NF-kB and Chromatin Changes in Human Sepsis

描述（由申请人提供）：人类败血症的初始促炎表型迅速被IL-1-P、TNF-a和其他先天免疫基因转录的持续抑制所取代。血液白细胞对脂多糖内毒素（LPS）的耐受性是这种获得性免疫抑制的典型表现。本提案的目的是阐明负责LPS耐受的核过程。我们最近发表了一项新的观察结果，即LPS耐受性与NF？而在人类THP-1细胞中，染色质重塑和NFKB p65/RelA:p50组装的破坏发生在IL-1-P启动子的天然DMA上，这是一种忠实于人类败血症白细胞表型的模型。我们的新初步数据为验证以下机制假设提供了理论依据：LPS对IL-1¿基因和其他被抑制的先天免疫基因耐受的启动子破坏涉及一个负反馈循环，在这个负反馈循环中，LPS诱导的RelB表达参与限制了一个有能力的转录复合物的组装。这一过程进一步依赖于IKK-a启动子功能的中断，IKK-a启动子功能干扰组蛋白H3 S10磷酸化。目的1将使用LPS响应和LPS耐受的THP-1细胞来验证我们的假设。Aim 2旨在将Aim 1中获得的机制发现转化为人类败血症特征的LPS耐受表型，最初的重点是RelB和IKK-a破坏范式。为了实现目标2，我们将从重症监护病房脓毒症患者中获得纯化的人血中性粒细胞（PMN）制剂。Aims 1或2中采用的实验方法将包括：Western blots，实时mRNA分析，THP-1细胞转染，siRNA敲低，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¿的S10的磷酸化。将评估TNF-a、IL- 8和ikB- a启动子的特异性。公共卫生相关性：本研究将确定某些免疫基因在严重的人类感染多器官衰竭期间是如何被调节的。这种感染是美国重症监护病房死亡和发病的主要原因。这项研究的结果可以指导设计新的致命感染治疗方法。

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.