G protein-coupled receptor 35 attenuates nonalcoholic steatohepatitis by reprogramming cholesterol homeostasis in hepatocytes.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide. Fat accumulation "sensitizes" the liver to insult and leads to nonalcoholic steatohepatitis (NASH). G protein-coupled receptor 35 (GPR35) is involved in metabolic stresses, but its role in NAFLD is unknown. We report that hepatocyte GPR35 mitigates NASH by regulating hepatic cholesterol homeostasis. Specifically, we found that GPR35 overexpression in hepatocytes protected against high-fat/cholesterol/fructose (HFCF) diet-induced steatohepatitis, whereas loss of GPR35 had the opposite effect. Administration of the GPR35 agonist kynurenic acid (Kyna) suppressed HFCF diet-induced steatohepatitis in mice. Kyna/GPR35 induced expression of StAR-related lipid transfer protein 4 (STARD4) through the ERK1/2 signaling pathway, ultimately resulting in hepatic cholesterol esterification and bile acid synthesis (BAS). The overexpression of STARD4 increased the expression of the BAS rate-limiting enzymes cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and CYP8B1, promoting the conversion of cholesterol to bile acid. The protective effect induced by GPR35 overexpression in hepatocytes disappeared in hepatocyte STARD4-knockdown mice. STARD4 overexpression in hepatocytes reversed the aggravation of HFCF diet-induced steatohepatitis caused by the loss of GPR35 expression in hepatocytes in mice. Our findings indicate that the GPR35-STARD4 axis is a promising therapeutic target for NAFLD.

Hepatic CYP2B10 is highly induced by binge ethanol and contributes to acute-on-chronic alcohol-induced liver injury.

BACKGROUND: The chronic-plus-binge model of ethanol consumption, where chronically (8-week) ethanol-fed mice are gavaged a single dose of ethanol (E8G1), is known to induce steatohepatitis in mice. However, how chronically ethanol-fed mice respond to multiple binges of ethanol remains unknown. METHODS: We extended the E8G1 model to three gavages of ethanol (E8G3) spaced 24 h apart, sacrificed each group 9 h after the final gavage, analyzed liver injury, and examined gene expression changes using microarray analyses in each group to identify mechanisms contributing to liver responses to binge ethanol. RESULTS: Surprisingly, E8G3 treatment induced lower levels of liver injury, steatosis, inflammation, and fibrosis as compared to mice after E8G1 treatment. Microarray analyses identified several pathways that may contribute to the reduced liver injury after E8G3 treatment compared to E8G1 treatment. The gene encoding cytochrome P450 2B10 (Cyp2b10) was one of the top upregulated genes in the E8G1 group and was further upregulated in the E8G3 group, but only moderately induced after chronic ethanol consumption, as confirmed by RT-qPCR and western blot analyses. Genetic disruption of Cyp2b10 worsened liver injury in E8G1 and E8G3 mice with higher blood ethanol levels compared to wild-type control mice, while in vitro experiments revealed that CYP2b10 did not directly promote ethanol metabolism. Metabolomic analyses revealed significant differences in hepatic metabolites from E8G1-treated Cyp2b10 knockout and WT mice, and these metabolic alterations may contribute to the reduced liver injury in Cyp2b10 knockout mice. CONCLUSION: Hepatic Cyp2b10 expression is highly induced after ethanol binge, and such upregulation reduces acute-on-chronic ethanol-induced liver injury via the indirect modification of ethanol metabolism.

Hepatocyte-specific deletion of cellular repressor of E1A-stimulated genes 1 exacerbates alcohol-induced liver injury by activating stress kinases.

Alcohol-associated liver disease (ALD) encompasses a wide range of pathologies from simple steatosis to cirrhosis and hepatocellular carcinoma and is a global health problem. Currently, there are no effective pharmacological treatments for ALD. We have previously demonstrated that aging exacerbates the pathogenesis of ALD, but the underlying mechanisms are still poorly understood. Cellular repressor of E1A-stimulated genes 1 protein (CREG1) is a recently identified small glycoprotein that has been implicated in aging process by promoting cellular senescence and activating stress kinases. Thus, the current study aimed to explore the role of aging associated CREG1 in ALD pathogenesis and CREG1 as a potential therapeutic target. Hepatic and serum CREG1 protein levels were elevated in ALD patients. Elevation of hepatic CREG1 protein and mRNA was also observed in a mouse model of Gao-binge alcohol feeding. Genetic deletion of the Creg1 gene in hepatocytes (Creg1∆hep ) markedly exacerbated ethanol-induced liver injury, apoptosis, steatosis and inflammation. Compared to wild-type mice, Creg1∆hep mice had increased phosphorylation of hepatic stress kinases such as apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 but not TGF-ß-activated kinase 1 (TAK1) or extracellular signal-regulated kinase (ERK) after alcohol feeding. In vitro, ethanol treatment elevated the phosphorylation of ASK1, JNK, and p38 in mouse hepatocyte AML-12 cells. This elevation was further enhanced by CREG1 knockdown but alleviated by CREG1 overexpression. Last, treatment with an ASK1 inhibitor abolished ethanol-induced liver injury and upregulated hepatic lipogenesis, proinflammatory genes and stress kinases in Creg1∆hep mice. Taken together, our data suggest that CREG1 protects against alcoholic liver injury and inflammation by inhibiting the ASK1-JNK/p38 stress kinase pathway and that CREG1 is a potential therapeutic target for ALD.

Hepatic NCoR1 deletion exacerbates alcohol-induced liver injury in mice by promoting CCL2-mediated monocyte-derived macrophage infiltration.

Nuclear receptor corepressor 1 (NCoR1) is a corepressor of the epigenetic regulation of gene transcription that has important functions in metabolism and inflammation, but little is known about its role in alcohol-associated liver disease (ALD). In this study, we developed mice with hepatocyte-specific NCoR1 knockout (NCoR1Hep-/-) using the albumin-Cre/LoxP system and investigated the role of NCoR1 in the pathogenesis of ALD and the underlying mechanisms. The traditional alcohol feeding model and NIAAA model of ALD were both established in wild-type and NCoR1Hep-/- mice. We showed that after ALD was established, NCoR1Hep-/- mice had worse liver injury but less steatosis than wild-type mice. We demonstrated that hepatocyte-specific loss of NCoR1 attenuated liver steatosis by promoting fatty acid oxidation by upregulating BMAL1 (a circadian clock component that has been reported to promote peroxisome proliferator activated receptor alpha (PPARα)-mediated fatty ß-oxidation by upregulating de novo lipid synthesis). On the other hand, hepatocyte-specific loss of NCoR1 exacerbated alcohol-induced liver inflammation and oxidative stress by recruiting monocyte-derived macrophages via C-C motif chemokine ligand 2 (CCL2). In the mouse hepatocyte line AML12, NCoR1 knockdown significantly increased ethanol-induced CCL2 release. These results suggest that hepatocyte NCoR1 plays distinct roles in controlling liver inflammation and steatosis, which provides new insights into the development of treatments for steatohepatitis induced by chronic alcohol consumption.

MicroRNA-29b ameliorates hepatic inflammation via suppression of STAT3 in alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is induced by chronic excessive alcohol consumption resulting in the clinical manifestations of steatosis, inflammation, and cirrhosis. MicroRNA-29b (miR-29b) is mainly expressed in hepatic nonparenchymal cells, and its expression level varies in different diseases. In this study, we aimed to determine the role of miR-29b in a mouse model of alcohol-associated liver disease. Wild-type (WT) and miR-29b knockout (miR-29b-/-) mice were fed a Lieber-DeCarli liquid diet containing 5% alcohol for 10 days, followed by gavage of a single dose of ethanol (5 g/kg body weight). Histology, immunoblotting, and biochemical analyses were then conducted for comparison. miR-29b expression was decreased in the livers of chronic-plus-binge ethanol-fed mice. Further analysis revealed that alcohol exposure exacerbated hepatic injury by significantly increasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, with decreased survival rates for miR-29b-/- mice. Results from the luciferase assay indicated that miR-29b negatively regulated the signal transducer and activator of transcription 3 (STAT3). Depletion of miR-29b led to an increase in STAT3 and more noticeable inflammation in the liver, whereas overexpression of miR-29b downregulated STAT3 and proinflammatory cytokine expression in primary mouse peritoneal macrophages. Taken together, these results demonstrate a novel association between miR-29b and ALD. miR-29b plays a hepatoprotective role in alcohol-induced inflammation and liver injury by targeting STAT3.

Aging exaggerates acute-on-chronic alcohol-induced liver injury in mice and humans by inhibiting neutrophilic sirtuin 1-C/EBPα-miRNA-223 axis.

BACKGROUND AND AIMS: Aging exacerbates liver neutrophil infiltration and alcohol-associated liver disease (ALD) in mice and humans, but the underlying mechanisms remain obscure. This study aimed to examine the effect of aging and alcohol consumption on neutrophilic Sirtuin 1 (SIRT1) and microRNA-223 (miR-223), and their contribution to ALD pathogeneses. APPROACH AND RESULTS: Young and aged myeloid-specific Sirt1 knockout mice were subjected to chronic-plus-binge ethanol feeding. Blood samples from healthy controls and patients with chronic alcohol drinking who presented with acute intoxication were analyzed. Neutrophilic Sirt1 and miR-223 expression were down-regulated in aged mice compared with young mice. Deletion of the Sirt1 gene in myeloid cells including neutrophils exacerbated chronic-plus-binge ethanol-induced liver injury and inflammation and down-regulated neutrophilic miR-223 expression. Immunoprecipitation experiments revealed that SIRT1 promoted C/EBPα deacetylation by directly interacting with C/EBPα, a key transcription factor that controls miR-223 biogenesis, and subsequently elevated miR-223 expression in neutrophils. Importantly, down-regulation of SIRT1 and miR-223 expression was also observed in circulating neutrophils from middle-aged and elderly subjects compared with those from young individuals. Chronic alcohol users with acute intoxication had a reduction in neutrophilic SIRT1 expression in young and middle-aged patients, with a greater reduction in the latter group. The neutrophilic SIRT1 expression correlated with neutrophilic miR-223 and serum alanine transaminase levels in those patients. CONCLUSIONS: Aging increases the susceptibility of alcohol-induced liver injury in mice and humans through the down-regulation of the neutrophilic SIRT1-C/EBPα-miR-223 axis, which could be a therapeutic target for the prevention and/or treatment of ALD.

Kupffer cell restoration after partial hepatectomy is mainly driven by local cell proliferation in IL-6-dependent autocrine and paracrine manners.

Kupffer cells (KCs), which are liver-resident macrophages, originate from the fetal yolk sac and represent one of the largest macrophage populations in the body. However, the current data on the origin of the cells that restore macrophages during liver injury and regeneration remain controversial. Here, we address the question of whether liver macrophage restoration results from circulating monocyte infiltration or local KC proliferation in regenerating livers after partial hepatectomy (PHx) and uncover the underlying mechanisms. By using several strains of genetically modified mice and performing immunohistochemical analyses, we demonstrated that local KC proliferation mainly contributed to the restoration of liver macrophages after PHx. Peak KC proliferation was impaired in Il6-knockout (KO) mice and restored after the administration of IL-6 protein, whereas KC proliferation was not affected in Il4-KO or Csf2-KO mice. The source of IL-6 was identified using hepatocyte- and myeloid-specific Il6-KO mice and the results revealed that both hepatocytes and myeloid cells contribute to IL-6 production after PHx. Moreover, peak KC proliferation was also impaired in myeloid-specific Il6 receptor-KO mice after PHx, suggesting that IL-6 signaling directly promotes KC proliferation. Studies using several inhibitors to block the IL-6 signaling pathway revealed that sirtuin 1 (SIRT1) contributed to IL-6-mediated KC proliferation in vitro. Genetic deletion of the Sirt1 gene in myeloid cells, including KCs, impaired KC proliferation after PHx. In conclusion, our data suggest that KC repopulation after PHx is mainly driven by local KC proliferation, which is dependent on IL-6 and SIRT1 activation in KCs.

Neutrophil-to-hepatocyte communication via LDLR-dependent miR-223-enriched extracellular vesicle transfer ameliorates nonalcoholic steatohepatitis.

Neutrophil infiltration around lipotoxic hepatocytes is a hallmark of nonalcoholic steatohepatitis (NASH); however, how these 2 types of cells communicate remains obscure. We have previously demonstrated that neutrophil-specific microRNA-223 (miR-223) is elevated in hepatocytes to limit NASH progression in obese mice. Here, we demonstrated that this elevation of miR-223 in hepatocytes was due to preferential uptake of miR-223-enriched extracellular vesicles (EVs) derived from neutrophils as well other types of cells, albeit to a lesser extent. This selective uptake was dependent on the expression of low-density lipoprotein receptor (LDLR) on hepatocytes and apolipoprotein E (APOE) on neutrophil-derived EVs, which was enhanced by free fatty acids. Once internalized by hepatocytes, the EV-derived miR-223 acted to inhibit hepatic inflammatory and fibrogenic gene expression. In the absence of this LDLR- and APOE-dependent uptake of miR-223-enriched EVs, the progression of steatosis to NASH was accelerated. In contrast, augmentation of this transfer by treatment with an inhibitor of proprotein convertase subtilisin/kexin type 9, a drug used to lower blood cholesterol by upregulating LDLR, ameliorated NASH in mice. This specific role of LDLR and APOE in the selective control of miR-223-enriched EV transfer from neutrophils to hepatocytes may serve as a potential therapeutic target for NASH.

Myeloid-Cell-Specific IL-6 Signaling Promotes MicroRNA-223-Enriched Exosome Production to Attenuate NAFLD-Associated Fibrosis.

BACKGROUND ANDS AIMS: NAFLD is associated with elevation of many cytokines, particularly IL-6; however, the role of IL-6 in NAFLD remains obscure. The aim of this study was to examine how myeloid-specific IL-6 signaling affects NAFLD by the regulation of antifibrotic microRNA-223 (miR-223) in myeloid cells. APPROACH AND RESULTS: Patients with NAFLD or NASH and healthy controls were recruited, and serum IL-6 and soluble IL-6 receptor α (sIL-6Rα) were measured. Compared to controls, serum IL-6 and sIL-6Rα levels were elevated in NAFLD/NASH patients. IL-6 levels correlated positively with the number of circulating leukocytes and monocytes. The role of IL-6 in NAFLD was investigated in Il6 knockout (KO) and Il6 receptor A (Il6ra) conditional KO mice after high-fat diet (HFD) feeding. HFD-fed Il6 KO mice had worse liver injury and fibrosis, but less inflammation, compared to wild-type mice. Hepatocyte-specific Il6ra KO mice had more steatosis and liver injury, whereas myeloid-specific Il6ra KO mice had a lower number of hepatic infiltrating macrophages (IMs) and neutrophils with increased cell death of these cells, but greater liver fibrosis (LF), than WT mice. Mechanistically, the increased LF in HFD-fed, myeloid-specific Il6ra KO mice was attributable to the reduction of antifibrotic miR-223 and subsequent up-regulation of the miR-223 target gene, transcriptional activator with PDZ-binding motif (TAZ), a well-known factor to promote NASH fibrosis. In vitro, IL-6 treatment up-regulated exosome biogenesis-related genes and subsequently promoted macrophages to release miR-223-enriched exosomes that were able to reduce profibrotic TAZ expression in hepatocytes by exosomal transfer. Finally, serum IL-6 and miR-223 levels were elevated and correlated with each other in NAFLD patients. CONCLUSIONS: Myeloid-specific IL-6 signaling inhibits LF through exosomal transfer of antifibrotic miR-223 into hepatocytes, providing therapeutic targets for NAFLD therapy.

Emerging Roles of SIRT1 in Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the most prevalent type of chronic liver disease worldwide with a wide spectrum of liver pathologies ranging from simple steatosis to steatohepatitis, cirrhosis, and even hepatocellular carcinoma. It has been demonstrated that ALD is mediated in whole or in part by a central signaling molecule sirtuin 1 (SIRT1), a conserved class III histone deacetylase.SIRT1 plays beneficial roles in regulating hepatic lipid metabolism, inhibiting hepatic inflammation, controlling hepatic fibrosis and mediating hepatocellular carcinoma in ALD. However, underlying molecular mechanisms are complex and remain incompletely understood. The aim of this review was to highlight the latest advances in understanding of SIRT1 regulatory mechanisms in ALD and discuss their unique potential role as novel therapeutic target for ALD treatment.

Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms.

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA-enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.

Colon cancer cells secrete exosomes to promote self-proliferation by shortening mitosis duration and activation of STAT3 in a hypoxic environment.

Colon-cancer-cell-derived exosomes (CDEs) are emerging mediators of tumorigenesis and serve as messengers of intercellular communication; however, whether the CDEs affect the proliferation of colon cancer cells themselves remains unknown. In the current study, the CDEs isolated from human colon cancer cell line SW480 and HCT116 showed a size range of 60-150 nm, typical bilayer-encapsulated vesicles, and expressed the exosomal markers CD81 and CD63. Incubation of SW480 cells with CDEs labelled with PKH67 fluorescent markers revealed that SW480 cells were able to absorb CDEs, which were mostly distributed around the nucleus. Hypoxic conditions promoted colon cancer cells to release a greater number of CDEs than normoxic conditions. MTT cell proliferation assay demonstrated CDEs promoted the proliferation of colon cancer cells in a time- and dose-dependent manner. Mechanistically, CDEs promoted colon cancer cell growth mainly through shortening mitosis duration. Meanwhile, the levels of phosphorylated STAT3 in colon cancer cells was up-regulated with the treatment of CDEs derived from hypoxic tumor cells. Our data suggests that colon cancer cells are able to promote self-growth through the secretion of exosomes, especially under hypoxic conditions, which shortens mitosis duration and activates STAT3.

Enhanced Regeneration and Hepatoprotective Effects of Interleukin 22 Fusion Protein on a Predamaged Liver Undergoing Partial Hepatectomy.

Liver ischemia-reperfusion injury (IRI) and regeneration deficiency are two major challenges for surgery patients with chronic liver disease. As a survival factor for hepatocytes, interleukin 22 (IL-22) plays an important role in hepatoprotection and the promotion of regeneration after hepatectomy. In this study, we aim to investigate the roles of an interleukin 22 fusion protein (IL-22-FP) in mice with a predamaged liver after a two-third partial hepatectomy (PHx). Predamaged livers in mice were induced by concanavalin A (ConA)/carbon tetrachloride (CCl4) following PHx with or without IL-22-FP treatment. A hepatic IRI mouse model was also used to determine the hepatoprotective effects of IL-22-FP. In the ConA/CCl4 model, IL-22-FP treatment alleviated liver injury and accelerated hepatocyte proliferation. Administration of IL-22-FP activated the hepatic signal transducer and activator of transcription 3 (STAT3) and upregulated the expression of many mitogenic proteins. IL-22-FP treatment prior to IRI effectively reduced liver damage through decreased aminotransferase and improved liver histology. In conclusion, IL-22-FP promotes liver regeneration in mice with predamaged livers following PHx and alleviates IRI-induced liver injury. Our study suggests that IL-22-FP may represent a promising therapeutic drug against regeneration deficiency and liver IRI in patients who have undergone PHx.