NF-kB and Chromatin Changes in Human Sepsis

该提案继续了我们的目标，即阐明控制急性全身炎症的分子事件，这是一个高度致命的过程。在之前的捐赠周期中，我们在先天免疫的巨噬细胞和中性粒细胞中发现了一个由Toll样受体(TLR)驱动的、临时定义的表观遗传学程序，它指导着急性全身炎症的进程。这个过程改变染色质结构，以抑制或激活不同功能的基因集，从而产生不同的表型阶段。其他的，我们已经表明，在急性全身炎症期间，生物能量的转移也发生在先天性免疫细胞中。这一应用发展了一个统一的概念，即生物能量学中的修饰与表观遗传学相结合，以指导急性炎症的相变。我们提出了两个具体目标：目标1)定义生物能源和表观遗传学之间的联系。我们将使用一个重现TLR4依赖的相移的细胞模型来：a)描绘基因特定的染色质修饰；b)确定蛋白质-蛋白质相互作用；c)通过质谱学评估代谢谱；以及c)将我们的概念转化为人类正常和脓毒症血白细胞。目的2)检测生物能修饰和表观遗传转换对临床结局的影响。我们将使用脓毒症的小鼠模型：a)定义生物能量和表观遗传相变，使用生化和遗传学方法分析血浆和从脾、腹膜和骨髓中分离的白细胞；以及b)使用药理学和遗传学方法来确定修改到适应期是否改变微血管炎症和生存。我们的结果将广泛影响炎症领域，为急性炎症是如何组织的提供了新的见解，并为新的治疗方法提供了信息。

The proposal continues our objective to elucidate the molecular events that control acute systemic inflammation, a highly lethal process. In the previous grant cycle, we discovered a Toll like receptor (TLR) - driven, temportally-defined, epigenetic program in innate immunity macrophages and neutrophis, which directs the course of acute systemic inflammation. This process modifies chromatin structure to repress or activate distinct functional sets of genes, thus generating distinct phenotypic phases. Others and we have shown that bioenergy shifts also occur in innate immunity cells during acute systemic inflammation. This application develops the unified concept that modifications in bioenergetics integrate with epigenetics to direct the phase shifts of acute inflammation. We propose two specific aims: Aim 1) To define the connections between bio-energy and epigenetics. We will use a cell model that reproduces TLR4-dependent phase shifts to: a) delineate gene-specific chromatin modifications; b) determine protein-protein interactions; c) assess metabolic profiles by mass spectroscopy; and c) translate our concept to human normal and sepsis blood leukocytes. Aim 2) To test effects of modifying bio-energy and epigenetic shift on clinical outcomes. We will us a murine model of sepsis to: a) define bio-energy and epigenetic phase shifts, using biochemical and genetic methods to analyze plasma and isolated leukocytes from spleen, peritoneum, and bone marrow; and b) use pharmacologic and genetic approaches to determine whether modifying to adaptive phase alters microvascular inflammation and survival. Our result will broadly impact the field of inflammation by providing new insight on how acute inflammation is orchestrated, and inform novel therapies.