The Role of Sleep in Innate Immune Homeostasis: Toward Mechanistic Understanding Through Genome-Wide Enhancer Analysis

睡眠在先天免疫稳态中的作用：通过全基因组增强子分析实现机制理解

• 批准号: 10589548
• 负责人: Michael Tun Yin Lam
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-10-01 至 2027-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589548
• 关键词: ATAC-seq; Activities of Daily Living; Acute; Address; Affect; Animals; Automobile Driving; Binding; Bone Marrow; Caring; Cells; ChIP-seq; Child Care; Chromatin; Chronic; Clinical; Complex; Coupled; Critical Care; Critical Illness; DNA Sequence; Data; Disease; Drowsiness; Endotoxins; Enhancers; Enzymes; Exhibits; Financial Hardship; Foundations; Gene Expression; Generations; Genetic Transcription; Genomics; Global Change; Goals; Health; Homeostasis; Immune; Immune System Diseases; Immune response; Immune system; Immunosuppression; Infection; Inflammation; Inflammatory; Inflammatory Response; Innate Immune System; Kinetics; Life; Link; Low Back Pain; Mediating; Medical; Medicine; Mental Depression; Mental Health; Modeling; Modernization; Molecular; Mus; Neutrophil Activation; Obstructive Sleep Apnea; Outcome; Pathway interactions; Patients; Peritoneal; Peritonitis; Peroxidases; Phagocytosis; Phenotype; Physicians; Population; Post-Traumatic Stress Disorders; Procedures; Production; Reactive Oxygen Species; Recovery; Regulator Genes; Regulatory Element; Reporting; Research; Risk; Role; Sepsis; Septic Shock; Shock; Signal Pathway; Signal Transduction; Sleep; Sleep Deprivation; Sleep Disorders; Sleep Fragmentations; Sleep disturbances; Sleeplessness; Societal Factors; Societies; Techniques; Testing; Time; Transcriptional Regulation; Translating; Vaccines; Veterans; adequate sleep; adverse outcome; chronic inflammatory disease; clinical practice; cytokine release syndrome; experience; experimental study; extracellular; genome-wide; genome-wide analysis; genomic tools; high risk; immune function; immunoregulation; improved; infection risk; innovation; insight; military veteran; mimetics; mortality; mouse model; neutrophil; new therapeutic target; novel; novel therapeutic intervention; pathogen; pharmacologic; poor sleep; preservation; programs; septic; shift work; single-cell RNA sequencing; sleep regulation; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; translational potential

The sleep-immune axis is highly complex, and the negative impact of inadequate sleep on immune dysfunction is well recognized. Unfortunately, sleep disorders are highly prevalent among Veterans; as a result, understanding how sleep influences immune regulation offers novel therapeutic strategies for inflammatory diseases associated with poor sleep. The transcription factors (TFs) and gene regulatory programs involved in the sleep-immune axis are not fully understood. Analysis of genome-wide changes in the activity of cis-regulatory elements, or cistrome, offers an unbiased assessment of TF activity to identify key pathways regulating the sleep- immune axis. The studies I propose in this CDA-2 application will identify and test TF network pathways contributing to alterations in neutrophil function during sleep disruption. Because of my clinical practice in critical care medicine, I am focusing on understanding the mechanistic impact of sleep on sepsis. Using an established murine model of sleep fragmentation, I have identified phenotypes of immune dysfunction related to sleep disruption. Previous studies showed sleep disrupted mice had worse outcomes in pathogen-based sepsis models. Whether the mortality is a consequence of an early hyperinflammatory response or a sequala of poor pathogen clearance is uncertain. In this proposal, I specifically tested for the impact of sleep on the early inflammatory response to sepsis using the pathogen-free, endotoxin-induced peritonitis model. Mice with sleep disruption had less mortality than matched controls upon LPS-induced peritonitis, suggesting a dampened acute inflammatory response. Consistent with this notion, peritoneal neutrophils from sleep-disrupted septic mice have lower expression of Myeloperoxidase (Mpo), a critical enzyme in reactive oxygen species (ROS) production. Single-cell RNA sequencing from the bone marrow of sleep-disrupted mice showed global transcriptomic changes in mature neutrophils with activity signature of Stat3, a TF that mediates immunosuppressive response. These findings support my hypothesis that neutrophil phenotype and function are sensitive to sleep perturbation. Using the innovative approach of cistrome analysis, I aim to identify the mechanistic TF pathways by which sleep disruption impacts neutrophil function. The overarching goal is to identify pathways that could be targeted to modulate neutrophil activation to treat Veterans suffering from the adverse consequences of sleep disruption. This proposal addresses the impact of sleep on neutrophil function through transcriptional regulation. First, I will investigate whether sleep disruption reduces the fundamental capabilities of neutrophils, including ROS production, phagocytosis, and NETosis (AIM1). I will then use cistromic tools to test whether the sleep disruption affects the transcriptional activity of Stat3 (AIM2). Using ChIP-seq, I expect a global change in Stat3 binding in cis-regulatory elements at a genome-wide scale. I will corroborate the Stat3 analysis with an unbiased activity profiling of regulatory elements using a novel technique, capped-short RNA sequencing. Identifying where Stat3 binds and profiling the activity of these regulatory elements is a powerful strategy to determine Stat3 activity under sleep disruption. Finally, I will test the impact of sleep recovery on sepsis survival, neutrophil phenotype, and the kinetic changes in cistromic activity (AIM3). Understanding which TFs are activated during sleep recovery has the translational potential for restoring neutrophil function when sleep is persistently disrupted. The findings of these experiments will have a significant impact on the care of Veterans and advance the field by 1) identifying fundamental mechanisms of how healthy versus disrupted sleep affects neutrophils 2) refining the model of sleep-immune homeostasis during acute inflammatory responses; and 3) identifying new therapeutic targets and management strategies to modulate immune dysfunction due to sleep disruption, especially in situations where sleep is difficult to preserve or restore.

睡眠免疫轴高度复杂，睡眠不足对免疫的负面影响 功能障碍已得到广泛认可。不幸的是，在退伍军人中，睡眠障碍非常普遍。因此， 了解睡眠如何影响免疫调节为炎症提供新颖的治疗策略 与睡眠不佳有关的疾病。涉及的转录因子（TFS）和基因调节程序 睡眠免疫轴未完全了解。整个基因组调节活性的变化分析 元素或Cistrome提供了对TF活动的公正评估，以识别调节睡眠的关键途径 - 免疫轴。我在此CDA-2应用中提出的研究将识别和测试TF网络途径 在睡眠中断过程中有助于中性粒细胞功能的改变。因为我的临床实践 护理医学，我专注于了解睡眠对败血症的机理影响。 使用已建立的睡眠碎片模型，我已经确定了免疫的表型 与睡眠中断有关的功能障碍。先前的研究表明，睡眠中断的小鼠的预后较差 基于病原体的败血症模型。死亡率是否是早期过度炎症反应的结果 或病原体清除不良的序列尚不确定。在此提案中，我专门测试了睡眠的影响 使用无病原体，内毒素诱导的腹膜炎模型对败血症的早期炎症反应。老鼠 在LPS诱发的腹膜炎时，睡眠中断的死亡率比匹配的对照较少，这表明是 急性炎症反应抑制。与这个概念一致，腹膜中性粒细胞来自睡眠 化粪池小鼠的骨过氧化物酶（MPO）的表达较低，这是活性氧中的临界酶（ROS） 生产。来自睡眠干扰小鼠的骨髓的单细胞RNA测序显示全球 具有STAT3活性特征的成熟中性粒细胞的转录组变化，STAT3的活性特征是介导的TF 免疫抑制反应。这些发现支持我的假设，即中性粒细胞表型和功能是 对睡眠扰动敏感。使用Cistrome分析的创新方法，我旨在确定 睡眠破坏会影响中性粒细胞功能的机械TF途径。总体目标是 确定可以针对可调节嗜中性粒细胞激活的途径，以治疗患有苦难的退伍军人 睡眠中断的不利后果。 该提案通过转录调节解决了睡眠对中性粒细胞功能的影响。 首先，我将调查睡眠中断是否降低了中性粒细胞的基本能力，包括 ROS产生，吞噬作用和Netosis（AIM1）。然后，我将使用Cistromic工具来测试是否睡眠 破坏会影响STAT3的转录活性（AIM2）。使用芯片序列，我预计STAT3的全球变化 在整个基因组量表中结合顺式调节元件。我将以公正的证实STAT3分析 使用一种新技术，限制的RNA测序对调节元件的活性分析。识别 STAT3结合和分析这些调节元素的活动是确定STAT3的有力策略 睡眠中断的活动。最后，我将测试睡眠恢复对败血症生存的影响，中性粒细胞 表型以及Cistromic活性的动力学变化（AIM3）。了解哪些TF在此期间被激活 当睡眠持续破坏时，睡眠恢复具有恢复中性粒细胞功能的转化潜力。 这些实验的发现将对退伍军人的护理产生重大影响，并促进 1）识别健康与破坏睡眠的基本机制影响中性粒细胞2） 在急性炎症反应过程中完善睡眠免疫体内稳态模型； 3）确定新的 治疗目标和管理策略，以调节由于睡眠中断而调节免疫功能障碍， 特别是在难以保存或恢复睡眠的情况下。