Inhibition of IRAK4 kinase activity improves ethanol-induced liver injury in mice.
Wang Han,Zhou Hao,Zhang Quanri,Poulsen Kyle L,Taylor Vanessa,McMullen Megan R,Czarnecki Doug,Dasarathy Dhweeja,Yu Minjia,Liao Yun,Allende Daniela S,Chen Xing,Hong Lingzi,Zhao Junjie,Yang Jinbo,Nagy Laura E,Li Xiaoxia
Journal of hepatology
BACKGROUNDS & AIMS:Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. METHODS:IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1β-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. RESULTS:Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1β synergistically exacerbate ethanol-induced cell death ex vivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. CONCLUSIONS:Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. LAY SUMMARY:Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death ex vivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.
Alcohol-induced Hsp90 acetylation is a novel driver of liver sinusoidal endothelial dysfunction and alcohol-related liver disease.
Yang Yilin,Sangwung Panjamaporn,Kondo Reiichiro,Jung Yirang,McConnell Matthew J,Jeong Jain,Utsumi Teruo,Sessa William C,Iwakiri Yasuko
Journal of hepatology
BACKGROUND:It is unknown whether liver sinusoidal endothelial cells (LSECs) metabolize alcohol. Chronic alcohol consumption decreases endothelial nitric oxide synthase (eNOS)-derived NO production typical of LSEC dysfunction. Heat shock protein 90 (Hsp90) interacts with eNOS to increase its activity. Cytochrome P450 2E1 (CYP2E1) is a key enzyme in alcohol metabolism and facilitates protein acetylation via acetyl-CoA, but its expression in LSECs is unknown. This study investigates alcohol metabolism by LSECs, the mechanism of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury. METHODS:Primary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs via adeno-associated virus (AAV)-mediated gene delivery to decrease Hsp90 acetylation in ethanol-fed mice. RESULTS:LSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with eNOS along with a decrease in NO production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyper-acetylation mutant decreased NO production. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. AAV8-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90's interaction with eNOS and ameliorated alcohol-induced liver injury in mice. CONCLUSION:Restoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy.
Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro-RNA 155 in Alcoholic Liver Disease.
Babuta Mrigya,Furi Istvan,Bala Shashi,Bukong Terence N,Lowe Patrick,Catalano Donna,Calenda Charles,Kodys Karen,Szabo Gyongyi
Hepatology (Baltimore, Md.)
Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3-II levels. Alcohol reduced autophagy flux in vivo in chloroquine-treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol-related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy-related protein 7 (Atg7), proteins involved in phagophore-autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal-associated membrane protein 1 (LAMP1) and lysosomal-associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro-RNA 155 (miR-155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR-155-deficient mice were protected from alcohol-induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down-regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol-induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR-155 as a mediator of alcohol-related regulation of autophagy and exosome production in hepatocytes and macrophages.
Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression.
Heo Mi Jeong,Kim Tae Hyun,You Jueng Soo,Blaya Delia,Sancho-Bru Pau,Kim Sang Geon
OBJECTIVE:Alcoholic liver disease (ALD) is a leading cause of death among chronic liver diseases. However, its pathogenesis has not been completely established. MicroRNAs (miRNAs) are key contributors to liver diseases progression. This study investigated hepatocyte-abundant miRNAs dysregulated by ALD, its impact on hepatocyte injury and the underlying basis. DESIGN:Alcoholic hepatitis (AH) human and animal liver samples and hepatocytes were used to assess miR-148a levels. Pre-miR-148a was delivered specifically to hepatocytes in vivo using lentivirus. Immunoblottings, luciferase reporter assays, chromatin immunoprecipitation and immunofluorescence assays were carried out in cell models. RESULTS:The miRNA profile and PCR analyses enabled us to find substantial decrease of miR-148a in the liver of patients with AH. In mice subjected to Lieber-DeCarli alcohol diet or binge alcohol drinking, miR-148a levels were also markedly reduced. In cultured hepatocytes and mouse livers, alcohol exposure inhibited forkhead box protein O1 (FoxO1) expression, which correlated with miR-148a levels and significantly decreased in human AH specimens. FoxO1 was identified as a transcription factor for transactivation. MiR-148a directly inhibited thioredoxin-interacting protein (TXNIP) expression. Consequently, treatment of hepatocytes with ethanol resulted in TXNIP overexpression, activating NLRP3 inflammasome and caspase-1-mediated pyroptosis. These events were reversed by miR-148a mimic or TXNIP small-interfering RNA transfection. Hepatocyte-specific delivery of miR-148a to mice abrogated alcohol-induced TXNIP overexpression and inflammasome activation, attenuating liver injury. CONCLUSION:Alcohol decreases miR-148a expression in hepatocytes through FoxO1, facilitating TXNIP overexpression and NLRP3 inflammasome activation, which induces hepatocyte pyroptosis. Our findings provide information on novel targets for reducing incidence and progression of ALD.
Development of Capsular Fibrosis Beneath the Liver Surface in Humans and Mice.
Balog Steven,Li Yuchang,Ogawa Tomohiro,Miki Toshio,Saito Takeshi,French Samuel W,Asahina Kinji
Hepatology (Baltimore, Md.)
Glisson's capsule is the connective tissue present in the portal triad as well as beneath the liver surface. Little is known about how Glisson's capsule changes its structure in capsular fibrosis (CF), which is characterized by fibrogenesis beneath the liver surface. In this study, we found that the human liver surface exhibits multilayered capsular fibroblasts and that the bile duct is present beneath the mesothelium, whereas capsular fibroblasts are scarce and no bile ducts are present beneath the mouse liver surface. Patients with cirrhosis caused by alcohol abuse or hepatitis C virus infection show development of massive CF. To examine the effect of alcohol on CF in mice, we first injected chlorhexidine gluconate (CG) intraperitoneally and then fed alcohol for 1 month. The CG injection induces CF consisting of myofibroblasts beneath the mesothelium. One month after CG injection, the fibrotic area returns to the normal structure. In contrast, additional alcohol feeding sustains the presence of myofibroblasts in CF. Cell lineage tracing revealed that mesothelial cells give rise to myofibroblasts in CF, but these myofibroblasts disappear 1 month after recovery with or without alcohol feeding. Capsular fibroblasts isolated from the mouse liver spontaneously differentiated into myofibroblasts and their differentiation was induced by transforming growth factor beta 1 (TGF-β1) or acetaldehyde in culture. In alcohol-fed mice, infiltrating CD11b Ly-6C monocytes had reduced mRNA expression of matrix metalloproteinase 13 and matrix metalloproteinase 9 and increased expression of tissue inhibitor of matrix metalloproteinase 1, Tgfb1, and interleukin-10 during resolution of CF. Conclusion: The present study revealed that the structure of Glisson's capsule is different between human and mouse livers and that alcohol impairs the resolution of CF by changing the phenotype of Ly-6C monocytes.
DNA-PKcs promotes alcohol-related liver disease by activating Drp1-related mitochondrial fission and repressing FUNDC1-required mitophagy.
Zhou Hao,Zhu Pingjun,Wang Jin,Toan Sam,Ren Jun
Signal transduction and targeted therapy
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a novel housekeeper of hepatic mitochondrial homeostasis outside the DNA repair process. In this study, DNA-PKcs was upregulated in the livers of mice that were exposed to alcohol; the expression of DNA-PKcs positively correlated with hepatic steatosis, fibrosis, apoptosis, and mitochondrial damage. Functional studies revealed that liver-specific DNA-PKcs knockout (DNA-PKcs ) mice were protected from chronic ethanol-induced liver injury and mitochondrial damage. Mechanistic investigations established that DNA-PKcs promoted p53 activation, which elevated dynamin-related protein 1 (Drp1)-related mitochondrial fission but repressed FUN14 domain containing 1 (FUNDC1)-required mitophagy. Excessive fission and defective mitophagy triggered mtDNA damage, mitochondrial respiratory inhibition, mROS overproduction, cardiolipin oxidation, redox imbalance, calcium overload, and hepatic mitochondrial apoptosis. In contrast, the deletion of DNA-PKcs rescued these phenotypic alterations, which alleviated the susceptibility of hepatocytes to alcohol-induced cytotoxicity. Additionally, we also showed that orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) was the upstream signal for DNA-PKcs activation and that the genetic ablation of NR4A1 ameliorated the progression of alcohol-related liver disease (ARLD); these results were similar to those obtained in DNA-PKcs knockout mice. Collectively, our results identified the NR4A1/DNA-PKcs/p53 axis as a novel signaling pathway responsible for ARLD pathogenesis that acts by activating Drp1-related mitochondrial fission and restricting FUNDC1-required mitophagy. The findings have potential implications for new approaches for ARLD therapy.
Mitochondrial Double-Stranded RNA in Exosome Promotes Interleukin-17 Production Through Toll-Like Receptor 3 in Alcohol-associated Liver Injury.
Lee Jun-Hee,Shim Young-Ri,Seo Wonhyo,Kim Myung-Ho,Choi Won-Mook,Kim Hee-Hoon,Kim Ye Eun,Yang Keungmo,Ryu Tom,Jeong Jong-Min,Choi Hei-Gwon,Eun Hyuk Soo,Kim Seok-Hwan,Mun Hyejin,Yoon Je-Hyun,Jeong Won-Il
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Mitochondrial double-stranded RNA (mtdsRNA) and its innate immune responses have been reported previously; however, mtdsRNA generation and its effects on alcohol-associated liver disease (ALD) remain unclear. Here, we report that hepatic mtdsRNA stimulates toll-like receptor 3 (TLR3) in Kupffer cells through the exosome (Exo) to enhance interleukin (IL)-17A (IL-17A) production in ALD. APPROACH AND RESULTS:Following binge ethanol (EtOH) drinking, IL-17A production primarily increased in γδ T cells of wild-type (WT) mice, whereas the production of IL-17A was mainly facilitated by CD4 T cells in acute-on-chronic EtOH consumption. These were not observed in TLR3 knockout (KO) or Kupffer cell-depleted WT mice. The expression of polynucleotide phosphorylase, an mtdsRNA-restricting enzyme, was significantly decreased in EtOH-exposed livers and hepatocytes of WT mice. Immunostaining revealed that mtdsRNA colocalized with the mitochondria in EtOH-treated hepatocytes from WT mice and healthy humans. Bioanalyzer analysis revealed that small-sized RNAs were enriched in EtOH-treated Exos (EtOH-Exos) rather than EtOH-treated microvesicles in hepatocytes of WT mice and humans. Quantitative real-time PCR and RNA sequencing analyses indicated that mRNA expression of mitochondrial genes encoded by heavy and light strands was robustly increased in EtOH-Exos from mice and humans. After direct treatment with EtOH-Exos, IL-1β expression was significantly increased in WT Kupffer cells but not in TLR3 KO Kupffer cells, augmenting IL-17A production of γδ T cells in mice and humans. CONCLUSIONS:EtOH-mediated generation of mtdsRNA contributes to TLR3 activation in Kupffer cells through exosomal delivery. Consequently, increased IL-1β expression in Kupffer cells triggers IL-17A production in γδ T cells at the early stage that may accelerate IL-17A expression in CD4 T cells in the later stage of ALD. Therefore, mtdsRNA and TLR3 may function as therapeutic targets in ALD.
Microbiota tryptophan metabolism induces aryl hydrocarbon receptor activation and improves alcohol-induced liver injury.
Wrzosek Laura,Ciocan Dragos,Hugot Cindy,Spatz Madeleine,Dupeux Margot,Houron Camille,Lievin-Le Moal Vanessa,Puchois Virginie,Ferrere Gladys,Trainel Nicolas,Mercier-Nomé Françoise,Durand Sylvere,Kroemer Guido,Voican Cosmin Sebastian,Emond Patrick,Straube Marjolène,Sokol Harry,Perlemuter Gabriel,Cassard Anne-Marie
OBJECTIVE:Chronic alcohol consumption is an important cause of liver-related deaths. Specific intestinal microbiota profiles are associated with susceptibility or resistance to alcoholic liver disease in both mice and humans. We aimed to identify the mechanisms by which targeting intestinal microbiota can improve alcohol-induced liver lesions. DESIGN:We used human associated mice, a mouse model of alcoholic liver disease transplanted with the intestinal microbiota of alcoholic patients and used the prebiotic, pectin, to modulate the intestinal microbiota. Based on metabolomic analyses, we focused on microbiota tryptophan metabolites, which are ligands of the aryl hydrocarbon receptor (AhR). Involvement of the AhR pathway was assessed using both a pharmacological approach and AhR-deficient mice. RESULTS:Pectin treatment modified the microbiome and metabolome in human microbiota-associated alcohol-fed mice, leading to a specific faecal signature. High production of bacterial tryptophan metabolites was associated with an improvement of liver injury. The AhR agonist Ficz (6-formylindolo (3,2-b) carbazole) reduced liver lesions, similarly to prebiotic treatment. Conversely, inactivation of the gene in alcohol-fed AhR mice abrogated the beneficial effects of the prebiotic. Importantly, patients with severe alcoholic hepatitis have low levels of bacterial tryptophan derivatives that are AhR agonists. CONCLUSIONS:Improvement of alcoholic liver disease by targeting the intestinal microbiota involves the AhR pathway, which should be considered as a new therapeutic target.
Induction of SIRT1 by melatonin improves alcohol-mediated oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway.
Lee Sung-Eun,Koh Hong,Joo Dong Jin,Nedumaran Balachandar,Jeon Hwang-Ju,Park Chul-Seung,Harris Robert A,Kim Yong Deuk
Journal of pineal research
Alcoholic liver disease is the most prevalent chronic liver disease. Melatonin is known to control many vital processes. Here, we explored a novel molecular mechanism by which melatonin-induced SIRT1 signaling protects against alcohol-mediated oxidative stress and liver injury. Gene expression profiles and metabolic changes were measured in liver specimens of mice and human subjects. Expression levels of Cb1r, Crbn, Btg2, Yy1, pro-inflammatory cytokines, and Cyp2e1 were significantly enhanced in chronic alcohol-challenged mice and human subjects. Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic CYP2E1 protein, and reactive oxygen species (ROS) were elevated in alcohol-fed WT mice but not in Cb1r antagonist-treated, Crbn null, or Yy1-silenced mice. Importantly, alcohol-induced Yy1 and Cyp2e1 expression, ROS amount, and liver injury were markedly diminished by melatonin treatment and the transduction of Sirt1 in mice, whereas this phenomenon was prominently ablated by silencing of Sirt1. Notably, SIRT1 physically interacted with YY1 and attenuated YY1 occupancy on the Cyp2e1 gene promoter. Melatonin-SIRT1 signaling ameliorates alcohol-induced oxidative liver injury by disrupting the CRBN-YY1-CYP2E1 signaling pathway. The manipulation of CRBN-YY1-CYP2E1 signaling network by the melatonin-SIRT1 pathway highlights a novel entry point for treating alcoholic liver disease.
Inhibition of Sphingosine-1-Phosphate-Induced Th17 Cells Ameliorates Alcohol-Associated Steatohepatitis in Mice.
Chu Shenghui,Sun Rui,Gu Xuemei,Chen Liang,Liu Min,Guo HaiXun,Ju Songwen,Vatsalya Vatsalya,Feng Wenke,McClain Craig J,Deng Zhongbin
Hepatology (Baltimore, Md.)
BACKGROUND AND AIMS:Chronic alcohol consumption is accompanied by intestinal inflammation. However, little is known about how alterations to the intestinal immune system and sphingolipids contribute to the pathogenesis of alcohol-associated liver disease (ALD). APPROACH AND RESULTS:We used wild-type mice, retinoid-related orphan receptor gamma t (RORγt)-deficient mice, sphingosine kinase-deficient mice, and local gut anti-inflammatory, 5-aminosalicyclic acid-treated mice in a chronic-binge ethanol feeding model. Targeted lipidomics assessed the sphingolipids in gut and liver samples. Gut immune cell populations, the amounts of sphingolipids, and the level of liver injury were examined. Alcohol intake induces a pro-inflammatory shift in immune cell populations in the gut, including an increase in Th17 cells. Using RORγt-deficient mice, we found that Th17 cells are required for alcohol-associated gut inflammation and the development of ALD. Treatment with 5-aminosalicyclic acid decreases alcohol-induced liver injury and reverses gut inflammation by the suppression of CD4 /RORγt /interleukin-17A cells. Increased Th17 cells were due to up-regulation of sphingosine kinase 1 activity and RORγt activation. We found that S1P/S1PR1 signaling is required for the development of Th17 cell-mediated ALD. Importantly, in vivo intervention blocking of S1P/S1PR1 signaling markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. CONCLUSIONS:Gut inflammation is a functional alteration of immune cells in ALD. Reducing gut Th17 cells leads to reduced liver damage. S1P signaling was crucial in the pathogenesis of ALD in a Th17 cell-dependent manner. Furthermore, our findings suggest that compounds that reduce gut inflammation locally may represent a unique targeted approach in the treatment of ALD.
Alpha-1 antitrypsin governs alcohol-related liver disease in mice and humans.
Grander Christoph,Schaefer Benedikt,Schwärzler Julian,Grabherr Felix,de Graaf Dennis M,Enrich Barbara,Oberhuber Georg,Mayr Lisa,Sangineto Moris,Jaschke Nikolai,Adolph Timon E,Effenberger Maria,Moschen Alexander R,Dinarello Charles A,Zoller Heinz,Tilg Herbert
OBJECTIVE:Alcohol-related liver disease (ALD) is a global healthcare problem with limited treatment options. Alpha-1 antitrypsin (AAT, encoded by ) shows potent anti-inflammatory activities in many preclinical and clinical trials. In our study, we aimed to explore the role of AAT in ALD. DESIGN:An unselected cohort of 512 patients with cirrhosis was clinically characterised. Survival, clinical and biochemical parameters including AAT serum concentration were compared between patients with ALD and other aetiologies of liver disease. The role of AAT was evaluated in experimental ALD models. RESULTS:Cirrhotic ALD patients with AAT serum concentrations less than 120 mg/dL had a significantly higher risk for death/liver transplantation as compared with patients with AAT serum concentrations higher than 120 mg/dL. Multivariate Cox regression analysis showed that low AAT serum concentration was a NaMELD-independent predictor of survival/transplantation. Ethanol-fed wild-type (wt) mice displayed a significant decline in hepatic AAT compared with pair-fed mice. Therefore, mice were ethanol-fed, and these mice displayed protection from liver injury associated with decreased steatosis, hepatic neutrophil infiltration and abated expression of proinflammatory cytokines. To test the therapeutic capability of AAT, ethanol-fed wt mice were treated with human AAT. Administration of AAT ameliorated hepatic injury, neutrophil infiltration and steatosis. CONCLUSION:Cirrhotic ALD patients with AAT concentrations less than 120 mg/dL displayed an increased risk for death/liver transplantation. Both mice and AAT-treated wt animals showed protection from ethanol-induced liver injury. AAT could reflect a treatment option for human ALD, especially for alcoholic hepatitis.
Stress-responsive gene FKBP5 mediates alcohol-induced liver injury through the hippo pathway and CXCL1 signaling.
Kusumanchi Praveen,Liang Tiebing,Zhang Ting,Ross Ruth Ann,Han Sen,Chandler Kristina,Oshodi Adepeju,Jiang Yanchao,Dent Alexander L,Skill Nicholas J,Huda Nazmul,Ma Jing,Yang Zhihong,Liangpunsakul Suthat
Hepatology (Baltimore, Md.)
Chronic alcohol drinking is a major risk factor for alcohol-associated liver disease (ALD). FK506-binding protein 51 (FKBP5), a co-chaperone protein, is involved in many key regulatory pathways. It is known to be involved in stress-related disorders but there are no reports regarding its role in ALD. This present study aimed to examine the molecular mechanism of FKBP5 in ALD. We found a significant increase in hepatic FKBP5 transcripts and protein expression in patients with ALD and mice fed with chronic-plus-single binge ethanol. Loss of Fkbp5 in mice protected against alcohol-induced hepatic steatosis and inflammation. Transcriptomic analysis revealed a significant reduction of Tead1 and Cxcl1 mRNA in ethanol-fed Fkbp5 mice. Ethanol-induced Fkbp5 expression was secondary to downregulation of methylation level at its 5' UTR promoter region. The increase in Fkbp5 expression led to induction in transcription factor Tead1 through Hippo signaling pathway. Fkbp5 can interact with YAP upstream kinase, MST1, affecting its ability to phosphorylate YAP and the inhibitory effect of hepatic YAP phosphorylation by ethanol leading to YAP nuclear translocation and TEAD1 activation. Activation of TEAD1 led to increased expression of its novel target, CXCL1, a chemokine-mediated neutrophil recruitment, causing hepatic inflammation and neutrophil infiltration in our mouse model. CONCLUSION: We identified a novel FKBP5-YAP-TEAD1-CXCL1 axis in the pathogenesis of ALD. Loss of FKBP5 ameliorates alcohol-induced liver injury through the Hippo pathway and CXCL1 signaling, suggesting its potential role as a target for the treatment of ALD.
Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.
Ambade Aditya,Lowe Patrick,Kodys Karen,Catalano Donna,Gyongyosi Benedek,Cho Yeonhee,Iracheta-Vellve Arvin,Adejumo Adeyinka,Saha Banishree,Calenda Charles,Mehta Jeeval,Lefebvre Eric,Vig Pamela,Szabo Gyongyi
Hepatology (Baltimore, Md.)
Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. 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 (alanine aminotransferase [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 tumor necrosis factor (TNF)-α, interleukin (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 fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.
The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis.
Bala Shashi,Csak Timea,Saha Banishree,Zatsiorsky James,Kodys Karen,Catalano Donna,Satishchandran Abhishek,Szabo Gyongyi
Journal of hepatology
BACKGROUND & AIMS:Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD. METHODS:Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks. RESULTS:We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)α (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naïve and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and α-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet. CONCLUSIONS:Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.
Inflammation is independent of steatosis in a murine model of steatohepatitis.
Wang Wei,Xu Ming-Jiang,Cai Yan,Zhou Zhou,Cao Haixia,Mukhopadhyay Partha,Pacher Pal,Zheng Shusen,Gonzalez Frank J,Gao Bin
Hepatology (Baltimore, Md.)
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 (PPARγ) 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 PPARγ 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 PPARγ with a PPARγ 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 PPARγ agonist and antagonist, respectively. Finally, acute ethanol binge markedly attenuated HFD-induced hepatic PPARγ activation, which contributed to the up-regulation of hepatic Cxcl1 expression post-HFD-plus-binge ethanol. CONCLUSION:Hepatic PPARγ plays an opposing role in controlling steatosis and neutrophil infiltration, leading to dissociation between steatosis and inflammation; acute ethanol gavage attenuates hepatic PPARγ activation and subsequently up-regulates hepatic CXCL1/interleukin-8 expression, thereby exacerbating hepatic neutrophil infiltration. (Hepatology 2017;66:108-123).
Aging aggravates alcoholic liver injury and fibrosis in mice by downregulating sirtuin 1 expression.
Ramirez Teresa,Li Yong-Mei,Yin Shi,Xu Ming-Jiang,Feng Dechun,Zhou Zhou,Zang Mengwei,Mukhopadhyay Partha,Varga Zoltan V,Pacher Pal,Gao Bin,Wang Hua
Journal of hepatology
BACKGROUND & AIMS:Aging is known to exacerbate the progression of alcoholic liver disease (ALD), but the underlying mechanisms remain obscure. The aim of this study was to use a chronic plus binge ethanol feeding model in mice to evaluate the effects of aging on alcohol-induced liver injury. METHODS:C57BL/6 mice were subjected to short-term (10days) ethanol plus one binge or long-term (8weeks) ethanol plus multiple binges of ethanol. Liver injury and fibrosis were determined. Hepatic stellate cells (HSCs) were isolated and used in in vitro studies. RESULTS:Middle-aged (12-14months) and old-aged (>16months) mice were more susceptible to liver injury, inflammation, and oxidative stress induced by short-term plus one binge or long-term plus multiple binges of ethanol feeding when compared to young (8-12weeks) mice. Long-term plus multiple binges of ethanol feeding induced greater liver fibrosis in middle-aged mice than that in young mice. Hepatic expression of sirtuin 1 (SIRT1) protein was downregulated in the middle-aged mice compared to young mice. Restoration of SIRT1 expression via the administration of adenovirus-SIRT1 vector ameliorated short-term plus binge ethanol-induced liver injury and fibrosis in middle-aged mice. HSCs isolated from middle-aged mice expressed lower levels of SIRT1 protein and were more susceptible to spontaneous activation in in vitro culture than those from young mice. Overexpression of SIRT1 reduced activation of HSCs from middle-aged mice in vitro with downregulation of PDGFR-α and c-Myc, while deletion of SIRT1 activated HSCs isolated from young mice in vitro. Finally, HSC-specific SIRT1 knockout mice were more susceptible to long-term chronic-plus-multiple binges of ethanol-induced liver fibrosis with upregulation of PDGFR-α expression. CONCLUSIONS:Aging exacerbates ALD in mice through the downregulation of SIRT1 in hepatocytes and HSCs. Activation of SIRT1 may serve as a novel target for the treatment of ALD. LAY SUMMARY:Aged mice are more susceptible to alcohol-induced liver injury and fibrosis, which is, at least in part, due to lower levels of sirtuin 1 protein in hepatocytes and hepatic stellate cells. Our findings suggest that sirtuin 1 activators may have beneficial effects for the treatment of alcoholic liver disease in aged patients.
Alcohol inhibits T-cell glucose metabolism and hepatitis in ALDH2-deficient mice and humans: roles of acetaldehyde and glucocorticoids.
Gao Yanhang,Zhou Zhou,Ren Tianyi,Kim Seung-Jin,He Yong,Seo Wonhyo,Guillot Adrien,Ding Yanhua,Wu Ruihong,Shao Shuang,Wang Xiaomei,Zhang Hong,Wang Wei,Feng Dechun,Xu Mingjiang,Han Elaine,Zhong Wei,Zhou Zhanxiang,Pacher Pal,Niu Junqi,Gao Bin
OBJECTIVE:Aldehyde dehydrogenase 2 (ALDH2), a key enzyme to detoxify acetaldehyde in the liver, exists in both active and inactive forms in humans. Individuals with inactive ALDH2 accumulate acetaldehyde after alcohol consumption. However, how acetaldehyde affects T-cell hepatitis remains unknown. DESIGN:Wild-type (WT) and knockout ( ) mice were subjected to chronic ethanol feeding and concanavalin A (ConA)-induced T-cell hepatitis. Effects of acetaldehyde on T-cell glucose metabolism were investigated in vitro. Human subjects were recruited for binge drinking and plasma cortisol and corticosterone measurement. RESULTS:Ethanol feeding exacerbated ConA-induced hepatitis in WT mice but surprisingly attenuated it in mice despite higher acetaldehyde levels in mice. Elevation of serum cytokines and their downstream signals in the liver post-ConA injection was attenuated in ethanol-fed mice compared to WT mice. In vitro exposure to acetaldehyde inhibited ConA-induced production of several cytokines without affecting their mRNAs in mouse splenocytes. Acetaldehyde also attenuated interferon-γ production in phytohaemagglutinin-stimulated human peripheral lymphocytes. Mechanistically, acetaldehyde interfered with glucose metabolism in T cells by inhibiting aerobic glycolysis-related signal pathways. Finally, compared to WT mice, ethanol-fed mice had higher levels of serum corticosterone, a well-known factor that inhibits aerobic glycolysis. Blockade of corticosterone partially restored ConA-mediated hepatitis in ethanol-fed mice. Acute alcohol drinking elevated plasma cortisol and corticosterone levels in human subjects with higher levels in those with inactive ALDH2 than those with active ALDH2. CONCLUSIONS:ALDH2 deficiency is associated with elevated acetaldehyde and glucocorticoids post-alcohol consumption, thereby inhibiting T-cell activation and hepatitis.
Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis.
Zhong Fudi,Hu Zhigao,Jiang Keqing,Lei Biao,Wu Zhan,Yuan Guandou,Luo Hongliang,Dong Chunqiang,Tang Bo,Zheng Chaowen,Yang Shuai,Zeng Yonglian,Guo Zhenya,Yu Shuiping,Su Huizhao,Zhang Guo,Qiu Xiaoqiang,Tomlinson Stephen,He Songqing
Complement is known to play a role in alcoholic fatty liver disease (AFLD), but the underlying mechanisms are poorly understood, thereby constraining the development of a rational approach for therapeutic intervention in the complement system. C3 deficiency has been shown to impart protective effects against ethanol-induced hepatic steatosis and inflammation. Here we demonstrate a protection effect in wild-type mice by treatment with CR2-Crry, a specific inhibitor of C3 activation. The expression of glycine transfer (t) RNA-derived fragments (Gly-tRFs) is upregulated in ethanol-fed mice and inhibition of Gly-tRFs in vivo decreases chronic ethanol feeding-induced hepatosteatosis without affecting inflammation. The expression of Gly-tRF was downregulated in C3-deficient or CR2-Crry-treated mice, but not in C5-deficient mice; Gly-tRF expression was restored by the C3 activation products C3a or Asp (C3a-des-Arg) via the regulation of CYP2E1. Transcriptome profiling of hepatic tissues showed that Gly-tRF inhibitors upregulate the expression of sirtuin1 (Sirt1) and subsequently affect downstream lipogenesis and β-oxidation pathways. Mechanistically, Gly-tRF interacts with AGO3 to downregulate Sirt1 expression via sequence complementarity in the 3' UTR. Notably, the expression levels of C3d, CYP2E1 and Gly-tRF are upregulated, whereas Sirt1 is decreased in AFLD patients compared to healthy controls. Collectively, our findings suggest that C3 activation products contribute to hepatosteatosis by regulating the expression of Gly-tRF. Complement inhibition at the C3 activation step and treatment with Gly-tRF inhibitors may be potential and precise therapeutic approaches for AFLD.
MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6-p47-oxidative stress pathway in neutrophils.
Li Man,He Yong,Zhou Zhou,Ramirez Teresa,Gao Yueqiu,Gao Yanhang,Ross Ruth A,Cao Haixia,Cai Yan,Xu Mingjiang,Feng Dechun,Zhang Ping,Liangpunsakul Suthat,Gao Bin
OBJECTIVES:Chronic-plus-binge ethanol feeding activates neutrophils and exacerbates liver injury in mice. This study investigates how recent excessive drinking affects peripheral neutrophils and liver injury in alcoholics, and how miR-223, one of the most abundant microRNAs (miRNAs) in neutrophils, modulates neutrophil function and liver injury in ethanol-fed mice. DESIGNS:Three hundred alcoholics with (n=140) or without (n=160) recent excessive drinking and 45 healthy controls were enrolled. Mice were fed an ethanol diet for 10 days followed by a single binge of ethanol. RESULTS:Compared with healthy controls or alcoholics without recent drinking, alcoholics with recent excessive drinking had higher levels of circulating neutrophils, which correlated with serum levels of alanine transaminase (ALT) and aspartate transaminase (AST). miRNA array analysis revealed that alcoholics had elevated serum miR-223 levels compared with healthy controls. In chronic-plus-binge ethanol feeding mouse model, the levels of miR-223 were increased in both serum and neutrophils. Genetic deletion of the miR-223 gene exacerbated ethanol-induced hepatic injury, neutrophil infiltration, reactive oxygen species (ROS) and upregulated hepatic expression of interleukin (IL)-6 and phagocytic oxidase (phox) p47. Mechanistic studies revealed that miR-223 directly inhibited IL-6 expression and subsequently inhibited p47 expression in neutrophils. Deletion of the p47 gene ameliorated ethanol-induced liver injury and ROS production by neutrophils. Finally, miR-223 expression was downregulated, while IL-6 and p47 expression were upregulated in peripheral blood neutrophils from alcoholics compared with healthy controls. CONCLUSIONS:miR-223 is an important regulator to block neutrophil infiltration in alcoholic liver disease and could be a novel therapeutic target for the treatment of this malady.
Fecal microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice.
Ferrere Gladys,Wrzosek Laura,Cailleux Frédéric,Turpin Williams,Puchois Virginie,Spatz Madeleine,Ciocan Dragos,Rainteau Dominique,Humbert Lydie,Hugot Cindy,Gaudin Françoise,Noordine Marie-Louise,Robert Véronique,Berrebi Dominique,Thomas Muriel,Naveau Sylvie,Perlemuter Gabriel,Cassard Anne-Marie
Journal of hepatology
BACKGROUND & AIMS:Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS:Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS:Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS:Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY:Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.
The Candida albicans exotoxin candidalysin promotes alcohol-associated liver disease.
Chu Huikuan,Duan Yi,Lang Sonja,Jiang Lu,Wang Yanhan,Llorente Cristina,Liu Jinyuan,Mogavero Selene,Bosques-Padilla Francisco,Abraldes Juan G,Vargas Victor,Tu Xin M,Yang Ling,Hou Xiaohua,Hube Bernhard,Stärkel Peter,Schnabl Bernd
Journal of hepatology
BACKGROUND & AIMS:Alcohol-associated liver disease is a leading indication for liver transplantation and a leading cause of mortality. Alterations to the gut microbiota contribute to the pathogenesis of alcohol-associated liver disease. Patients with alcohol-associated liver disease have increased proportions of Candida spp. in the fecal mycobiome, yet little is known about the effect of intestinal Candida on the disease. Herein, we evaluated the contributions of Candida albicans and its exotoxin candidalysin in alcohol-associated liver disease. METHODS:C. albicans and the extent of cell elongation 1 (ECE1) were analyzed in fecal samples from controls, patients with alcohol use disorder and those with alcoholic hepatitis. Mice colonized with different and genetically manipulated C. albicans strains were subjected to the chronic-plus-binge ethanol diet model. Primary hepatocytes were isolated and incubated with candidalysin. RESULTS:The percentages of individuals carrying ECE1 were 0%, 4.76% and 30.77% in non-alcoholic controls, patients with alcohol use disorder and patients with alcoholic hepatitis, respectively. Candidalysin exacerbates ethanol-induced liver disease and is associated with increased mortality in mice. Candidalysin enhances ethanol-induced liver disease independently of the β-glucan receptor C-type lectin domain family 7 member A (CLEC7A) on bone marrow-derived cells, and candidalysin does not alter gut barrier function. Candidalysin can damage primary hepatocytes in a dose-dependent manner in vitro and is associated with liver disease severity and mortality in patients with alcoholic hepatitis. CONCLUSIONS:Candidalysin is associated with the progression of ethanol-induced liver disease in preclinical models and worse clinical outcomes in patients with alcoholic hepatitis. LAY SUMMARY:Candidalysin is a peptide toxin secreted by the commensal gut fungus Candida albicans. Candidalysin enhances alcohol-associated liver disease independently of the β-glucan receptor CLEC7A on bone marrow-derived cells in mice without affecting intestinal permeability. Candidalysin is cytotoxic to primary hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease. Candidalysin might be an effective target for therapy in patients with alcohol-associated liver disease.
MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease.
Satishchandran Abhishek,Ambade Aditya,Rao Sitara,Hsueh Ying-Chao,Iracheta-Vellve Arvin,Tornai David,Lowe Patrick,Gyongyosi Benedek,Li Jia,Catalano Donna,Zhong Li,Kodys Karen,Xie Jun,Bala Shashi,Gao Guangping,Szabo Gyongyi
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 polymerase chain reaction 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 with 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 liver tissues from mice. Levels of GRHL2 also were 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 alanine aminotransferase compared with mice given a control vector. Levels of hypoxia-inducible factor 1 alpha (HIF1α) 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 Hif1α 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 following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.
DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.
Chen Hanqing,Shen Feng,Sherban Alex,Nocon Allison,Li Yu,Wang Hua,Xu Ming-Jiang,Rui Xianliang,Han Jinyan,Jiang Bingbing,Lee Donghwan,Li Na,Keyhani-Nejad Farnaz,Fan Jian-Gao,Liu Feng,Kamat Amrita,Musi Nicolas,Guarente Leonard,Pacher Pal,Gao Bin,Zang Mengwei
Hepatology (Baltimore, Md.)
Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. CONCLUSION:The dysregulation of SIRT1-DEPTOR-mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1-S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).
Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice.
Hendrikx Tim,Duan Yi,Wang Yanhan,Oh Jee-Hwan,Alexander Laura M,Huang Wendy,Stärkel Peter,Ho Samuel B,Gao Bei,Fiehn Oliver,Emond Patrick,Sokol Harry,van Pijkeren Jan-Peter,Schnabl Bernd
OBJECTIVE:Antimicrobial C-type lectin regenerating islet-derived 3 gamma (REG3G) is suppressed in the small intestine during chronic ethanol feeding. Our aim was to determine the mechanism that underlies REG3G suppression during experimental alcoholic liver disease. DESIGN:Interleukin 22 (IL-22) regulates expression of REG3G. Therefore, we investigated the role of IL-22 in mice subjected to chronic-binge ethanol feeding (NIAAA model). RESULTS:In a mouse model of alcoholic liver disease, we found that type 3 innate lymphoid cells produce lower levels of IL-22. Reduced IL-22 production was the result of ethanol-induced dysbiosis and lower intestinal levels of indole-3-acetic acid (IAA), a microbiota-derived ligand of the aryl hydrocarbon receptor (AHR), which regulates expression of IL-22. Importantly, faecal levels of IAA were also found to be lower in patients with alcoholic hepatitis compared with healthy controls. Supplementation to restore intestinal levels of IAA protected mice from ethanol-induced steatohepatitis by inducing intestinal expression of IL-22 and REG3G, which prevented translocation of bacteria to liver. We engineered to produce IL-22 (/IL-22) and fed them to mice along with the ethanol diet; these mice had reduced liver damage, inflammation and bacterial translocation to the liver compared with mice fed an isogenic control strain and upregulated expression of REG3G in intestine. However, /IL-22 did not reduce ethanol-induced liver disease in mice. CONCLUSION:Ethanol-associated dysbiosis reduces levels of IAA and activation of the AHR to decrease expression of IL-22 in the intestine, leading to reduced expression of REG3G; this results in bacterial translocation to the liver and steatohepatitis. Bacteria engineered to produce IL-22 induce expression of REG3G to reduce ethanol-induced steatohepatitis.
MicroRNA 181b-3p and its target importin α5 regulate toll-like receptor 4 signaling in Kupffer cells and liver injury in mice in response to ethanol.
Saikia Paramananda,Bellos Damien,McMullen Megan R,Pollard Katherine A,de la Motte Carol,Nagy Laura E
Hepatology (Baltimore, Md.)
Increased inflammatory signaling by Kupffer cells contributes to alcoholic liver disease (ALD). Here we investigated the impact of small, specific-sized hyaluronic acid of 35 kD (HA35) on ethanol-induced sensitization of Kupffer cells, as well as ethanol-induced liver injury in mice. Unbiased analysis of microRNA (miRNA) expression in Kupffer cells identified miRNAs regulated by both ethanol and HA35. Toll-like receptor 4 (TLR4)-mediated signaling was assessed in primary cultures of Kupffer cells from ethanol- and pair-fed rats after treatment with HA35. Female C57BL6/J mice were fed ethanol or pair-fed control diets and treated or not with HA35. TLR4 signaling was increased in Kupffer cells by ethanol; this sensitization was normalized by ex vivo treatment with HA35. Next generation sequencing of Kupffer cell miRNA identified miRNA 181b-3p (miR181b-3p) as sensitive to both ethanol and HA35. Importin α5, a protein involved in p65 translocation to the nucleus, was identified as a target of miR181b-3p; importin α5 protein was increased in Kupffer cells from ethanol-fed rats, but decreased by HA35 treatment. Overexpression of miR181b-3p decreased importin α5 expression and normalized lipopolysaccharide-stimulated tumor necrosis factor α expression in Kupffer cells from ethanol-fed rats. In a mouse model of ALD, ethanol feeding decreased miR181b-3p in liver and increased expression of importin α5 in nonparenchymal cells. Treatment with HA35 normalized these changes and also protected mice from ethanol-induced liver and intestinal injury. CONCLUSION:miR181b-3p is dynamically regulated in Kupffer cells and mouse liver in response to ethanol and treatment with HA35. miR181b-3p modulates expression of importin α5 and sensitivity of TLR4-mediated signaling. This study identifies a miR181b-3p-importin α5 axis in regulating inflammatory signaling pathways in hepatic macrophages. (Hepatology 2017;66:602-615).
SNX10 mediates alcohol-induced liver injury and steatosis by regulating the activation of chaperone-mediated autophagy.
You Yan,Li Wan-Zhen,Zhang Sulin,Hu Bin,Li Yue-Xuan,Li Hai-Dong,Tang Huan-Huan,Li Qian-Wen,Guan Yun-Yun,Liu Li-Xin,Bao Wei-Lian,Shen Xiaoyan
Journal of hepatology
BACKGROUND & AIMS:Alcoholic liver disease (ALD) is a major cause of morbidity and mortality worldwide. However, the cellular defense mechanisms underlying ALD are not well understood. Recent studies highlighted the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Sorting nexin (SNX)-10 has a regulatory function in endolysosomal trafficking and stabilisation. Here, we investigated the roles of SNX10 in CMA activation and in the pathogenesis of alcohol-induced liver injury and steatosis. METHODS:Snx10 knockout (Snx10 KO) mice and their wild-type (WT) littermates fed either the Lieber-DeCarli liquid alcohol diet or a control liquid diet, and primary cultured WT and Snx10 KO hepatocytes stimulated with ethanol, were used as in vivo and in vitro ALD models, respectively. Activation of CMA, liver injury parameters, inflammatory cytokines, oxidative stress and lipid metabolism were measured. RESULTS:Compared with WT littermates, Snx10 KO mice exhibited a significant amelioration in ethanol-induced liver injury and hepatic steatosis. Both in vivo and in vitro studies showed that SNX10 deficiency upregulated lysosome-associated membrane protein type 2A (LAMP-2A) expression and CMA activation, which could be reversed by SNX10 overexpression in vitro. LAMP-2A interference confirmed that the upregulation of Nrf2 and AMPK signalling pathways induced by SNX10 deficiency relied on CMA activation. Pull-down assays revealed an interaction between SNX10 and cathepsin A (CTSA), a key enzyme involved in LAMP-2A degradation. Deficiency in SNX10 inhibited CTSA maturation and increased the stability of LAMP-2A, resulting in an increase in CMA activity. CONCLUSIONS:SNX10 controls CMA activity by mediating CTSA maturation, and, thus, has an essential role in alcohol-induced liver injury and steatosis. Our results provide evidence for SNX10 as a potential promising therapeutic target for preventing or ameliorating liver injury in ALD. LAY SUMMARY:Alcoholic liver disease is a major cause of morbidity and mortality worldwide. Recent studies highlight the involvement of chaperone-mediated autophagy (CMA) in regulating hepatic lipid metabolism. Our study reveals that deficiency of sorting nexin (SNX) 10 increases the stability of LAMP-2A by inhibiting cathepsin A maturation, resulting in the increase of CMA activity and, thus, alleviates alcohol-induced liver injury and steatosis.
Brown fat activation mitigates alcohol-induced liver steatosis and injury in mice.
Shen Hong,Jiang Lin,Lin Jiandie D,Omary M Bishr,Rui Liangyou
The Journal of clinical investigation
Chronic alcohol consumption causes liver injury, inflammation and fibrosis, thereby increasing morbidity and mortality. Paradoxically, modest drinking is believed to confer metabolic improvement, but the underlying mechanism remains elusive. Here, we have identified a novel hepatoprotective brain/brown adipose tissue (BAT)/liver axis. Alcohol consumption or direct alcohol administration into the brain stimulated hypothalamic neural circuits and sympathetic nerves innervating BAT, and dramatically increased BAT uncoupling protein 1 (Ucp1) expression and activity in a BAT sympathetic nerve-dependent manner. BAT and beige fat oxidized fatty acids to fuel Ucp1-mediated thermogenesis, thereby inhibiting lipid trafficking into the liver. BAT also secreted several adipokines, including adiponectin that suppressed hepatocyte injury and death. Genetic deletion of Ucp1 profoundly augmented alcohol-induced liver steatosis, injury, inflammation and fibrosis in male and female mice. Conversely, activation of BAT and beige fat through cold exposure suppressed alcoholic liver disease development. Our results unravel an unrecognized brain alcohol-sensing/sympathetic nerve/BAT/liver axis that counteracts liver steatosis and injury.
Recovery of ethanol-induced depletion ameliorates alcoholic liver disease.
Grander Christoph,Adolph Timon E,Wieser Verena,Lowe Patrick,Wrzosek Laura,Gyongyosi Benedek,Ward Doyle V,Grabherr Felix,Gerner Romana R,Pfister Alexandra,Enrich Barbara,Ciocan Dragos,Macheiner Sophie,Mayr Lisa,Drach Matthias,Moser Patrizia,Moschen Alexander R,Perlemuter Gabriel,Szabo Gyongyi,Cassard Anne Marie,Tilg Herbert
OBJECTIVE:Alcoholic liver disease (ALD) is a global health problem with limited therapeutic options. Intestinal barrier integrity and the microbiota modulate susceptibility to ALD. , a Gram-negative intestinal commensal, promotes barrier function partly by enhancing mucus production. The aim of this study was to investigate microbial alterations in ALD and to define the impact of administration on the course of ALD. DESIGN:The intestinal microbiota was analysed in an unbiased approach by 16S ribosomal DNA (rDNA) sequencing in a Lieber-DeCarli ALD mouse model, and faecal abundance was determined in a cohort of patients with alcoholic steatohepatitis (ASH). The impact of on the development of experimental acute and chronic ALD was determined in a preventive and therapeutic setting, and intestinal barrier integrity was analysed. RESULTS:Patients with ASH exhibited a decreased abundance of faecal when compared with healthy controls that indirectly correlated with hepatic disease severity. Ethanol feeding of wild-type mice resulted in a prominent decline in abundance. Ethanol-induced intestinal depletion could be restored by oral supplementation. Furthermore, administration when performed in a preventive setting decreased hepatic injury, steatosis and neutrophil infiltration. also protected against ethanol-induced gut leakiness, enhanced mucus thickness and tight-junction expression. In already established ALD, used therapeutically ameliorated hepatic injury and neutrophil infiltration. CONCLUSION: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.
Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients.
Marin Veronica,Poulsen Kyle,Odena Gemma,McMullen Megan R,Altamirano Jose,Sancho-Bru Pau,Tiribelli Claudio,Caballeria Juan,Rosso Natalia,Bataller Ramon,Nagy Laura E
Journal of hepatology
BACKGROUND & AIMS:Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. METHODS:MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). RESULTS:HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. CONCLUSIONS:Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.
The epigenetic regulator SIRT6 protects the liver from alcohol-induced tissue injury by reducing oxidative stress in mice.
Kim Hyeong Geug,Huang Menghao,Xin Yue,Zhang Yang,Zhang Xinge,Wang Gaihong,Liu Sheng,Wan Jun,Ahmadi Ali Reza,Sun Zhaoli,Liangpunsakul Suthat,Xiong Xiwen,Dong Xiaocheng Charlie
Journal of hepatology
BACKGROUND & AIMS:As a nicotinamide adenine dinucleotide-dependent deacetylase and a key epigenetic regulator, sirtuin 6 (SIRT6) has been implicated in the regulation of metabolism, DNA repair, and inflammation. However, the role of SIRT6 in alcohol-related liver disease (ALD) remains unclear. The aim of this study was to investigate the function and mechanism of SIRT6 in ALD pathogenesis. METHODS:We developed and characterized Sirt6 knockout (KO) and transgenic mouse models that were treated with either control or ethanol diet. Hepatic steatosis, inflammation, and oxidative stress were analyzed using biochemical and histological methods. Gene regulation was analyzed by luciferase reporter and chromatin immunoprecipitation assays. RESULTS:The Sirt6 KO mice developed severe liver injury characterized by a remarkable increase of oxidative stress and inflammation, whereas the Sirt6 transgenic mice were protected from ALD via normalization of hepatic lipids, inflammatory response, and oxidative stress. Our molecular analysis has identified a number of novel Sirt6-regulated genes that are involved in antioxidative stress, including metallothionein 1 and 2 (Mt1 and Mt2). Mt1/2 genes were downregulated in the livers of Sirt6 KO mice and patients with alcoholic hepatitis. Overexpression of Mt1 in the liver of Sirt6 KO mice improved ALD by reducing hepatic oxidative stress and inflammation. We also identified a critical link between SIRT6 and metal regulatory transcription factor 1 (Mtf1) via a physical interaction and functional coactivation. Mt1/2 promoter reporter assays showed a strong synergistic effect of SIRT6 on the transcriptional activity of Mtf1. CONCLUSIONS:Our data suggest that SIRT6 plays a critical protective role against ALD and it may serve as a potential therapeutic target for ALD. LAY SUMMARY:The liver, the primary organ for ethanol metabolism, can be damaged by the byproducts of ethanol metabolism, including reactive oxygen species. In this study, we have identified a key epigenetic regulator SIRT6 that plays a critical role in protecting the liver from oxidative stress-induced liver injury. Thus, our data suggest that SIRT6 may be a potential therapeutic target for alcohol-related liver disease.
Impaired TFEB-Mediated Lysosome Biogenesis and Autophagy Promote Chronic Ethanol-Induced Liver Injury and Steatosis in Mice.
Chao Xiaojuan,Wang Shaogui,Zhao Katrina,Li Yuan,Williams Jessica A,Li Tiangang,Chavan Hemantkumar,Krishnamurthy Partha,He Xi C,Li Linheng,Ballabio Andrea,Ni Hong-Min,Ding Wen-Xing
BACKGROUND & AIMS:Defects in lysosome function and autophagy contribute to the pathogenesis of alcoholic liver disease. We investigated the mechanisms by which alcohol consumption affects these processes by evaluating the functions of transcription factor EB (TFEB), which regulates lysosomal biogenesis. METHODS:We performed studies with GFP-LC3 mice, mice with liver-specific deletion of TFEB, mice with disruption of the transcription factor E3 gene (TFE3-knockout mice), mice with disruption of the Tefb and Tfe3 genes (TFEB and TFE3 double-knockout mice), and Tfeb albumin cre-negative mice (controls). TFEB was overexpressed from adenoviral vectors or knocked down with small interfering RNAs in mouse livers. Mice were placed on diets of regular ethanol feeding plus an acute binge to induce liver damage (ethanol diet); some mice also were given injections of torin-1, an inhibitor of the kinase activity of the mechanistic target of rapamycin (mTOR). Liver tissues were collected and analyzed by immunohistochemistry, immunoblots, and quantitative real-time polymerase chain reaction to monitor lysosome biogenesis. We analyzed levels of TFEB in liver tissues from patients with alcoholic hepatitis and from healthy donors (controls) by immunohistochemistry. RESULTS:Liver tissues from mice on the ethanol diet had lower levels of total and nuclear TFEB compared with control mice, and hepatocytes had decreased lysosome biogenesis and autophagy. Hepatocytes from mice on the ethanol diet had increased translocation of mTOR into lysosomes, resulting in increased mTOR activation. Administration of torin-1 increased liver levels of TFEB and decreased steatosis and liver injury induced by ethanol. Mice that overexpressed TFEB in the liver developed less severe ethanol-induced liver injury and had increased lysosomal biogenesis and mitochondrial bioenergetics compared with mice carrying a control vector. Mice with knockdown of TFEB and TFEB-TFE3 double-knockout mice developed more severe liver injury in response to the ethanol diet than control mice. Liver tissues from patients with alcohol-induced hepatitis had lower nuclear levels of TFEB than control tissues. CONCLUSIONS:We found that ethanol feeding plus an acute binge decreased hepatic expression of TFEB, which is required for lysosomal biogenesis and autophagy. Strategies to block mTOR activity or increase levels of TFEB might be developed to protect the liver from ethanol-induced damage.
β-Hydroxybutyrate protects from alcohol-induced liver injury via a Hcar2-cAMP dependent pathway.
Chen Yonglin,Ouyang Xinshou,Hoque Rafaz,Garcia-Martinez Irma,Yousaf Muhammad Nadeem,Tonack Sarah,Offermanns Stefan,Dubuquoy Laurent,Louvet Alexandre,Mathurin Philippe,Massey Veronica,Schnabl Bernd,Bataller Ramon Alberola,Mehal Wajahat Zafar
Journal of hepatology
BACKGROUND & AIMS:Sterile inflammation resulting in alcoholic hepatitis (AH) occurs unpredictably after many years of excess alcohol intake. The factors responsible for the development of AH are not known but mitochondrial damage with loss of mitochondrial function are common features. Hcar2 is a G-protein coupled receptor which is activated by β-hydroxybutyrate (BHB). We aimed to determine the relevance of the BHB-Hcar2 pathway in alcoholic liver disease. METHODS:We tested if loss of BHB production can result in increased liver inflammation. We further tested if BHB supplementation is protective in AH through interaction with Hcar2, and analyzed the immune and cellular basis for protection. RESULTS:Humans with AH have reduced hepatic BHB, and inhibition of BHB production in mice aggravated ethanol-induced AH, with higher plasma alanine aminotransferase levels, increased steatosis and greater neutrophil influx. Conversely supplementation of BHB had the opposite effects with reduced alanine aminotransferase levels, reduced steatosis and neutrophil influx. This therapeutic effect of BHB is dependent on the receptor Hcar2. BHB treatment increased liver Il10 transcripts, and promoted the M2 phenotype of intrahepatic macrophages. BHB also increased the transcriptional level of M2 related genes in vitro bone marrow derived macrophages. This skewing towards M2 related genes is dependent on lower mitochondrial membrane potential (Δψ) induced by BHB. CONCLUSIONS:Collectively, our data shows that BHB production during excess alcohol consumption has an anti-inflammatory and hepatoprotective role through an Hcar2 dependent pathway. This introduces the concept of metabolite-based therapy for AH. LAY SUMMARY:Alcoholic hepatitis is a life-threatening condition with no approved therapy that occurs unexpectedly in people who consume excess alcohol. The liver makes many metabolites, and we demonstrate that loss of one such metabolite β-hydroxybutyrate occurs in patients with alcoholic hepatitis. This loss can increase alcohol-induced liver injury, and β-hydroxybutyrate can protect from alcohol-induced liver injury via a receptor on liver macrophages. This opens the possibility of metabolite-based therapy for alcoholic hepatitis.
Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury.
Cho Young-Eun,Yu Li-Rong,Abdelmegeed Mohamed A,Yoo Seong-Ho,Song Byoung-Joon
Journal of hepatology
BACKGROUND & AIMS:Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness. METHODS:The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses. RESULTS:Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (β-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels. CONCLUSIONS:These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease. LAY SUMMARY:Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.