Immunologic Mechanisms of Alcoholic Liver Disease

本实验室一直在积极研究酒精性肝病的发病机制，重点关注PPAR γ和细胞外囊泡在酒精性肝病中的作用。 在脂肪性肝炎的鼠模型中，炎症与脂肪变性无关。 肥胖和饮酒协同促进脂肪性肝炎，中性粒细胞浸润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起着复杂的作用;因此，本研究的目的是在3个月高脂饮食（HFD）喂养加酒精狂欢（HFD+酒精狂欢）的临床相关小鼠脂肪性肝炎模型中剖析其在调节脂肪变性和中性粒细胞浸润中的作用。肝细胞特异性Pparg破坏减少了肝脏脂肪变性，但令人惊讶的是，在HFD加酒精狂欢后增加了肝脏中性粒细胞浸润。在该模型中，敲除或敲低PPAR靶基因（脂肪特异性蛋白27）可减少脂肪变性，而不影响中性粒细胞浸润。此外，肝细胞特异性缺失Pparg基因，而不是脂肪特异性蛋白27基因，显着上调肝细胞水平的趋化因子（C-X-C基序）配体1（Cxcl 1，中性粒细胞浸润的趋化因子）在HFD加酗酒喂养小鼠。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Cxcl 1的诱导。相反，用PPAR激动剂激活PPAR可减弱肝细胞中Cxcl 1的表达。棕榈酸还上调人肝细胞中白细胞介素-8（人中性粒细胞募集的关键趋化因子）的表达，分别通过与PPAR激动剂和拮抗剂的共处理来减弱和增强。最后，急性酒精狂欢显着减弱HFD诱导的肝脏过氧化物酶体增殖物激活受体的激活，这有助于上调肝Cxcl 1表达后，HFD加酒精狂欢。结论：肝脏PPAR在控制脂肪变性和中性粒细胞浸润中起相反的作用，导致脂肪变性和炎症之间的分离;急性乙醇灌胃减弱肝脏PPAR激活，随后上调肝脏CXCL 1/白细胞介素-8表达，从而加剧肝脏中性粒细胞浸润 线粒体DNA富集微粒促进慢性酒精性嗜酸性粒细胞增多症和肝毒性。 在过去的几年里，酒精性肝病研究领域的一个主要进展是发现，在长期乙醇喂养的小鼠和过度饮酒的人类受试者（EAU）中，酗酒会诱导嗜中性粒细胞和肝脏中性粒细胞浸润;然而，潜在的机制仍然不清楚。在这里，我们证明了有近期过量饮酒史的慢性EAU患者（EAU + RD）的血清线粒体DNA（mtDNA）富集微粒（MP）水平高于近期无饮酒史的EAU（EAU-RD）和健康对照组，其与循环中性粒细胞呈正相关。类似地，慢性加狂欢（E10 d + 1B）乙醇喂养的小鼠也具有显著升高的血清mtDNA富集MP水平，并激活肝脏ER应激和炎症反应。通过基因KO或抑制剂抑制ER应激可减弱乙醇诱导的富含mtDNA的MP升高、嗜中性粒细胞增多和肝损伤。来自小鼠和培养的肝细胞中蛋白激酶RNA样ER激酶（Perk）基因的肝细胞特异性缺失的研究数据表明，肝细胞是乙醇喂养后富含mtDNA的MP的主要来源。最后，从E10 d +1B喂养的小鼠中分离的富含mtDNA的MP的给药引起小鼠的嗜中性粒细胞。总之，E10 d + 1B乙醇消耗激活肝脏ER应激依赖性mtDNA富集MP释放，导致嗜中性粒细胞和肝损伤。

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.