Role of the Gut Microbiota and TLR4 in Alcoholic Hepatocarcinogenesis

描述（由申请人提供）：肝细胞癌（HCC）是全球第五大常见癌症，每年造成约50万人死亡。在美国，HCC的发病率在过去三十年中几乎翻了一番。尽管HCV和非酒精性脂肪性肝病引起的HCC增加，酒精仍然被认为是HCC发展的主要危险因素。现已确定：(i)脂多糖水平在酒精性肝病（ALD）的所有阶段都高度升高，（ii）早期ALD的炎症和损伤主要由脂多糖（LPS）及其受体TLR4介导。最近，NF-kB、IL-1、IL-6和淋巴蛋白α和β等炎症通路已被确定为非酒精性肝癌发生的关键因素。我们假设炎症信号在酒精性肝癌发生中起重要作用，LPS和TLR4是ALD中炎症驱动增殖和肝癌发生的关键促进因子。我们假设LPS和TLR4代表炎症前癌信号级联的最上游调节因子，如NF-kB、JNK、IL-1、IL-6、IL-17、淋巴毒素α和β。本应用的长期目标是建立LPS和TLR4作为促进酒精性肝癌发生的炎症性肠-肝轴的关键因素，并为预防或治疗酒精性HCC开发新的概念。TLR4在酒精性肝癌发生中的作用将在TLR4ko和wt小鼠中进行测试，这些小鼠接受了启动剂量的二乙基亚硝胺（DEN）和随后的含酒精饮食（AIM 1）。肠道微生物群对酒精性HCC的贡献将被评估，用DEN和含酒精的饮食治疗肠道消毒和无菌小鼠（AIM 2）。LPS和TLR4促进酒精性HCC的靶细胞和分子机制将在Kupffer细胞、肝细胞或肝星状细胞中TLR4条件缺失的骨髓嵌合小鼠和小鼠中确定（AIM 3）。TLR4的下游靶点将在全肝和分离细胞群中通过芯片识别，并在敲除小鼠中研究候选基因如IL-6、IL-1、TNF、LTα、LTß或IL-17的功能参与。本研究的结果可能会确立LPS和TLR4在ALD炎症和前癌性肠-肝轴通路中的重要作用，并可能导致针对肠道微生物群、TLR4或特定下游介质的新型治疗策略的发展。

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) is the fifth most common cancer causing about 500,000 deaths/year world-wide. In the US, the incidence of HCC has almost doubled over the last three decades. Despite increases in HCV- and non-alcoholic fatty liver disease-induced HCC, alcohol is still considered a leading risk factor for the development of HCC. It is well established that (i) LPS levels are highly elevated in all stages of alcoholic liver disease (ALD), and that (ii) inflammation and injury in early stages of ALD are largely mediated by lipopolysaccharide (LPS) and its receptor TLR4. Recently, inflammatory pathways such as NF-kB, IL-1, IL-6 and lymphotoxin α and ß have been identified as key contributors to non-alcoholic hepatocarcinogenesis. We hypothesize that inflammatory signals play an essential role in alcoholic hepatocarcinogenesis, and that LPS and TLR4 act as key promoters of inflammation-driven proliferation and hepatocarcinogenesis in ALD. We hypothesize that LPS and TLR4 represent the most upstream regulators of inflammatory procarcinogenic signaling cascades such as NF-kB, JNK, IL-1, IL-6, IL-17, lymphotoxin α and ß. The long-term goal of this application is to establish LPS and TLR4 as key contributors to an inflammatory gut-liver axis that promotes alcoholic hepatocarcinogenesis, and to develop new concepts for the prevention or treatment of alcoholic HCC. The role of TLR4 in alcoholic hepatocarcinogenesis will be tested in TLR4ko and wt mice subjected to a priming dose of diethylnitrosamine (DEN) and subsequent alcohol-containing diets (AIM 1). The contribution of the gut microbiota to alcoholic HCC will be assessed gut-sterilized and germ-free mice treated with DEN and alcohol-containing diets (AIM 2). Target cells and molecular mechanisms by which LPS and TLR4 promote alcoholic HCC will be determined in bone marrow-chimeric mice and mice with conditional deletion of TLR4 in Kupffer cells, hepatocytes or hepatic stellate cells (AIM 3). Downstream targets of TLR4 will be identified by microarray in whole liver and isolated cell populations, and the functional involvement of candidate genes such as IL-6, IL-1, TNF, LTα, LTß or IL-17 will be investigated in knockout mice. Results from this study are likely to establish an important contribution of LPS and TLR4 to an inflammatory and procarcinogenic gut-liver axis pathway in ALD, and may lead to the development of novel therapeutic treatment strategies targeting the gut microbiota, TLR4 or specific downstream mediators.