Ethanol exposure diminishes intestinal abundance in both mice and humans and can be recovered in experimental ALD by oral supplementation. promotes intestinal barrier integrity and ameliorates experimental ALD. Our data suggest that patients with ALD might benefit from supplementation.

Edited by Jeffrey PessinFibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon regulatory factor 3 (IRF3) regulates hepatocyte apoptosis and production of type I IFNs. In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the endoplasmic reticulum (ER) adapter, stimulator of interferon genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically induced liver fibrogenesis. To test this, we performed acute or chronic CCl 4 administration to WT and IRF3-, Toll/ Interleukin-1R (TIR) domain-containing adapter-inducing interferon-␤ (TRIF)-, TRIF-related adaptor molecule (TRAM)-, and STING-deficient mice. We report that acute CCl 4 administration to WT mice resulted in early ER stress, activation of IRF3, and type I IFNs, followed by hepatocyte apoptosis and liver injury, accompanied by liver fibrosis upon repeated administration of CCl 4 . Deficiency of IRF3 or STING prevented hepatocyte death and fibrosis both in acute or chronic CCl 4 . In contrast, mice deficient in type I IFN receptors or in TLR4 signaling adaptors, TRAM or TRIF, upstream of IRF3, were not protected from hepatocyte death and/or fibrosis, suggesting that the proapoptotic role of IRF3 is independent of TLR signaling in fibrosis. Hepatocyte death is required for liver fibrosis with causal involvement of STING and IRF3. Thus, our results identify that IRF3, by its association with STING in the presence of ER stress, couples hepatocyte apoptosis with liver fibrosis and indicate that innate immune signaling regulates outcomes of liver fibrosis via modulation of hepatocyte death in the liver.

Kupffer cell (KC) and macrophage (MØ) activation contribute to steatosis, inflammation and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells and expression of Ccr2 and Ccr5 in the livers of patients with ALD and increased circulating chemokines, CCL2 and CCL5 in alcoholic hepatitis patients. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (ALT) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (Sirius-red, hydroxyproline and collagen-1) were normalized by both modes of CVC administration. We found that "prevention" and "treatment" with CVC reversed alcohol-related increases in liver mRNA and protein expression of TNFα, IL-1β, IL-6 and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80 CD11b ) and reduced proinflammatory Ly6C MØ in livers of alcohol-fed mice. CVC increased liver T cell numbers and attenuated Il-2 expression without an effect on CD69 or CD25 T cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte Fasn and Adrp while it augmented Acox-1, Pgc1α and Ucp-2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to LPS-induced liver injury (TNFα, ALT and LDH release). Alcohol feeding induced apoptosis (PARP and caspase-3 cleavage) and pyroptosis (gasdermin D cleavage) in livers and CVC prevented both these forms of cell death. Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage. This article is protected by copyright. All rights reserved.

Background & Aims The inflammasome is a well-characterized inducer of inflammation in ASH. Inflammasome activation requires two signals for mature interleukin (IL)-1β production. Here we asked whether metabolic danger signals trigger inflammasome activation in ASH. Methods Wild-type mice, ATP receptor 2×7 (P2rx7)-KO mice, or mice overexpressing uricase were fed Lieber-DeCarli ethanol or control diet. We also implemented a pharmacological approach in which mice were treated with probenecid or allopurinol. Results The sterile danger signals, ATP and uric acid, were increased in the serum and liver of alcohol-fed mice. Depletion of uric acid or ATP, or lack of ATP signaling attenuated ASH and prevented inflammasome activation and its major downstream cytokine, IL-1β. Pharmacological depletion of uric acid with allopurinol provided significant protection from alcohol-induced inflammatory response, steatosis and liver damage, and additional protection was achieved in mice treated with probenecid, which depletes uric acid and blocks ATP-induced P2rx7 signaling. We found that alcohol-damaged hepatocytes released uric acid and ATP in vivo and in vitro and that these sterile danger signals activated the inflammasome in LPS-exposed liver mononuclear cells. Conclusions Our data indicate that the second signal in inflammasome activation and IL-1β production in ASH results from the endogenous danger signals, uric acid and ATP. Inhibition of signaling triggered by uric acid and ATP may have therapeutic implications in ASH.

Background & Aims Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. Methods We obtained liver samples from 12 patients with ALD and cirrhosis and 9 healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8 weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8 weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative PCR analyses in Huh-7 cells. Results Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared to mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in livers tissues from mice. Levels of GRHL2 were also increased in liver tissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of ALT compared to mice given a control vector. Levels of hypoxia inducible factor 1 alpha (HIF1A) mRNA, a target of MIR122, were increased in liver tissues from patients and mice with ALD, compared with controls. Mice with hepatocyte-specific disruption of Hif1a developed less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. Conclusions Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage followin...