Alcohol, ROS, and Macrophage Epigenetics

描述(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证据表明，急性乙醇导致巨噬细胞肿瘤坏死因子-α(TNF)分泌抑制，从而导致免疫抑制。然而，慢性乙醇与刺激的肿瘤坏死因子反应增强有关，这与肝脏损伤有关。巨噬细胞系和Kupffer细胞的初步数据已经证实，慢性乙醇通过氧化还原依赖的表观遗传机制增强了肿瘤坏死因子的转录，以响应乙醇。这在一定程度上是由于增加了核转录因子-kB的核转位。然而，使用染色质免疫沉淀分析的初步研究也表明，AP-1家族成员FosB和干扰素调节剂-3与肿瘤坏死因子启动子的结合增加，这种结合对于增强肿瘤坏死因子转录是至关重要的。此外，初步数据表明，慢性酒精还导致转化生长因子-β对肿瘤坏死因子-α转录的缺陷抑制，部分原因是在慢性酒精环境中存在缺陷的Smad3/4核转位。 基于这些数据，我们假设慢性酒精暴露通过改变细胞的氧化还原状态导致巨噬细胞中肿瘤坏死因子转录增加，从而导致AP-1和IRF-3与肿瘤坏死因子启动子结合增加，以及降低作为肿瘤坏死因子抑制因子的Smad3和4的核水平。我们将通过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与人肿瘤坏死因子启动子结合的氧化还原依赖性增加，导致肿瘤坏死因子基因转录增加。2.探讨Smad3和Smad4的抑制缺失与慢性乙醇所致的肿瘤坏死因子产生增加有关的假说。3.探讨AP-1、Trif/IRF-3、Smad3和Smad4在调节慢性酒精中毒小鼠单个核细胞产生肿瘤坏死因子中的作用。通过了解乙醇对肿瘤坏死因子转录的复杂调控，我们希望促进对酒精对免疫系统影响的了解，并加强对酒精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.