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，睡眠不足对免疫系统的负面影响， 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了新的治疗策略。 与睡眠不佳有关的疾病。转录因子（TF）和基因调控程序参与 睡眠免疫轴还没有被完全理解。顺式调节蛋白活性的全基因组变化分析 元素，或顺式，提供了一个公正的评估TF活动，以确定关键途径调节睡眠- 免疫轴我在CDA-2申请中提出的研究将识别和测试TF网络通路 导致睡眠中断期间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械影响。 使用已建立的睡眠片段化小鼠模型，我已经确定了免疫表型， 与睡眠中断有关的功能障碍。以前的研究表明，睡眠中断的小鼠在睡眠中的结果更糟。 基于病原体的脓毒症模型。死亡率是否是早期炎症反应过度的结果 或病原体清除不良的后遗症是不确定的。在这个提案中，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脓毒症的早期炎症反应的影响。小鼠 睡眠中断的患者在LPS诱导的腹膜炎中的死亡率低于匹配的对照组，这表明 抑制了急性炎症反应与这一观点一致，睡眠中断引起的腹膜中性粒细胞 脓毒症小鼠的髓过氧化物酶（Mpo）表达较低，Mpo是活性氧（ROS）的关键酶 生产睡眠中断小鼠骨髓的单细胞RNA测序显示， 成熟中性粒细胞转录组的变化与Stat3的活性签名，TF介导 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是 对睡眠干扰很敏感使用顺反子分析的创新方法，我的目标是确定 睡眠中断影响中性粒细胞功能的机制性TF途径。总体目标是 确定可以靶向调节中性粒细胞活化的途径，以治疗患有 睡眠中断的不良后果。 该建议通过转录调节解决睡眠对中性粒细胞功能的影响。 首先，我将研究睡眠中断是否会降低中性粒细胞的基本功能，包括 ROS产生、吞噬作用和NETosis（AIM 1）。然后，我将使用顺时针工具来测试睡眠是否 破坏影响Stat3（AIM 2）的转录活性。使用ChIP-seq，我预计Stat3会发生全局变化 在全基因组范围内与顺式调控元件结合。我会用一个公正的 使用一种新的技术，加帽短RNA测序的调控元件的活性谱。识别 Stat3结合的位置和这些调控元件的活性分析是确定Stat3 睡眠中断下的活动。最后，我将测试睡眠恢复对脓毒症生存率的影响，中性粒细胞 表型和顺式活性的动力学变化（AIM 3）。了解哪些TF在 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化潜力。 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 1）确定健康睡眠与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的睡眠免疫稳态模型;以及3）鉴定新的 调节由于睡眠中断引起的免疫功能障碍的治疗靶点和管理策略， 特别是在睡眠难以保持或恢复的情况下。

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.