NF-kappaB and Chromatin Changes in Human Sepsis

描述（由申请人提供）：人脓毒症的初始促炎表型迅速被IL-1-P、TNF-α和其他先天免疫基因转录的持续抑制所取代。血液白细胞中的脂多糖内毒素（LPS）耐受性代表这种获得性免疫抑制。该建议的目的是阐明负责LPS耐受性的核过程。我们最近发表了新的观察，LPS耐受性与NF？B在胞质溶胶中，而染色质重塑和NF κ B p65/RelA：p50装配的破坏发生在人THP-1细胞中IL-1-P启动子的天然DMA上，这是一种忠实于人脓毒症白细胞表型的模型。我们的新的初步数据提供了基本原理，以测试的机制假设，启动子破坏LPS耐受性的IL-1基因和其他抑制先天免疫基因涉及一个负反馈回路，其中LPS诱导的表达RelB参与限制组装的一个主管转录复合物。该过程进一步依赖于IKK-a启动子功能的干扰，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。目的2旨在将目的1中获得的机制发现转化为表征人类脓毒症的LPS耐受表型，最初关注RelB和IKK-破坏范例。为了实现目标2，我们将从重症监护病房的脓毒症患者中获得人血中性粒细胞（PMN）的纯化制剂。在目标1或2中采用的实验方法将包括：蛋白质印迹、真实的时间mRNA分析、THP-1细胞的转染、siRNA敲低、以及ChIP和双ChIP测定以评估RelB、IKK-a、RelA/p65、p50和组蛋白H3 K9的甲基化以及天然IL-1的S10的磷酸化。将评估TNF-α、IL- 8和ikB-a启动子的特异性。公共卫生相关性：这项研究将确定某些免疫基因在严重的人类感染与多个器官衰竭期间是如何调节的。在美国，此类感染是重症监护病房中死亡和发病的主要原因。这项研究的结果可能会指导致命感染的新治疗方法的设计。

DESCRIPTION (provided by applicant): 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 NF?B 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¿ 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¿. 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.