessio, have designed a specific training plan that will afford Dr. Singer new knowledge and research skills in the pathobiology of acute lung inflammation and acute respiratory distress syndrome (ARDS), which is a lung condition that causes tremendous morbidity and mortality in the United States. Despite extensive research into the initial injury and inflammation that drive ARDS, no targeted therapies accelerate its resolution. Experimental studies established that committed regulatory T cells (Tregs)-immune system cells that limit inflammation and orchestrate repair of damaged tissues-resolve inflammation in mouse models of lung injury. However, the mechanisms that cause Tregs to execute their pro-repair program following lung injury remain unknown. Our preliminary data identify DNA methylation, which involves a DNA methyltransferase adapter protein known as Uhrf1, as a critical phenomenon limiting expression of the main protein that directs Treg pro- repair function: Foxp3. Thus, we hypothesize that Uhrf1 deficiency in committed Tregs will lead to Foxp3 locus hypomethylation, increased Foxp3 expression, and enhanced Treg pro-repair function that facilitates resolution of acute lung injury. To test this hypothesis we propose the following Specific Aims: 1) define the role of Uhrf1 in promoting DNA methylation at the Foxp3 locus in committed Tregs following lung injury, and 2) define the role of Uhrf1 on committed Treg pro-repair function and immunoregulatory phenotype. To specifically test our hypothesis we are breeding novel mice that have Uhrf1 deficiency only within Tregs. We have also designed an RNA interference strategy to acutely knock down Uhrf1 in cultured Tregs. Major methods for this proposal include an established mouse model of acute lung injury (intratracheal lipopolysaccharide administration), DNA methylation sequencing techniques, and multicolor flow cytometry. Accomplishment of these aims will provide a rigorous training program for Dr. Singer and uncover mechanisms controlling Treg function during resolution of acute lung injury that could be translated for therapeutic benefit in ARDS.

 描述（由申请人提供）：这项 K08 提案的主要目标有两个：1）促进基本科学和专业技能的发展，使他的候选人 Benjamin Singer 博士能够实现其长期目标，成为一名独立的医师科学家，专注于将表观遗传修饰作为肺部病理学的治疗目标；2）研究指导免疫系统解决严重肺部疾病的机制。通过实验室经验、彭博公共卫生学院的课程以及同行评审过程，辛格博士将获得实验设计、实验室程序、数据分析和科学交流方面的专业知识。辛格博士和他在约翰霍普金斯大学的导师，博士。 Srinivasan Yegnasubramanian 和 Franco D'Alessio 设计了一项具体的培训计划，该计划将为 Singer 博士提供急性肺部炎症和急性呼吸窘迫综合征 (ARDS) 病理学方面的新知识和研究技能，ARDS 是一种在美国造成巨大发病率和死亡率的肺部疾病。尽管对导致 ARDS 的初始损伤和炎症进行了广泛的研究，但没有靶向治疗可以加速其解决。实验研究表明，定向调节性 T 细胞 (Treg) 是限制炎症并协调受损组织修复的免疫系统细胞，可以解决小鼠肺损伤模型中的炎症。然而，导致Tregs在肺损伤后执行其促修复程序的机制仍然未知。我们的初步数据确定 DNA 甲基化（涉及称为 Uhrf1 的 DNA 甲基转移酶接头蛋白）是限制指导 Treg 修复功能的主要蛋白 Foxp3 表达的关键现象。因此，我们假设定型Treg中的Uhrf1缺陷将导致Foxp3基因座低甲基化、Foxp3表达增加以及Treg促修复功能增强，从而促进急性肺损伤的解决。为了检验这一假设，我们提出以下具体目标：1) 定义 Uhrf1 在促进肺损伤后定型 Treg 中 Foxp3 位点 DNA 甲基化中的作用，2) 定义 Uhrf1 对定型 Treg 促修复功能和免疫调节表型的作用。为了具体检验我们的假设，我们正在培育仅在 Tregs 中存在 Uhrf1 缺陷的新型小鼠。我们还设计了一种 RNA 干扰策略来急性敲低培养的 Tregs 中的 Uhrf1。该提案的主要方法包括建立小鼠急性肺损伤模型（气管内脂多糖给药）、DNA甲基化测序技术和多色流式细胞术。这些目标的实现将为 Singer 博士提供严格的培训计划，并揭示在急性肺损伤缓解过程中控制 Treg 功能的机制，这些机制可以转化为 ARDS 的治疗益处。