Lung Injury Repair by Regulatory T cell Dhx58/LGP2

调节性 T 细胞 Dhx58/LGP2 修复肺损伤

• 批准号: 9324420
• 负责人: Franco R D'Alessio
• 金额: $ 40.75万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324420
• 关键词: Acute; Acute Lung Injury; Adoptive Transfer; Adult Respiratory Distress Syndrome; Affect; Alveolar; Attenuated; Biological Assay; CD4 Positive T Lymphocytes; Cell Therapy; Cells; Complex; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Data; Deoxycytidine; Disease; Double-Stranded RNA; Epigenetic Process; Epithelial; Flow Cytometry; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genomics; Glucocorticoids; Goals; Guanine; Healed; Homeostasis; IL2RA gene; Immune; Immune system; In Vitro; Inflammation; Inflammatory Response; Influenza; Injury; Interferons; Investigation; Lead; Life; Lipopolysaccharides; Lung; Lung Inflammation; Mediating; Methods; Methylation; Morbidity - disease rate; Mus; Outcome; Pathway interactions; Pattern; Phase; Phenotype; Play; Proteins; Pseudomonas; Pseudomonas aeruginosa; RNA Interference; Regulation; Regulatory T-Lymphocyte; Repression; Research; Research Personnel; Resolution; Role; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; Syndrome; Techniques; Testing; Therapeutic; Transgenic Mice; Translating; Translations; Universities; Viral; Virus Diseases; Wild Type Mouse; Work; bisulfite; design; epigenetic regulation; experience; genetic regulatory protein; healing; improved; improved outcome; in vitro testing; in vivo; inflammatory lung disease; inhibitor/antagonist; injured; injury and repair; insight; knock-down; lung injury; lung repair; macrophage; methylation pattern; mortality; mouse model; mutant; new therapeutic target; novel; novel therapeutics; outcome forecast; overexpression; plasmid DNA; protein expression; public health relevance; repaired; response; small molecule; targeted treatment; therapy design; transcription factor; transcriptome sequencing

 DESCRIPTION (provided by applicant): Lung Injury Repair by Regulatory T cell Dhx58/LGP2 The overall objective of this R01 proposal is to investigate mechanisms that direct the immune system to resolve severe acute lung inflammation. Our team, comprising expert investigators from two leading universities, has designed a research strategy that will validate new therapeutic targets acute respiratory distress syndrome (ARDS), which is a devastating condition that causes significant morbidity and mortality in the U.S. Despite thorough investigation into the injury and inflammation that drive ARDS, no targeted therapies promote its resolution. Regulatory T cells (Tregs) - a subset of CD4+ lymphocytes that suppress exuberant immune system activation - resolve inflammation in mouse models of lung injury. However, the mechanisms that promote Treg function following lung injury remain unknown. Our preliminary data identified in Tregs the Dhx58 locus, which encodes the immune regulatory protein LGP2, as a novel site that augments Treg responses to inflammation. We determined that DNA methylation at this site is dynamic and contributes to repression of the Dhx58 locus following lung injury. Thus, we hypothesize that Treg DNA hypomethylation at the Dhx58 locus will increase LGP2 protein levels, enhance Treg pro-resolution function, and promote resolution of acute lung inflammation. To test this hypothesis we propose the following Specific Aims: 1. To define the role of the DNA methylating complex Dnmt1-Uhrf1 in promoting DNA methylation at the Dhx58 locus to regulate LGP2 protein expression in Tregs during lung injury resolution; 2. To examine the role of LGP2 in regulating Treg suppressive phenotype and function; and 3. To determine the requirement for Treg LGP2 and its therapeutic potential in resolution of lung injury. To specifically test our hypothesis we will employ transgenic mice including strains with Dhx58 deficiency as well as Treg-specific Uhrf1 deficiency. We have also designed an RNA interference strategy to acutely knock down Dhx58 in cultured Tregs and boost Dhx58 expression with mutant DNA plasmids. Major methods for this project include established mouse models of acute lung injury (intratracheal lipopolysaccharide and Pseudomonas aeruginosa administration), DNA methylation sequencing techniques, and multicolor flow cytometry. Accomplishment of these aims will 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）：调节性T细胞Dhx 58/LGP 2的肺损伤修复本R 01提案的总体目标是研究指导免疫系统解决严重急性肺部炎症的机制。我们的团队由来自两所顶尖大学的专家研究人员组成，他们设计了一种研究策略，将验证新的治疗目标急性呼吸窘迫综合征（ARDS），这是一种在美国导致严重发病率和死亡率的破坏性疾病。调节性T细胞（Tcells）-抑制旺盛的免疫系统激活的CD 4+淋巴细胞亚群-解决肺损伤小鼠模型中的炎症。然而，肺损伤后促进Treg功能的机制仍不清楚。我们的初步数据确定在Tendon的Dhx 58基因座，它编码的免疫调节蛋白LGP 2，作为一个新的网站，增强Treg反应炎症。我们确定该位点的DNA甲基化是动态的，并有助于肺损伤后Dhx 58位点的抑制。因此，我们假设Dhx 58位点的Treg DNA低甲基化将增加LGP 2蛋白水平，增强Treg促消退功能，并促进急性肺部炎症的消退。为了验证这一假设，我们提出了以下具体目标：1。目的：1.明确肺损伤消退过程中DNA甲基化复合物Dnmt 1-Uhrf 1在促进Dhx 58基因座DNA甲基化以调节肺组织LGP 2蛋白表达中的作用。研究LGP 2在调节Treg抑制性表型和功能中的作用;和3.确定Treg LGP 2的需求及其在解决肺损伤中的治疗潜力。为了具体检验我们的假设，我们将采用转基因小鼠，包括Dhx 58缺陷以及Treg特异性Uhrf 1缺陷的品系。我们还设计了一种RNA干扰策略，以急性敲低Dhx 58在培养的大肠杆菌和提高Dhx 58的表达与突变的DNA质粒。本项目的主要方法包括建立小鼠急性肺损伤模型（经气管内注射脂多糖和铜绿假单胞菌）、DNA甲基化测序技术和流式细胞术。这些目标的实现将揭示在急性肺损伤的解决过程中控制Treg功能的机制，这些机制可以转化为ARDS的治疗益处。