The Role of IL-17 in Alcoholic Liver Disease and Cancer

描述（由申请人提供）：酒精性肝病（ALD）从正常肝脏发展为酒精性脂肪性肝炎、纤维化和肝细胞癌（HCC）。尽管有大量的研究，但由于缺乏模拟ALD进展阶段的动物模型，对ALD的发病机制知之甚少。此外，IL-17在ALD中的作用尚未评估。我们最近已经证明，IL-17信号在肝纤维化和癌症的发展中起着关键作用。根据我们的初步数据，ALD也需要IL-17信号传导，缺乏IL-17信号传导的小鼠发生脂肪性肝炎和纤维化较少。在这里，我们建议使用改良的小鼠ALD模型进一步探索IL-17在ALD中的作用，该模型是Karin，Gao，Tsukamoto和Kisseleva博士合作开发的。该模型最接近地再现了ALD的阶段，并反映了患者从脂肪性肝炎到酒精诱导的肝纤维化和HCC的生理ALD进展。我们的中心假设是IL-17加剧了ALD从脂肪性肝炎到纤维化和HCC的进展。这项研究的目的是确定阻断IL-17的策略是否对ALD有治疗作用。在本研究中，将使用IL-17-/-、IL-17 RA-/-和几种细胞特异性敲除小鼠来测试IL-17信号传导在ALD中的作用以及IL-17调节的途径， 确定IL- 17信号传导的作用：1）模拟脂肪性肝炎早期阶段的慢性暴食模型（AIM 1），2）模拟酒精性脂肪性肝炎和纤维化的胃内乙醇喂养模型（AIM 2），和3）模拟酒精性肝癌的二乙基亚硝胺（DEN）+酒精模型（AIM 3）。具体而言，这些模型将使我们能够在ALD的不同阶段剖析特定的IL-17功能。因此，对于每种模型，1）将通过测量酒精代谢酶脱氢酶（ADH）、细胞色素、P4502 E1（CYP 2 E1）、成脂基因（PPARγ、PPARα、CEBP 1）的表达、脂质过氧化、ROS产生来确定酒精诱导的肝损伤的发生。2)将确定IL-17在炎症细胞募集、枯否细胞/巨噬细胞活化中的作用。3)将鉴定IL-17的细胞来源。4)将确定关键介导ALD进展的IL-17靶细胞，分离特定细胞类型并进行离体分析。5)我们将在枯否细胞、肝细胞和肝星状细胞（HSC）中产生条件性IL-17 RA-/-敲除小鼠，以研究它们对ALD的贡献。6)将确定每种类型的敲除小鼠的总体细胞因子表达谱。7)将确定IL-23、IL-25和IL-27细胞因子在Th 17细胞（或其它产生IL-17的细胞）的IL-17中的调节作用。这些独特的遗传学研究将提供IL-17抑制剂作为治疗ALD患者的新方法的有用性的见解。

DESCRIPTION (provided by applicant): Alcoholic liver disease (ALD) progresses from a normal liver, to alcoholic steatohepatitis, fibrosis and hepatocellular carcinoma (HCC). Despite of intensive studies, the pathogenesis of ALD is poorly understood due to the lack of animal models which mimic the stages of ALD progression. Furthermore, the role of IL-17 in ALD has not been evaluated. We have recently demonstrated that IL-17 signaling plays a critical role in development of liver fibrosis and cancer. Based on our preliminary data, IL-17 signaling also required for ALD, and mice devoid of IL-17 signaling develop less steatohepatitis and fibrosis. Here we propose to further explore the role of IL-17 in ALD, using an improved model of ALD in mice, which was developed as a result of a collaborative effort of Drs. Karin, Gao, Tsukamoto and Kisseleva. This model most closely reproduces the stages of ALD and reflects physiological ALD progression from steatohepatitis to alcohol-induced liver fibrosis and HCC in patients. Our central hypothesis is that IL-17 exacerbates progression of ALD from steatohepatitis to fibrosis and HCC. The goal of the study is to determine if the strategy of blocking IL-17 will have a therapeutic effect on ALD. The role of IL-17 signaling in ALD, and pathways of IL-17 regulation will be tested in this study using IL-17-/-, IL-17RA-/-, and several cell-specific knockout mice to determine the role of IL- 17 signaling 1) chronic-binge model that mimics early stages of steatohepatitis (AIM 1), 2) intragastric ethanol feeding model that mimics alcoholic steatohepatitis and fibrosis (AIM 2), and 3) diethylnitrosamine (DEN)+alcohol model that mimics alcoholic liver cancer (AIM 3). Specifically, these models will allow us to dissect specific IL-17 functions at different stages of ALD. Thus, for each model, 1) development of alcohol- induced liver injury will be determined by measuring alcohol-metabolizing enzymes dehydrogenase (ADH), cytochrome, P4502E1 (CYP2E1), expression of adipogenic genes (PPARγ, PPARα, CEBP1), lipid peroxidation, ROS production. 2) The role of IL-17 in recruitment of inflammatory cells, Kupffer cells/macrophage activation will be determined. 3) The cellular sources of IL-17 will be identified. 4) The IL-17 target cells that critically mediate ALD progression will be determined, specific cell types will be isolated and ex vivo analyzed. 5) We will generate conditional IL-17RA-/- knockout mice in Kupffer cells, hepatocytes and Hepatic Stellate Cells (HSCs) to study their contribution to ALD. 6) The global cytokine expression profile will determined for each type of knockout mice. 7) The regulatory role of IL-23, IL-25 and IL-27 cytokines in IL-17 by Th17 cells (or other IL-17 producing cells) will be determined. These unique genetic studies will provide an insight into usefulness of IL-17 inhibitors as a novel approach to treat ALD in patients.