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Nonalcoholic steatohepatitis (NASH) and alcoholic hepatitis (AH) affect a large part of the general population worldwide. Dysregulation of lipid metabolism and alcohol toxicity drive disease progression by the activation of hepatic stellate cells and the capillarization of liver sinusoidal endothelial cells. Collagen deposition, along with sinusoidal remodeling, alters sinusoid structure, resulting in hepatic inflammation, portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for NASH and AH. However, the success of such treatments is limited and unpredictable. We report a strategy for NASH and AH treatment involving the induction of integrin α v β 3 -mediated cell apoptosis using a rationally designed protein (ProAgio). Integrin α v β 3 is highly expressed in activated hepatic stellate cells (αHSCs), the angiogenic endothelium, and capillarized liver sinusoidal endothelial cells (caLSECs). ProAgio induces the apoptosis of these disease-driving cells, therefore decreasing collagen fibril, reversing sinusoid remodeling, and reducing immune cell infiltration. The reversal of sinusoid remodeling reduces the expression of leukocyte adhesion molecules on LSECs, thus decreasing leukocyte infiltration/activation in the diseased liver. Our studies present a novel and effective approach for NASH and AH treatment.

Neutrophil infiltration occurs in a variety of liver diseases, but it is unclear how neutrophils and hepatocytes interact. Neutrophils generally use granule proteases to digest phagocytosed bacteria and foreign substances or neutralize them in neutrophil extracellular traps. In certain pathological states, granule proteases play a destructive role against the host as well. More recently, nondestructive actions of neutrophil granule proteins have been reported, such as modulation of tissue remodeling and metabolism. Here, we report a completely different mechanism by which neutrophils act nondestructively, by inserting granules directly into hepatocytes. Specifically, elastase-containing granules were transferred to hepatocytes where elastase selectively degraded intracellular calcium channels to reduce cell proliferation without cytotoxicity. In response, hepatocytes increased expression of Serpin E2 and A3, which inhibited elastase activity. Elastase insertion was seen in patient specimens of alcohol-associated hepatitis, and the relationship between elastase-mediated ITPR2 degradation and reduced cell proliferation was confirmed in mouse models. Moreover, neutrophils from patients with alcohol-associated hepatitis were more prone to degranulation and more potent in reducing calcium channel expression than neutrophils from healthy individuals. This nondestructive and reversible action on hepatocytes defines a previously unrecognized role for neutrophils in the transient regulation of epithelial calcium signaling mechanisms.

Acute liver failure caused by alcoholic hepatitis (AH) is only effectively treated with liver transplantation. Livers of patients with AH show a unique molecular signature characterized by defective hepatocellular redox metabolism, concurrent to hepatic infiltration of neutrophils that express myeloperoxidase (MPO) and form neutrophil extracellular traps (NETs). Exacerbated NET formation and MPO activity contribute to liver damage in mice with AH and predicts poor prognosis in AH patients. The identification of pathways that maladaptively exacerbate neutrophilic activity in liver could inform of novel therapeutic approaches to treat AH. Whether the redox defects of hepatocytes in AH directly exacerbate neutrophilic inflammation and NET formation is unclear. Here we identify that the protein content of the mitochondrial biliverdin exporter ABCB10, which increases hepatocyte-autonomous synthesis of the ROS-scavenger bilirubin, is decreased in livers from humans and mice with AH. Increasing ABCB10 expression selectively in hepatocytes of mice with AH is sufficient to decrease MPO gene expression and histone H3 citrullination, a specific marker of NET formation. These anti-inflammatory effects can be explained by ABCB10 function reducing ROS-mediated actions in liver. Accordingly, ABCB10 gain-of-function selectively increased the mitochondrial GSH/GSSG ratio and decreased hepatic 4-HNE protein adducts, without elevating mitochondrial fat expenditure capacity, nor mitigating steatosis and hepatocyte death. Thus, our study supports that ABCB10 function regulating ROS-mediated actions within surviving hepatocytes mitigates the maladaptive activation of infiltrated neutrophils in AH. Consequently, ABCB10 gain-of-function in human hepatocytes could potentially decrease acute liver failure by decreasing the inflammatory flare caused by excessive neutrophil activity., Competing Interests: Declaration of competing interest M.L. is a co-founder of Enspire Bio and was a consultant of Enspire Bio and Capacity Bio. O.S.S is a co-founder and SAB member of Enspire Bio, Senergy-Bio and Capacity-Bio, and when this study was conducted he was serving as a consultant to LUCA-Science, IMEL, Epirium, Johnson & Johnson, Pfizer, and Stealth Biotherapeutics., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: The aim of this study was to investigate the effect and mechanism of kaempferol on alcoholic steatohepatitis., Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for 6 weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1β, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1β and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling pathway activation., Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumor necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect., Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine., (© 2024 S. Karger AG, Basel.)

Aims: The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of Alcoholic Hepatitis (AH) and its relationship with inflammation., Background: Chronic alcohol consumption induces CYP2E1, which is involved in the development of Alcoholic Hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H 2 O 2 ), the concentration of which is elevated under inflammatory conditions., Objective: The mechanisms underlying the induction of CYP2E1 by H 2 O 2 were examined with a focus on Keap1, a target factor of H 2 O 2 ., Methods: We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H 2 O 2 , and evaluated changes in the expression of the NF-kB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH., Results: The induction of CYP2E1 by H 2 O 2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H 2 O 2 , markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients., Conclusion: We herein demonstrated that the expression of CYP2E1 was induced by H 2 O 2 . The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H 2 O 2 did not after the knockdown of RelA. These results suggest that H 2 O 2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H 2 O 2 -Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Alcohol-associated hepatitis (AH) is often diagnosed at advanced stages, and severe AH usually carries poor prognosis and high short-term mortality. In addition, it is challenging to understand the molecular mechanisms of immune dysregulation and inflammation in AH due to the cellular complexity and heterogeneity. Using single-cell RNA sequencing, previous studies found that AH causes dysfunctional innate immune response in monocytes, involving activation of pattern recognition receptors (PRRs) and cytokine signaling pathways. To better understand the coordinated systemic immune response in AH patients, we performed combined single-cell transcriptome, cell-surface protein, and lymphocyte antigen receptor analysis of peripheral blood mononuclear cell (PBMC) samples. Our results showed inflammatory cytokines and chemokines were highly expressed in AH, including IL-2, IL-32, CXC3R1 and CXCL16 in monocytes and NK cells, whereas HLA-DR genes were reduced in monocytes. In addition, we also found altered differentiation of T-helper cells (T H 1 and T H 17), which could further lead to neutrophil recruitment and macrophage activation. Lastly, our results also suggest impaired NK-cell activation and differentiation in AH with reduced gene expression of KLRC2 and increased gene expression of KLRG1. Our findings indicate different mechanisms may be involved in impaired immune and inflammatory responses for the cellular subtypes of the PBMCs in AH., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Changes in the expression of cyto- and chemokines due to alcohol-associated liver disease (ALD) have been reported to be both protective and pathogenic. This study examined plasma levels of two key cytokines, Il-17 and Il-22, which construct the proinflammatory vs. anti-inflammatory axes across the spectrum of alcohol use disorder (AUD) and ALD including alcohol-associated hepatitis (AH) to determine the underlying status of the inflammation., Methods: Forty-two males and females aged 25-63 yrs. were grouped as healthy controls (HV[n=8]), AUD with no liver injury (AUDNLI [n=8]), AUD with liver injury (AUDLI [n=8]), non-severe alcohol-associated hepatitis (NSAH [n=9]), and severe alcohol-associated hepatitis (SAH [n=9]). Demographic, drinking, and clinical data were collected. Blood samples were collected at baseline (BL, all subjects) and during week 4 (W4, only patients) for IL-17 and IL-22; and statistically analyzed., Results: IL-17 was highly elevated in the SAH group both at BL and post-SOC. LTDH and BL IL-22 in non-severe AH patients were associated significantly. LTDH significantly predicted W4 IL-22 levels, positively (increasing) in NSAH and inversely (lowering) in SAH patients. BL and W4 IL-22 levels were significantly higher (4-fold, p≤0.001) in all AH patients compared to all AUD patients (AUROC=0.988, p≤0.001). IL-22 showed significant affinity with AST, AST: ALT ratio, total bilirubin, INR, and PT both at BL and W4. IL-22 was inversely associated with IL-1β; and positively with TNF-α and IL-8 both at BL, and W4. BL IL-17 showed a positive correlation with MELD (p=0.017) in all AH patients. In SAH, > 2-fold W4 IL-17 level compared to BL showed significant within subjects' effects, p=0.006. In AUD patients without AH, the drop in IL-17 at W4 vs. BL showed a significant within subjects' effect, p=0.031., Discussion: Drinking chronicity predicted opposite effects in IL-22 levels in NSAH (antiinflammatory) and SAH (pro-inflammatory) patients at post-SOC. BL IL-22 levels differentiated AH patients robustly from the AUD patients (with or without liver injury); and showed corresponding increases stepwise with the stages of ALD. IL-22 was closely associated with progression and injury markers of the liver; and response to the cytokines of pro-inflammatory nature. Pro-inflammatory indicator of IL-17 cell axis, IL-17 showed a strong positive association with MELD, a severity indicator of AH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sagaram, Frimodig, Jayanty, Hu, Royer, Bruner, Kong, Schwandt and Vatsalya.)

Background: Chronic alcohol consumption impairs gut barrier function and perturbs the gut microbiome. Although shifts in bacterial communities in patients with alcohol-associated liver disease (ALD) have been characterized, less is known about the interactions between host metabolism and circulating microbe-derived metabolites during the progression of ALD., Methods: A large panel of gut microbiome-derived metabolites of aromatic amino acids was quantified by stable isotope dilution liquid chromatography with online tandem mass spectrometry in plasma from healthy controls (n = 29), heavy drinkers (n = 10), patients with moderate (n = 16) or severe alcohol-associated hepatitis (n = 40), and alcohol-associated cirrhosis (n = 10)., Results: The tryptophan metabolites, serotonin and indole-3-propionic acid, and tyrosine metabolites, p-cresol sulfate, and p-cresol glucuronide, were decreased in patients with ALD. Patients with severe alcohol-associated hepatitis and alcohol-associated cirrhosis had the largest decrease in concentrations of tryptophan and tyrosine-derived metabolites compared to healthy control. Western blot analysis and interrogation of bulk RNA sequencing data from patients with various liver pathologies revealed perturbations in hepatic expression of phase II metabolism enzymes involved in sulfonation and glucuronidation in patients with severe forms of ALD., Conclusions: We identified several metabolites decreased in ALD and disruptions of hepatic phase II metabolism. These results indicate that patients with more advanced stages of ALD, including severe alcohol-associated hepatitis and alcohol-associated cirrhosis, had complex perturbations in metabolite concentrations that likely reflect both changes in the composition of the gut microbiome community and the ability of the host to enzymatically modify the gut-derived metabolites., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Background & Aims: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocyte markers and showing immature features. However, the mechanisms and impact of hepatocyte dedifferentiation in liver disease are poorly understood., Methods: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ArLD). Hepatocyte-specific overexpression or deletion of C-X-C motif chemokine receptor 4 (CXCR4), and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity., Results: Here, we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness, and cancer gene programs. The CXCR4 pathway was highly enriched in HB cells and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with a biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased the hepatocyte-specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression., Conclusions: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH., Impact and Implications: Here, we show that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis. Therefore, this study reveals the importance of preserving strict control over hepatocyte plasticity in order to preserve liver function and promote tissue repair., (Copyright © 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Metabolic-associated fatty liver disease (MAFLD) and alcoholic hepatitis (AH) are among the most prevalent liver diseases worldwide, and their coexistence is common in clinical practice. However, currently established models of MAFLD-AH coexistence do not fully replicate their pathological characteristics and require sophisticated experimental techniques. Therefore, we aimed to develop an easily replicable model that mimics obesity-induced MAFLD-AH in patients. Our goal was to establish a murine model that replicates MAFLD and AH coexistence, resulting in significant liver injury and inflammation. To this end, we administered a single ethanol gavage dose to ob/ob mice on a chow diet. The administration of a single dose of ethanol led to elevated serum transaminase levels, increased liver steatosis, and apoptosis in ob/ob mice. Furthermore, ethanol binge caused a significant increase in oxidative stress in ob/ob mice, as measured via 4-hydroxynonenal. Importantly, the single dose of ethanol also markedly exacerbated liver neutrophil infiltration and upregulated the hepatic mRNA expression of several chemokines and neutrophil-related proteins, including Cxcl1, Cxcl2, and Lcn2. Whole-liver transcriptomic analysis revealed that ethanol-induced changes in gene expression profile shared similar features with AH and MAFLD. In ob/ob mice, a single dose of ethanol binge caused significant liver injury and neutrophil infiltration. This easy-to-replicate murine model successfully mimics the pathological and clinical features of patients with coexisting MAFLD and AH and closely resembles the transcriptional regulation seen in human disease.

Background and Aims: Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3)-/- mice are completely protected from ethanol-induced liver injury, Mlkl-/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury., Approach and Results: Bone marrow transplants between Mlkl-/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl-/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→ Mlkl-/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1-mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl-/- mice phagocytosed fewer bioparticles than KCs of WT mice., Conclusion: Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of American Association for the Study of Liver Diseases.)

Objective: Alcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide. Interleukin-11 (IL-11) is a profibrotic, proinflammatory cytokine with increasingly recognised toxicities in parenchymal and epithelial cells. We explored IL-11 serum levels and their prognostic value in patients suffering from AH and cirrhosis of various aetiology and experimental ALD., Design: IL-11 serum concentration and tissue expression was determined in a cohort comprising 50 patients with AH, 110 patients with cirrhosis and 19 healthy volunteers. Findings were replicated in an independent patient cohort (n=186). Primary human hepatocytes exposed to ethanol were studied in vitro. Ethanol-fed wildtype mice were treated with a neutralising murine IL-11 receptor-antibody (anti-IL11RA) and examined for severity signs and markers of ALD., Results: IL-11 serum concentration and hepatic expression increased with severity of liver disease, mostly pronounced in AH. In a multivariate Cox-regression, a serum level above 6.4 pg/mL was a model of end-stage liver disease independent risk factor for transplant-free survival in patients with compensated and decompensated cirrhosis. In mice, severity of alcohol-induced liver inflammation correlated with enhanced hepatic IL-11 and IL11RA expression. In vitro and in vivo, anti-IL11RA reduced pathogenic signalling pathways (extracellular signal-regulated kinases, c-Jun N-terminal kinase, NADPH oxidase 4) and protected hepatocytes and murine livers from ethanol-induced inflammation and injury., Conclusion: Pathogenic IL-11 signalling in hepatocytes plays a crucial role in the pathogenesis of ALD and could serve as an independent prognostic factor for transplant-free survival. Blocking IL-11 signalling might be a therapeutic option in human ALD, particularly AH., Competing Interests: Competing interests: SC, SS and AAW are coinventors of the patent application US 2020/0262910 and other patents relating to IL11 biology. SC and SS are coshareholders of Enleofen Bio PTE., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

The present study was to investigate the therapeutical effects and mechanisms of Asiatic acid from Potentilla chinensis against alcoholic hepatitis. Rats were intragastrically fed with alcohol for 12 weeks to induce alcoholic hepatitis and then treated with various drugs for further 12 weeks. The results showed that Asiatic acid significantly alleviated liver injury caused by alcohol in rats, as evidenced by the improved histological changes and the lower levels of AST, ALT, and TBIL. Besides, Asiatic acid significantly enhanced the activity of ADH and ALDH, promoting alcohol metabolism. Asiatic acid suppressed CYP2E1 activity and NADP + /NADPH ratio, resulting in low ROS production. Further study revealed that Asiatic acid markedly reduced hepatocyte apoptosis by regulating the expression levels of apoptosis-related protein. Moreover, Asiatic acid could regulate the Nrf2 and NF-κB signaling pathway, attenuating oxidative stress and inflammation as a result. Interestingly, the comprehensive analysis of transcriptomics and metabolomics indicated that Asiatic acid inhibited the gene expression of Gpat3 and thereby affected the biosynthesis of the metabolites (1-acyl-Sn-glycerol-3-phosphocholine, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine), regulating the glycerophospholipid metabolism pathway and ultimately ameliorating hepatocyte damage. In conclusion, this study demonstrates that Asiatic acid can ameliorate alcoholic hepatitis by modulating the NF-κB and Nrf2 signaling pathways and the glycerophospholipid metabolism pathway, which may be developed as a potential medicine for the treatment of alcoholic hepatitis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intrahepatic neutrophil infiltration has been implicated in severe alcoholic hepatitis (SAH) pathogenesis; however, the mechanism underlying neutrophil-induced injury in SAH remains obscure. This translational study aims to describe the patterns of intrahepatic neutrophil infiltration and its involvement in SAH pathogenesis. Immunohistochemistry analyses of explanted livers identified two SAH phenotypes despite a similar clinical presentation, one with high intrahepatic neutrophils (Neuhi), but low levels of CD8+ T cells, and vice versa. RNA-Seq analyses demonstrated that neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with hepatic inflammation and disease progression. To study specifically the mechanisms related to Neuhi in AH patients and liver injury, we used the mouse model of chronic-plus-binge ethanol feeding and found that myeloid-specific deletion of the Ncf1 gene abolished ethanol-induced hepatic inflammation and steatosis. RNA-Seq analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic hepatitis (AH) by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key antiinflammatory and antifibrotic microRNA). In conclusion, two distinct histopathological phenotypes based on liver immune phenotyping are observed in SAH patients, suggesting a separate mechanism driving liver injury and/or failure in these patients.

Alcoholic pancreatitis and hepatitis are frequent, potentially lethal diseases with limited treatment options. Our previous study reported that the expression of CFTR Cl - channel is impaired by ethanol in pancreatic ductal cells leading to more severe alcohol-induced pancreatitis. In addition to determining epithelial ion secretion, CFTR has multiple interactions with other proteins, which may influence intracellular Ca 2+ signaling. Thus, we aimed to investigate the impact of ethanol-mediated CFTR damage on intracellular Ca 2+ homeostasis in pancreatic ductal epithelial cells and cholangiocytes. Human and mouse pancreas and liver samples and organoids were used to study ion secretion, intracellular signaling, protein expression and interaction. The effect of PMCA4 inhibition was analyzed in a mouse model of alcohol-induced pancreatitis. The decreased CFTR expression impaired PMCA function and resulted in sustained intracellular Ca 2+ elevation in ethanol-treated and mouse and human pancreatic organoids. Liver samples derived from alcoholic hepatitis patients and ethanol-treated mouse liver organoids showed decreased CFTR expression and function, and impaired PMCA4 activity. PMCA4 co-localizes and physically interacts with CFTR on the apical membrane of polarized epithelial cells, where CFTR-dependent calmodulin recruitment determines PMCA4 activity. The sustained intracellular Ca 2+ elevation in the absence of CFTR inhibited mitochondrial function and was accompanied with increased apoptosis in pancreatic epithelial cells and PMCA4 inhibition increased the severity of alcohol-induced AP in mice. Our results suggest that improving Ca 2+ extrusion in epithelial cells may be a potential novel therapeutic approach to protect the exocrine pancreatic function in alcoholic pancreatitis and prevent the development of cholestasis in alcoholic hepatitis., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

This study aimed to develop a well-characterized mouse model of alcoholic hepatitis (AH) regression. Male C57BL/6J mice were fed a Lieber-DeCarli (LD) control diet or LD containing 5% ethanol for ten days followed by one binge, which is the chronic-binge model of AH developed by the National Institute on Alcohol Abuse and Alcoholism. To determine AH regression, mice previously exposed to ethanol were put on LD control diet and metabolic and inflammatory features were monitored weekly for three weeks. Serum alcohol, total cholesterol, and alanine transaminase levels were increased in ethanol-fed mice, which declined to those of no ethanol controls within one and three weeks after ethanol withdrawal, respectively. Serum malondialdehyde was increased with ethanol feeding, but it was restored to no ethanol control levels within one week. Ethanol-induced changes in the hepatic expression of genes involved in lipogenesis, fatty acid oxidation, ethanol metabolism, and antioxidant response were restored to those of no ethanol controls after 3 weeks of ethanol withdrawal. Also, ethanol-induced hepatic inflammation was gradually decreased during the 3 weeks of ethanol withdrawal. Hepatic nicotinamide adenine dinucleotide (NAD + ) levels and the expression of enzymes involved in the NAD + salvage pathway were decreased by ethanol feeding, which was mitigated after ethanol withdrawal. Ethanol significantly lowered hepatic sirtuin 1 expression, but its levels were restored with ethanol cessation. This study established a mouse model of AH regression, which can be used as a preclinical model to study the potential of dietary bioactives or therapeutic agents on AH regression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.

Acute alcoholic liver injury (AALI) is a threat to human health. Dendrobium officinale polysaccharide (DOP) has the potential to protect the liver by enhancing the anti-oxidative system to maintain the relative balance of ROS (active oxygen species) and antioxidants in AALI mice. However, the dynamic improvement effect of DOP on AALI is still not clear and accurate medication guidance is not available, which limits the clinical application of DOP. Because of the advantages of high sensitivity, noninvasiveness, and visualization, near-infrared (NIR) fluorescence imaging has been widely studied in biochemistry and biomedicine. As the glutathione (GSH) level in the liver is closely related to the progression of AALI, herein, an NIR fluorescent probe for GSH, HCG was used to dynamically evaluate the effect of DOP on AALI mice. In this study, DOP was proven to maintain the relative balance of GSH content in the liver to protect it from damage. To the best of our knowledge, it is the first time to assess the effect of DOP on AALI mice through a NIR fluorescence imaging technique. This study may also provide a potential NIR imaging agent for the clinical research to improve the management of liver injury-related diseases., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: In alcoholic hepatitis (AH), dysfunctional T lymphocytes may contribute to the high mortality from infections. T lymphocyte activation is governed by the expression of co-stimulatory receptors such as 4-1BB balanced by inhibitory receptors such as Programmed Death receptor 1 (PD-1). 4-1BB expression is unaccounted for in AH, while PD-1 is elevated. We characterized expression of 4-1BB and PD-1 and the associated T lymphocyte functional status in AH and investigated whether these were associated with short-term mortality., Methods: Thirty-five patients with AH (at diagnosis and days 7 and 90) were compared with healthy controls (HC). Spontaneous and in vitro stimulated receptor expression were quantified by flow cytometry, and plasma proteins by ELISA., Results: At diagnosis, the patients showed increased stimulated 4-1BB responses of CD4+ T lymphocytes. Also, the frequencies of PD-1+ T lymphocytes both with and without co-expressed 4-1BB were increased. Further, interferon-gamma was predominantly produced in T lymphocytes co-expressing 4-1BB. A decrease in the frequency of spontaneous 4-1BB+ T lymphocytes and an increase in soluble 4-1BB during the first week after diagnosis were associated with higher mortality at day 90 in AH. PD-1 expression showed no systematic dynamics related to mortality., Conclusions: We found an increased stimulated 4-1BB response of T lymphocytes in AH and early loss of these lymphocytes was associated with a higher short-term mortality. This suggests a role of T lymphocyte 4-1BB expression in the progression of AH., Competing Interests: The authors have declared that no competing interests exist.

The alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs) play critical roles in alcoholism development and alcohol toxicology; however, few studies have focused on the miRNA-mediated mechanisms underlying the expressions of alcohol-metabolizing enzymes. In the present study, we showed the expression changes of each alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver samples of alcoholic hepatitis (AH) patients, and predicted the miRNAs targeting the dysregulated alcohol-metabolizing genes by a systematic in silico analysis. 13 miRNAs were predicted to regulate the expressions of ADH1A, ADH4, and ALDH2, respectively, with hsa-miR-148a-3p (miR-148a) showing the most significant down-regulation in AH patients. Following experimental evidence using HepG2 cells proved that miR-148a promoted ADH4 expression by directly binding to the coding sequence of ADH4 and increasing the mRNA stability via an AGO1-dependent manner. Additional assays showed that secondary structure of ADH4 transcript affected the target accessibility and binding of miR-148a-3p. In sum, our results suggest that the expressions of key alcohol-metabolizing enzymes are repressed in AH patients, and the non-canonical positive regulation of miR-148a on ADH4 reveals a new regulationary mechanism for ADH genes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction and Objectives: Alcoholic hepatitis (AH) is characterized by high morbidity and mortality. MicroRNA-494-3p is possibly involved in the regulation of cancers, but its role in AH has been rarely studied., Materials and Methods: AH mice model and primarily cultured mice hepatic stellate cells (HSCs) model were constructed. Levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed by ELISA. Expressions of miRNAs, HSC activation-related proteins and fibrosis-related protein were analyzed by qRT-PCR and Western blot. Cell counting kit, colony formation and flow cytometry assays were used to detect cell viability, proliferation and apoptosis, respectively. The relationship between TNF receptor-associated factor 3 (TRAF3) and miR-494-3p was predicted and verified by TargetScan and dual-luciferase assay, respectively. Results of the above experiments were verified by rescue experiments using TRAF3., Results: Liver damage and miRNA expression were observed in AH mice, and AST and ALT levels were increased in serum of AH mice. MiR-494-3p was reduced in AH liver tissues, and it decreased the levels of α-SMA and fibrosis-related proteins. HSCs were isolated, and activating HSCs or upregulating miR-494-3p had a regulatory effect on the levels of miR-494-3p, HSC activation-related proteins and fibrosis-related proteins as well as cell viability, proliferation and apoptosis. In addition, miR-494-3p targeted TRAF3 and inhibited TRAF3 expression, while overexpressed TRAF3 promoted TRAF3 expression and rescued the regulatory effect of miR-494-3p on the levels of related proteins as well as cell viability, proliferation and apoptosis., Conclusions: This study provided a novel mechanistic comprehension of the anti-fibrotic effect of miR-494-3p., (Copyright © 2021 Fundación Clínica Médica Sur, A.C. Published by Elsevier España, S.L.U. All rights reserved.)

Background & Aims: Interleukin (IL)-20 and IL-22 belong to the IL-10 family. IL-10 is a well-documented anti-inflammatory cytokine while IL-22 is well known for epithelial protection and its antibacterial function, showing great therapeutic potential for organ damage; however, the function of IL-20 remains largely unknown., Methods: Il20 knockout (Il20 -/- ) mice and wild-type littermates were generated and injected with Concanavalin A (ConA) and Klebsiella pneumoniae (K.P.) to induce acute hepatitis and bacterial infection, respectively., Results: Il20 -/- mice were resistant to acute hepatitis and exhibited selectively elevated levels of the hepatoprotective cytokine IL-6. Such selective inhibition of IL-6 by IL-20 was due to IL-20 targeting hepatocytes that produce high levels of IL-6 but a limited number of other cytokines. Mechanistically, IL-20 upregulated NAD(P)H: quinone oxidoreductase 1 (NQO1) expression and subsequently promoted the protein degradation of transcription factor IκBζ, resulting in selective downregulation of the IκBζ-dependent gene Il6 as well several other IκBζ-dependent genes including lipocalin-2 (Lcn2). Given the important role of IL-6 and LCN2 in limiting bacterial infection, we examined the effect of IL-20 on bacterial infection and found Il20 -/- mice were resistant to K.P. infection and exhibited elevated levels of hepatic IκBζ-dependent antibacterial genes. Moreover, IL-20 upregulated hepatic NQO1 by binding to IL-22R1/IL-20R2 and activating ERK/p38MAPK/NRF2 signaling pathways. Finally, the levels of hepatic IL1B, IL20, and IκBζ target genes were elevated, and correlated with each other, in patients with severe alcoholic hepatitis., Conclusions: IL-20 selectively inhibits hepatic IL-6 production rather than exerting IL-10-like broad anti-inflammatory properties. Unlike IL-22, IL-20 aggravates acute hepatitis and bacterial infection. Thus, anti-IL-20 therapy could be a promising option to control acute hepatitis and bacterial infection., Lay Summary: Several interleukin (IL)-20 family cytokines have been shown to play important roles in controllimg inflammatory responses, infection and tissue damage, but the role of IL-20 remains unclear. Herein, we elucidated the role of IL-20 in liver disease and bacterial infection. We show that IL-20 can aggravate hepatitis and bacterial infection; thus, targeting IL-20 holds promise for the treatment of patients with liver disease., Competing Interests: Conflict of interest No conflicts of interest exist for any of the authors. Please refer to the accompanying ICMJE disclosure forms for further details., (Published by Elsevier B.V.)

The chemokine system of ligands and receptors is implicated in the progression of alcohol-associated hepatitis (AH). Finding upstream regulators could lead to novel therapies. This study involved coordinated expression of chemokines in livers of healthy controls (HC) and patients with AH in 2 distinct cohorts of patients with various chronic liver diseases. Studies in cultured hepatocytes and in tissue-specific KO were used for mechanistic insight into a potential upstream regulator of chemokine expression in AH. Selected C-X-C chemokine members of the IL-8 chemokine family and C-C chemokine CCL20 were highly associated with AH compared with HC but not in patients with liver diseases of other etiologies (nonalcoholic fatty liver disease [NAFLD] and hepatitis C virus [HCV]). Our previous studies implicate macrophage migration inhibitory factor (MIF) as a pleiotropic cytokine/chemokine with the potential to coordinately regulate chemokine expression in AH. LPS-stimulated expression of multiple chemokines in cultured hepatocytes was dependent on MIF. Gao-binge ethanol feeding to mice induced a similar coordinated chemokine expression in livers of WT mice; this was prevented in hepatocyte-specific Mif-KO (MifΔHep) mice. This study demonstrates that patients with AH exhibit a specific, coordinately expressed chemokine signature and that hepatocyte-derived MIF might drive this inflammatory response.

Cytochrome P450 (CYP) enzymes play critical roles in drug transformation, and the total CYPs are markedly decreased in alcoholic hepatitis (AH), a fatal alcoholic liver disease. miRNAs are endogenous small noncoding RNAs that regulate many essential biological processes. Knowledge concerning miRNA regulation of CYPs in AH disease is limited. Here we presented the changes of key CYPs in liver samples of AH patients retrieved from GEO database, performed in silico prediction of miRNAs potentially targeting the dysregulated CYP transcripts, and deciphered a novel mechanism underlying miRNA mediated CYPs expression in liver cells. Nine miRNAs were predicted to regulate CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2J2, and CYP3A4, among which hsa-miR-148a-3p was selected as a case study. Biochemical and molecular evidences demonstrated that miR-148a promoted CYP2B6 expression by increasing mRNA stability via directly binding to the 3'UTR sequence, and that this positive posttranscriptional regulation was AGO1/2-dependent. Further, luciferase reporter gene assay and RNA secondary structure analysis illustrated that the seedless target site, not the seed target site, controlled miR-148a-mediated CYP2B6 upregulation. Moreover, we identified HNF4A as a liver-specific transcription factor of MIR-148A through EMSA and chromatin immunoprecipitation experiments. In conclusion, ethanol downregulated miR-148a in hepatocytes through HNF4A regulation, which eventually decreased CYP2B6 expression. Our finding will benefit the understanding of dysregulated drug metabolism in AH patients and highlight an unconventional mechanism for epigenetic regulation of CYP gene expression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acute alcoholic microvesicular steatosis (MIC) may complicate heavy alcohol intake and present as alcoholic hepatitis (AH) syndrome. However, detailed clinical, biological, and histologic data associated with MIC are scarce. We compared the clinical presentation, histologic features, and hepatic transcriptomic of patients presenting with AH due to either MIC or severe alcoholic steatohepatitis (ASH). In this case-control study, patients who drank heavily (>100 g/day) with the AH syndrome were included either in the MIC group (>50% severe microvesicular steatosis, no inflammation) or in the severe ASH group (polynuclear neutrophil infiltration, macrosteatosis, ballooned hepatocytes). All patients received standard supportive care plus steroids for those with severe ASH and were followed up for 3 months. Whole-liver transcriptome profiling was performed on liver snap-frozen biopsies. Compared to ASH (n = 24, mean age 49.3 years), patients in the MIC group (n = 12, mean age 49.1 years) had a higher reported alcohol intake ( P  < 0.01), lower Model for End-Stage Liver Disease score ( P  < 0.05), lower hepatic venous pressure gradient ( P  < 0.01), higher alanine aminotransferase ( P  < 0.02) and gamma-glutamyltransferase ( P  < 0.001), higher triglycerides ( P  < 0.001) and total cholesterol ( P  < 0.002), but similar bilirubin levels ( P  = 0.54). At histology, patients with MIC had a lower fibrotic stage compared to those with ASH ( P  < 0.001). A higher density of megamitochondria was seen in MIC compared to ASH ( P  < 0.05). During follow-up, death or transplantation occurred in 4/12 (33%) patients with MIC and 7/24 (29%) patients with severe ASH. Differential hepatic gene expression in MIC compared to ASH included down-regulation of genes related to inflammation and fibrosis and up-regulation of genes involved in lipid metabolism and mitochondrial function. Conclusion: MIC is an acute, noninflammatory, potentially severe alcoholic liver injury mimicking ASH, is associated with a lower fibrosis stage, and has a distinct gene expression profile., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Alcoholic hepatitis (AH) is the dramatic acute presentation of alcoholic liver disease, with a 15% mortality rate within 28 days in severe cases. Research into AH has been hampered by the lack of effective and reproducible murine models that can be operated under different regulatory frameworks internationally. The liquid Lieber-deCarli (LdC) diet has been used as a means of ad libitum delivery of alcohol but without any additional insult, and is associated with relatively mild liver injury. The transcription factor nuclear factor-erythroid 2-related factor 2 ( Nrf2 ) protects against oxidative stress, and mice deficient in this molecule are suggested to be more sensitive to alcohol-induced injury. We have established a novel model of AH in mice and compared the nature of liver injury in C57/BL6 wild-type (WT) versus Nrf2 -/- mice. Our data showed that both WT and Nrf2 -/- mice demonstrate robust weight loss, and an increase in serum transaminase, steatosis and hepatic inflammation when exposed to diet and ethanol. This is accompanied by an increase in peripheral blood and hepatic myeloid cell populations, fibrogenic response and compensatory hepatocyte regeneration. We also noted characteristic disturbances in hepatic carbohydrate and lipid metabolism. Importantly, use of Nrf2 -/- mice did not increase hepatic injury responses in our hands, and female WT mice exhibited a more-reproducible response. Thus, we have demonstrated that this simple murine model of AH can be used to induce an injury that recreates many of the key human features of AH - without the need for challenging surgical procedures to administer ethanol. This will be valuable for understanding of the pathogenesis of AH, for testing new therapeutic treatments or devising metabolic approaches to manage patients whilst in medical care.This article has an associated First Person interview with the joint first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Prominin 1 (PROM1) is one of a few clinically relevant progenitor markers in human alcoholic hepatitis (AH) and hepatocellular carcinoma (HCC), and mouse liver tumor initiating stem cell-like cells (TICs). However, the origin, fate and functions of PROM1 +  cells in AH and HCC are unknown. Here we show by genetic lineage tracing that PROM1 +  cells are derived in part from hepatocytes in AH and become tumor cells in mice with diethyl nitrosamine (DEN)-initiated, Western alcohol diet-promoted liver tumorigenesis. Our RNA sequencing analysis of mouse PROM1 +  cells, reveals transcriptomic landscapes indicative of their identities as ductular reaction progenitors (DRPs) and TICs. Indeed, single-cell RNA sequencing reveals two subpopulations of Prom1 +  Afp - DRPs and Prom1 +  Afp +  TICs in the DEN-WAD model. Integrated bioinformatic analysis identifies Discodin Domain Receptor 1 (DDR1) as a uniquely upregulated and patient-relevant gene in PROM1 +  cells in AH and HCC. Translational relevance of DDR1 is supported by its marked elevation in HCC which is inversely associated with patient survival. Further, knockdown of Ddr1 suppresses the growth of TICs and TIC-derived tumor growth in mice. These results suggest the importance of PROM1 +  cells in the evolution of liver cancer and DDR1 as a potential driver of this process.

Alcoholic hepatitis (AH) is identified as an inflammatory syndrome with high morbidity and mortality as a result of severe hepatocellular dysfunction and liver injury. Accumulated studies indicated that miRNAs are involved in AH. The potential effect of miR-451a in AH mice was examined in the current study. A mice AH model was established and the miR-451a expression in AH mice compared with the sham group was tested by real-time polymerase chain reaction (qRT-PCR). AH mice were injected with pre-miR-451a lentivirus for miR-451a overexpression and histone deacetylase (HDAC8) lentivirus for HDAC8 overexpression in AH mice. The underlying mechanisms were explored by searching the potential target genes of miR-451a in miRanda database and then we confirmed this. We found that miR-451a expression was significantly decreased in AH mice compared with the sham group. Moreover, miR-451a overexpression alleviated alcohol-induced liver inflammation and injuries of AH mice. Additionally, further mechanism exploration disclosed that HDAC8 was a target of miR-451a. The protective effect of miR-451a on AH in AH mice was abolished by HDAC8 overexpression. In summary, miR-451a ameliorates AH via repressing HDAC8-mediated proinflammatory response., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Alcoholic hepatitis (AH) is a clinical syndrome characterized by jaundice and progressive inflammatory liver injury in patients with a history of prolonged periods of excess alcohol consumption and recent heavy alcohol abuse. Severe AH is a life-threatening form of alcohol-associated liver disease with a high short-term mortality rate around 30-50% at one month from the initial presentation. A large number of pro-inflammatory mediators, metabolic pathways, transcriptional factors and epigenetic factors have been suggested to be associated with the development and progression of AH. Several factors may contribute to liver failure and mortality in patients with severe AH including hepatocyte death, inflammation, and impaired liver regeneration. Although the pathogeneses of AH have been extensively investigated and many therapeutic targets have been identified over the last five decades, no new drugs for AH have been successfully developed. In this review, we discuss interleukin-22 (IL-22) biology and its roles of anti-apoptosis, anti-fibrosis, anti-oxidation, anti-bacterial infection and regenerative stimulation in protecting against liver injury in many preclinical models including several recently developed models such as chronic-plus-binge ethanol feeding, acute-on-chronic liver failure, C-X-C motif chemokine ligand 1 plus high-fat diet-induced nonalcoholic steatohepatitis. Finally, clinical trials of IL-22 for the treatment of AH are also discussed, which showed some promising benefits for AH patients.

Alcoholic hepatitis (AH) is an important form of alcoholic liver disease (ALD), and its incidence is continuously increasing leading to advanced disease burden. The NOD-like receptors (NLRs) are a specialized group of intracellular pattern recognition receptors, which participate in inflammatory diseases. However, the role of NLRs in the pathogenesis of AH still remain obscure. The animal model of alcoholic hepatitis in mice was established according to National Institute on Alcohol Abuse and Alcoholism (NIAAA) method. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of NLR family members in liver tissues of the ethanol-fed(EtOH-fed)group and pair-fed group. NLRP6 was overexpressed in mice by injecting Recombinant Adeno-Associated Virus into the tail vein. Mouse Cytokines and Chemokines RT 2 Profiler PCR Array was used to analyze the related cytokines and chemokines involved in the development of alcoholic hepatitis. Among the NLR family members, the expression of NLRP6 decreased most significantly in the animal model of AH. Our results demonstrated that overexpression of NLRP6 in vivo obviously alleviated steatosis, inflammation and fibrosis in liver. Meanwhile, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice also decreased. Besides, Chemokine (C-C motif) ligand 20(CCL20) was one of the most significantly up-regulated chemokines in the mouse AH model and CCL20 was participated in NLRP6-mediated AH. NLRP6 could inhibit the activation of nuclear factor (NF)-κB signaling pathway in vitro and in vivo. Furthermore, the activation, proliferation, and migration of hepatic stellate cells was enhanced after downregulation of NLRP6. In summary, NLRP6 may play a protective role in the development of AH. NLRP6 could inhibit activation of NF-κB signaling pathway in AH., Competing Interests: Declaration of competing interest The authors claimed to have no conflict of interest with the manuscript., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A 4 (LXA 4 ), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA 4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15 +/+ and Alox12/15 -/- mice, with or without supplementation of LXA 4 . Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA 4 significantly lowered transaminase levels only in Alox12/15 -/- mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA 4 injection attenuated selected parameters of disease progression in Alox12/15 -/- mice, its beneficial impact on immunity was also apparent in Alox12/15 +/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis., (Copyright © 2020 Queck, Fink, Sirait-Fischer, Rüschenbaum, Thomas, Snodgrass, Geisslinger, Baba, Trebicka, Zeuzem, Weigert, Lange and Brüne.)

Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The numbers of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared with control mice. The miRNA (miR)-seq analysis detected 20 upregulated and 4 downregulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced α-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7 and Nr1d2 genes, which were downregulated in HSCs from the AH mice, were predicted targets of 20 miRs upregulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, indeed repressed Nr1d2, the quiescent HSC marker. AH-HC-EVs were also enriched with organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1β and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting the quiescent HSC transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1β and IL-17 by HMs. KEY MESSAGES: • Circulating EVs and HC-EVs were increased in AH mice compared with control mice • AH-HC-EVs were enriched in miRNAs, organelle proteins, and mitochondrial DNA • AH-HC-EVs increased cytokine production by AH-HMs in a TLR9-dependent manner.

Background: Extracellular vesicles (EVs) are implicated in intercellular communication in liver diseases. An EV-associated fraction of the macrophage biomarker soluble CD163, denoted EV-CD163, was recently identified. EV-CD163 may be released during later phases of the inflammatory response as opposed to the acute shedding of CD163 ectodomain (Ecto-CD163). Total sCD163 is a well-described biomarker in liver inflammation, and we investigated the distribution of CD163 fractions along with EV-associated soluble CD206 (EV-CD206) in patients with acute and chronic alcoholic liver inflammation. Methods: Patients with acute alcoholic hepatitis (AH) ( n =  48) and alcoholic cirrhosis (AC) ( n =  26) were enrolled. Patients with AH were followed for 30 days after diagnosis. Healthy blood donors ( n =  30) served as a reference group. Fractions of sCD163 and sCD206 were separated using ExoQuick™ and measured by ELISA. Results: We demonstrated a possible EV-associated fraction of CD206 in plasma, correlating with levels of EV-CD163 (r s = 0.46, p  < .001). The distribution of biomarker fractions was skewed toward EVs in chronic cirrhosis for both biomarkers (median: 35.8% EV-CD163, 58.8% EV-CD206) as compared to AH patients (median: 26.2% EV-CD163 p  < .0001, 48.8% EV-CD206, p  < .01). In AH patients, total sCD163 and Ecto-CD163 at inclusion were related to survival, whereas EV-CD163 was not. Conclusion: Extracellular vesicles of macrophage origin associated with membrane receptors CD163 and CD206 are present in liver disease. We observed a shift in the distribution towards an increased EV fraction in chronic liver cirrhosis. These data support that Ecto and EV fractions may be markers of different inflammatory processes, possibly resulting from a switch in macrophage phenotype.

Severe alcoholic hepatitis (SAH) is a deadly liver disease without an effective medical therapy. Although SAH mortality is known to correlate with hepatic accumulation of immature liver cells, why this occurs and how it causes death are unclear. Here, we demonstrate that expression of epithelial splicing regulatory protein 2 (ESRP2), an RNA-splicing factor that maintains the nonproliferative, mature phenotype of adult hepatocytes, was suppressed in both human SAH and various mouse models of SAH in parallel with the severity of alcohol consumption and liver damage. Inflammatory cytokines released by excessive alcohol ingestion reprogrammed adult hepatocytes into proliferative, fetal-like cells by suppressing ESRP2. Sustained loss of ESRP2 permitted reemergence of a fetal RNA-splicing program that attenuates the Hippo signaling pathway and thus allows fetal transcriptional regulators to accumulate in adult liver. We further showed that depleting ESRP2 in mice exacerbated alcohol-induced steatohepatitis, enabling surviving hepatocytes to shed adult hepatocyte functions and become more regenerative, but threatening overall survival by populating the liver with functionally immature hepatocytes. Our findings revealed a mechanism that explains why liver failure develops in patients with the clinical syndrome of SAH, suggesting that recovery from SAH might be improved by limiting adult-to-fetal reprogramming in hepatocytes.

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., (Published by Elsevier B.V.)

Mesenchymal stem cells (MSCs) are a population of pluripotent cells that have been tested for the treatment of many inflammatory diseases. It remains unclear whether MSCs were effective in treating mice with alcoholic hepatitis (AH) and its underlying mechanism. In the present study, MSCs were isolated from bone marrow of 4-6 week-old C57BL/6N male mice. AH was induced in female mice by chronic-binge ethanol feeding for 10 days. Intraperitoneal (i.p.) transplantation of MSCs or saline were performed in mice on day 10. Blood samples and hepatic tissues were harvested on day 11. Biochemical, liver histological and flow cytometric analyses were performed. Compared to the control mice, the AH mice had significantly increased liver/body weight ratio, serum alanine aminotransferase (ALT) and aspartate aminotransferases (AST), hepatic total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), hepatic neutrophil and macrophage infiltration (P<0.001), which were markedly reduced by i.p. transplantation of MSCs (P<0.01). Compared to the control mice, the hepatic glutathione (GSH) was prominently lower in the AH mice (P<0.001), which was markedly enhanced after i.p. injection of MSCs (P<0.001). MSCs were effective for the treatment of AH mice, which might be associated with their ability in inhibiting hepatic neutrophil and macrophage infiltration, and alleviating oxidative stress., Competing Interests: The authors have declared that no competing interests exist.

Background: Endothelial progenitor cells (EPCs) help in neovascularization and endothelial repair during injury. Patients with cirrhosis show increased number and function of EPCs in circulation., Methods: Since natural killer (NK) cells regulate EPCs, we investigated the relationship between the 2 in alcoholic cirrhosis (AC, n = 50) and severe alcoholic hepatitis (SAH, n = 18) patients and compared with nonalcoholic cirrhosis (n = 15) and healthy controls (HC, n = 30). Levels of systemic inflammatory cytokines were measured, and coculture assays were performed between EPCs and NK cells in contact-dependent and contact-independent manner. NK cell-mediated killing of EPCs was evaluated, and expression of receptors including fractalkine (FKN) on EPCs and its cognate receptor CX3CR1 on NK cells was studied by RT-PCR assays., Results: Patients with SAH had higher regulated on activation, normal T cell expressed and secreted (RANTES) (p = 0.01), vascular endothelial growth factor (VEGF) (p = 0.04), IL-1β (p = 0.04), and IL-6 (p = 0.00) growth factors and proinflammatory cytokines as compared to AC and HC. Distinct populations of CD31+ CD34+ EPCs with low and high expression of CD45 were significantly lower in SAH than HC (CD45 low , p = 0.03; CD45 hi , p = 0.04) and AC (CD45 low , p = 0.05; CD45 hi , p = 0.02). SAH patients, however, showed increased functional capacity of EPCs including colony formation and LDL uptake. NK cells were reduced in SAH compared with AC (p = 0.002), however with higher granzyme ability (p < 0.001 and p = 0.04, respectively). In SAH, EPC-NK cell interaction assays showed that NK cells lysed the EPCs in both contact-dependent and contact-independent assays. Expression of interaction receptor CX3CR1 was significantly higher on NK cells (p = 0.0005), while its cognate receptor, FKN, was increased on EPCs in SAH patients as compared to HC (p = 0.0055)., Conclusion: We conclude that in SAH, NK cells induce killing of EPCs via CX3CR1/FKN axis that may be one of the key events contributing to disease severity and proinflammatory responses in SAH., (© 2019 by the Research Society on Alcoholism.)

Background: Alcohol-related liver disease is the main cause of liver-related mortality worldwide. The development of novel targeted therapies for patients with advanced forms (i.e., alcoholic hepatitis, AH) is hampered by the lack of suitable animal models. Here, we developed a novel mouse model of acute-on-chronic alcohol liver injury with cholestasis and fibrosis and performed an extensive molecular comparative analysis with human AH., Methods: For the mouse model of acute-on-chronic liver injury, we used 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, 0.05% w/w) diet for 8 weeks to establish cholestatic liver fibrosis. After 1-week washout period, male mice were fed intragastrically for 4 weeks with up to 24 g/kg of ethyl alcohol in a high-fat diet. This animal model was phenotyped using histopathology, clinical chemistry, microbiome, and gene expression approaches. Data were compared to the phenotypes of human alcohol-related liver disease, including AH., Results: Mice with cholestatic liver fibrosis and subsequent alcohol exposure (DDC + EtOH) exhibited exacerbated liver fibrosis with a pericellular pattern, increased neutrophil infiltration, and ductular proliferation, all characteristics of human AH. DDC administration had no effect on urine alcohol concentration or liver steatosis. Importantly, DDC- and alcohol-treated mice showed a transcriptomic signature that resembled that of patients with AH. Finally, we show that mice in the DDC + EtOH group had an increased gut barrier dysfunction, mimicking an important pathophysiological mechanism of human AH., Conclusions: We developed a novel mouse model of acute-on-chronic cholestatic alcoholic liver injury that has considerable translational potential and can be used to test novel therapeutic modalities for AH., (© 2019 by the Research Society on Alcoholism.)

In humans, the three main circulating monocyte subsets are defined by their relative cell surface expression of CD14 and CD16. They are all challenging to study because their characteristics are strongly context specific, and this has led to a range of conflicting reports about their function, which is especially so for CD14 ++ CD16 + (intermediate) monocytes. Ex vivo cultures are also often confounded by the concomitant use of immunosuppressive drugs. We therefore sought to characterize the phenotype and function of intermediate monocytes in the setting of acute inflammation prior to treatment in a cohort of 41 patients with acute alcoholic hepatitis (AH). Circulating intermediate monocytes were enriched in patients with AH and had an activated phenotype with enhanced expression of CCR2 and CD206 compared with healthy controls. Proinflammatory cytokine expression, including IL-1β and IL-23, was also higher than in healthy controls, but both classical (CD14 ++ CD16 - ) and intermediate monocytes in AH were refractory to TLR stimulation. Compared with healthy controls, both AH monocyte subsets had greater phagocytic capacity, enhanced ability to drive memory T cell proliferation in coculture, and skewed CD4 + T cells to express an increased ratio of IL-17/IFN-γ. Furthermore, liver tissue from AH patients demonstrated an enrichment of monocytes including the intermediate subset compared with controls. These data demonstrate that intermediate monocytes are expanded, functionally activated, induce CD4 + T cell IL-17 expression, and are enriched in the liver of patients with AH., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background & Aims: Activation of TGFB (transforming growth factor β) promotes liver fibrosis by activating hepatic stellate cells (HSCs), but the mechanisms of TGFB activation are not clear. We investigated the role of ECM1 (extracellular matrix protein 1), which interacts with extracellular and structural proteins, in TGFB activation in mouse livers., Methods: We performed studies with C57BL/6J mice (controls), ECM1-knockout (ECM1-KO) mice, and mice with hepatocyte-specific knockout of EMC1 (ECM1 Δhep ). ECM1 or soluble TGFBR2 (TGFB receptor 2) were expressed in livers of mice after injection of an adeno-associated virus vector. Liver fibrosis was induced by carbon tetrachloride (CCl 4 ) administration. Livers were collected from mice and analyzed by histology, immunohistochemistry, in situ hybridization, and immunofluorescence analyses. Hepatocytes and HSCs were isolated from livers of mice and incubated with ECM1; production of cytokines and activation of reporter genes were quantified. Liver tissues from patients with viral or alcohol-induced hepatitis (with different stages of fibrosis) and individuals with healthy livers were analyzed by immunohistochemistry and in situ hybridization., Results: ECM1-KO mice spontaneously developed liver fibrosis and died by 2 months of age without significant hepatocyte damage or inflammation. In liver tissues of mice, we found that ECM1 stabilized extracellular matrix-deposited TGFB in its inactive form by interacting with αv integrins to prevent activation of HSCs. In liver tissues from patients and in mice with CCl 4 -induced liver fibrosis, we found an inverse correlation between level of ECM1 and severity of fibrosis. CCl 4 -induced liver fibrosis was accelerated in ECM1 Δhep mice compared with control mice. Hepatocytes produced the highest levels of ECM1 in livers of mice. Ectopic expression of ECM1 or soluble TGFBR2 in liver prevented fibrogenesis in ECM1-KO mice and prolonged their survival. Ectopic expression of ECM1 in liver also reduced the severity of CCl 4 -induced fibrosis in mice., Conclusions: ECM1, produced by hepatocytes, inhibits activation of TGFB and its activation of HSCs to prevent fibrogenesis in mouse liver. Strategies to increase levels of ECM1 in liver might be developed for treatment of fibrosis., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Alcoholic hepatitis (AH) is a severe condition developed in patients with underlying alcoholic liver disease. Epithelial cell adhesion molecule (EPCAM) plays a role in hepatitis. Therefore, the current study aimed to explore the effect of EPCAM and its potential mechanism in AH. Bioinformatic analysis was performed to screen differentially expressed genes associated with AH. AH mouse models were established through a Lieber-DeCarli liquid diet containing 4% ethanol, which were co-treated with siRNA against EPCAM or the PI3K/Akt/mTOR signaling pathway inhibitor in order to investigate the effects of EPCAM and the PI3K/Akt/mTOR signaling pathway on hepatic fibrosis, hepatic stellate cell (HSC) proliferation and apoptosis. The relationship between EPCAM and the PI3K/Akt/mTOR signaling pathway was investigated for the purposes of elucidating the potential mechanism of EPCAM in AH. EPCAM was predicted to regulate AH progression through the PI3K/Akt/mTOR signaling pathway. Silencing EPCAM or inhibition of the PI3K/Akt/mTOR signaling pathway inhibited the hepatic fibrosis and HSC proliferation yet induced HSC apoptosis. Moreover, silencing EPCAM was found to repress the PI3K/Akt/mTOR signaling pathway as evidenced by decreased levels of Bcl2 yet increased levels of caspase-3. Collectively, silencing EPCAM could hinder AH progression by inhibiting the PI3K/Akt/mTOR signaling pathway, which might serve as a potential therapeutic target for AH treatment.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14 ++ CD16 + intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX 3 CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16 + monocytes, leading to apparent "loss" of intermediate monocytes. Reduction in CD14 ++ CD16 - monocytes and increased CD14 ++ CD16 + is associated with altered responses in functional assays ex vivo . Patients with AP and AAH had reduced proportions of CD14 ++ CD16 + monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

Alcoholic liver diseases are classified as one of the major reasons for worldwide morbidity and mortality. Curcuminoids exhibit a wide range of pharmacological activities that are beneficial for health, including hepatoprotective effects, but its clinical significance is limited due to poor oral bioavailability. In the present study, a novel formulation of curcumin as curcumin-galactomannosides (CGM) with enhanced oral bioavailability alleviated alcohol-induced liver damage in wistar rats with an increased potency compared to the unformulated natural curcuminoids (CM). Ethanol administration significantly elevated liver toxicity markers, lipid peroxidation and inflammatory markers with a simultaneous reduction in antioxidant defenses. Supplementation of CGM reversed all of the pathological effects of alcohol administration, almost close to the normal level, when compared with CM. Histopathology of liver tissue also confirmed the better protective effect of CGM, indicating the enhancement in antioxidant and anti-inflammatory effects as a function of bioavailability., (© 2019 Wiley Periodicals, Inc.)

4-Methylcoumarin-[5,6-g]-hesperetin (4-MCH) is a hesperidin derivative produced by the structural modification of hesperetin. Alcoholic hepatitis (AH) is the origin of many serious liver diseases that are accompanied by hepatic inflammation. In this study, we detected the anti-inflammatory activity of 4-MCH in EtOH fed mice and examined the potential molecular mechanism of this activity. We found that 4-MCH suppressed the release of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in primary liver macrophages isolated from mice and in EtOH-treated RAW264.7 cells. In addition, we showed that the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) was down-regulated in vivo and in vitro in AH. Furthermore, 4-MCH acted as an activator of PPAR-γ, which could therefore ameliorate the inhibitory effects of EtOH on the expression of PPAR-γ. The impairment of PPAR-γ function (T0070907 or PPAR-γ siRNA treatment) resulted in greater inflammation than that in the control group. Conversely, over-expression of PPAR-γ further reduced the release of inflammatory cytokines from EtOH-stimulated RAW264.7 cells. Additional investigations showed that 4-MCH significantly inhibited the phosphorylation of p65. Collectively, these results indicate that 4-MCH alleviated the inflammatory reaction through PPAR-γ activation via the NF-κB-p65 signaling pathway, which regulates the expression of IL-6 and TNF-α in AH., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chronic liver diseases are characterized by the expansion of ductular reaction (DR) cells and the expression of liver progenitor cell (LPC) markers. In alcoholic hepatitis (AH), the degree of DR expansion correlates with disease progression and short-term survival. However, little is known about the biological properties of DR cells, their impact on the pathogenesis of human liver disease, and their contribution to tissue repair. In this study, we have evaluated the transcriptomic profile of DR cells by laser capture microdissection in patients with AH and assessed its association with disease progression. The transcriptome analysis of cytokeratin 7-positive (KRT7 + ) DR cells uncovered intrinsic gene pathways expressed in DR and genes associated with alcoholic liver disease progression. Importantly, DR presented a proinflammatory profile with expression of neutrophil recruiting C-X-C motif chemokine ligand (CXC) and C-C motif chemokine ligand chemokines. Moreover, LPC markers correlated with liver expression and circulating levels of inflammatory mediators such as CXCL5. Histologically, DR was associated with neutrophil infiltration at the periportal area. In order to model the DR and to assess its functional role, we generated LPC organoids derived from patients with cirrhosis. Liver organoids mimicked the transcriptomic and proinflammatory profile of DR cells. Conditioned medium from organoids induced neutrophil migration and enhanced cytokine expression in neutrophils. Likewise, neutrophils promoted the proinflammatory profile and the expression of chemokines of liver organoids. Conclusion: Transcriptomic and functional analysis of KRT7 + cells indicate that DR has a proinflammatory profile and promote neutrophil recruitment. These results indicate that DR may be involved in the liver inflammatory response in AH, and suggest that therapeutic strategies targeting DR cells may be useful to mitigate the inflammatory cell recruitment in AH., (© 2018 by the American Association for the Study of Liver Diseases.)

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 MIR148A 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., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Diabetes mellitus (DM) and alcohol consumption is still one of the important research models that simulate variable clinical conditions and metabolic diseases, such as alcoholic liver diseases., Objectives: The aim of this study was to evaluate the long-term cumulative effects of low alcohol consumption on the liver tissue, biochemical assays and some inflammatory cytokines in experimentally-induced DM rats., Material and Methods: Ethanol was administered in the drinking water (3% v/v) for 30 days to adult male Sprague-Dawley rats, with or without DM induced by streptozocin injection. Histological and biochemical parameters as well as some inflammatory cytokines - interleukin (IL)-4, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α) - were measured., Results: A significant increase in blood glucose level in the combination group was accompanied by a significant decrease in plasma insulin (p < 0.001 vs controls). Hepatic histopathology of the combination group revealed steatosis and fibrosis in addition to a significant increase in the gamma-glutamyltransferase (γ-GT) and alkaline phosphatase (ALP) levels (p < 0.05 and p < 0.001, respectively). A non-high-density lipoprotein (HDL) lipid profile (total cholesterol (TC), triglycerides (TG) and low-density lipoprotein (LDL)) revealed a significant increase in comparison to controls (p < 0.05), while HDL showed no significant change. The IL-4 and IL-6 levels were significantly higher (p < 0.05), while IL-10 and TNF-α revealed non-significant changes., Conclusions: Depletion of the hyperglycemic response in the case of low alcohol consumption in DM rats was associated with elevated plasma cytokines, especially IL-6 and IL-4, which could be a part of a host defense mechanism to repair the hepatic and pancreatic damage through this inflammatory process. The severe liver damage under insult of low alcohol consumption and DM could serve as inhibitory factors in gluconeogenesis and glycogenolysis, with little or no impact on insulin levels.