Role of the Gut Microbiota and TLR4 in Alcoholic Hepatocarcinogenesis

描述（由申请人提供）：肝细胞癌（HCC）是第五大常见癌症，全球每年约有500，000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和非酒精性脂肪肝疾病诱导的HCC增加，但酒精仍然被认为是HCC发展的主要危险因素。已经确定的是，（i）LPS水平在酒精性肝病（ALD）的所有阶段都高度升高，并且（ii）ALD早期阶段的炎症和损伤主要由脂多糖（LPS）及其受体TLR 4介导。近年来，炎症通路如NF-kB、IL-1、IL-6、α-光毒素和β-光毒素已被确定为非酒精性肝癌发生的关键因素。我们推测炎症信号在酒精性肝癌发生中起重要作用，LPS和TLR 4在ALD中作为炎症驱动的增殖和肝癌发生的关键促进剂。我们假设LPS和TLR 4代表炎性前致癌信号级联的最上游调节因子，如NF-κ B、JNK、IL-1、IL-6、IL-17、α-光敏素和TNF α。本申请的长期目标是建立LPS和TLR 4作为促进酒精性肝癌发生的炎症性肠-肝轴的关键贡献者，并开发预防或治疗酒精性HCC的新概念。将在接受引发剂量的二乙基亚硝胺（DEN）和随后的含酒精饮食（AIM 1）的TLR 4ko和wt小鼠中测试TLR 4在酒精性肝癌发生中的作用。肠道微生物群对酒精性HCC的贡献将在用DEN和含酒精饮食处理的肠道灭菌和无菌小鼠中进行评估（AIM 2）。LPS和TLR 4促进酒精性HCC的靶细胞和分子机制将在骨髓嵌合小鼠和库普弗细胞、肝细胞或肝星状细胞（AIM 3）中条件性缺失TLR 4的小鼠中确定。TLR 4的下游靶标将通过微阵列在全肝和分离的细胞群中鉴定，并且候选基因如IL-6、IL-1、TNF、LTα、LT β或IL-17的功能参与将在敲除小鼠中研究。这项研究的结果可能会建立一个重要的贡献LPS和TLR 4的炎症和前致癌肠肝轴途径在ALD，并可能导致新的治疗策略的发展，针对肠道微生物群，TLR 4或特定的下游介质。

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.