Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol‐induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol‐induced hepatocyte injury ...or inflammation. Because receptor‐interacting protein kinase (RIP) 3–mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol‐induced liver injury is RIP3‐dependent and RIP1‐independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)‐deficient mice, indicating CYP2E1‐mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol‐induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin‐1 did not attenuate ethanol‐induced hepatocyte injury. Ethanol‐induced apoptosis, assessed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling–positive nuclei and accumulation of cytokeratin‐18 fragments in the liver, was independent of RIP3. Conclusion: CYP2E1‐dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol‐induced hepatocyte injury is RIP3‐dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy. (HEPATOLOGY 2013)
Background and Aim
Impaired gut‐liver axis is a potential factor contributing to alcoholic liver disease. Ethanol depletes intestinal integrity and causes gut dysbiosis. Butyrate, a fermentation ...byproduct of gut microbiota, is altered negatively following chronic ethanol exposure. This study aimed to determine whether prophylactic tributyrin could protect the intestinal barrier and liver in mice during combined chronic‐binge ethanol exposure.
Methods
C57BL/6J mice exposed to 5% v/v ethanol‐containing diet for 10 days received a single ethanol gavage (5 g/kg) 9 h before euthanasia. Control mice were isocalorically pair‐fed maltose dextrin for ethanol. Diets were supplemented (5 mM) with tributyrin or glycerol. Intestine and liver disease activity was assessed histologically. Protein and mRNA expression of tight junction (TJ) proteins, toll‐like receptors, and tumor necrosis factor‐alpha were assessed. Caco‐2 monolayers with or without ethanol exposure and/or sodium butyrate were used to test butyrate's direct effects on intestinal integrity.
Results
Chronic‐binge ethanol feeding impaired intestinal TJ protein co‐localization staining; however, tributyrin co‐treatment mitigated these effects. Ethanol depleted TJ and transepithelial electrical resistance in Caco‐2 monolayers, but butyrate co‐treatment reduced these effects. Hepatic toll‐like receptor mRNA expression and tumor necrosis factor‐alpha protein expression was induced by ethanol; however, the response was significantly dampened in mice co‐treated with tributyrin. Tributyrin altered localization of both neutrophils and single hepatocyte death: Leukocytes and apoptotic hepatocytes localized predominantly around the portal tract in ethanol‐only treated mice, whereas localization predominated around the central vein in ethanol‐tributyrin mice.
Conclusions
Prophylactic tributyrin supplementation mitigated effects of combined chronic‐binge ethanol exposure on disruption of intestinal TJ localization and intestinal permeability and liver injury.
Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a ...key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy.
Rip3+/+, Rip3−/−, Mlkl+/+ and Mlkl−/− mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA).
The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC diet also induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl−/− mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes.
Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy.
Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.
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•MLKL-mediated signaling contributes to FFC diet-induced liver injury.•FFC diet or palmitic acid treatment induces MLKL expression in hepatocytes.•Palmitic acid drives MLKL translocation to autophagosomes independently of Rip3.•Mlkl, but not Rip3, regulates autophagic flux in a murine model of NAFL/NASH.•Pharmacologic inhibition of autophagy induces MLKL expression.
Multiple pathways of programmed cell death are important in liver homeostasis. Hepatocyte death is associated with progression of nonalcoholic fatty liver disease, and inhibition of apoptosis ...partially protects against liver injury in response to a high‐fat diet (HFD). However, the contribution of necroptosis, a caspase‐independent pathway of cell death, to HFD‐induced liver injury is not known. Wild‐type C57BL/6 and receptor interacting protein (RIP) 3−/− mice were randomized to chow or HFD. HFD‐fed C57BL/6 mice increased expression of RIP3, the master regulator of necroptosis, as well as phosphorylated mixed lineage kinase domain‐like, an effector of necroptotic cell death, in liver. HFD did not increase phosphorylated mixed lineage kinase domain‐like in RIP3−/− mice. HFD increased fasting insulin and glucose, as well as glucose intolerance, in C57BL/6 mice. RIP3−/− mice were glucose‐intolerant even on the chow diet; HFD further increased fasting glucose and insulin but not glucose intolerance. HFD also increased hepatic steatosis, plasma alanine aminotransferase activity, inflammation, oxidative stress, and hepatocellular apoptosis in wild‐type mice; these responses were exacerbated in RIP3−/− mice. Importantly, increased inflammation and injury were associated with early indicators of fibrosis in RIP3−/− compared to C57BL/6 mice. Culture of AML12 hepatocytes with palmitic acid increased cytotoxicity through apoptosis and necrosis. Inhibition of RIP1 with necrostatin‐1 or small interfering RNA knockdown of RIP3 reduced palmitic acid‐induced cytotoxicity. Conclusion: Absence of RIP3, a key mediator of necroptosis, exacerbated HFD‐induced liver injury, associated with increased inflammation and hepatocyte apoptosis, as well as early fibrotic responses; these findings indicate that shifts in the mode of hepatocellular death can influence disease progression and have therapeutic implications because manipulation of hepatocyte cell death pathways is being considered as a target for treatment of nonalcoholic fatty liver disease. (Hepatology 2016;64:1518‐1533)
Complement plays a crucial role in microbial defense and clearance of apoptotic cells. Emerging evidence suggests complement is an important contributor to alcoholic liver disease. While complement ...component 1, Q subcomponent (C1q)-dependent complement activation contributes to ethanol-induced liver injury, the role of the alternative pathway in ethanol-induced injury is unknown. Activation of complement via the classical and alternative pathways was detected in alcoholic hepatitis patients. Female C57BL/6J wild type (WT), C1q-deficient ( C1qa
, lacking classical pathway activation), complement protein 4-deficient ( C4
, lacking classical and lectin pathway activation), complement factor D-deficient ( FD
, lacking alternative pathway activation), and C1qa/FD
(lacking classical and alternative pathway activation) mice were fed an ethanol-containing liquid diet or pair-fed control diet for 4 or 25 days. Following chronic ethanol exposure, liver injury, steatosis, and proinflammatory cytokine expression were increased in WT but not C1qa
, C4
, or C1qa/FD
mice. In contrast, liver injury, steatosis, and proinflammatory mediators were robustly increased in ethanol-fed FD
mice compared with WT mice. Complement activation, assessed by hepatic accumulation of C1q and complement protein 3 (C3) cleavage products (C3b/iC3b/C3c), was evident in livers of WT mice in response to both short-term and chronic ethanol. While C1q accumulated in ethanol-fed FD
mice (short term and chronic), C3 cleavage products were detected after short-term but not chronic ethanol. Consistent with impaired complement activation, chronic ethanol induced the accumulation of apoptotic cells and fibrogenic responses in the liver of FD
mice. These data highlight the protective role of complement factor D (FD) and suggest that FD-dependent amplification of complement is an adaptive response that promotes hepatic healing and recovery in response to chronic ethanol. NEW & NOTEWORTHY Complement, a component of the innate immune system, is an important pathophysiological contributor to ethanol-induced liver injury. We have identified a novel role for factor D, a component of the alternative pathway, in protecting the liver from ethanol-induced inflammation, accumulation of apoptotic hepatocytes, and profibrotic responses. These data indicate a dual role of complement with regard to inflammatory and protective responses and suggest that accumulation of apoptotic cells impairs hepatic healing/recovery during alcoholic liver disease.
Background
Macrophage migration inhibitory factor (MIF), a pluripotent immune regulator, is an emerging mediator in alcohol‐related liver disease (ALD). MIF is associated with ALD progression through ...its chemokine‐ and cytokine‐like activities.
Methods
Mechanistic studies into the role of MIF in ethanol (EtOH)‐induced liver injury were performed in Mif−/− mice and in C57BL/6J mice treated with a small‐molecule MIF antagonist, MIF098, after Gao‐Binge (acute‐on‐chronic) EtOH feeding, an EtOH feeding protocol associated with hepatic neutrophilia and induction of the unfolded protein response (UPR).
Results
The MIF axis, for example, MIF and MIF receptors invariant polypeptide of major histocompatibility complex, class II antigen‐associated (CD74), CXCR2, CXCR4, and CXCR7, was enhanced in the livers of alcoholic hepatitis (AH) patients as compared to healthy controls. Mif−/− mice were protected from hepatocellular injury after Gao‐Binge feeding, independent of neutrophilia and inflammation, but were associated with the UPR. Interestingly, the UPR signature in AH patients and in mice following Gao‐Binge feeding was biased toward cell death with increased expression of pro‐cell death CCAAT–enhancer‐binding protein homologous protein (CHOP) and decreased prosurvival GRP78. The UPR and liver injury 6 hours after binge were prevented both in Mif−/− mice and in MIF098‐treated mice. However, both MIF interventions led to increased liver injury and exacerbated the hepatic UPR 9 hours after binge. Induction of upstream UPR signaling and expression of CHOP protein by thapsigargin in alpha mouse liver 12 hepatocytes were blunted by coexposure to MIF098, directly connecting MIF to UPR in hepatocytes.
Conclusions
The current study revealed that, in addition to its cytokine/chemokine functions, MIF is an upstream regulator of UPR in response to EtOH feeding in mice. Importantly, both MIF and UPR can either protect or contribute to liver injury, dependent upon the stage or severity of EtOH‐induced liver injury.
MIF is a pivotal regulator of innate immunity in alcohol‐related liver disease and in models of ethanol feeding in mice. In the current study, MIF was discovered to control aspects of the UPR following Gao‐Binge ethanol feeding in mice independent of inflammation. Global Mif deletion (A, B) and a MIF inhibitor, MIF098 (C, D), prevented ethanol‐induced eIF2α phosphorylation and expression of CHOP protein in livers of mice. MIF098 also decreased UPR activation in thapsigargin‐challenged hepatocytes (E–F) linking MIF directly to the UPR.
Protein synthesis and autophagy are regulated by cellular ATP content. We tested the hypothesis that mitochondrial dysfunction, including generation of reactive oxygen species (ROS), contributes to ...impaired protein synthesis and increased proteolysis resulting in tissue atrophy in a comprehensive array of models. In myotubes treated with ethanol, using unbiased approaches, we identified defects in mitochondrial electron transport chain components, endogenous antioxidants, and enzymes regulating the tricarboxylic acid (TCA) cycle. Using high sensitivity respirometry, we observed impaired cellular respiration, decreased function of complexes I, II, and IV, and a reduction in oxidative phosphorylation in ethanol-treated myotubes and muscle from ethanol-fed mice. These perturbations resulted in lower skeletal muscle ATP content and redox ratio (NAD+/NADH). Ethanol also caused a leak of electrons, primarily from complex III, with generation of mitochondrial ROS and reverse electron transport. Oxidant stress with lipid peroxidation (thiobarbituric acid reactive substances) and protein oxidation (carbonylated proteins) were increased in myotubes and skeletal muscle from mice and humans with alcoholic liver disease. Ethanol also impaired succinate oxidation in the TCA cycle with decreased metabolic intermediates. MitoTEMPO, a mitochondrial specific antioxidant, reversed ethanol-induced mitochondrial perturbations (including reduced oxygen consumption, generation of ROS and oxidative stress), increased TCA cycle intermediates, and reversed impaired protein synthesis and the sarcopenic phenotype. We show that ethanol causes skeletal muscle mitochondrial dysfunction, decreased protein synthesis, and increased autophagy, and that these perturbations are reversed by targeting mitochondrial ROS.
Overall schema of ethanol-induced mitochondrial function in skeletal muscle. Ethanol impairs ETC function with leak of electrons to generate superoxide (O2-) that causes oxidative injury to tissue and decreases TCA cycle intermediates. Display omitted
•Unbiased approaches showed that ethanol altered muscle mitochondrial regulatory proteins.•Mitochondrial functional studies in situ showed defects in electron transport chain components.•Ethanol increased mitochondrial ROS and oxidative stress in myotubes, human and mouse muscle.•Tricarboxylic acid cycle intermediates were reduced by ethanol in muscle and myotubes.•MitoTempo reversed ethanol induced perturbations in myotubes.
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).
Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we ...investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.
Lipopolysaccharide (LPS)‐mediated activation of Toll‐like receptors (TLRs) in hepatic macrophages and injury to hepatocytes are major contributors to the pathogenesis of alcoholic liver disease. ...However, the mechanisms by which TLR‐dependent inflammatory responses and alcohol‐induced hepatocellular damage coordinately lead to alcoholic liver disease are not completely understood. In this study, we found that mice deficient in interleukin‐1 receptor‐associated kinase M (IRAKM), a proximal TLR pathway molecule typically associated with inhibition of TLR signaling, were actually protected from chronic ethanol‐induced liver injury. In bone marrow‐derived macrophages challenged with low concentrations of LPS, which reflect the relevant pathophysiological levels of LPS in both alcoholic patients and ethanol‐fed mice, the IRAKM Myddosome was preferentially formed. Further, the IRAKM Myddosome mediated the up‐regulation of Mincle, a sensor for cell death. Mincle‐deficient mice were also protected from ethanol‐induced liver injury. The endogenous Mincle ligand spliceosome‐associated protein 130 (SAP130) is a danger signal released by damaged cells; culture of hepatocytes with ethanol increased the release of SAP130. Ex vivo studies in bone marrow‐derived macrophages suggested that SAP130 and LPS synergistically activated inflammatory responses, including inflammasome activation. Conclusion: This study reveals a novel IRAKM‐Mincle axis that contributes to the pathogenesis of ethanol‐induced liver injury. (Hepatology 2016;64:1978‐1993).
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