NF-kB and Chromatin Changes in Human Sepsis

人脓毒症的初始促炎表型迅速被持续抑制所取代， IL-1-β、TNF-α和其它先天免疫基因的转录。脂多糖内毒素耐受性 在血液白细胞中，典型的是这种获得性免疫抑制。本提案的目的是阐明 负责LPS耐受性的核过程。我们最近发表了一项新的观察， 耐受性与胞质中NF xB的正常激活有关，而染色质的破坏 重构和NF κ B p65/RelA：p50装配发生在人THP-1中IL-1-P启动子的天然DNA上 细胞，忠实于人脓毒症白细胞表型的模型。我们新的初步数据提供了理论依据 为了检验IL-1-β基因和其他基因的LPS耐受性中启动子破坏的机制假说， 被抑制的先天免疫基因涉及负反馈回路，其中LPS诱导的 RelB参与限制感受态转录复合物的组装。这一进程进一步 依赖于IKK-a启动子功能的中断，其干扰组蛋白H3 S10磷酸化。 目的1将使用LPS应答和LPS耐受的THP-1细胞来测试我们的前提。目标2寻求翻译 目的1中获得的机制发现与表征人脓毒症的LPS耐受表型相关， 最初的重点是RelB和IKK-一个破坏范式。为了实现目标2，我们将获得纯化的 制备人血中性粒细胞（PMN）从参与者与败血症在我们的重症监护病房。的 目的1或2中使用的实验方法包括：蛋白质印迹，真实的时间mRNA分析， 转染THP-1细胞，siRNA敲低，和ChIP和双ChIP测定以评估RelB，IKK-a， RelA/p65、p50和组蛋白H3 K9的甲基化和天然IL-1-（3. TNF-α、IL-1、IL-2、IL-3、IL-4、IL-6、IL-10、IL 8，并且评估ikB-a启动子的特异性。公共卫生相关性：这项研究将确定 在严重的人类感染导致多器官衰竭期间，某些免疫基因是如何调节的。 这类感染是美国重症监护病房死亡率和发病率的主要原因。 这项研究可能会指导致命感染的新治疗方法的设计。

The initial proinflammatory phenotype of human sepsis is rapidly replaced by sustained repression in transcription of IL-1-P, TNF-a and other innate immunity genes. Lipopolysacaride endotoxin (LPS) tolerance in blood leukocytes typifies this acquired immunosuppression. The objective of this proposal is to elucidate nuclear processes responsible of LPS tolerance. We recently published the novel observation that LPS tolerance is associated with normal activation of NFxB in the cytosol, while disruption of chromatin remodeling and NFKB p65/RelA:p50 assembly occurs on native DMA of the IL-1-P promoter in human THP-1 cells, a model faithful to the human sepsis leukocyte phenotype. Our new preliminary data provide rationale to test the mechanistic hypothesis that promoter disruption in LPS tolerance of the IL-1-(3 gene and other repressed innate immunity genes involves a negative feedback loop in which LPS inducible expression of RelB participates in limiting the assembly of a competent transcription complex. This process further depends on interuption of IKK-a promoter function, which interferes with Histone H3 S10 phosphorylation. Aim 1 will test our premise using LPS responsive and LPS tolerant THP-1 cells. Aim 2 seeks to translate mechanistic findings obtained in Aim 1 to the LPS tolerant phenotype that characterizes human sepsis, with initial focus on the RelB and IKK-a disruption paradigm. To achieve Aim 2, we will obtain purified preparations of human blood neutrophils (PMN) from participants with sepsis in our critical care units. The experimental approaches employed in Aims 1 or 2 will include: Western blots, real time mRNA analysis, transfection of THP-1 cells, siRNA knockdown, and ChIP and double ChIP assays to assess RelB, IKK-a, RelA/p65, p50 and methylation of Histone H3 K9 and phosphorylationof S10 of native IL-1-(3. The TNF-a, IL- 8, and ikB-a promoters will be assessed for specificity. Public Health Relevance: This research will identify how certain immune genes are regulated during severe human infections with failure of multiple organs. Such infections are the major cause of mortality and morbidity in intensive care units in the USA.Results from this research may guide the design of new treatments for lethal infection.