The global morbidity and mortality of alcoholic

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1 AMERICAN ASSOCIATION FOR THE STUDY OFLIVERD I S E ASES NEW HORIZONS HEPATOLOGY, VOL. 64, NO. 4, 2016 Alcoholic Hepatitis: Translational Approaches to Develop Targeted Therapies Pranoti Mandrekar, 1 Ramon Bataller, 2 Hidekazu Tsukamoto, 3 and Bin Gao 4 Alcoholic liver disease is a leading cause of liver-related mortality worldwide. In contrast to recent advances in therapeutic strategies for patients with viral hepatitis, there is a significant lack of novel therapeutic options for patients with alcoholic liver disease. In particular, there is an urgent need to focus our efforts on effective therapeutic interventions for alcoholic hepatitis (AH), the most severe form of alcoholic liver disease. AH is characterized by an abrupt development of jaundice and complications related to liver insufficiency and portal hypertension in patients with heavy alcohol intake. The mortality of patients with AH is very high (20%-50% at 3 months). Available therapies are not effective in many patients, and targeted approaches are imminently needed. The development of such therapies requires translational studies in human samples and suitable animal models that reproduce the clinical and histological features of AH. In recent years, new animal models that simulate some of the features of human AH have been developed, and translational studies using human samples have identified potential pathogenic factors and histological parameters that predict survival. Conclusion: This review summarizes the unmet needs for translational studies on the pathogenesis of AH, preclinical translational tools, and emerging drug targets to benefit the AH patient. (HEPATOLOGY 2016;64: ) The global morbidity and mortality of alcoholic liver disease (ALD) are associated with alcohol abuse over prolonged periods. Recent epidemiologic studies show that liver cirrhosis is the twelfth leading cause of death, and approximately 50% of the total fatalities are alcohol-related (National Institute on Alcohol Abuse and Alcoholism Surveillance report 100). ALD encompasses different stages as a consequence of susceptibility factors, duration, and intensity of alcohol consumption. Some of the histological stages can coexist and include steatosis, alcoholic steatohepatitis (ASH), and progressive fibrosis to cirrhosis and superimposed hepatocellular carcinoma. Moreover, patients with underlying ALD and heavy alcohol intake can develop a form of acute-on-chronic liver injury called alcoholic hepatitis (AH). Severe AH has very high short-term mortality (20%-50% at 3 months) and represents one of the deadliest diseases in clinical hepatology. (1) Despite its economical and health burden, ALD has received very limited attention from health policy makers, pharmaceutical companies, and private funding agencies. While research has made significant progress, the pathogenesis of ALD remains incompletely understood, and there is little or no impact on development of new therapies. Limitations in patient characterization due to poor Abbreviations: ABIC score, age1serum bilirubin1international normalized ratio1serum creatinine score; AH, alcoholic hepatitis; ALD, alcoholic liver disease; ASH, alcoholic steatohepatitis; CCl 4, carbon tetrachloride; DAMP, damage-associated molecular pattern; DF, discriminant function; IL, interleukin; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; SIRS, systemic inflammatory response syndrome; TNFa, tumor necrosis factor-a. Received July 23, 2015; accepted February 21, Supported by RO1 #2AA and Department of Defense grants PRMRP #W81XWH and PRMRP#W81XWH (to P.M.); grants 1U01AA and 1U01AA (to R.B.); National Institute on Alcohol Abuse and Alcoholism grants 5P50AA011999, 1U01AA018663, and 5R24AA and Department of Veterans Affairs Merit Review award 5I01BX (to H.T.); and the National Institute on Alcohol Abuse and Alcoholism Intramural Program (to B.G.). Copyright VC 2016 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI /hep Potential conflict of interest: Dr. Tsukamoto consults for Ajinomoto, Gilead, and Suntory. Dr. Bataller consults for Oncozyme. 1343

2 MANDREKAR ET AL. HEPATOLOGY, October 2016 diagnostic and prognostic measures, inadequate models of disease, and insufficient preclinical/translational approaches have stunted progress in the field of ALD. One of the most urgent needs in this field is to identify new targeted therapies for patients with AH. This need is further confirmed by the recent Steroid or Pentoxifylline for Alcoholic Hepatitis (STOPAH) trial, which showed limited efficacy of prednisolone. (2) In this article, we review the molecular pathogenesis of AH and highlight translational studies that might lead to targeted approaches for its treatment. Discussion of entry criteria and endpoints for clinical trials is beyond the scope of this review. AH: Clinical Aspects and Unmet Needs Until it reaches advanced stages, ALD is mostly asymptomatic. Patients with prolonged alcohol misuse develop steatosis and signs of hepatocellular damage (ballooning and Mallory-Denk bodies) and inflammatory infiltrate (typically neutrophils), which define ASH. ASH is a histological concept that can be present both in subclinical patients (defined as subclinical ASH) and in patients with significant clinical syndrome (defined as AH). Patients with subclinical ASH can be asymptomatic for prolonged periods of time (fully compensated and preserved hepatic function). (1,3) This subclinical stage of disease is poorly characterized in humans, and there is an unmet need to delineate its natural history and prognostic factors as well as to develop noninvasive markers. Eventually, a proportion of patients with subclinical ASH may develop cirrhosis (8%-20%), which confers a high risk of complications. Some patients with ASH can also present a form of acute-on-chronic liver failure called AH, which often occurs after recent excessive alcohol binge drinking. (1) AH is characterized by an abrupt rise in serum bilirubin levels, jaundice, coagulopathy, and liver-related complications. Traditionally, it was thought that AH could occur in patients with mild underlying ALD. However, more recent studies using tru-cut needles to obtain liver biopsy reveal that the vast majority of patients with AH have underlying cirrhosis. (4) The episode of AH may occur as the first manifestation in patients who have experienced silent ALD, while in other patients it is an exacerbation of preexisting alcoholic cirrhosis. Therefore, AH should be distinguished from subclinical ASH in fully compensated patients (Fig. 1). Importantly, patients with ALD can present an episode of jaundice and liver decompensation for reasons other than superimposed AH. These conditions include severe sepsis, biliary obstruction, diffuse hepatocellular carcinoma, drug-induced liver injury, and ischemic hepatitis (i.e., due to massive bleeding or cocaine use). Therefore, not all episodes of jaundice in patients with underlying ALD should be attributed to AH. In patients with other potential causes of jaundice, with severe forms of ALD, or involved in clinical trials, a transjugular liver biopsy is recommended to confirm the existence of AH. (5,6) Future studies, including clarification of the nomenclature to define subclinical ASH as well as moderate and severe AH, are needed to further understand the heterogeneity and stages of human ALD. ARTICLE INFORMATION: From the 1 Department of Medicine, University of Massachusetts Medical School, Worcester, MA; 2 Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3 Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, University of Southern California, Greater Los Angeles Department of Veterans Affairs Healthcare System, Los Angeles, CA; 4 Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Pranoti Mandrekar, Ph.D., F.AASLD Gastrointestinal Division Department of Medicine University of Massachusetts Medical Center LRB Rm # Plantation Street Worcester, MA Pranoti.Mandrekar@umassmed.edu Tel: or Ramon Bataller, M.D. University of North Carlina at Chapel Hill ramon_bataller@med.unc.edu or Hidekazu Tsukamoto, DMV, Ph.D., F.AASLD University of South California htsukamo@med.usc.edu or Bin Gao, M.D., Ph.D., NIAAA, NIH bgao@mail.nih.gov 1344

3 HEPATOLOGY, Vol. 64, No. 4, 2016 MANDREKAR ET AL. FIG. 1. Histology, pathogenesis, and experimental models for various stages of ALD, including steatosis, subclinical ASH with or without fibrosis, and AH. Histology and pathogenesis of steatosis, subclinical ASH with or without fibrosis, and AH are briefly summarized with more details in the text. Several animal models currently used in the field could represent the different stages of ALD. Chronic ab libitum feeding of the Lieber-DeCarli ethanol diet is a model to study the early stage of ALD such as steatosis. (56) Several models have been used to investigate subclinical ASH, with the strongest liver damage in Western diet ab libitum 1 intragastric alcohol 1 weekly binge, (25) followed by Western diet ab libitum 1 intragastric alcohol, (25) intragastric alcohol with high-fat diet, (57) Lieber-DeCarli 1 multiple binges, (23) and Lieber-DeCarli 1 single binge. (23,49) The Western diet ab libitum 1 intragastric alcohol 1 weekly binge model recapitulates some of histologic and clinical features of human AH, (25) and the Lieber-DeCarli 1 multiple binge model also partially reproduces these features but to a lesser degree. (23) Models using second hits such as LPS or CCl 4 exhibit coagulative necrosis, failing to recapitulate clinical AH features. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, c-glutamyltransferase; HFD, high-fat diet; INR, international normalized ratio; MDB, Mallory-Denk body; NKT, natural killer cell. The incidence of AH is not well known, and it is likely that many cases are undiagnosed. Populationbased studies estimate approximately 4.5 hospitalizations for AH per 100,000 persons each year, with a slight male predominance in Western countries. (7) AH patients typically present with symptoms such as rapidly progressive jaundice, which can be accompanied by fever, abdominal pain, anorexia, and weight loss. There are several clinical scoring systems to assess the severity of AH: Maddrey s discriminant function (DF), Model for End-Stage Liver Disease, age1serum bilirubin1international normalized ratio1serum creatinine [ABIC], and Glasgow. (4,8,9) Among them, Maddrey s DF is the most widely used. Short-term mortality in patients with severe AH is due to sepsis, liver failure, and multiorgan dysfunction. (10) Moreover, a recent multicentric study developed a histological scoring system capable of predicting short-term survival in patients with AH. (4) The resulting Alcoholic Hepatitis Histological Score comprises four parameters that are independently associated with patient survival: fibrosis stage, neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria. By combining these parameters in a semiquantitative manner, patients can be stratified as being at low, intermediate, or high risk for death within 90 days. (4) The management of patients with AH has not evolved substantially over the last decades. In severe AH (e.g., poor mental status or hypotension), patients may need admission to an intensive care unit. Prevention of alcohol withdrawal symptoms and Wernicke s encephalopathy are recommended. Daily protein intake of 1.5 g/kg body weight is also advised. Because 1345

4 MANDREKAR ET AL. HEPATOLOGY, October 2016 patients with AH are predisposed to severe infections, which may threaten survival, early diagnosis and empiric antibiotic treatment are advised. On discharge, patients should follow alcohol counseling to reach sustained abstinence, a major determinant of long-term survival. (11) In addition to these general measures, pharmacological therapy with prednisolone for 4 weeks has been shown to improve short-term survival in patients with severe AH. The efficacy of pentoxifylline is still questionable. (1,2,12) A recent study has shown some beneficial effects of N-acetylcysteine, a potent but cell-impermeant antioxidant. (13) Unfortunately, many patients do not respond to prednisolone; thus, novel targeted therapies are urgently needed. Recently, liver transplantation, in highly selected AH patients, has been shown to improve survival significantly (14) ; but a number of clinical and ethical considerations, particularly recidivism, limit eligibility. Furthermore, the relative shortage of donor organs adds to the ethical challenges before consideration of patients for liver transplant. There are several important unmet needs in the diagnosis, management, and therapy of AH. (15) First, patients often present in the emergency room with very advanced disease, severe sepsis, and poor physical status, having high mortality within few days. Informative campaigns in primary care centers, addiction centers, and the general population about the clinical relevance of jaundice in patients with alcohol abuse should be emphasized. Second, the specific mechanisms, outcome, and responses to therapy of the most common secondary complications (encephalopathy, infections, renal failure, etc.) are largely unknown. There are only few studies characterizing these complications in this particular population, and additional studies to understand their underlying mechanisms are warranted. (16,17) Third, the extrahepatic consequences of AH that lead to multiorgan dysfunction and death are not well defined. The existence of systemic inflammatory response syndrome (SIRS) is a major predictor of acute organ failure in these patients, suggesting that the extrahepatic consequences of AH play a major role in disease severity. (10,17) Finally, a meaningful molecular classification of these patients could guide the use of targeted therapies. Not all patients are seen at the same stage of the disease. It is likely that different types and patterns of hepatic inflammation play defining roles for subclinical ASH and for moderate and severe AH, whereas impaired liver regeneration could be the major event in patients with severe AH. The development of a molecular classification for these patients is urgently needed and will certainly help in developing tailored therapies for these different spectra of the deadly disease. Pathogenesis and Molecular Targets of AH The cellular and molecular mechanisms of chronic ALD have been extensively studied in animal models over the last 40 years. A wide variety of inflammatory mediators and their downstream signaling pathways as well as several types of inflammatory cells have been identified as contributing to the pathogenesis of steatosis, hepatocellular damage, inflammation, and fibrosis in chronic ALD, which are summarized in several recent reviews. (3,18-21) In the current article, we mainly discuss the potential mechanisms initiated by persistent alcohol drinking causing liver failure as well as multiorgan failure in AH patients (Fig. 2). In addition, potential molecular targets of AH are discussed. Many patients with underlying ALD develop AH after excessive and/or binge alcohol intake, suggesting that excessive binge drinking may contribute to the development of AH. This notion is supported by several recent studies from the chronic-plus-binge ethanol feeding model showing that acute ethanol binge markedly exacerbates liver injury and hepatic neutrophil infiltration in chronically ethanol-fed mice (22,23) or in high-fat diet-fed mice. (24) Moreover, recent efforts to recapitulate the histologic and clinical features of AH in mice have resulted in the development of diffuse hepatic neutrophil infiltration, fibrosis, ductular reaction, splenomegaly, hypoalbuminemia, and hyperbilirubinemia. (25) This model is based on intragastric feeding, which assures heavy alcohol intake with sustained blood alcohol levels, the most important requirement for reproduction of AH patient behavior, and a combination of common AH patient lifestyle factors such as Western diet and weekly binge drinking. (25) Interestingly, without weekly binge, this hybrid feeding model (intragastric and ad libitum feeding) produces chronic ASH with macrophage inflammation and liver fibrosis. When weekly binge is added, inflammation shifts to neutrophil infiltration and histologic and clinical features of clinical AH ensue despite the same overall alcohol intake, (25) further highlighting the critical role of binge drinking in inducing the AH phenotype. At present, how binge drinking initiates AH is not clear. It is likely that excessive binge drinking induces massive hepatocellular damage in patients with underlying chronic ALD who are prone to liver injury 1346

5 HEPATOLOGY, Vol. 64, No. 4, 2016 MANDREKAR ET AL. FIG. 2. Pathogenesis and molecular mechanisms that trigger liver failure, SIRS, and multiorgan failure in AH. Excessive drinking in alcoholics with underlying ALD induces hepatocellular damage, induces systemic inflammation, and impairs liver regeneration, resulting in liver failure and other complications in AH. Abbreviations: APC, antigenpresenting cell; DAMP, damageassociated molecular pattern; PAMP, pathogen-associated molecular pattern; PMN, polymorphonuclear neutrophil. and that these damaged hepatocytes subsequently release a variety of damage-associated molecular patterns (DAMPs) to promote SIRS. (20,26) In addition to DAMPs, pathogen-associated molecular patterns (PAMPs) likely play an important role in inducing SIRS in AH patients. Chronic alcohol consumption is known to suppress antibacterial immune responses and increase bacterial infection, resulting in elevation of PAMPs in the liver and circulation. (27) In addition, chronic alcohol intake induces gut dysbiosis and increases gut permeability, resulting in translocation of PAMPs such as lipopolysaccharide (LPS) into the portal circulation. (28,29) These elevated PAMPs then activate Kupffer cells and other inflammatory cells to produce a variety of proinflammatory cytokines such as tumor necrosis factor-a (TNFa), monocyte chemoattractant protein 1, and interleukin-1b (IL-1b), which contribute to the pathogenesis of ALD. (20) Superimposing binge drinking aggravates gut permeability and bacterial and PAMP translocation, contributing to the manifestation of SIRS (30) and multiple organ failure in alcoholics with underlying chronic ALD, resulting in AH (Fig. 2). So far, a large number of inflammatory mediators have been identified from studies of animal models and human AH biopsies, and some of them are listed in Table 1. Many of these inflammatory mediators are elevated as a consequence of PAMPs, bacterial translocation, and severe hepatocellular damage in AH and may further promote liver injury or deteriorate liver regeneration. Among these inflammatory mediators, TNFa has received the greatest attention, in the early 1990s, and has been shown to play a critical role in promoting liver injury in a rodent model of mild ALD. (31) Consequently, TNFa-blocking agents were tested in patients with AH, albeit with disappointing results due to the development of severe bacterial infections. (32) This serves as an important lesson that TNFa has pleiotropic effects and that inflammation stimulated by TNFa or its downstream mediators may have protective antimicrobial and proregenerative roles that should be preserved. Later, a variety of chemokines including IL-8, chemokine (C-X-C motif) ligand 5, Gro-c, chemokine (C-X-C motif) ligand 6, IL-1, osteopontin, and monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 were found to be up-regulated, contributing to macrophage activation and neutrophil recruitment in AH and in some cases correlating with AH patient survival. (33-35) Another report identified molecular chaperone heat shock protein 90, increased in AH, as a potential therapeutic target to modulate inflammatory responses. (36) Activation 1347

6 MANDREKAR ET AL. HEPATOLOGY, October 2016 TABLE 1. Molecular Targets for AH: Anti-inflammatory and Hepatoprotective Targets Potential Mechanisms Beneficial Effects Detrimental Effects or Obscure Functions in AH Anti-inflammatory targets Steroids Broad immunosuppression Inhibits systemic inflammation; improves short-term survival rates in AH patents Modulation of gut microbiome Gut leakage-lps-tnf-a IL-1 inhibitor Chemokines and their receptors Heat shock protein (Hsp90) Complements Hepatoprotective agents IL-22 Caspase inhibitors HGF IL-6 Antioxidants ALD is associated with intestinal bacterial overgrowth and dysbiosis (28,58) Chronic alcohol drinking increases gut leakage and elevates LPS and TNFa IL-1 promotes liver injury and inflammation in animal models (34) Human AH is associated with upregulation of a variety of chemokines and their receptors, which promote inflammation (35,53) Hsp90 functions as an important chaperone of LPS signaling and is required for the production of proinflammatory cytokines Activation of complements plays a role in promoting alcoholic liver injury in mice Promotes hepatocyte survival and proliferation; inhibits bacterial infection AH is associated with hepatocyte apoptosis Promotes hepatocyte proliferation and survival Promotes hepatocyte survival and proliferation Oxidative stress plays an important role in the pathogenesis of ALD Probiotics improve liver functions in patients with ALD (59) Inhibition of gut leakage, LPS, and TNFa improves liver functions in animal models of ALD Inhibition of IL-1 improves liver functions in animal models of ALD (34) Inhibition of various chemokines and their receptors ameliorates liver injury in animal models of ALD Hsp90 inhibitor attenuates liver inflammation and injury induced by LPS and alcohol in animal models (36) Inhibition of complement activation ameliorates chronic alcoholic liver injury in mice (60) Ameliorates steatosis, liver injury, bacterial infection, and kidney injury and promotes liver repair (45) Prevents hepatocyte apoptosis Ameliorates steatosis and liver injury and promotes liver repair Ameliorates steatosis and liver injury and promotes liver repair Treatment with antioxidants shows beneficial effects in animal models of chronic ALD and in patients with NASH Increases infection; inhibits liver regeneration (44) ; exacerbates neutrophilia Clinical trial using probiotics for the treatment of AH is under consideration Anti-TNFa therapy did not improve survival rate in AH patients (32) ; anti-lps trial and zinc nutritional supplement trial for the treatment of AH are under consideration Anti-IL-1 trial for the treatment of AH is under consideration Targeting multiple chemokines or their receptors may be required for AH therapy because of chemokine receptor redundancy Many Hsp90 inhibitors are currently undergoing clinical evaluation in various types of diseases; Hsp90 inhibitors have not been tested in AH The role of complement activation in AH has not been tested A clinical trial of IL-22 for AH is under consideration (45) A clinical trial using caspase inhibitors for the treatment of AH was stopped (Dr. Vijay Shah, personal communication) Promotes liver cancer cell proliferation Promotes inflammation and liver cancer cell proliferation; clinical application was halted due to many side effects Treatment with antioxidants did not improve survival rate in patients with severe AH (48) Abbreviations: HGF, hepatocyte growth factor; NASH, nonalcoholic steatohepatitis. of the canonical inflammasome pathway, required for proteolytic cleavage of pro-il-1b and pro-il-18, is also implicated in experimental ALD (34,37) ; and inhibition of IL-1 is currently under investigation for the treatment of AH patients. Although the importance of liver resident macrophages (Kupffer cells) in chronic ALD is well documented, recent studies suggest that infiltrating macrophages derived from monocytes contribute to the pathogenesis of ASH. (38) The migration of monocytederived macrophages is dependent on Notch-1 and plays an important role in promoting M1 macrophage inflammation in experimental subclinical ASH. (39) However, the precise biologic or pathologic significance of resident versus infiltrating macrophages during progression to AH or whether M1 macrophage activation is required for AH is still unclear. Neutrophil infiltration is a hallmark of subclinical ASH and is believed to 1348

7 HEPATOLOGY, Vol. 64, No. 4, 2016 MANDREKAR ET AL. induce hepatocellular damage and inflammation in AH. However, a recent study reported that infiltration of neutrophils is associated with better prognosis in AH, suggesting that neutrophils may also play beneficial roles in promoting liver repair and controlling bacterial infection in these patients. (4) To this end, an ideal therapeutic agent may need to suppress the release of cytotoxic mediators by neutrophils while preserving their efficient bacteria killing properties. (40) In addition to inflammation, poor hepatic regenerative response is probably another important mechanism contributing to liver failure in some AH patients. A detailed analysis of liver explants from AH patients who underwent liver transplantation revealed that patients who failed to respond to medical therapy had reduced hepatic expression of liver regeneration-related cytokines and a lack of proliferative hepatocytes. (41) This observation was confirmed by another study, which showed that the presence of proliferating hepatocytes in AH is associated with a better prognosis. (42) In addition, a massive expansion of liver progenitor cells called ductular reaction is often observed in AH patients, but these liver progenitor cells fail to differentiate into mature hepatocytes and correlate positively with severity of liver disease and short-term mortality in these patients. (43) At present, the mechanisms leading to inefficient liver regeneration in AH are unknown and deserve further investigation. In addition, the treatment of AH patients with steroids may further block liver regeneration in these patients because steroids suppress inflammation and subsequently inflammation-mediated liver regeneration. (44) In summary, in overt AH patients, a large number of inflammatory mediators are activated that most likely contribute to SIRS, which together with impaired liver regeneration causes liver failure and multiple organ failure (Fig. 2). These inflammatory mediators cooperatively promote liver inflammation and injury in AH. Thus, it may be difficult to demonstrate clinical efficacy for the treatment of AH by targeting a single inflammatory mediator. Because many inflammatory mediators, including TNFa, also play important roles in promoting liver regeneration, anti-inflammatory modalities for AH patients must preserve effective liver regeneration and anti-inflammatory drugs may be combined with hepatoprotective agents. Indeed, a combination therapy with anti-inflammatory drugs (such as steroids) plus hepatoprotective drugs (such as IL-22) has been proposed and is currently under consideration for AH. (45) In addition to inflammation and poor hepatic regenerative response, other challenges associated with AH patients include bacterial infections, hepatic encephalopathy, renal failure or hepatorenal syndrome, portal hypertension, ascites, bleeding tendencies, and malnutrition. Those patients likely require combination therapies such as immunosuppressive drugs plus hepatoprotective and renalprotective drugs as well as antibacterial drugs. (45) In AH, macrophages and T cells are often defective in their antibacterial functions, (27) which in turn increases the risk of bacterial infection in these patients. Identifying new approaches to correct these defects and restore normal immune responses, rather than to eliminate or suppress, appears a most logical and important future direction. Translational Approaches in AH: Human Specimens and Experimental Models The traditional approach to develop new targets for therapy in the field of AH consists of identifying molecular drivers in preclinical omics studies, in vitro and in vivo, that are ultimately tested in randomized clinical trials in patients (Fig. 3). Early human studies identified molecular drivers such as TNFa and macrophage-derived reactive oxygen species in AH patients. (46,47) Later studies in patients with AH using TNFa blocking agents and antioxidant cocktails were carried out with minimal positive outcomes. (32,48) Thus, a more rational approach would be to perform integrative omics studies in human samples from patients with AH and use pathway/ network computational analysis to identify cellular and molecular mediators that correlate with disease outcome (i.e., short-term mortality or scoring systems that predict survival like Maddrey s DF, ABIC score, etc.). Such studies could enable identification of disease-specific pathways, correlating expression or activation of molecular targets to clinical outcomes. Ideally, the functional role of such potential disease mediators should then be tested in preclinical models of overt AH. As discussed below, while recent animal models of subclinical ASH offer some promise, they do not show all the features of overt AH (i.e., jaundice and cirrhosis). Developing a reliable model that closely reproduces the histological and clinical features of AH will advance this field. However, ALD is a disease of humans and not other animals, and the inherent anatomical and biological differences among the species likely dictate this gap; 1349

8 MANDREKAR ET AL. HEPATOLOGY, October 2016 FIG. 3. Clinical and translational methods in AH. Description of the step-wise process to test plausible drug targets is briefly described. The method involves omics analysis in human samples and studies in animal models in preclinical drug development culminating in testing drugs systematically in phase I-IV clinical trials in AH patients. Abbreviations: MELD, Model for End-Stage Liver Disease; PBMC, peripheral blood mononuclear cell. thus, development of a true AH model may be too idealistic. For this reason, subsequent studies should test plausible molecular targets in preclinical models based on clear pathologic criteria. Because patients with AH are severely ill and have profound hepatic and renal dysfunctions, careful pharmacokinetic and pharmacodynamics studies as well as good laboratory practice toxicology studies are mandatory. Moreover, as detailed later, phase 1 studies in healthy controls and ideally in patients with advanced liver disease are required before phase 2 trials can be carried out. Human studies in patients with AH can be performed systematically using different types of biospecimens followed by correlation of function or expression of a molecule/pathway with the clinical outcome or phenotype (Fig. 3). The first step consists of obtaining prospectively anthropometric, clinical, analytical, and histological data. When a liver biopsy is not possible due to hemodynamic instability or unavailability in the research center, a precise clinical diagnosis should be established. In patients with confounding factors such as sepsis at admission, massive bleeding or hypotension, uncertain alcohol assessment, or recent intake of hepatotoxic drugs, a liver biopsy is required to establish a proper diagnosis of AH. The second step is to define the phenotype of the patient; 1-month and 3-month mortality rates are the most widely used parameters to define disease severity. Alternatively, existing scoring systems (i.e., Maddrey s DF, ABIC, or Model for End-Stage Liver Disease) can be used to establish prognosis. Then, genetic signatures or the expression of genes or proteins found in human biospecimens can be correlated with clinical or histological features or patient outcome to identify potential molecular drivers and/or pathways. The types of biospecimens that can be used for translational studies include liver tissue (typically obtained through a transjugular approach), suprahepatic and peripheral blood (total blood, serum, or plasma), and peripheral blood mononuclear cells and neutrophils. These biospecimens can be used for genomic, proteomic, or metabolomic studies. Due to increasing evidence that gut-derived bacterial products play a significant role in pathophysiology, collecting stools can be useful for translational studies in patients with AH. Overall, initiation of translational research should involve hypothesis-generating omics studies, 1350

9 HEPATOLOGY, Vol. 64, No. 4, 2016 MANDREKAR ET AL. in which disease-associated pathways or signatures linked to clinical outcome are identified. Key molecules of disease pathways should be further tested in preclinical in vivo and in vitro models to identify druggable molecular drivers. To overcome the complexity of studies assessing different biological levels (i.e., DNA, RNA, proteins, metabolites), an integrative analysis by systems biology experts could further aid in understanding interdisciplinary complex interactions within biological systems of AH. Over the last four decades, chronic ethanol feeding studies in rodents using either ad libitum or intragastric feeding models have significantly enhanced our understanding of the pathogenesis of ALD (Table 2). However, these models may produce some features of chronic ALD but not acute-on-chronic liver injury observed in AH patients. Recently, a new mouse model of chronicplus-binge ethanol feeding was developed. (23,49) The feeding protocol in this model is similar to the drinking patterns of many AH patients: a history of chronic drinking superimposed by episodic excessive alcohol consumption. Chronic-plus-binge ethanol feeding synergistically induced steatosis, liver injury, and hepatic neutrophil infiltration in mice, (23,49) which may be useful for the study of neutrophils and early stages of alcoholic liver injury in AH patients. Using this chronic-plus-binge alcohol model, researchers have begun to identify novel mechanisms that participate in the pathogenesis of alcoholic liver injury, thereby revealing novel therapeutic targets. (22,23,36,50,51) However, hepatocellular damage and inflammation caused by chronic-plus-binge ethanol feeding are moderate and transient. (23,49) Further, many AH patients are on a Western diet and binge drink weekly on top of heavy daily alcohol intake, and it is important to study how alcohol drinking and obesity synergistically induce liver injury. Indeed, a single acute binge of ethanol induces acute steatohepatitis in high-fat diet-fed mice, resulting in steatohepatitis with neutrophil infiltration and liver fibrosis. (24) However, these mouse models do not exhibit sustained high blood alcohol levels, which exemplifies human alcoholics. To circumvent these weaknesses, a hybrid feeding model was developed by allowing ad libitum feeding of a solid Western diet at 40% of their caloric intake while assuring high alcohol intake and bloodconcentrationbyintragastricinfusionofanethanol liquid diet at 60% calories. (25) This model reproduces chronic ASH characterized by ballooned cell degeneration, macrophage activation and infiltration, and progression of liver fibrosis. Further, addition of weekly binge to the model produces diffuse neutrophil infiltration and ductular reaction similar to AH in patients, as well as clinical features of AH such as splenomegaly, hypoalbuminemia, and hyperbilirubinemia. However, this model has not achieved decompensation such as jaundice and ascites seen in severe AH, although mild abdominal effusion and hyperbilirubinemia are noted. (25) Thus, our challenge now is to manipulate this model to tip the pathologic progression toward decompensated AH. In summary, the chronic-plus-binge model of ethanol feeding and the hybrid feeding model represent moderate and advanced ASH, respectively (Fig. 1). These models are beginning to highlight potential differences in the significance of inflammation and inflammatory mediators in subclinical ASH and to reinforce the notion that the pathogenesis of ALD is complex, multifactorial, and stage-dependent (Fig. 1). Although we are far from achieving a true model which reproduces overt AH features, these subclinical ASH models still prove useful for comparative omic studies with AH patient samples, identification of potential molecular drivers that initiate the pathogenesis of ASH, and screening for therapeutic drugs targeting these molecules. The second or multiple hits used experimentally include nutritional modifications, pharmacologic agents (e.g., concanavalin A or carbon tetrachloride [CCl 4 ], hormones, cytochrome P450 inducers, toll-like receptor ligands),geneticmanipulation,andviralinfections. (52) When introducing these second or multiple hits, it becomes important to assess whether the hits are clinically relevant and whether the liver pathology produced mimics clinical ALD. For example, alcohol feeding is used to prime and sensitize the liver for the second hit such as single injection of LPS or CCl 4.Inthesemodels, coagulative necrosis and accompanying inflammation occur, but this is a primary consequence of the second hit, deviating from balloon cell degeneration seen in ALD. Recently, Affo et al. (53) developed a model of acute-on-chronic liver injury by chronically administering mice with CCl 4 followed by injecting LPS, and this model has some features of liver inflammation observed in AH, though no alcohol was involved. Investigators using the second hit model must be cautious when interpreting the results because it may be difficult to determine whether the observed mechanisms are a consequence of the ethanol feeding or the second hit. Challenges and Future Perspectives AH was first described in 1961 as an acute disease that often ensues due to episodic excessive drinking in 1351

10 MANDREKAR ET AL. HEPATOLOGY, October 2016 TABLE 2. Commonly Used Animal Models for ALD Models (References) Characteristics Mechanisms Represent Features of Human ALD Deficiencies Acute binge ethanol Mild elevation of serum ALT and AST feeding model (61) Low levels of liver inflammation with a decrease in hepatic macrophages Requires stomach gavage Chronic ad libitum Mild elevation of serum ALT and AST ethanol feeding (56) Low levels of liver inflammation with an increase in macrophages but not neutrophils Easy to perform Intragastric chronic Moderate elevation of serum ALT and AST ethanol feeding (57) Moderate liver inflammation with an increase in macrophages but low levels of neutrophils Requires surgical skills Chronic-plus-binge feeding model (23,49) High-fat diet-plusbinge ethanol model (24) Hybrid model with Western diet ad libitum plus intragastric ethanol and binge (25) Second hit or multiple hits model (52) Moderate and transient elevation of serum ALT and AST Moderate and transient liver inflammation with neutrophils Requires stomach gavage Significant elevation of serum ALT and AST Significant liver inflammation with an increase in neutrophils Mild liver fibrosis Requires stomach gavage Significant elevation of serum ALT, AST, bile acids Ballooned cell degeneration of hepatocytes Intense inflammation shifting from M1 macrophages to intense neutrophils by binge Moderate liver fibrosis Splenomegaly, hypoalbuminemia, hyperbilirubinemia Ductular reaction Requires surgical skills Moderate to significant elevation of serum ALT, AST, and liver inflammation dependent on second hit Damages hepatocyte mitochondrial functions and produces oxidative stress Increases gut permeability and activates LPS-TLR4-Kupffer cells Damages hepatocyte mitochondrial functions and produces oxidative stress Severe mitochondrial GSH depletion Accentuated ER, mitochondrial, and lysosomal stress Defective AMPK pathway Dysbiosis and bacterial translocation M1 macrophage activation Increases hepatic neutrophil infiltration, induces liver injury Damages hepatocyte mitochondrial functions and produces oxidative stress Damages hepatocyte mitochondrial functions Promotes hepatic neutrophil infiltration and induces acute steatohepatitis A shift from M1 macrophage inflammation to neutrophil infiltration by repeated binge Macrophage depletion or dysfunction by repeated binge Enhanced bacterial translocation Chronic ethanol feeding increases susceptibility of livers to second or multiple hit(s)-induced liver injury and inflammation Represents mild acute changes: transient endotoxemia and fatty liver Represents early stages of and mild chronic human ALD Useful for the study of ALD steatosis and macrophage activation Achieves heavy alcohol intake and sustained blood alcohol levels as seen in ALD patients Represents moderate chronic human ALD Useful for the study of ALD steatosis, macrophage activation, and mild fibrosis Useful for the study of transient neutrophil infiltration and recovery from it Represents a model to study the interaction between obesity and binge drinking on acute steatohepatitis Represents moderate/severe human AH Useful for the study of AH steatosis, neutrophil infiltration, macrophage activation, and mild fibrosis Depends on the nature of the second or multiple hits Does not produce chronic ALD process Only produces fatty liver but not hepatocellular death, inflammation, or fibrosis Advanced cirrhosis only combined with iron supplementation Does not produce AH Does not produce sustained neutrophil infiltration Advanced fibrosis is not produced No clinical features of AH are produced Cirrhosis and decompensation are not produced Risk of producing primary injury from the second hit, deviating from natural ALD pathology Abbreviations: ALT, alanine aminotransferase; AMPK, adenosine monophosphate-activated protein kinase; AST, aspartate aminotransferase; ER, estrogen receptor; GSH, glutathione; TLR, Toll-like receptor. 1352

11 HEPATOLOGY, Vol. 64, No. 4, 2016 MANDREKAR ET AL. chronic alcoholics and is characterized by jaundice with severe clinical syndromes such as anorexia, nausea, upper abdominal pain, hepatomegaly, and fever. (54) Today, it is generally accepted that AH is a form of acute-on-chronic liver failure in patients with underlying ALD. Because the mechanisms underlying AH pathogenesis remain unknown and current therapeutic options for this severe disease are largely ineffective, there is an urgent need for systematic translational research to identify therapeutic targets. Many investigators have collaborated and started to use integrative approaches to investigate the pathogenesis of AH and explore the novel therapeutic targets for AH by analyzing human AH biopsy samples and animal models. For example, a recent collaborative study compared transcriptome data from a clinically relevant chronicplus-binge model and biopsy-proven AH and identified similar alterations in expression of many hepatic genes in this animal model and human AH samples. (23) Among these genes, fat-specific protein 27/ CIDEC, which was highly up-regulated in this animal model and in AH samples, plays an important role in promoting ASH in mice and possibly in humans. (23) In addition, the National Institute on Alcohol Abuse and Alcoholism released a major initiative to support four large multi-institutional consortia for conducting translational research on the pathogenesis of AH, identifying new therapeutic targets, and performing clinical trials of plausible drugs for AH therapy. These efforts will likely enhance our understanding of AH pathogenesis and help identify novel therapies for AH. Despite these recent efforts, many challenges remain for the research and therapy of this severe disease. (15) First, the current models do not fully reproduce all features of severe human AH, and many of these models represent subclinical ASH. The continued development of appropriate in vivo models that closely mimic human AH is warranted. Second, chronic ethanol feeding models in rodents that have been used for the last 40 years have revealed therapeutic targets, and these targets may be relevant to early stages of ALD but not acute-on-chronic liver injury in AH. It is essential to reexamine these targets in chronic-plusbinge ethanol feeding models or severe multihit hybrid models before testing in AH patients. Third, so far, many therapeutic targets have been identified from studies of human AH samples and animal models. Here, it is important to recognize that targets identified in mild versus severe ASH models may have different functional significance and that targeting such presumed culprits may yield completely different or even opposite outcomes, as recently noted for osteopontin. (25,55) Carefully designed clinical trials to test these targets in AH patients are urgently needed. Finally, AH is a major severe form of liver disease worldwide, with no effective drugs for this disease. Therefore, there is an urgent need to train young hepatologists and basic/discovery scientists and foster global collaborations in both clinical and basic research related to ALD, particularly AH. Acknowledgment: We apologize to the colleagues whose work was not mentioned or cited in this article because of space constraints. REFERENCES 1) Lucey MR, Mathurin P, Morgan TR. Alcoholic hepatitis. N Engl J Med 2009;360: ) Thursz MR, Richardson P, Allison M, Austin A, Bowers M, Day CP, et al. Prednisolone or pentoxifylline for alcoholic hepatitis. N Engl J Med 2015;372: ) Bataller R, Gao B. Liver fibrosis in alcoholic liver disease. Semin Liver Dis 2015;35: ) Altamirano J, Miquel R, Katoonizadeh A, Abraldes JG, Duarte- Rojo A, Louvet A, et al. A histologic scoring system for prognosis of patients with alcoholic hepatitis. Gastroenterology 2014; 146: ) O Shea RS, Dasarathy S, McCullough AJ; Practice Guideline Committee of the American Association for the Study of Liver Diseases, Practice Parameters Committee of the American College of Gastroenterology. Alcoholic liver disease. HEPATOLOGY 2010;51: ) European Association for the Study of Liver. EASL clinical practical guidelines: management of alcoholic liver disease. J Hepatol 2012;57: ) Sandahl TD, Jepsen P, Thomsen KL, Vilstrup H. Incidence and mortality of alcoholic hepatitis in Denmark : a nationwide population based cohort study. J Hepatol 2011;54: ) Papastergiou V, Tsochatzis EA, Pieri G, Thalassinos E, Dhar A, Bruno S, et al. Nine scoring models for short-term mortality in alcoholic hepatitis: cross-validation in a biopsy-proven cohort. Aliment Pharmacol Ther 2014;39: ) Louvet A, Labreuche J, Artru F, Boursier J, Kim DJ, O Grady J, et al. Combining data from liver disease scoring systems better predicts outcomes of patients with alcoholic hepatitis. Gastroenterology 2015;149: ) Michelena J, Altamirano J, Abraldes JG, Affo S, Morales- Ibanez O, Sancho-Bru P, et al. Systemic inflammatory response and serum lipopolysaccharide levels predict multiple organ failure and death in alcoholic hepatitis. HEPATOLOGY 2015;62: ) Potts JR, Goubet S, Heneghan MA, Verma S. Determinants of long-term outcome in severe alcoholic hepatitis. Aliment Pharmacol Ther 2013;38: ) Singh S, Murad MH, Chandar AK, Bongiorno CM, Singal AK, Atkinson SR, et al. Comparative effectiveness of pharmacological interventions for severe alcoholic hepatitis: a systematic review and network meta-analysis. Gastroenterology 2015;149:

12 MANDREKAR ET AL. HEPATOLOGY, October ) Nguyen-Khac E, Thevenot T, Piquet MA, Benferhat S, Goria O, Chatelain D, et al. Glucocorticoids plus N-acetylcysteine in severe alcoholic hepatitis. N Engl J Med 2011;365: ) Mathurin P, Moreno C, Samuel D, Dumortier J, Salleron J, Durand F, et al. Early liver transplantation for severe alcoholic hepatitis. N Engl J Med 2011;365: ) Sanyal AJ, Gao B, Szabo G. Gaps in knowledge and research priorities for alcoholic hepatitis. Gastroenterology 2015;149: ) Louvet A, Wartel F, Castel H, Dharancy S, Hollebecque A, Canva-Delcambre V, et al. Infection in patients with severe alcoholic hepatitis treated with steroids: early response to therapy is the key factor. Gastroenterology 2009;137: ) Altamirano J, Fagundes C, Dominguez M, Garcia E, Michelena J, Cardenas A, et al. Acute kidney injury is an early predictor of mortality for patients with alcoholic hepatitis. Clin Gastroenterol Hepatol 2012;10: ) Gao B, Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets. Gastroenterology 2011;141: ) Szabo G. Gut-liver axis in alcoholic liver disease. Gastroenterology 2015;148: ) Mandrekar P, Szabo G. Signalling pathways in alcohol-induced liver inflammation. J Hepatol 2009;50: ) Wang HJ, Gao B, Zakhari S, Nagy LE. Inflammation in alcoholic liver disease. Annu Rev Nutr 2012;32: ) Bertola A, Park O, Gao B. Chronic plus binge ethanol feeding synergistically induces neutrophil infiltration and liver injury in mice: a critical role for E-selectin. HEPATOLOGY 2013;58: ) Xu MJ, Cai Y, Wang H, Altamirano J, Chang B, Bertola A, et al. Fat-specific protein 27/CIDEC promotes development of alcoholic steatohepatitis in mice and humans. Gastroenterology 2015;149: ) Chang B, Xu MJ, Zhou Z, Cai Y, Li M, Wang W, et al. Short- or long-term high-fat diet feeding plus acute ethanol binge synergistically induce acute liver injury in mice: an important role for CXCL1. HEPATOLOGY 2015;62: ) Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, et al. Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. HEPATOLOGY 2015;61: ) Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology 2012;143: ) Markwick LJ, Riva A, Ryan JM, Cooksley H, Palma E, Tranah TH, et al. Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis. Gastroenterology 2015;148: ) Chen P, Torralba M, Tan J, Embree M, Zengler K, Starkel P, et al. Supplementation of saturated long-chain fatty acids maintains intestinal eubiosis and reduces ethanol-induced liver injury in mice. Gastroenterology 2015;148: ) Chen P, Starkel P, Turner JR, Ho SB, Schnabl B. Dysbiosisinduced intestinal inflammation activates tumor necrosis factor receptor I and mediates alcoholic liver disease in mice. HEPATO- LOGY 2015;61: ) Bala S, Marcos M, Gattu A, Catalano D, Szabo G. Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals. PLoS One 2014;9:e ) Yin M, Wheeler MD, Kono H, Bradford BU, Gallucci RM, Luster MI, et al. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 1999;117: ) Boetticher NC, Peine CJ, Kwo P, Abrams GA, Patel T, Aqel B, et al. A randomized, double-blinded, placebo-controlled multicenter trial of etanercept in the treatment of alcoholic hepatitis. Gastroenterology 2008;135: ) Mandrekar P, Ambade A, Lim A, Szabo G, Catalano D. An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice. HEPATOLOGY 2011;54: ) Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, et al. IL-1 receptor antagonist ameliorates inflammasomedependent alcoholic steatohepatitis in mice. J Clin Invest 2012; 122: ) Dominguez M, Miquel R, Colmenero J, Moreno M, Garcia- Pagan JC, Bosch J, et al. Hepatic expression of CXC chemokines predicts portal hypertension and survival in patients with alcoholic hepatitis. Gastroenterology 2009;136: ) Ambade A, Catalano D, Lim A, Kopoyan A, Shaffer SA, Mandrekar P. Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury. J Hepatol 2014;61: ) DeSantis DA, Ko CW, Liu Y, Liu X, Hise AG, Nunez G, et al. Alcohol-induced liver injury is modulated by Nlrp3 and Nlrc4 inflammasomes in mice. Mediators Inflamm 2013;2013: ) Wang M, You Q, Lor K, Chen F, Gao B, Ju C. Chronic alcohol ingestion modulates hepatic macrophage populations and functions in mice. J Leukoc Biol 2014;96: ) Xu J, Chi F, Guo T, Punj V, Lee WN, French SW, et al. NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation. J Clin Invest 2015;125: ) Ruchaud-Sparagano MH, Mills R, Scott J, Simpson AJ. MPLA inhibits release of cytotoxic mediators from human neutrophils while preserving efficient bacterial killing. Immunol Cell Biol 2014;92: ) Dubuquoy L, Louvet A, Lassailly G, Truant S, Boleslawski E, Artru F, et al. Progenitor cell expansion and impaired hepatocyte regeneration in explanted livers from alcoholic hepatitis. Gut 2015;64: ) Lanthier N, Rubbia-Brandt L, Lin-Marq N, Clement S, Frossard JL, Goossens N, et al. Hepatic cell proliferation plays a pivotal role in the prognosis of alcoholic hepatitis. J Hepatol 2015;63: ) Sancho-Bru P, Altamirano J, Rodrigo-Torres D, Coll M, Millan C, Jose Lozano J, et al. Liver progenitor cell markers correlate with liver damage and predict short-term mortality in patients with alcoholic hepatitis. HEPATOLOGY 2012;55: ) Kwon HJ, Won YS, Park O, Feng D, Gao B. Opposing effects of prednisolone treatment on T/NKT cell- and hepatotoxinmediated hepatitis in mice. HEPATOLOGY 2014;59: ) Gao B, Shah VH. Combination therapy: new hope for alcoholic hepatitis? Clin Res Hepatol Gastroenterol 2015;39(Suppl. 1): S7-S11. 46) Diluzio NR. Prevention of the acute ethanol-induced fatty liver by the simultaneous administration of antioxidants. Life Sci 1964;3: ) Bird GL, Sheron N, Goka AK, Alexander GJ, Williams RS. Increased plasma tumor necrosis factor in severe alcoholic hepatitis. Ann Intern Med 1990;112: ) Phillips M, Curtis H, Portmann B, Donaldson N, Bomford A, O Grady J. Antioxidants versus corticosteroids in the treatment of severe alcoholic hepatitis a randomised clinical trial. J Hepatol 2006;44: ) Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc 2013;8: ) Williams JA, Ni HM, Ding Y, Ding WX. Parkin regulates mitophagy and mitochondrial function to protect against alcoholinduced liver injury and steatosis in mice. Am J Physiol Gastrointest Liver Physiol 2015;309:G324-G