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，重点研究PPARγ和细胞外小泡在酒精性肝病中的作用。 在脂肪性肝炎的小鼠模型中，炎症与脂肪变性无关。 肥胖和饮酒协同促进脂肪性肝炎，中性粒细胞渗透被认为与脂肪变性有关。然而，潜在的机制仍然不清楚。过氧化物酶体增殖物激活受体γ(PPAR)在脂质代谢和炎症中扮演着复杂的角色；因此，本研究的目的是在临床相关的高脂饮食(HFD)喂养+酗酒(HFD+酗酒)的临床相关小鼠脂肪性肝炎模型中分析PPAR在调节脂肪变性和中性粒细胞渗透中的作用。肝细胞特异性PPARG阻断减少了肝脏脂肪变性，但令人惊讶的是，在HFD加狂饮酒精后，肝脏中性粒细胞浸润增加。在这个模型中，PPAR靶基因脂肪特异性蛋白27的敲除或敲除可以减少脂肪变性，而不影响中性粒细胞的渗透。此外，肝细胞特异性PPARG基因的缺失，而不是脂肪特异性蛋白27基因的缺失，显著上调了高脂饮食加酗酒小鼠肝脏趋化因子(C-X-C基序)配体1(CXCL1，一种促进中性粒细胞渗透的趋化因子)基因的水平。在体外，PPARG基因的缺失也高度增强了棕榈酸或肿瘤坏死因子α对小鼠肝细胞中CXCL1的诱导。相反，用PPAR激动剂激活PPAR可减弱肝细胞中CXCL1的表达。棕榈酸还上调人肝细胞白介素8(人中性粒细胞募集的关键趋化因子)的表达，该作用分别被PPAR激动剂和拮抗剂共同处理而减弱和增强。最后，急性酗酒显著减弱HFD诱导的肝脏PPAR的激活，这有助于在HFD加酗酒后上调肝脏CXCL1的表达。结论：肝脏PPAR在控制脂肪变性和中性粒细胞浸润方面起着相反的作用，导致脂肪变性和炎症之间的分离；急性乙醇灌胃抑制肝脏PPAR的激活，进而上调肝脏CXCL1/IL-8的表达，从而加剧肝脏中性粒细胞的浸润。 线粒体DNA丰富的微粒促进急性-慢性酒精中性粒细胞增多和肝毒性。 在过去的几年里，酒精性肝病研究领域的主要进展之一是发现酗酒导致慢性酒精喂养的小鼠和过度饮酒的人(EAU)的中性粒细胞增多和肝脏中性粒细胞的浸润；然而，其潜在的机制仍然不清楚。在这里，我们证明了有近期过度饮酒史的慢性EAU患者(EAU+RD)的血清线粒体DNA(MtDNA)丰富的微粒(MPS)水平高于不饮酒的EAU(EAU-RD)和健康对照组，这与循环中的中性粒细胞呈正相关。同样，长期暴饮暴食(E10d+1B)酒精的小鼠血清mtDNA丰富的MPS水平也显著升高，肝脏ER应激和炎症反应被激活。用KO基因或抑制剂抑制内质网应激可减轻乙醇诱导的mtDNA丰富的MPS升高、中性粒细胞增多和肝损伤。小鼠肝细胞特异性缺失蛋白激酶类核糖核酸激酶(PERK)基因和培养肝细胞的研究结果表明，乙醇喂养后肝细胞是mtDNA富含MPS的主要来源。最后，给予从E10d+1B喂养的小鼠中分离出来的mtDNA浓缩的MPS会引起小鼠的中性粒细胞增多。综上所述，E10d+1B酒精摄入可激活肝脏内质网应激依赖的线粒体DNA富集型MP的释放，导致中性粒细胞增多和肝损伤。

Our laboratory has been actively studying the pathogenesis of alcoholic liver disease, focusing on the role of PPAR gamma and extracellular vesicle in alcoholic liver disease. Inflammation is independent of steatosis in a murine model of steatohepatitis. Obesity and alcohol consumption synergistically promote steatohepatitis, and neutrophil infiltration is believed to be associated with steatosis. However, the underlying mechanisms remain obscure. Peroxisome proliferator-activated receptor gamma (PPAR) plays a complex role in lipid metabolism and inflammation; therefore, the purpose of this study was to dissect its role in regulating steatosis and neutrophil infiltration in a clinically relevant mouse steatohepatitis model of 3-month high-fat diet (HFD) feeding plus a binge of ethanol (HFD-plus-binge ethanol). Hepatocyte-specific Pparg disruption reduced liver steatosis but surprisingly increased hepatic neutrophil infiltration after HFD-plus-binge ethanol. Knockout or knockdown of the PPAR target gene, fat-specific protein 27, reduced steatosis without affecting neutrophil infiltration in this model. Moreover, hepatocyte-specific deletion of the Pparg gene, but not the fat-specific protein 27 gene, markedly up-regulated hepatic levels of the gene for chemokine (C-X-C motif) ligand 1 (Cxcl1, a chemokine for neutrophil infiltration) in HFD-plus-binge ethanol-fed mice. In vitro, deletion of the Pparg gene also highly augmented palmitic acid or tumor necrosis factor alpha induction of Cxcl1 in mouse hepatocytes. In contrast, activation of PPAR with a PPAR agonist attenuated Cxcl1 expression in hepatocytes. Palmitic acid also up-regulated interleukin-8 (a key chemokine for human neutrophil recruitment) expression in human hepatocytes, which was attenuated and enhanced by cotreatment with a PPAR agonist and antagonist, respectively. Finally, acute ethanol binge markedly attenuated HFD-induced hepatic PPAR activation, which contributed to the up-regulation of hepatic Cxcl1 expression post-HFD-plus-binge ethanol. CONCLUSION: Hepatic PPAR plays an opposing role in controlling steatosis and neutrophil infiltration, leading to dissociation between steatosis and inflammation; acute ethanol gavage attenuates hepatic PPAR activation and subsequently up-regulates hepatic CXCL1/interleukin-8 expression, thereby exacerbating hepatic neutrophil infiltration Mitochondrial DNA-enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity. Over the last several years, one of the major advances in the field of alcoholic liver disease research was the discovery that binge alcohol consumption induced neutrophilia and hepatic neutrophil infiltration in chronically ethanol-fed mice and human subjects with excessive alcohol use (EAU); however, the underlying mechanisms remain obscure. Here, we demonstrated that chronic EAU patients with a history of recent excessive drinking (EAU + RD) had higher serum levels of mitochondrial DNA (mtDNA)-enriched microparticles (MPs) than EAU without recent drinking (EAU - RD) and healthy controls, which correlated positively with circulating neutrophils. Similarly, mice with chronic-plus-binge (E10d + 1B) ethanol feeding also had markedly elevated serum levels of mtDNA-enriched MPs, with activation of hepatic ER stress and inflammatory responses. Inhibition of ER stress by gene KO or inhibitors attenuated ethanol-induced elevation of mtDNA-enriched MPs, neutrophilia, and liver injury. The data from the study of hepatocyte-specific deletion of the protein kinase RNA-like ER kinase (Perk) gene in mice and of cultured hepatocytes demonstrated that hepatocytes were the main source of mtDNA-enriched MPs after ethanol feeding. Finally, administration of mtDNA-enriched MPs isolated from E10d+1B-fed mice caused neutrophilia in mice. In conclusion, E10d + 1B ethanol consumption activates hepatic ER stress-dependent mtDNA-enriched MP release, leading to neutrophilia and liver injury.