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

睡眠免疫轴是高度复杂的，睡眠不足对免疫的负面影响 功能障碍是公认的。不幸的是，睡眠障碍在退伍军人中非常普遍；因此， 了解睡眠如何影响免疫调节为炎症性疾病提供新的治疗策略 与睡眠不佳有关的疾病。参与的转录因子(TF)和基因调控程序 对睡眠免疫轴的了解还不完全。全基因组顺式调控活性变化的分析 元素，或Cystrome，提供了对TF活性的公正评估，以确定调节睡眠的关键途径- 免疫轴。我在CDA-2应用程序中提出的研究将识别和测试TF网络路径 在睡眠中断期间导致中性粒细胞功能的改变。因为我在危重病中的临床实践 护理医学，我的重点是了解睡眠对脓毒症的机制影响。 使用已建立的小鼠睡眠碎片模型，我已经确定了免疫表型 与睡眠障碍有关的功能障碍。先前的研究表明，睡眠障碍的小鼠的预后更差 病原体为基础的败血症模型。死亡是否是早期高炎症反应的结果 或者，病原体清除不良的后遗症还不确定。在这个提案中，我专门测试了睡眠的影响 用无病原体、内毒素诱导的腹膜炎模型研究脓毒症的早期炎症反应。老鼠 在内毒素诱导的腹膜炎中，睡眠障碍患者的死亡率比对照组低，这表明 抑制急性炎症反应。与这一概念一致的是，睡眠中断后的腹膜中性粒细胞 脓毒症小鼠的髓过氧化物酶(MPO)表达较低，这是活性氧(ROS)的关键酶 制作。从睡眠障碍小鼠骨髓中提取的单细胞RNA测序结果显示 具有STAT3活性特征的成熟中性粒细胞转录水平的变化 免疫抑制反应。这些发现支持了我的假设，即中性粒细胞的表型和功能 对睡眠干扰敏感。使用创新的循环分析方法，我的目标是识别 睡眠中断影响中性粒细胞功能的机械性转铁蛋白途径。首要目标是 确定可靶向调节中性粒细胞激活的途径来治疗退伍军人 睡眠中断的不良后果。 这项建议通过转录调控来解决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会降低中性粒细胞的基本能力，包括 ROS产生、吞噬和网织作用(AIM1)。然后我会用脑电图仪测试睡眠是否 干扰会影响STAT3(AIM2)的转录活性。使用CHIP-SEQ，我预计STAT3将发生全球变化 在全基因组范围内结合顺式调控元件。我会不偏不倚地证实STAT3的分析 使用一种新的技术，封端-短RNA测序，对调节元件的活性进行分析。识别 STAT3在哪里绑定并分析这些调控元件的活动是确定STAT3的一个强有力的策略 在睡眠中断情况下的活动。最后，我将测试睡眠恢复对败血症存活率、中性粒细胞的影响。 表型和细胞周期活动的动态变化(AIM3)。了解哪些TF在过程中被激活 当睡眠持续中断时，睡眠恢复具有恢复中性粒细胞功能的转换潜力。 这些实验的结果将对退伍军人的护理产生重大影响，并推动 通过1)确定睡眠健康和睡眠中断如何影响中性粒细胞的基本机制2) 改进急性炎症反应期间睡眠免疫稳态的模型；以及3)识别新的 调节睡眠紊乱所致免疫功能障碍的治疗目标和管理策略， 尤其是在睡眠难以维持或恢复的情况下。

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.