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

项目摘要/摘要 NRSA个人奖学金的建议围绕两个主要目标：1)为候选人提供 发展成为独立的内科科学家所需的必要时间和资源，以及2)调查 实验性流感病毒诱导调节性T细胞稳定性的机制 肺炎。候选人和他的导师概述了一项全面和进步的研究计划，以 实现这些目标，同时为成功的、独立的研究事业奠定基础。尽管 几十年的临床经验和研究，严重的病毒性肺炎和ARDS仍然是导致 世界范围内的发病率和死亡率。调节性T细胞是对维持免疫至关重要的CD4+T细胞的一个亚群 动态平衡和协调肺组织损伤后的修复。Treg需要稳定表达Foxp3 转录因子。来自胸腺的稳定、长寿的FoxP3+Treg被称为天然的 Tregs(NTregs)。转化生长因子-β在体外诱导一过性foxp3表达并发挥暂时性抑制作用 培养幼稚的CD4+T细胞，产生被定义为iTregs的细胞。ITreg表型和功能的内在不稳定性 作为一种细胞疗法，作为一种回复到CD4+效应T细胞表型的临床应用，引起了人们的关注 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，由DNA甲基转移酶及其适配蛋白uhrf1介导的过程。ITregs 由uhrf1缺失的幼稚CD4+T细胞产生的T细胞具有增强的抑制功能。因此，我们 假设uhrf1在iTregs中的表达破坏了获得性抑制和修复的稳定性 转录程序，导致病毒性肺炎后恢复功能的丧失。长的- 这项提议的长期希望是确定维持转录和功能稳定性的决定因素 ITregs并验证其在病毒性肺炎患者中的细胞治疗作用 阿兹。 在特定的目标1中，我们将确定uhrf1是否是破坏iTreg抑制和修复的稳定所必需的 通过串联RNA-seq和DNA甲基化分析在体外和体内的转录程序。具体而言 目的2，我们将确定iTregs中uhrf1的缺失是否足以促进病毒感染后的恢复。 肺炎。我们将使用尖端技术进行过继细胞移植，肺损伤严重程度评估， 基于他莫昔芬的小鼠诱导系统，流式细胞术，RNA测序转录图谱，以及 以改良还原代表性亚硫酸氢盐测序为主要方法的DNA甲基化分析 支持这一提议的实验设计。

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.