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

项目概要/摘要 这项 NRSA 个人奖学金提案集中于两个主要目标：1）为候选人提供 发展成为一名独立的医师科学家所需的必要时间和资源，以及 2) 调查 实验性流感病毒中诱导性调节性 T 细胞 (iTreg) 稳定性的调控机制 肺炎。候选人和他的导师概述了一项全面且渐进的研究计划 实现这些目标，同时为成功的独立研究生涯奠定基础。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 全世界的发病率和死亡率。调节性 T 细胞是 CD4 T 细胞的一个子集，对于维持免疫至关重要 稳态和协调损伤后肺组织的修复。 Tregs 需要 Foxp3 的稳定表达 转录因子。源自胸腺的稳定、长寿的 FoxP3 Tregs 被称为天然的 Tregs（nTreg）。在体外，TGF-β 诱导短暂的 Foxp3 表达并赋予暂时的抑制功能 幼稚 CD4 T 细胞，产生定义为 iTreg 的细胞。 iTreg表型和功能固有的不稳定性 由于逆转 CD4 效应 T 细胞表型，因此对其作为细胞疗法的临床应用提出了担忧 促进炎症。 Treg 特异性转录程序的稳定性已知受 DNA 调节 甲基化，由 DNA 甲基转移酶及其接头蛋白 UHRF1 介导的过程。 iTregs 由 UHRF1 缺失的幼稚 CD4 T 细胞产生的具有增强的抑制功能。因此，我们 假设 iTreg 中的 UHRF1 表达破坏了获得性抑制和修复的稳定性 转录程序，导致病毒性肺炎后促恢复功能丧失。长- 该提案的长期希望是确定维持转录和功能稳定性的决定因素 iTregs 并为它们作为病毒性肺炎引起的患者的细胞疗法的用途提供了验证 急性呼吸窘迫综合征。 在具体目标 1 中，我们将确定 UHRF1 是否是破坏 iTreg 抑制和修复稳定性所必需的 通过串联 RNA-seq 和 DNA 甲基化分析进行体外和体内转录程序。具体来说 目标 2，我们将确定 iTreg 中 UHRF1 的丢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、 小鼠中基于他莫昔芬的诱导系统、流式细胞术、RNA测序转录分析，以及 以改良简化代表性亚硫酸氢盐测序为主要方法的 DNA 甲基化分析 支持本提案的实验设计。

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.