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

 描述(由申请人提供)：这份K08提案的广泛目标有两个：1)促进基本科学和专业技能的发展，使候选人Benjamin Singer博士能够实现他的长期目标，即成为一名独立的内科医生和科学家，专注于将表观遗传修饰作为肺部病理的治疗目标，以及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工作和同行评审过程，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。Singer博士和他在约翰·霍普金斯大学的导师斯里尼瓦桑·耶格纳苏布拉马尼亚博士和Franco D‘Alessio博士设计了一项专门的培训计划，将为Singer博士提供急性肺部炎症和急性呼吸窘迫综合征(ARDS)的病理生物学方面的新知识和研究技能。ARDS是一种在美国导致巨大发病率和死亡率的肺部疾病。尽管对导致ARDS的最初损伤和炎症进行了广泛的研究，但没有针对性的治疗方法加速其解决。实验研究证实，致力于调节T细胞(Tregs)--限制炎症并协调受损组织修复的免疫系统细胞--可以在小鼠肺损伤模型中化解炎症。然而，导致Tregs在肺损伤后执行促进修复计划的机制仍不清楚。我们的初步数据发现，DNA甲基化涉及一种名为uhrf1的DNA甲基转移酶适配器蛋白，这是一种关键现象，限制了指导Treg前修复功能的主要蛋白质Foxp3的表达。因此，我们假设，在承诺的Treg中，uhrf1缺乏将导致Foxp3基因位点低甲基化，增加Foxp3的表达，并增强Treg修复功能，从而促进急性肺损伤的解决。为了验证这一假设，我们提出了以下具体目标：1)确定uhrf1在肺损伤后承诺Treg中促进Foxp3基因甲基化的作用；2)确定uhrf1在承诺Treg修复功能和免疫调节表型中的作用。为了具体测试我们的假设，我们培育了仅在Tregs内存在uhrf1缺陷的新型小鼠。我们还设计了一种RNA干扰策略来敏锐地敲除培养的Tregs中的uhrf1。这一建议的主要方法包括建立小鼠急性肺损伤模型(气管内注射脂多糖)、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辛格博士提供严格的培训计划，并揭示在急性肺损伤解决过程中控制Treg功能的机制，这些机制可以转化为ARDS的治疗效益。

 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.