Pyroptosis by Caspase11/4-Gasdermin-D Pathway in Alcoholic Hepatitis. Mus musculus

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH, is largely unknown. To address this question, unbiased RNA-seq and proteomic analyses were performed on livers of the recently developed AH mouse model which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared with gene expression profiling data from AH patients. This cross-analysis has identified Casp11 (CASP4 in man) as a commonly upregulated gene known to be involved in non-canonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice but not in chronic ASH. Gasdermin-D (GSDMD) which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and the severity of AH. Conversely, the deficiency of IL-18, the key anti-microbial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Further, hepatocyte-specific expression of constitutively active GSDMD worsens hepatocellular lytic death and PMN inflammation. These results implicate pyroptosis induced by CASP11/4-GSDMD pathway in the pathogenesis of AH. Overall design: 9 total samples = 3 AH liver, 3 ASH liver, 3 control

酒精性肝炎（Alcoholic hepatitis, AH）仍是一类高死亡率且缺乏有效内科治疗手段的疾病。尽管重度酒精性肝炎表现为慢性基础上的急性肝衰竭，但从慢性酒精性脂肪性肝炎（chronic alcoholic steatohepatitis, ASH）向AH的转变机制仍未明确。为阐明这一科学问题，研究团队对新近构建的AH小鼠模型的肝脏组织开展了无偏倚RNA测序（RNA-seq）与蛋白质组学分析；该模型在每周酒精暴饮后可由慢性ASH进展为AH，并将上述结果与AH患者的基因表达谱数据进行了交叉比对。
通过交叉分析，研究人员将半胱天冬酶11（人类中为CASP4）鉴定为共同上调基因，该基因已被证实参与非经典炎性体通路。免疫印迹实验证实，AH小鼠肝脏中存在CASP11/4的激活，而慢性ASH小鼠肝脏中则无此现象。Gasdermin-D（GSDMD）作为CASP11/4激活下游的效应蛋白，可介导焦亡（pyroptosis，即细菌感染引发的溶解性细胞死亡），其在AH小鼠肝脏中同样被激活。
敲除Casp11可降低GSDMD的激活水平、肝脏细菌载量以及AH的疾病严重程度。与之相反，敲除IL-18（关键的抗微生物细胞因子）则会加重肝脏细菌载量、GSDMD激活程度与AH病情。进一步实验显示，肝细胞特异性表达组成型激活的GSDMD会加剧肝细胞溶解性死亡与中性粒细胞（PMN）炎症反应。上述结果表明，CASP11/4-GSDMD通路介导的焦亡参与了AH的发病机制。
整体实验设计：本次研究共纳入9份样本，包括3份AH模型小鼠肝脏组织、3份慢性ASH模型小鼠肝脏组织及3份对照样本。