The role of epigenetic regulator UHRF1 in stability of induced regulatory T-cell function during influenza A virus-induced lung injury

表观遗传调节因子 UHRF1 在甲型流感病毒诱导的肺损伤过程中诱导调节 T 细胞功能稳定性中的作用

• 批准号: 10389878
• 负责人: Anthony Joudi
• 金额: $ 7.62万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389878
• 关键词: 2019-nCoV; Acute Respiratory Distress Syndrome; Adaptor Signaling Protein; Adoptive Cell Transfers; Adoptive Transfer; Blood Component Removal; CD4 Positive T Lymphocytes; Cause of Death; Cell Therapy; Cell physiology; Cells; Clinical; DNA Methylation; DNA Modification Methylases; DNA methylation profiling; Data; Environment; Epigenetic Process; Experimental Designs; FOXP3 gene; Failure; Fellowship; Flow Cytometry; Foundations; Gene Expression Profiling; Generations; Genetic Transcription; Goals; Homeostasis; Immune; Immune system; In Vitro; Individual; Infection; Inflammation; Inflammatory; Influenza; Influenza A virus; Injury; Intensive Care Units; Knowledge; Laboratories; Maintenance; Mediating; Mentors; Mentorship; Methods; Methylation; Morbidity - disease rate; Mus; National Research Service Awards; Patient-Focused Outcomes; Patients; Pattern; Phenotype; Physicians; Positioning Attribute; Process; Publishing; Pulmonary Inflammation; Reagent; Recovery; Recovery of Function; Regulatory T-Lymphocyte; Reporting; Research; Resolution; Resources; Risk; Role; Scientist; Severities; Structure of parenchyma of lung; Supportive care; System; T-Lymphocyte; Tamoxifen; Technical Expertise; Techniques; Testing; Therapeutic; Thymus Gland; Time; Training; Transforming Growth Factor beta; Umbilical Cord Blood; Validation; Viral; Viral Pneumonia; Work; base; bisulfite sequencing; career; cell type; effector T cell; experience; genomic locus; in vivo; insight; lung injury; lung repair; methylation pattern; mortality; programs; repair function; repaired; respiratory virus; skills; tissue repair; transcription factor; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT This proposal for an NRSA Individual Fellowship is centered on two principal goals: 1) afford the candidate the necessary time and resources required to develop into an independent physician-scientist, and 2) investigate the mechanisms governing the stability of induced regulatory T cells (iTregs) in experimental influenza viral pneumonia. The candidate and his mentors have outlined a comprehensive and progressive research plan to achieve these objectives while laying the foundation for a successful, independent research career. Despite decades of clinical experience and research, severe viral pneumonia and ARDS remain a leading cause of morbidity and mortality worldwide. Regulatory T-cells are a subset of CD4+ T-cells critical to maintaining immune homeostasis and coordinating lung tissue repair after injury. Tregs require stable expression of the Foxp3 transcription factor. Stable, long-lived FoxP3+ Tregs that originate from the thymus are referred to as natural Tregs (nTregs). In vitro, TGF-β induces transient Foxp3 expression and imparts temporary suppressive function to naïve CD4+ T-cells, generating cells defined as iTregs. The inherent instability of iTreg phenotype and function poses a concern for their clinical use as a cellular therapy, as reversion to a CD4+ effector T-cell phenotype promotes inflammation. The stability of Treg-specific transcriptional programs is known to be regulated by DNA methylation, a process mediated by DNA methyltransferases and their adapter protein, UHRF1. iTregs generated from UHRF1-null naïve CD4+ T-cells possess enhanced suppressive function. Hence, we hypothesize that UHRF1 expression in iTregs destabilizes acquired suppressive and reparative transcriptional programs, leading to loss of pro-recovery function following viral pneumonia. The long- term hope of this proposal is to identify determinants of maintenance of transcriptional and functional stability in iTregs and provide validation for their use as cellular therapy in patients suffering from viral pneumonia-induced ARDS. In Specific Aim 1, we will determine whether UHRF1 is necessary to destabilize iTreg suppressive and reparative transcriptional programs both in vitro and in vivo via tandem RNA-seq and DNA methylation analysis. In Specific Aim 2, we will determine whether loss of UHRF1 in iTregs is sufficient to promote recovery following viral pneumonia. We will use cutting-edge techniques for adoptive cell transfer, severity assessment of lung injury, tamoxifen-based inducible systems in mice, flow cytometry, transcriptional profiling with RNA-sequencing, and DNA methylation profiling with modified reduced representation bisulfite sequencing as the primary methods to support the experimental design of this proposal.

项目总结/摘要 NRSA个人奖学金的建议集中在两个主要目标：1）为候选人提供 必要的时间和资源，发展成为一个独立的医生，科学家，和2）调查 实验性流感病毒感染中诱导调节性T细胞（iTcells）稳定性的机制 肺炎候选人和他的导师概述了一个全面和渐进的研究计划， 实现这些目标，同时奠定了成功的基础，独立的研究生涯。尽管 根据数十年的临床经验和研究，严重病毒性肺炎和ARDS仍然是导致 发病率和死亡率。调节性T细胞是CD 4 + T细胞的一个子集，对维持免疫系统至关重要。 内环境稳定和协调损伤后肺组织修复。转录因子需要Foxp 3的稳定表达， 转录因子来源于胸腺的稳定、长寿命的FoxP 3 + T细胞被称为天然的 T（nT）。在体外，TGF-β诱导瞬时Foxp 3表达并赋予暂时的抑制功能 幼稚的CD 4 + T细胞，产生被定义为iT细胞的细胞。iTreg表型和功能的固有不稳定性 作为细胞疗法的临床应用引起了关注，如逆转为CD 4+效应T细胞表型 促进炎症。已知Treg特异性转录程序的稳定性受DNA调节 甲基化是由DNA甲基转移酶及其衔接蛋白UHRF 1介导的过程。iTregs 从UHRF 1缺失的幼稚CD 4 + T细胞产生的抗体具有增强的抑制功能。所以我们 假设在iT细胞中UHRF 1表达使获得性抑制性和修复性免疫不稳定， 转录程序，导致病毒性肺炎后促恢复功能的丧失。很长的- 这个建议的长期希望是确定维持转录和功能稳定性的决定因素， iTHBE并为它们在患有病毒性肺炎诱导的患者中作为细胞疗法的用途提供验证。 ARDS。 在具体目标1中，我们将确定UHRF 1是否是使iTreg抑制性和修复性细胞不稳定所必需的。 通过串联RNA-seq和DNA甲基化分析进行体外和体内转录程序。在特定 目的2，我们将确定iTHBV 1中UHRF 1的缺失是否足以促进病毒感染后的恢复。 肺炎我们将使用先进的技术进行过继细胞转移，肺损伤的严重程度评估， 小鼠中基于他莫昔芬的诱导系统、流式细胞术、RNA测序的转录谱分析，以及 DNA甲基化谱与改良的还原亚硫酸氢盐测序作为主要方法， 支持本提案的实验设计。