Immunologic Mechanisms of Alcoholic Liver Disease

我们实验室一直在积极研究酒精性肝病的发病机制，重点关注乙醛脱氢酶2（ALDH 2）和泼尼松龙在酒精性肝损伤中的作用，我们还开发了一种慢性加酗酒乙醇喂养的小鼠模型，该模型代表了人类酒精性脂肪性肝炎的早期阶段。 长期饮酒是全球慢性肝病的主要原因，导致肝硬化和肝细胞癌。目前，最广泛使用的酒精性肝损伤模型是自由进食含乙醇的Lieber-DeCarli液体饮食4-6周;然而，该模型在不添加二次损伤的情况下仅诱导轻度脂肪变性、血清丙氨酸转氨酶（ALT）轻微升高和很少或没有炎症。在此报告期间，我们开发了一个简单的酒精性肝损伤的小鼠模型，通过慢性乙醇喂养（10天自由口服饲料与Lieber-DeCarli乙醇液体饮食）加一个单一的酗酒乙醇喂养。这种慢性加单次饮酒的乙醇喂养协同诱导肝损伤，炎症和脂肪肝，这模拟了患者的急性慢性酒精性肝损伤。慢性酒精摄入导致血液中酒精水平升高;因此，这种简单的模型对于酒精性肝病（ALD）和其他因饮酒而受损的器官的研究非常有用。 乙醛脱氢酶2（ALDH 2）是代谢酒精代谢产生的乙醛的主要酶。大约40%至50%的东亚人携带无活性的ALDH 2基因，并在饮酒后表现出乙醛积累。然而，ALDH 2缺乏在酒精性肝损伤发病机制中的作用仍不清楚。在本报告期间，我们使用野生型（WT）和ALDH 2-/-小鼠通过使用乙醇喂养和/或四氯化碳（CCl 4）处理来检查ALDH 2在酒精性肝损伤和纤维化中的作用。与WT小鼠相比，乙醇喂养的ALDH 2-/-小鼠具有更高水平的丙二醛和乙醛（MAA）加合物和更大的肝脏炎症，具有更高的肝脏白细胞介素-6（IL-6）表达，但令人惊讶的是脂肪变性和血清ALT水平较低。在ALDH 2-/-小鼠的乙醇处理的精确切割的肝脏切片中以及从乙醇喂养的ALDH 2-/-小鼠分离的Kupffer细胞中也检测到比WT小鼠中的那些水平更高的IL-6水平。在体外孵育与MAA增强LPS介导的刺激库普弗细胞中的IL-6的产生。与这些发现一致，在乙醇喂养的ALDH 2-/-小鼠中，主要IL-6下游信号传导分子信号转导子和转录激活子3（STAT 3）的肝脏活化高于WT小鼠。肝脏STAT 3的额外缺失导致ALDH 2-/-小鼠脂肪变性和肝细胞损伤增加。最后，乙醇喂养的ALDH 2-/-小鼠比乙醇喂养的WT小鼠更容易发生CCl 4诱导的肝脏炎症和纤维化。结论：ALDH 2-/-小鼠对乙醇诱导的脂肪变性具有抗性，但易于通过库普弗细胞中MAA介导的IL-6旁分泌激活而发生炎症和纤维化。这些发现表明，ALDH 2缺乏的个体可能对脂肪变性有抵抗力，但在饮酒后容易发生肝脏炎症和纤维化。 泼尼松龙是一种皮质类固醇，已用于治疗炎症性肝脏疾病，例如自身免疫性肝炎和酒精性肝炎。然而，结果一直存在争议，泼尼松龙如何影响肝病进展仍然未知。在本报告期间，我们研究了泼尼松龙治疗对几种肝损伤模型的影响，包括刀豆球蛋白A（Con A）和半乳糖神经酰胺（-GalCer）诱导的T/NKT细胞肝炎，以及四氯化碳（CCl 4）诱导的肝毒素介导的肝炎。泼尼松龙给药减弱ConA和GalCer诱导的肝炎和全身炎症反应。在肝毒素（CCl 4）诱导的肝炎模型中，用泼尼松龙治疗小鼠也抑制了炎症反应，但令人惊讶的是，加重了肝损伤并延迟了肝修复。免疫组织化学和流式细胞术分析表明，泼尼松龙治疗抑制肝巨噬细胞和中性粒细胞浸润四氯化碳诱导的肝炎，并抑制其吞噬活性在体内和体外。巨噬细胞和/或中性粒细胞减少加重CCl 4诱导的肝损伤，并阻碍肝再生。最后，巨噬细胞和中性粒细胞中糖皮质激素受体的条件性破坏消除了泼尼松龙介导的肝毒素诱导的肝损伤加重。结论：泼尼松龙治疗可预防T/NKT细胞肝炎，但通过抑制巨噬细胞和嗜中性粒细胞介导的吞噬和再生功能，加剧了肝毒素诱导的肝损伤。这些发现不仅可以增加我们对类固醇治疗机制的理解，还可以帮助我们更好地管理肝脏疾病的类固醇治疗。 此外，我们还与来自NIAAA的乔治库诺斯和帕切尔博士合作，研究内源性大麻素系统在酒精性肝病中的作用。

Our laboratory has been actively studying the pathogenesis of alcoholic liver disease, focusing on the role of acetaldehyde dehydrogenase 2 (ALDH2) and prednisolone in alcoholic liver injury, and we have also developed a mouse model of chronic plus binge ethanol feeding model, which represents early stages of human alcoholic steatohepatitis. Chronic alcohol consumption is a leading cause of chronic liver disease worldwide, leading to cirrhosis and hepatocellular carcinoma. Currently, the most widely used model for alcoholic liver injury is ad libitum feeding with the Lieber-DeCarli liquid diet containing ethanol for 4-6 weeks; however, this model, without the addition of a secondary insult, only induces mild steatosis, slight elevation of serum alanine transaminase (ALT) and little or no inflammation. During this reporting period, we developed a simple mouse model of alcoholic liver injury by chronic ethanol feeding (10-d ad libitum oral feeding with the Lieber-DeCarli ethanol liquid diet) plus a single binge ethanol feeding. This chronic-plus-single-binge ethanol feeding synergistically induces liver injury, inflammation and fatty liver, which mimics acute-on-chronic alcoholic liver injury in patients. Chronic-binge ethanol feeding leads to high blood alcohol levels; thus, this simple model will be very useful for the study of alcoholic liver disease (ALD) and of other organs damaged by alcohol consumption. Aldehyde dehydrogenase 2 (ALDH2) is the major enzyme that metabolizes acetaldehyde produced from alcohol metabolism. Approximately 40 to 50% of East Asians carry an inactive ALDH2 gene and exhibit acetaldehyde accumulation after alcohol consumption. However, the role of ALDH2 deficiency in the pathogenesis of alcoholic liver injury remains obscure. During this reporting period, we have used wild-type (WT) and ALDH2-/- mice to examine the roles of ALDH2 in alcoholic liver injury and fibrosis by using ethanol feeding and/or carbon tetrachloride (CCl4) treatment. Compared with WT mice, ethanol-fed ALDH2-/- mice had higher levels of malondialdehyde and acetaldehyde (MAA) adduct and greater hepatic inflammation, with higher hepatic interleukin-6 (IL-6) expression but surprisingly lower levels of steatosis and serum ALT. Higher IL-6 levels were also detected in ethanol-treated precision-cut-liver slices from ALDH2-/- mice and in Kupffer cells isolated from ethanol-fed ALDH2-/- mice than those levels in WT mice. In vitro incubation with MAA enhanced the LPS-mediated stimulation of IL-6 production in Kupffer cells. In agreement with these findings, hepatic activation of the major IL-6 downstream signaling molecule signal transducer and activator of transcription 3 (STAT3) was higher in ethanol-fed ALDH2-/- mice than in WT mice. An additional deletion of hepatic STAT3 resulted in increased steatosis and hepatocellular damage in ALDH2-/- mice. Finally, ethanol-fed ALDH2-/- mice were more prone to CCl4-induced liver inflammation and fibrosis than ethanol-fed WT mice. CONCLUSIONS: ALDH2-/- mice are resistant to ethanol-induced steatosis but prone to inflammation and fibrosis via MAA-mediated paracrine activation of IL-6 in Kupffer cells. These findings suggest that ALDH2-deficient individuals may be resistant to steatosis, but are prone to liver inflammation and fibrosis following alcohol consumption. Prednisolone is a corticosteroid that has been used to treat inflammatory liver diseases, such as autoimmune hepatitis and alcoholic hepatitis. However, the results have been controversial, and how prednisolone affects liver disease progression remains unknown. During this reporting period, we examined the effect of prednisolone treatment on several models of liver injury, including T/NKT cell hepatitis induced by concanavalin A (Con A) and -galactosylceramide (-GalCer), and hepatotoxin-mediated hepatitis induced by carbon tetrachloride (CCl4). Prednisolone administration attenuated ConA- and -GalCer-induced hepatitis and systemic inflammatory responses. Treating mice with prednisolone also suppressed inflammatory responses in a model of hepatotoxin (CCl4)-induced hepatitis, but surprisingly exacerbated liver injury and delayed liver repair. Immunohistochemical and flow cytometric analyses demonstrated that prednisolone treatment inhibited hepatic macrophage and neutrophil infiltration in CCl4-induced hepatitis and suppressed their phagocytic activities in vivo and in vitro. Macrophage and/or neutrophil depletion aggravated CCl4-induced liver injury and impeded liver regeneration. Finally, conditional disruption of glucocorticoid receptor in macrophages and neutrophils abolished prednisolone-mediated exacerbation of hepatotoxin-induced liver injury. Conclusion: Prednisolone treatment prevents T/NKT cell hepatitis but exacerbates hepatotoxin-induced liver injury by inhibiting macrophage- and neutrophil-mediated phagocytic and regenerative functions. These findings may not only increase our understanding of the steroid treatment mechanism but also help us to better manage steroid therapy in liver diseases. In addition, we are also collaborating with Drs. George Kunos and Pal Pacher from NIAAA to investigate the role of the endocannabinoid system in alcoholic liver disease.