MFG-E8 and progression of alcohol-induced tissue injury

描述(申请人提供)：酒精性肝病的特征是脂肪变性、炎症、纤维化和肝硬变。在慢性饮酒过程中，肠源性细菌产物如脂多糖、乙醇代谢产物和免疫细胞之间的相互作用导致了肝损伤从脂肪变性到肝硬变的进展。然而，这些相互作用及其相关效应背后的分子机制仍然知之甚少。已有研究表明，正常有效的组织修复是肝脏损伤后恢复的关键。此外，有效地去除死亡细胞和胶原纤维对于成功的组织修复至关重要。这一过程由多个介质控制，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所起作用的机械性认识还远未完成。乳脂球-表皮生长因子8(MFG-E8)是一种巨噬细胞来源的糖蛋白。在主要由上皮细胞组成的几个器官中，它在促进组织修复和减轻组织纤维化方面发挥着关键作用，但它对肝脏修复的影响尚不清楚。此前，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。我们的实验数据表明：(A)MFG-E8在肝脏中由巨噬细胞表达，(B)乙醇和内毒素的代谢产物协同抑制巨噬细胞中MFG-E8基因的表达，以及(C)体内炎症过程中缺乏MFG-E8会导致纤维化。基于这些初步研究，我们假设酒精及其代谢产物除了引起肝脏脂肪变性外，还与肠道细菌产物(如脂多糖)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转归。这一中心假说将通过三个特定的目的得到验证：(1)检测过量饮酒是否改变肝脏MFG-E8的表达，如果是，则研究TLR4(一种内毒素受体)和肠源性内毒素在慢性饮酒改变MFG-E8基因表达中的作用。本研究用含乙醇的液体饲料喂养野生型和TLR4突变小鼠，用多粘菌素B和新霉素处理消除肠源性内毒素，用实时荧光定量RT-PCR和Western印迹法检测MFG-E8基因在肝脏中的表达。(2)探讨乙醇和内毒素代谢产物调节巨噬细胞/枯否细胞MFG-E8基因表达的分子机制。将采用分析基因表达和启动子功能的标准方法。这项研究将通过与R01DK64240的共同努力完成。(3)研究MFG-E8水平的变化是否影响内毒素酒精中毒小鼠坏死性炎性肝损伤的转归。在这项研究中，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内毒素与巨噬细胞中MFG-E8基因的相互作用是否以及如何促进酒精诱导的肝损伤的进展提供洞察力。它们可以极大地提高我们对酒精性肝病发病机制的认识。 公共卫生相关性：酒精性肝病在美国影响着200多万人。这是一个重大的公共卫生问题。在这个项目中，我们将使用新的研究工具来研究慢性饮酒导致肝损伤进展的分子机制。这一探索性项目的结果将有可能显著促进我们对进行性酒精性肝损伤潜在的病理生理原因的理解。这将最终导致新的分子靶点的确定和新的潜在的酒精性肝病治疗药物的开发。

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.