Impact of Chronic Alcohol Consumption on the Functional and Epigenetic Landscapes of Monocytes and Their Progenitors

项目总结： 饮酒在美国很普遍，约7%的饮酒者从事 酗酒。众所周知，长期大量饮酒(CHD)与 易受感染，伤口愈合和组织修复受损，导致术后情况不佳 结果。有证据表明，这些缺陷中的许多是由过度的炎症反应引起的。 起源于髓系细胞，特别是循环单核细胞和组织驻留的巨噬细胞。然而，许多人 目前的研究依赖于健康捐赠者或细胞系的单核细胞体外暴露于高剂量的 乙醇。由于缺乏使用可靠的体内模型的研究，我们对潜在机制的理解 冠心病背景下的异常炎症反应仍然不完整。为了解决这些知识 ，我们建议利用恒河猴自愿乙醇自我给药的模型 反映了人类的生理，并概括了复杂的人类饮酒行为。使用这种模型，我们的实验室已经 最近证实，CHD导致循环单核细胞转录和表观遗传重新连接，并 脾巨噬细胞，导致对内毒素刺激的异常反应。然而，功能上的影响 以及控制这种重新编程的表观遗传机制仍不清楚。重要的是，因为 单核细胞是在骨髓不断繁殖的情况下短暂的循环细胞，这些 观察表明，造血生态位受到了干扰。造血细胞单细胞分析的初步研究 祖细胞指出，随着酒精的作用，分化潜能向更成熟的髓系祖细胞转移。 然而，祖细胞的这种表型和它们在血液中的分化状态之间的联系仍然存在。 不清楚。在这一应用中，我们建议测试长期饮酒重新编程的假设 单核细胞祖细胞在骨髓中引起循环的表观遗传格局 单核细胞倾向于高炎症反应。我们将首先研究CHD对 循环单核细胞的功能重新编程，进行检测以测试其迁移能力， 吞噬，并产生适当的代谢反应。然后我们将研究刺激对 通过评估染色质可及性和组蛋白的差异结合来评价单核细胞表观遗传景观 用酒精进行修饰。最后，我们将确定CHD对分化潜能的影响， 转录组激活和骨髓髓系祖细胞的表观遗传学重排。我们将执行功能 粒细胞/单核细胞祖细胞体外来源的单核细胞的检测及其与 从外周血单核细胞中获得。此外，对髓系的scRNA-Seq分析和表观遗传学评估 祖细胞将使我们能够确定酒精对骨髓室的特定影响以及如何 这会导致外周血单核细胞过度炎症，表观基因重新编程。完成这项工作 这项提议将扩大我们对饮酒对造血的免疫影响的认识。

Project Summary: Alcohol consumption is widespread in the United States with ~7% of alcohol-consuming individuals engaging in heavy alcohol use. It is well established that chronic heavy drinking (CHD) is associated with increased susceptibility to infections as well as impaired wound healing and tissue repair resulting in poor post-operative outcomes. Evidence suggests that many of these defects are mediated by excessive inflammatory responses originating from myeloid cells, notably circulating monocytes and tissue-resident macrophages. However, many of the current studies rely on in vitro exposure of monocytes from healthy donors or cell lines to high doses of ethanol. Due to a lack of studies utilizing reliable in vivo models, our understanding of the mechanisms underlying aberrant inflammatory responses in the context of CHD remains incomplete. In order to address these knowledge gaps, we propose to leverage a rhesus macaque model of voluntary ethanol self-administration that accurately mirrors human physiology and recapitulates complex human drinking behavior. Using this model, our lab has recently demonstrated that CHD results in transcriptional and epigenetic rewiring of circulating monocytes and splenic macrophages, resulting in aberrant responses to LPS stimulation. However, the functional implications of and the epigenetic mechanisms controlling this reprogramming remain unknown. Importantly, because monocytes are short-lived circulating cells under constant repopulation from the bone marrow, these observations suggest perturbations of the hematopoietic niche. Preliminary single-cell analyses of hematopoietic progenitors point to a shift in differentiation potential towards more mature myeloid progenitors with alcohol. However, a link between this phenotype in progenitor cells and their differentiated states in blood remains unclear. In this application, we propose to test the hypothesis that chronic alcohol consumption reprograms the epigenetic landscape of monocyte progenitors in the bone marrow giving rise to circulating monocytes poised towards a hyper-inflammatory response. We will first examine the impact of CHD on functional reprogramming of circulating monocytes, implementing assays to test their ability to migrate, phagocytose, and generate proper metabolic responses. We will then examine the effect of stimulation on the monocyte epigenetic landscape through assessment of chromatin accessibility and differential binding of histone modifications with alcohol. Finally, we will determine the effect of CHD on the differentiation potential, transcriptome activation, and epigenetic rewiring of bone marrow myeloid progenitors. We will perform functional assays on monocytes derived in vitro from granulocyte/monocyte progenitors and integrate these data with those obtained from peripheral monocytes. Further, scRNA-Seq analysis and epigenetic assessment of the myeloid progenitors will allow us to determine the specific effects of alcohol on the bone marrow compartment and how this leads to hyper-inflammatory, epigenetically reprogrammed peripheral monocytes. Completion of this proposal will expand our knowledge of the immunological effects of alcohol consumption on hematopoiesis.