Alcohol, ROS, and Macrophage Epigenetics

描述（由申请人提供）：乙醇是美国最常见的滥用物质，也是细菌性肺炎等感染的重要风险因素，细菌性肺炎是酗酒者死亡的主要原因。我们和其他人已经积累了大量的证据表明，急性乙醇诱导抑制巨噬细胞肿瘤坏死因子-α（TNF）的分泌，从而导致免疫抑制。然而，慢性乙醇与刺激的TNF反应的增强有关，这与肝损伤有关。在巨噬细胞系以及枯否细胞中的初步数据已经确定，慢性EtOH通过氧化还原依赖性表观遗传机制增强响应于EtOH的TNF转录。部分原因是NF-kB的核转位增加。然而，使用染色质免疫沉淀测定的初步研究也显示AP-1家族成员FosB和干扰素调节-3与TNF启动子的结合增加，并且这种结合对于增强的TNF转录是关键的。此外，初步数据表明，慢性乙醇也导致有缺陷的TGF-β抑制TNF-α转录，部分原因是由于有缺陷的Smad 3/4核转位在慢性乙醇的设置。 基于这些数据，我们假设慢性乙醇暴露通过改变细胞氧化还原状态导致AP-1和IRF-3与TNF启动子的结合增加以及作为TNF阻遏物的Smad 3和4的核水平降低而导致巨噬细胞中TNF转录增加。我们将用以下具体目标来检验这一假设。1.1.我们的假设预测，慢性乙醇导致AP-1和IRF-3与人TNF启动子结合的氧化还原依赖性增加，导致TNF基因转录增加。2.研究Smad 3和4抑制的丧失导致慢性乙醇增加TNF产生的假设。3.研究AP-1、Trif/IRF-3和Smad 3和4在慢性乙醇小鼠模型中调节单核细胞TNF产生增加的作用。通过了解乙醇对TNF转录的复杂调节，我们希望能够进一步了解乙醇对免疫系统的影响，并增强乙醇相关疾病的潜在治疗策略。

DESCRIPTION (provided by applicant): Ethanol is the most common substance of abuse in the US and an important risk-factor for infections such as bacterial pneumonias which are a leading cause of mortality in alcoholics. We, and others, have accumulated a body of evidence that acute ethanol induces suppression of macrophage tumor necrosis factor-alpha (TNF) secretion, which leads to immunosuppression. Chronic ethanol however, has been associated with an augmentation of stimulated TNF responses, which is associated with liver injury. Preliminary data in macrophage cell lines as well as Kupffer cells has identified that chronic EtOH trough a redox dependent epigenetic mechanism augments TNF transcription in response to EtOH. In part this is due to increase nuclear translocation of NF-kB. However preliminary studies using chromatin immunoprecipitation assays also show increased binding of the AP-1 family member FosB and interferon regulatory-3 to the TNF promoter and this binding is critical for augmented TNF transcription. Moreover, preliminary data suggest that chronic ethanol also results in defective TGF-beta repression of TNF-alpha transcription in part due to defective Smad3/4 nuclear translocation in the setting of chronic ethanol. Based on these data, we hypothesize that chronic ethanol exposure results in increased TNF transcription in macrophages by altering the cellular redox state which results in increased binding of both AP-1 and IRF-3 to the TNF promoter as well as reducing the nuclear level of Smad 3 and 4 which function as a TNF represser. We will test this hypothesis with the following Specific Aims. 1.1. Our hypothesis predicts that chronic ethanol results in a redox-dependent increase in AP-1 and IRF-3 binding to the human TNF promoter resulting in increased transcription of the TNF gene. 2. Investigate the hypothesis that loss of repression by Smad 3 and 4 contributes to increased TNF production by chronic ethanol. 3. Investigate the contributions of AP-1, Trif/IRF-3, and Smad 3 and 4 in regulating increased mononuclear cell TNF production in a mouse model of chronic ethanol. By understanding the complex regulation of ethanol on TNF transcription, we hope to advance the understanding of ethanol's effect on the immune system and enhance potential therapeutic strategies for ethanol related diseases.