Hypothermia enhances inflammatory cytokine expression via NF-kappa B

描述（由申请人提供）：拟议研究的目标是让费尔柴尔德博士转变为独立研究者，阐明低温对NF κ B依赖性基因表达的细胞、分子和体内影响。治疗性低温（HT）正在研究中的儿童创伤性脑损伤和新生儿缺氧缺血性脑病，有许多其他潜在的用途，这种疗法在儿科和新生儿重症监护。虽然HT在某些患者中似乎具有神经保护作用，但仍存在潜在的有害影响。费尔柴尔德博士已经表明，在临床试验中使用的范围内的HT增加了LPS刺激的人巨噬细胞中转录因子核因子κ B（NF-/κ B）的活化和促炎细胞因子TNF α、IL-1/3和IL-8的表达。当过表达时，这些细胞因子可以是细胞毒性的。在幼年小鼠中的初步实验表明，体内低温也显着改变细胞因子的产生。我们的总体目标是了解低温增强NF-/kB依赖性基因表达的机制，以便更好地预测和预防治疗性HT的不良反应。我们的初步数据表明，HT改变了关键的磷酸化和乙酰化事件，有利于协会的p65反式激活组件NF-/kB与细胞因子基因启动子染色质。在目的1中，我们将阐明改变NF-/kB p65反式激活的作用，在低温增强细胞因子的生产，使用Gal 4-p65报告基因的研究和慢病毒介导的RNA干扰。目标2中的实验将通过免疫印迹、体外磷酸酶和脱乙酰酶测定以及RNA干扰来确定关键磷酸化和乙酰化事件的改变如何有助于HT对促炎细胞因子的作用。使用染色质免疫沉淀，我们将阐明HT对磷酸化和乙酰化p65及其共激活因子和压缩因子在特定细胞因子基因启动子上的加载的影响。最后，在目标3中，我们将开发一个系统，用于研究HT对幼年转基因NF-/kB报告小鼠中NF-/kB活化和细胞因子表达的体内影响。HT作为儿童和新生儿脑损伤的神经保护疗法正在研究中，并且已经提出了HT的许多其他临床用途。最终，我们的研究可能会提供适当的适应症，协议和辅助策略，以优化接受治疗性HT患者的结果。

DESCRIPTION (provided by applicant): The goal of the proposed research is for Dr. Fairchild to transition into an independent investigator elucidating the cellular, molecular, and in vivo effects of hypothermia on NF kappa B-dependent gene expression. Therapeutic hypothermia (HT) is under investigation in children with traumatic brain injury and neonates with hypoxic ischemic encephalopathy, and there are many other potential uses of this therapy in pediatric and neonatal critical care. While HT appears to be neuroprotective in some patients, concerns remain over potential detrimental effects. Dr. Fairchild has shown that HT in the range used in clinical trials increases activation of the transcription factor nuclear factor kappa B (NF-/kB) and expression of the pro- inflammatory cytokines TNFa, IL-1/3, and IL-8 in LPS-stimulated human macrophages. When overexpressed, these cytokines can be cytotoxic. Preliminary experiments in juvenile mice show that in vivo hypothermia also significantly alters cytokine production. Our overall goal is to understand the mechanisms of hypothermic enhancement of NF-/kB-dependent gene expression in order to better anticipate and perhaps prevent adverse effects of therapeutic HT. Our preliminary data show that HT alters key phosphorylation and acetylation events which favor association of the p65 transactivation component of NF-/kB with cytokine gene promoters on chromatin. In Aim 1 we will elucidate the role of altered NF-/kB p65 transactivation in the hypothermic enhancement of cytokine production, using Gal4-p65 reporter gene studies and lentiviral mediated RNA interference. Experiments in Aim 2 will determine how alterations in critical phosphorylation and acetylation events contribute to the HT effect on pro-inflammatory cytokines, through immunoblotting, in vitro phosphatase and deacetylase assays, and RNA interference. Using chromatin immunoprecipitation, we will elucidate effects of HT on loading of phosphorylated and acetylated p65 and its coactivators and compressors on specific cytokine gene promoters. Finally, in Aim 3, we will develop a system for studying in vivo effects of HT on NF-/kB activation and cytokine expression in juvenile transgenic NF-/kB reporter mice. HT is under investigation as a neuroprotective therapy for children and neonates with brain injury, and many other clinical uses of HT have been proposed. Ultimately, our studies may provide insight into appropriate indications, protocols, and adjunct strategies to optimize outcome of patients undergoing therapeutic HT.