Hypothermia enhances inflammatory cytokine expression via NF-kappa B

低温通过 NF-κ B 增强炎症细胞因子的表达

• 批准号: 7141247
• 负责人: KAREN D FAIRCHILD
• 金额: $ 13.01万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-11 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7141247
• 关键词: Lentivirus; RNA interference; acetylation; brain injury; chromatin immunoprecipitation; cryotherapy; enzyme activity; gene induction /repression; genetically modified animals; inflammation; juvenile animal; laboratory mouse; molecular dynamics; neuroprotectants; nuclear factor kappa beta; pediatrics; phosphorylation; reporter genes; therapy adverse effect; trauma

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.

描述（由申请人提供）：拟议的研究的目的是使Fairchild博士过渡到独立研究者，阐明了体温过低对NF Kappa B依赖性基因表达的细胞，分子和体内影响。治疗性体温过低（HT）正在研究脑损伤和新生儿缺血性脑病的儿童中，并且这种疗法在小儿和新生儿重症监护中还有许多其他潜在用途。尽管HT在某些患者中似乎是神经保护作用，但担心仍然存在潜在的有害影响。 Fairchild博士表明，在临床试验中使用的HT增加了转录因子核因子KAPPA B（NF-/KB）的激活，以及在LPS刺激的人类巨噬细胞中促炎性细胞因子TNFA，IL-1/3和IL-8的表达。当过表达时，这些细胞因子可能是细胞毒性的。在少年小鼠中的初步实验表明，体内体温过低也显着改变了细胞因子的产生。我们的总体目标是了解NF-/KB依赖性基因表达的低温增强的机制，以便更好地预测并可能预防治疗性HT的不良影响。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，这些事件有利于NF-/KB的p65反式激活成分与染色质素上的细胞因子基因启动子的关联。在AIM 1中，我们将使用GAL4-P65报告基因研究和慢病毒介导的RNA干扰来阐明改变NF-/Kb p65反式激活在细胞因子产生的低温增强中的作用。 AIM 2中的实验将确定临界磷酸化和乙酰化事件的改变如何通过免疫印迹，体外磷酸酶和脱乙酰基酶测定以及RNA干扰对HT对促炎细胞因子的影响。使用染色质免疫沉淀，我们将阐明HT对磷酸化和乙酰化p65的负载及其共激活剂和压缩机对特定细胞因子基因启动子的负载。最后，在AIM 3中，我们将开发一个系统，用于研究HT对少年转基因NF-/KB报告小鼠NF-/Kb激活和细胞因子表达的体内影响。 HT正在研究为儿童和脑损伤的儿童的神经保护疗法，并提出了许多其他HT临床用途。最终，我们的研究可能会深入了解适当的适应症，方案和辅助策略，以优化接受治疗性HT的患者的结果。