These results implicate pyroptosis induced by the CASP11/4-GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737-1753).

Alcoholic hepatitis (AH) is a distinct spectrum of alcoholic liver disease (ALD) with intense neutrophilic (PMN) inflammation and high mortality. Although a recent study implicates osteopontin (SPP1) in AH, SPP1 is also shown to have protective effects on experimental ALD. To address this unsettled question, we examined the effects of SPP1 deficiency in male mice given 40% calories derived from ad libitum consumption of the Western diet high in cholesterol and saturated fat (HCFD) and the rest from intragastric feeding (iG) of alcohol diet without or with weekly alcohol binge. Weekly binge in this new hybrid feeding model shifts chronic ASH with macrophage inflammation and perisinusoidal and pericelluar fibrosis to AH in 57% (15/26) of the mice, accompanied by inductions of chemokines (Spp1, Cxcl1, Il-17a), progenitor genes (Cd133, Cd24, Nanog, Epcam), PMN infiltration, and clinical features of AH such as hypoalbuminemia, bilirubinemia, and splenomegaly. SPP1 deficiency does not reduce the AH incidence and inductions of progenitor and fibrogenic genes but rather enhances the Il-17a induction and PMN infiltration in some mice. Further, in the absence of SPP1, chronic ASH mice without weekly binge begin to develop AH. In conclusion, these results suggest SPP1 has a protective rather than causal role for experimental AH reproduced in our model.

Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin including specific microRNAs (miRNAs). Here we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with a miRNA signature that can be measured in circulation. A time course experiment showed that after two weeks of intragastric infusion, a time-point that results in isolated steatosis, there was no increase of blood EVs. After four weeks of infusion mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (p<0.05), as well as and in culture media of isolated HCs (p<0.001) and hepatic macrophages (p<0.001) with HCs being the predominant source of EVs. The transcriptome analysis of HC-EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up-regulated and four significantly down-regulated miRNAs. Target prediction and pathway analyses of the up-regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways such as NF-κB, EGF, Wnt, and Bcl2. Three miRNAs, let7f, miR-29a, and miR-340, were increased in blood EVs from ASH mice (p<0.05), but not in blood EVs from three other models of chronic liver injury including bile duct ligation, non-alcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. The blood EV level (p<0.01) and three miRNAs (p<0.05) were significantly increased in patients with ambulatory mild ALD as compared to non-alcoholics. Conclusions Our results reveal that damaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH-related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH.

Direct intragastric delivery of a diet, nutrient or test substance can be achieved in rodents (mice and rats) on a long-term (2–3 months) basis using a chronically implanted gastrostomy catheter and a flow-through swivel system. This rodent intragastric infusion (iG) model has broad applications in research on food intake, gastrointestinal (GI) physiology, GI neuroendocrinology, drug metabolism and toxicity, obesity and liver disease. It achieves maximal control over the rate and pattern of delivery and it can be combined with normal ad libitum feeding of solid diet if so desired. It may be adopted to achieve infusion at other sites of the GI system to test the role of a bypassed GI segment in neuroendocrine physiology, and its use in genetic mouse models facilitates the genetic analysis of a central question under investigation.