NF-kB and Chromatin Changes in Human Sepsis

描述(由申请人提供)：人类脓毒症最初的促炎表型迅速被IL-1-P、肿瘤坏死因子-α和其他先天免疫基因转录的持续抑制所取代。血白细胞对脂多糖内毒素(LPS)的耐受是这种获得性免疫抑制的典型表现。这项建议的目的是阐明导致内毒素耐受的核过程。我们最近发表了一项新的观察结果，即内毒素耐受与胞浆中核因子B的正常激活有关，而染色质重塑和NFKB p65/relA：P50的组装发生在人THP-1细胞中IL-1-P启动子的天然DMA上，这是一个忠实于人类脓毒症白细胞表型的模型。我们的新的初步数据为检验机械假说提供了理论基础，该假说认为启动子破坏IL-1基因和其他被抑制的先天免疫基因的内毒素耐受性涉及一个负反馈环，在该环中，内毒素诱导的RelB的表达参与限制具有活性的转录复合体的组装。这一过程还依赖于干扰组蛋白H3S10磷酸化的IKK-a启动子功能。目标1将使用内毒素反应和耐受内毒素的THP-1细胞来测试我们的假设。目的2试图将在目标1中获得的机制发现转化为表现为人类败血症特征的内毒素耐受表型，最初的重点是RelB和IKK-A破坏范式。为了达到目标2，我们将从我们重症监护病房的败血症患者那里获得人血中性粒细胞(PMN)的纯化制剂。在AIMS 1或2中采用的实验方法将包括：Western blotts、实时mRNA分析、THP-1细胞转染、siRNA敲除、芯片和双芯片分析以评估RelB、ikk-a、rela/p65、p50和组蛋白H3K9的甲基化和天然IL-1的S10的磷酸化。将评估肿瘤坏死因子-α、白介素8和IKB-a启动子的特异性。公共卫生相关性：这项研究将确定在严重的人类感染并伴有多个器官衰竭时，某些免疫基因是如何调节的。这种感染是美国重症监护病房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.