NF-kB and Chromatin Changes in Human Sepsis

人类脓毒症最初的促炎表型迅速被持续抑制所取代 IL-1-P、肿瘤坏死因子-α等天然免疫基因的转录。脂多糖内毒素耐受性 这种获得性免疫抑制是血液中白细胞的典型表现。这项提议的目的是澄清 与内毒素耐受有关的核过程。我们最近发表了一项新的观察，即内毒素 耐受性与胞浆中NFxB的正常激活有关，而染色质的破坏 人THP-1中IL-1-P启动子DNA重塑与NFKB p65/relA：P50组装 细胞，一个忠实于人类败血症白细胞表型的模型。我们新的初步数据提供了理论依据 为了验证启动子破坏IL-1-(3)基因和其他基因的内毒素耐受性的机制假设 被抑制的先天免疫基因涉及一个负反馈环，在这个环中，内毒素诱导表达 RelB参与限制有能力的转录复合体的组装。这一过程进一步 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。《目标2》寻求翻译 在目标1中获得的具有人类败血症特征的内毒素耐受表型的机械性发现， 最初的重点是RelB和IKK--一种颠覆范式。为了达到目标2，我们将获得纯净的 我们重症监护病房中败血症患者的血液中性粒细胞(PMN)的制备。这个 在AIMS 1或2中采用的实验方法包括：蛋白质印迹、实时信使核糖核酸分析， THP-1细胞的转染，siRNA敲除，芯片和双芯片检测以评估RelB，IKK-a， 天然IL-1组蛋白H3K9的RrelA/p65、p50和甲基化及S10的磷酸化(3.肿瘤坏死因子-α、白介素1- 8，将对IKB-a启动子进行特异性评估。公共卫生相关性：这项研究将确定 在多器官衰竭的严重人类感染期间，某些免疫基因是如何被调节的。 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.