Lysophosphatidylcholines modulate immunoregulatory checkpoints in peripheral monocytes and are associated with mortality in people with acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is a life-threatening disease characterised by high-grade inflammation and immunoparesis, which is associated with a high incidence of death from sepsis. Herein, we aimed to describe the metabolic dysregulation in ALF and determine whether systemic immune responses are modulated via the lysophosphatidylcholine (LPC)-autotaxin (ATX)-lysophosphatidylcholinic acid (LPA) pathway. METHODS: Ninety-six individuals with ALF, 104 with cirrhosis, 31 with sepsis and 71 healthy controls (HCs) were recruited. Pathways of interest were identified by multivariate statistical analysis of proton nuclear magnetic resonance spectroscopy and untargeted ultraperformance liquid chromatography-mass spectrometry-based lipidomics. A targeted metabolomics panel was used for validation. Peripheral blood mononuclear cells were cultured with LPA 16:0, 18:0, 18:1, and their immune checkpoint surface expression was assessed by flow cytometry. Transcript-level expression of the LPA receptor (LPAR) in monocytes was investigated and the effect of LPAR antagonism was also examined in vitro. RESULTS: LPC 16:0 was highly discriminant between ALF and HC. There was an increase in ATX and LPA in individuals with ALF compared to HCs and those with sepsis. LPCs 16:0, 18:0 and 18:1 were reduced in individuals with ALF and were associated with a poor prognosis. Treatment of monocytes with LPA 16:0 increased their PD-L1 expression and reduced CD155, CD163, MerTK levels, without affecting immune checkpoints on T and NK/CD56+T cells. LPAR1 and 3 antagonism in culture reversed the effect of LPA on monocyte expression of MerTK and CD163. MerTK and CD163, but not LPAR genes, were differentially expressed and upregulated in monocytes from individuals with ALF compared to controls. CONCLUSION: Reduced LPC levels are biomarkers of poor prognosis in individuals with ALF. The LPC-ATX-LPA axis appears to modulate innate immune response in ALF via LPAR1 and LPAR3. Further investigations are required to identify novel therapeutic agents targeting these receptors. IMPACT AND IMPLICATIONS: We identified a metabolic signature of acute liver failure (ALF) and investigated the immunometabolic role of the lysophosphatidylcholine-autotaxin-lysophosphatidylcholinic acid pathway, with the aim of finding a mechanistic explanation for monocyte behaviour and identifying possible therapeutic targets (to modulate the systemic immune response in ALF). At present, no selective immune-based therapies exist. We were able to modulate the phenotype of monocytes in vitro and aim to extend these findings to murine models of ALF as a next step. Future therapies may be based on metabolic modulation; thus, the role of specific lipids in this pathway require elucidation and the relative merits of autotaxin inhibition, lysophosphatidylcholinic acid receptor blockade or lipid-based therapies need to be determined. Our findings begin to bridge this knowledge gap and the methods used herein could be useful in identifying therapeutic targets as part of an experimental medicine approach.

Integrated Transcriptomic and Proteomic Analysis Identifies Plasma Biomarkers of Hepatocellular Failure in Alcohol-Associated Hepatitis.

Alcohol-associated hepatitis (AH) is a form of liver failure with high short-term mortality. Recent studies have shown that defective function of hepatocyte nuclear factor 4 alpha (HNF4a) and systemic inflammation are major disease drivers of AH. Plasma biomarkers of hepatocyte function could be useful for diagnostic and prognostic purposes. Herein, an integrative analysis of hepatic RNA sequencing and liquid chromatography-tandem mass spectrometry was performed to identify plasma protein signatures for patients with mild and severe AH. Alcohol-related liver disease cirrhosis, nonalcoholic fatty liver disease, and healthy subjects were used as comparator groups. Levels of identified proteins primarily involved in hepatocellular function were decreased in patients with AH, which included hepatokines, clotting factors, complement cascade components, and hepatocyte growth activators. A protein signature of AH disease severity was identified, including thrombin, hepatocyte growth factor α, clusterin, human serum factor H-related protein, and kallistatin, which exhibited large abundance shifts between severe and nonsevere AH. The combination of thrombin and hepatocyte growth factor α discriminated between severe and nonsevere AH with high sensitivity and specificity. These findings were correlated with the liver expression of genes encoding secreted proteins in a similar cohort, finding a highly consistent plasma protein signature reflecting HNF4A and HNF1A functions. This unbiased proteomic-transcriptome analysis identified plasma protein signatures and pathways associated with disease severity, reflecting HNF4A/1A activity useful for diagnostic assessment in AH.

A genetic risk score and diabetes predict development of alcohol-related cirrhosis in drinkers.

BACKGROUND & AIMS: Only a minority of excess alcohol drinkers develop cirrhosis. We developed and evaluated risk stratification scores to identify those at highest risk. METHODS: Three cohorts (GenomALC-1: n = 1,690, GenomALC-2: n = 3,037, UK Biobank: relevant n = 6,898) with a history of heavy alcohol consumption (≥80 g/day (men), ≥50 g/day (women), for ≥10 years) were included. Cases were participants with alcohol-related cirrhosis. Controls had a history of similar alcohol consumption but no evidence of liver disease. Risk scores were computed from up to 8 genetic loci identified previously as associated with alcohol-related cirrhosis and 3 clinical risk factors. Score performance for the stratification of alcohol-related cirrhosis risk was assessed and compared across the alcohol-related liver disease spectrum, including hepatocellular carcinoma (HCC). RESULTS: A combination of 3 single nucleotide polymorphisms (SNPs) (PNPLA3:rs738409, SUGP1-TM6SF2:rs10401969, HSD17B13:rs6834314) and diabetes status best discriminated cirrhosis risk. The odds ratios (ORs) and (95% CIs) between the lowest (Q1) and highest (Q5) score quintiles of the 3-SNP score, based on independent allelic effect size estimates, were 5.99 (4.18-8.60) (GenomALC-1), 2.81 (2.03-3.89) (GenomALC-2), and 3.10 (2.32-4.14) (UK Biobank). Patients with diabetes and high risk scores had ORs of 14.7 (7.69-28.1) (GenomALC-1) and 17.1 (11.3-25.7) (UK Biobank) compared to those without diabetes and with low risk scores. Patients with cirrhosis and HCC had significantly higher mean risk scores than patients with cirrhosis alone (0.76 ± 0.06 vs. 0.61 ± 0.02, p = 0.007). Score performance was not significantly enhanced by information on additional genetic risk variants, body mass index or coffee consumption. CONCLUSIONS: A risk score based on 3 genetic risk variants and diabetes status enables the stratification of heavy drinkers based on their risk of cirrhosis, allowing for the provision of earlier preventative interventions. LAY SUMMARY: Excessive chronic drinking leads to cirrhosis in some people, but so far there is no way to identify those at high risk of developing this debilitating disease. We developed a genetic risk score that can identify patients at high risk. The risk of cirrhosis is increased >10-fold with just two risk factors - diabetes and a high genetic risk score. Risk assessment using this test could enable the early and personalised management of this disease in high-risk patients.

Serum transferrin as a biomarker of hepatocyte nuclear factor 4 alpha activity and hepatocyte function in liver diseases.

BACKGROUND: Serum transferrin levels represent an independent predictor of mortality in patients with liver failure. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of hepatocyte functions. The aim of this study was to explore whether serum transferrin reflects HNF4α activity. METHODS: Factors regulating transferrin expression in alcoholic hepatitis (AH) were assessed via transcriptomic/methylomic analysis as well as chromatin immunoprecipitation coupled to DNA sequencing. The findings were corroborated in primary hepatocytes. Serum and liver samples from 40 patients with advanced liver disease of multiple etiologies were also studied. RESULTS: In patients with advanced liver disease, serum transferrin levels correlated with hepatic transferrin expression (r = 0.51, p = 0.01). Immunohistochemical and biochemical tests confirmed reduced HNF4α and transferrin protein levels in individuals with cirrhosis. In AH, hepatic gene-gene correlation analysis in liver transcriptome revealed an enrichment of HNF4α signature in transferrin-correlated transcriptome while transforming growth factor beta 1 (TGFß1), tumor necrosis factor α (TNFα), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6) negatively associated with transferrin signature. A key regulatory region in transferrin promoter was hypermethylated in patients with AH. In primary hepatocytes, treatment with TGFß1 or the HNF4α inhibitor BI6015 suppressed transferrin production, while exposure to TNFα, IL-1ß, and IL-6 had no effect. The correlation between hepatic HNF4A and transferrin mRNA levels was also seen in advanced liver disease. CONCLUSIONS: Serum transferrin levels constitute a prognostic and mechanistic biomarker. Consequently, they may serve as a surrogate of impaired hepatic HNF4α signaling and liver failure.

Assessment and Management of Infection in Alcoholic Hepatitis.

Severe alcoholic hepatitis (SAH) is a condition characterized by jaundice and liver failure that develops after heavy and prolonged alcohol consumption. Infection frequently complicates the natural history of the disease and is independently associated with mortality. Objective recognition and recording of infection are therefore essential in the evaluation of therapeutic interventions and for antibiotic stewardship. This review will evaluate infections that complicate SAH at admission and beyond. Factors that associate with the development of infection will be identified and clinical and laboratory techniques available to identify infection will be discussed. Common pathogens and frequently used antibiotics will be reviewed and recommendations will be made for the management of infection for SAH patients. New techniques to assess infection earlier and more precisely may improve diagnosis and treatment of this important driver of mortality in SAH.

In Severe Alcoholic Hepatitis, Serum Keratin-18 Fragments Are Diagnostic, Prognostic, and Theragnostic Biomarkers.

INTRODUCTION: Up to 40% of patients with severe alcoholic hepatitis (AH) die within 6 months of presentation, making prompt diagnosis and appropriate treatment essential. We determined the associations between serum keratin-18 (K18) and histological features, prognosis, and differential response to prednisolone in patients with severe AH. METHODS: Total (K18-M65) and caspase-cleaved K18 (K18-M30) were quantified in pretreatment sera from 824 patients enrolled in the Steroids or Pentoxifylline for Alcoholic Hepatitis trial (87 with suitable histological samples) and disease controls. RESULTS: K18 fragments were markedly elevated in severe AH and strongly predicted steatohepatitis (alcoholic steatohepatitis) on biopsy (area under receiver operating characteristics: 0.787 and 0.807). Application of published thresholds to predict alcoholic steatohepatitis would have rendered biopsy unnecessary in 84% of all AH cases. K18-M30 and M65 were associated with 90-day mortality, independent of age and Model for End-stage Liver Disease score in untreated patients. The association for K18-M65 was independent of both age and Model for End-stage Liver Disease in prednisolone-treated patients. Modelling of the effect of prednisolone on 90-day mortality as a function of pretreatment serum K18 levels indicated benefit in those with high serum levels of K18-M30. At low pretreatment serum K18 levels, prednisolone was potentially harmful. A threshold of K18-M30 5 kIU/L predicted therapeutic benefit from prednisolone above this level (odds ratio: 0.433, 95% confidence interval: 0.19-0.95, P = 0.0398), but not below (odds ratio: 1.271, 95% confidence interval: 0.88-1.84, P = 0.199). Restricting prednisolone usage to the former group would have reduced exposure by 87%. DISCUSSION: In a large cohort of patients with severe AH, serum K18 strongly correlated with histological severity, independently associated with 90-day mortality, and predicted response to prednisolone therapy. Quantification of serum K18 levels could assist in clinical decision-making.

Serum Transferrin Is an Independent Predictor of Mortality in Severe Alcoholic Hepatitis.

OBJECTIVES: Severe alcoholic hepatitis (sAH) confers substantial mortality, but the disease course is difficult to predict. As iron parameters are attractive outcome predictors in other liver diseases, we tested their prognostic ability in sAH. METHODS: Serum ferritin, transferrin, iron, transferrin saturation, nontransferrin-bound iron, soluble transferrin receptor, and hepcidin were measured in 828 patients with sAH recruited prospectively through the STOPAH trial. The cohort was randomly divided into exploratory (n = 200) and validation sets (n = 628). RESULTS: Patients with sAH had diminished serum transferrin but increased transferrin saturation. Among iron parameters, baseline transferrin was the best predictor of 28-day (area under the receiver operated characteristic 0.72 [95% confidence interval 0.67-0.78]) and 90-day survival (area under the receiver operated characteristic 0.65 [0.61-0.70]). Transferrin's predictive ability was comparable with the composite scores, namely model of end-stage liver disease, Glasgow alcoholic hepatitis score, and discriminant function, and was independently associated with survival in multivariable analysis. These results were confirmed in a validation cohort. Transferrin did not correlate with markers of liver synthesis nor with non-transferrin-bound iron or soluble transferrin receptor (as markers of excess unbound iron and functional iron deficiency, respectively). DISCUSSION: In patients with sAH, serum transferrin predicts mortality with a performance comparable with commonly used composite scoring systems. Hence, this routinely available parameter might be a useful marker alone or as a component of prognostic models.

Corticosteroids Reduce Risk of Death Within 28 Days for Patients With Severe Alcoholic Hepatitis, Compared With Pentoxifylline or Placebo-a Meta-analysis of Individual Data From Controlled Trials.

BACKGROUND & AIMS: We performed a meta-analysis of individual patient data from 11 randomized controlled trials comparing corticosteroids, pentoxifylline, or their combination in patients with severe alcoholic hepatitis. We compared the effects of the treatments on survival for 28 days or 6 months, and response to treatment based on the Lille model. METHODS: We searched PubMed for randomized controlled trials of pharmacologic therapy for severe alcoholic hepatitis. Our final analysis comprised 11 studies, of 2111 patients. We performed 4 meta-analyses of the effects of corticosteroids vs placebo or control, corticosteroids vs pentoxifylline, corticosteroids and pentoxifylline vs corticosteroids and placebo or control, and pentoxifylline vs placebo. In each meta-analysis, the effect of treatment on the primary outcome (overall survival at 28 days, defined as the period from the first day of assigned treatment to 28 days) was estimated using a Cox proportional hazards regression model, including trials as random effect. RESULTS: Corticosteroid treatment significantly decreased risk of death within 28 days compared with controls (hazard ratio [HR] 0.64; 95% confidence interval [CI] 0.48-0.86) or to pentoxifylline (HR 0.64; 95% CI 0.43-0.95). In multiple-imputation and complete case analyses, the effect of corticosteroids compared with controls remained significant. When we compared corticosteroids vs pentoxifylline, the corticosteroid effect remained significant in the complete case analysis (HR 0.66; P = .04) but not in multiple-imputation analysis (HR 0.71; P = .08). There was no difference in 28-day mortality when patients were given a combination of corticosteroids and pentoxifylline vs corticosteroids alone or between patients given pentoxifylline vs control. In our analysis of secondary outcomes, we found no significant differences in 6-month mortality when any treatments or controls were compared. Corticosteroids were significantly associated with increased response to therapy compared with controls (relative risk 1.24; 95% CI 1.10-1.41) or pentoxifylline (relative risk 1.43; 95% CI 1.20-1.68). We found no difference in response to therapy between patients given a combination of corticosteroids and pentoxifylline vs corticosteroids alone or pentoxifylline vs controls. CONCLUSIONS: In a meta-analysis of 4 controlled trials, we found corticosteroid use to reduce risk of death within 28 days of treatment, but not in the following 6 months. This loss of efficacy over time indicates a need for new therapeutic strategies to improve medium-term outcomes.

Application of prognostic scores in the STOPAH trial: Discriminant function is no longer the optimal scoring system in alcoholic hepatitis.

BACKGROUND & AIMS: 'Static' prognostic models in alcoholic hepatitis, using data from a single time point, include the discriminant function (DF), Glasgow alcoholic hepatitis score (GAHS), the age, serum bilirubin, international normalized ratio and serum creatinine (ABIC) score and the model of end-stage liver disease (MELD). 'Dynamic' scores, incorporating evolution of bilirubin at seven days, include the Lille score. The aim of this study was to assess these scores' performance in patients from the STOPAH trial. METHODS: Predictive performance of scores was assessed by area under the receiver operating curve (AUC). The effect of different therapeutic strategies upon survival was assessed by Kaplan-Meier analysis and tested using the log-rank test. RESULTS: A total of 1,068 patients were studied. The AUCs for the DF were significantly lower than for MELD, ABIC and GAHS for both 28- and 90-day outcomes: 90-day values were 0.670, 0.704, 0.726 and 0.713, respectively. 'Dynamic' scores and change in 'static' scores by Day 7 had similar AUCs. Patients with consistently low 'static' scores had low 28-day mortalities that were not improved with prednisolone (MELD <25: 8.6%; ABIC <6.71: 6.6%; GAHS <9: 5.9%). In patients with high 'static' scores without gastrointestinal bleeding or sepsis, prednisolone reduced 28-day mortality (MELD: 22.2% vs. 28.9%, p = 0.13; ABIC 14.6% vs. 21%, p = 0.02; GAHS 21% vs. 29.3%, p = 0.04). Overall mortality from treating all patients with a DF ≥32 and Lille assessment (90-day mortality 26.8%) was greater than combining newer 'static' and 'dynamic' scores (90-day mortality: MELD/Lille 21.8%; ABIC/Lille 23.7%; GAHS/Lille 20.6%). CONCLUSION: MELD, ABIC and GAHS are superior to the DF in alcoholic hepatitis. Consistently low scores have a favourable outcome not improved with prednisolone. Combined baseline 'static' and Day 7 scores reduce the number of patients exposed to corticosteroids and improve 90-day outcome. LAY SUMMARY: Alcoholic hepatitis is a life-threatening condition. Several scores exist to determine the outcome of these patients as well as to identify those who may benefit from treatment. This study looked at the performance of existing scores in patients who had been recruited to the largest alcoholic hepatitis clinical trial: STOPAH. 'Static' scores are calculable at the start of assessment. The three newer static scores (ABIC, GAHS and MELD) were shown to be superior to the oldest score (DF). ABIC and GAHS could also identify patients who had a survival benefit 28 days after starting prednisolone treatment. 'Dynamic' scores relate to the change in disease over the first week of treatment. Combination of the 'static' scores 'with the 'dynamic' scores or change in 'static' scores allowed identification of patients who could benefit from prednisolone up to 90 days.

In Patients With Severe Alcoholic Hepatitis, Prednisolone Increases Susceptibility to Infection and Infection-Related Mortality, and Is Associated With High Circulating Levels of Bacterial DNA.

BACKGROUND & AIMS: Infections are common in patients with severe alcoholic hepatitis (SAH), but little information is available on how to predict their development or their effects on patients. Prednisolone is advocated for treatment of SAH, but can increase susceptibility to infection. We compared the effects of infection on clinical outcomes of patients treated with and without prednisolone, and identified risk factors for development of infection in SAH. METHODS: We analyzed data from 1092 patients enrolled in a double-blind placebo-controlled trial to evaluate the efficacy of treatment with prednisolone (40 mg daily) or pentoxifylline (400 mg 3 times each day) in patients with SAH. The 2 × 2 factorial design led to 547 patients receiving prednisolone; 546 were treated with pentoxifylline. The trial was conducted in the United Kingdom from January 2011 through February 2014. Data on development of infection were collected at evaluations performed at screening, baseline, weekly during admission, on discharge, and after 90 days. Patients were diagnosed with infection based on published clinical and microbiologic criteria. Risk factors for development of infection and effects on 90-day mortality were evaluated separately in patients treated with prednisolone (n = 547) and patients not treated with prednisolone (n = 545) using logistic regression. Pretreatment blood levels of bacterial DNA (bDNA) were measured in 731 patients. RESULTS: Of the 1092 patients in the study, 135 had an infection at baseline, 251 developed infections during treatment, and 89 patients developed an infection after treatment. There was no association between pentoxifylline therapy and the risk of serious infection (P = .084), infection during treatment (P = .20), or infection after treatment (P = .27). Infections classified as serious were more frequent in patients treated with prednisolone (odds ratio [OR], 1.27; 95% confidence interval [CI], 1.27-2.92; P = .002). There was no association between prednisolone therapy and infection during treatment (OR, 1.04; 95% CI, 0.78-1.37; P = .80). However, a higher proportion (10%) of patients receiving prednisolone developed an infection after treatment than of patients not given prednisolone (6%) (OR, 1.70; 95% CI, 1.07-2.69; P = .024). Development of infection was associated with increased 90-day mortality in patients with SAH treated with prednisolone, independent of model for end-stage liver disease or Lille score (OR, 2.46; 95% CI, 1.41-4.30; P = .002). High circulating bDNA predicted infection that developed within 7 days of prednisolone therapy, independent of Model for End-Stage Liver Disease and white blood cell count (OR, 4.68; 95% CI, 1.80-12.17; P = .001). In patients who did not receive prednisolone, infection was not independently associated with 90-day mortality (OR, 0.94; 95% CI, 0.54-1.62; P = .82) or levels of bDNA (OR, 0.83; 95% CI, 0.39-1.75; P = .62). CONCLUSIONS: Patients with SAH given prednisolone are at greater risk for developing serious infections and infections after treatment than patients not given prednisolone, which may offset its therapeutic benefit. Level of circulating bDNA before treatment could identify patients at high risk of infection if given prednisolone; these data could be used to select therapies for patients with SAH. EudraCT no: 2009-013897-42; Current Controlled Trials no: ISRCTN88782125.

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase.

OBJECTIVE: In order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection. DESIGN: Monocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy. RESULTS: MOB, production of superoxide and bacterial killing in response to Escherichia coli were markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91 phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy. CONCLUSIONS: Monocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.

Homozygosity for rs738409:G in PNPLA3 is associated with increased mortality following an episode of severe alcoholic hepatitis.

BACKGROUND & AIMS: Carriage of rs738409:G in PNPLA3 is associated with an increased risk of developing alcohol-related cirrhosis and has a significant negative effect on survival. Short-term mortality in patients with severe alcoholic hepatitis is high; drinking behaviour is a major determinant of outcome in survivors. The aim of this study was to determine whether carriage of rs738409:G has an additional detrimental effect on survival in this patient group. METHODS: Genotyping was undertaken in 898 cases with severe alcoholic hepatitis, recruited through the UK Steroids or Pentoxifylline for Alcoholic Hepatitis (STOPAH) trial, and 1188 White British/Irish alcohol dependent controls with no liver injury, recruited via University College London. Subsequent drinking behaviour was classified, in cases surviving ≥90days, as abstinent or drinking. The relationship between rs738409 genotype, drinking behaviour and survival was explored. RESULTS: The frequency of rs738409:G was significantly higher in cases than controls (29.5% vs. 18.9%; p=2.15×10-15; odds ratio 1.80 [95% confidence interval (CI) 1.55-2.08]). Case-mortality at days 28, 90 and 450 was 16%, 25% and 41% respectively. There was no association between rs738409:G and 28-day mortality. Mortality in the 90 to 450-day period was higher in survivors who subsequently resumed drinking (hazard ratio [HR] 2.77, 95% CI 1.79-4.29; p<0.0001) and in individuals homozygous for rs738409:G (HR 1.69, 95% CI 1.02-2.81, p=0.04). CONCLUSION: Homozygosity for rs738409:G in PNPLA3 confers significant additional risk of medium-term mortality in patients with severe alcoholic hepatitis. Rs738409 genotype may be taken into account when considering treatment options for these patients. LAY SUMMARY: Individuals misusing alcohol who carry a particular variant of the gene PNPLA3 are more at risk of developing severe alcoholic hepatitis, a condition with a poor chance of survival. The longer-term outcome in people with this condition who survive the initial illness is strongly influenced by their ability to remain abstinent from alcohol. However, carriers of this gene variant are less likely to survive even if they are able to stop drinking completely. Knowing if someone carries this gene variant could influence the way in which they are managed. Clinical trial numbers: EudraCT reference number: 2009-013897-42; ISRCTN reference number: ISRCTN88782125. CLINICAL TRIAL NUMBERS: EudraCT reference number: 2009-013897-42; ISRCTN reference number: ISRCTN88782125.

Comparative Effectiveness of Pharmacological Interventions for Severe Alcoholic Hepatitis: A Systematic Review and Network Meta-analysis.

BACKGROUND & AIMS: Severe alcoholic hepatitis (AH) has high mortality. We assessed the comparative effectiveness of pharmacological interventions for severe AH, through a network meta-analysis combining direct and indirect treatment comparisons. METHODS: We conducted a systematic literature review, through February 2015, for randomized controlled trials of adults with severe AH (discriminant function ≥32 and/or hepatic encephalopathy) that compared the efficacy of active pharmacologic interventions (corticosteroids, pentoxifylline, and N-acetylcysteine [NAC], alone or in combination) with each other or placebo, in reducing short-term mortality (primary outcome) and medium-term mortality, acute kidney injury, and/or infections (secondary outcomes). We performed direct and Bayesian network meta-analysis for all treatments, and used Grading of Recommendations Assessment, Development and Evaluation criteria to appraise quality of evidence. RESULTS: We included 22 randomized controlled trials (2621 patients) comparing 5 different interventions. In a direct meta-analysis, only corticosteroids decreased risk of short-term mortality. In a network meta-analysis, moderate quality evidence supported the use of corticosteroids alone (relative risk [RR], 0.54; 95% credible interval [CrI], 0.39-0.73) or in combination with pentoxifylline (RR, 0.53; 95% CrI, 0.36-0.78) or NAC (RR, 0.15; 95% CI, 0.05-0.39), to reduce short-term mortality; low quality evidence showed that pentoxifylline also decreased short-term mortality (RR, 0.70; 95% CrI, 0.50-0.97). The addition of NAC, but not pentoxifylline, to corticosteroids may be superior to corticosteroids alone for reducing short-term mortality. No treatment was effective in reducing medium-term mortality. Imprecise estimates and the small number of direct trials lowered the confidence in several comparisons. CONCLUSIONS: In patients with severe AH, pentoxifylline and corticosteroids (alone and in combination with pentoxifylline or NAC) can reduce short-term mortality. No treatment decreases risk of medium-term mortality.

Therapeutic inhibition of monocyte recruitment prevents checkpoint inhibitor-induced hepatitis.

BACKGROUND: Checkpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and to therapeutically target key pathways driving this pathology. METHODS: C57BL/6 wild-type (WT) mice were dosed with toll-like receptor (TLR)9 agonist (TLR9-L) for hepatic priming combined with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) plus anti-programmed cell death 1 (PD-1) ("CPI") or phosphate buffered saline (PBS) control for up to 7 days. Flow cytometry, histology/immunofluorescence and messenger RNA sequencing were used to characterize liver myeloid/lymphoid subsets and inflammation. Hepatocyte damage was assessed by plasma alanine transaminase (ALT) and cytokeratin-18 (CK-18) measurements. In vivo investigations of CPI-hepatitis were carried out in Rag2-/- and Ccr2rfp/rfp transgenic mice, as well as following anti-CD4, anti-CD8 or cenicriviroc (CVC; CCR2/CCR5 antagonist) treatment. RESULTS: Co-administration of combination CPIs with TLR9-L induced liver pathology closely resembling human disease, with increased infiltration and clustering of granzyme B+perforin+CD8+ T cells and CCR2+ monocytes, 7 days post treatment. This was accompanied by apoptotic hepatocytes surrounding these clusters and elevated ALT and CK-18 plasma levels. Liver RNA sequencing identified key signaling pathways (JAK-STAT, NF-ΚB) and cytokine/chemokine networks (Ifnγ, Cxcl9, Ccl2/Ccr2) as drivers of CPI-hepatitis. Using this model, we show that CD8+ T cells mediate hepatocyte damage in experimental CPI-hepatitis. However, their liver recruitment, clustering, and cytotoxic activity is dependent on the presence of CCR2+ monocytes. The absence of hepatic monocyte recruitment in Ccr2rfp/rfp mice and CCR2 inhibition by CVC treatment in WT mice was able to prevent the development and reverse established experimental CPI-hepatitis. CONCLUSION: This newly established mouse model provides a platform for in vivo mechanistic studies of CPI-hepatitis. Using this model, we demonstrate the central role of liver infiltrating CCR2+ monocyte interaction with tissue-destructive CD8+ T cells in the pathogenesis of CPI-hepatitis and highlight CCR2 inhibition as a novel therapeutic target.