MFG-E8 and progression of alcohol-induced tissue injury

描述（由申请人提供）：酒精性肝病以脂肪变性、炎症、纤维化和肝硬化为特征。肠道来源的细菌产物如脂多糖（LPS）、乙醇代谢物和免疫细胞之间的相互作用有助于慢性饮酒期间肝损伤从脂肪变性到肝硬化的进展。然而，这些相互作用的分子机制及其相关效应仍然知之甚少。研究表明，正常有效的组织修复对于肝损伤后的恢复至关重要。此外，有效去除死细胞和胶原原纤维对成功的组织修复至关重要。这一过程受多种介质控制，并由巨噬细胞介导。然而，对这些介质和巨噬细胞在慢性饮酒引起的肝损伤修复中的作用机制的了解还远未完成。乳脂球egf因子8 （MFG-E8）是巨噬细胞衍生的糖蛋白。在一些主要由上皮细胞组成的器官中，它在促进组织修复和减少组织纤维化中起关键作用，但其对肝脏修复的影响尚不清楚。之前，我们和其他人已经证明严重的组织损伤通常与MFG-E8基因表达下调有关。我们的前期数据显示(a)肝脏巨噬细胞表达MFG-E8， (b)乙醇和LPS的代谢物协同抑制巨噬细胞中MFG-E8基因的表达，(c)体内炎症期间MFG-E8的缺乏导致纤维化。基于这些初步研究，我们假设除了引起肝脏脂肪变性外，酒精及其代谢物与肠道来源的细菌产物（如LPS）协同作用，调节巨噬细胞中MFG-E8基因的表达，从而影响酒精性肝病的预后。这一中心假设将在三个具体目标中得到验证：(1)研究过量饮酒是否会改变肝脏中MFG-E8的表达，如果是，研究TLR4（一种LPS受体）和肠道源性LPS在慢性饮酒改变MFG-E8基因表达中的作用。在本研究中，野生型和TLR4突变型小鼠喂食含乙醇的液体饲料，通过多粘菌素B和新霉素处理消除肠道来源的LPS，并通过实时RT-PCR和western blot检测肝脏中MFG-E8基因的表达。(2)明确乙醇和LPS代谢物调控巨噬细胞/库普弗细胞MFG-E8基因表达的分子机制。将采用标准方法分析基因表达和启动子功能。该研究将在R01DK64240的共同努力下完成。(3)研究MFG-E8水平的改变是否影响注射LPS的酒精暴露小鼠坏死性炎症性肝损伤的结局。在本研究中，将使用细胞特异性基因工程技术。总之，这些探索性数据的结果将为理解酒精和LPS与巨噬细胞中MFG-E8基因的相互作用是否以及如何促进酒精性肝损伤的进展提供见解。它们可以显著提高我们对酒精性肝病发病机制的认识。

DESCRIPTION (provided by applicant): Alcoholic liver disease is characterized by steatosis, inflammation, fibrosis and cirrhosis. Interactions among gut-derived bacterial products such as lipopolysaccharide (LPS), metabolites of ethanol, and immune cells contribute to progression of liver injury from steatosis to cirrhosis during chronic alcohol consumption. However, molecular mechanisms underlying these interactions and their associated effects remain poorly understood. It has been shown that normal and efficient tissue repair is critical for liver recovery from injury. Furthermore, efficient removal of dead cells and collagen fibrils is critical for successful tissue repair. This process is governed by several mediators and mediated by macrophages. However, mechanistic insight into the role of these mediators and macrophages in repair of liver injury caused by chronic alcohol consumption is far from complete. Milk fat globule-EGF factor 8 (MFG-E8) is a macrophage-derived glycoprotein. It plays a critical role in promoting tissue repair and diminishing tissue fibrosis in several organs that are composed largely of epithelial cells, but its impact on the liver repair remains unknown. Previously, we and others have demonstrated that severe tissue injury is often associated with down-regulation of MFG-E8 gene expression. Our pilot data showed that (a) MFG-E8 is expressed by macrophages in the liver, (b) metabolites of ethanol and LPS synergistically suppress MFG-E8 gene expression in macrophages, and (c) lack of MFG-E8 causes fibrosis during inflammation in vivo. Based on these preliminary studies, we hypothesized that in addition to causing hepatic steatosis, alcohol and its metabolites synergize with gut-derived bacterial products such as LPS to modulate MFG-E8 gene expression in macrophages, which subsequently influences the outcome of the alcoholic liver disease. This central hypothesis will be tested in three Specific Aims: (1) To examine whether excessive intake of alcohol alters MFG-E8 expression in the liver, if so, to investigate the role of TLR4 (a LPS receptor) and gut-derived LPS in alteration of MFG-E8 gene expression by chronic alcohol consumption. In this study, wild-type and TLR4 mutant mice are fed with ethanol-containing liquid diet, gut-derived LPS is eliminated by treatment with polymyxin B and neomycin, and MFG-E8 gene expression in the liver will be determined with real-time RT-PCR and western blot. (2) To define the molecular mechanism through which metabolites of ethanol and LPS modulate MFG-E8 gene expression in macrophages/Kupffer cells. The standard approach for analysis of the gene expression and promoter function will be applied. The study will be completed through efforts in conjunction with R01DK64240. (3) To study whether alteration of MFG-E8 levels influences the outcome of necroinflammatory liver injury in alcohol-exposed mice injected with LPS. In this study, the cell-specific genetic engineering technology will be used. Together, results from these exploratory data will provide insights into understanding whether and how interactions of alcohol and LPS with MFG-E8 gene in macrophages contribute to progression of alcohol-induced liver damage. They could significantly advance our knowledge about the pathogenesis of alcoholic liver disease. PUBLIC HEALTH RELEVANCE: Alcoholic liver disease affects more than 2 million people in the United States. It is a major public health concern. In this project, we will use novel research tools to investigate molecular mechanisms underlying progression of liver injury induced by chronic alcohol consumption. The results of this exploratory project will have the potential to markedly advance our understanding of underlying pathophysiological causes of progressive alcoholic liver injury. It will ultimately lead to the identification of novel molecular targets and the development of new potential therapeutic agents for patients with alcoholic liver disease.