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.

项目摘要/摘要 NRSA个人奖学金的这一建议集中在两个主要目标上：1）候选人负担 发展成独立的身体科学家所需的必要时间和资源，2）调查 在实验性影响力病毒中，管理诱导调节T细胞（ITREGS）稳定性的机制 肺炎。候选人及其导师概述了一项全面而进步的研究计划 在为成功的独立研究职业奠定基础的同时，实现这些目标。尽管 数十年的临床经验和研究，严重的病毒性肺炎和ARD仍然是 全球发病率和死亡率。调节性T细胞是CD4+ T细胞的子集，对于维持免疫 受伤后的稳态和协调肺组织修复。 Treg需要稳定的Foxp3表达 转录因子。稳定的，长寿命的Foxp3+ Tregs起源于胸腺被称为自然 tregs（ntregs）。在体外，TGF-β诱导瞬时FOXP3表达并赋予暂时的抑制功能 对于天真的CD4+ T细胞，生成定义为ITREGS的细胞。 ITREG表型和功能的固有不稳定性 对它们作为细胞疗法的临床用途提出了关注，与CD4+效应T细胞表型相反 促进炎症。已知TREG特异性转录程序的稳定性受DNA的调节 甲基化，这是由DNA甲基转移酶及其衔接蛋白UHRF1介导的过程。 itregs 由UHRF1-NULL幼稚的CD4+ T细胞产生的具有增强的抑制功能。因此，我们 假设在ITREGS中的UHRF1表达破坏了获得的抑制和修复 转录程序，导致病毒性肺炎后促进恢复功能的丧失。长期 该提案的期望是确定在 ITREGS并为其用作病毒性肺炎诱导的患者的细胞疗法提供验证 ARDS。 在特定目标1中，我们将确定UHRF1是否需要破坏ITREG抑制和修复的稳定性 在体外和体内转录程序都通过串联RNA-seq和DNA甲基化分析。具体 AIM 2，我们将确定ITREGS中UHRF1的丢失是否足以促进病毒后恢复 肺炎。我们将使用尖端技术进行自适应细胞转移，肺损伤的严重程度评估， 基于他莫昔芬的诱导系统，流式细胞术，带有RNA测序的转录分析，以及 DNA甲基化分析与修饰的减少表示亚硫酸盐测序作为主要方法 支持该建议的实验设计。