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.

Bariatric Surgery Is Associated with Alcohol-Related Liver Disease and Psychiatric Disorders Associated with AUD.

BACKGROUND/AIMS: Bariatric surgery can increase the risk of addictive disorders and nutritional deficiencies. The aim of this study was to evaluate the association between bariatric surgery and alcohol use disorder (AUD), alcohol-related liver disease (ALD), and psychiatric disorders associated with AUD. The impact of vitamin D deficiency in these associations was also investigated. METHODS: A cross-sectional study was performed using the National Inpatient Sample database and its ICD-9 codes information. Diagnostic and comorbidity data from hospital discharges were obtained from patients with bariatric surgery and other abdominal surgeries between 2005 and 2015. The two groups were then compared for alcohol-related outcomes after propensity-score matching. RESULTS: The final study cohort included 537,757 patients with bariatric surgery and 537,757 with other abdominal surgeries. The bariatric surgery group had an increased risk of AUD [odds ratio (OR): 1.90; 95% CI: 1.85-1.95], ALD [OR: 1.29; 95% CI: 1.22-1.37], cirrhosis [OR, 1.39; 95% CI: 1.37-1.42], and psychiatric disorders associated with AUD [OR, 3.59; 95% CI: 3.37-3.84]. Vitamin D deficiency did not impact in the association between bariatric surgery and AUD, ALD, or psychiatric disorders associated with AUD. CONCLUSIONS: Bariatric surgery is associated with an increased prevalence of AUD, ALD, and psychiatric disorders associated with AUD. These associations appear to be independent from vitamin D deficiency.

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.

Genetic and Environmental Susceptibility to Alcoholic Hepatitis.

Constitutional, environmental, and genetic risk factors influence the development of alcohol-related cirrhosis. The amount of alcohol consumed and whether excessive drinking continues after the identification of pre-cirrhotic liver damage are key risk factors. Female sex, ethnicity, obesity, coffee consumption, cigarette smoking, and exposure to other causes of liver injury also influence the risk of disease development. More recently several genetic loci have been robustly associated with the risk for developing significant alcohol-related liver disease. It remains unclear whether additional risk factors are involved in the development of the clinical syndrome of alcoholic hepatitis, but the genetic evidence is suggestive.

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.

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.

Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFß1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFß1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.

Identification of mutations in circulating cell-free tumour DNA as a biomarker in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is increasing globally. Prognostic biomarkers are urgently needed to guide treatment and reduce mortality. Tumour-derived circulating cell-free DNA (ctDNA) is a novel, minimally invasive means of determining genetic alterations in cancer. We evaluate the accuracy of ctDNA as a biomarker in HCC. METHODS: Plasma cell-free DNA, matched germline DNA and HCC tissue DNA were isolated from patients with HCC (n = 51) and liver cirrhosis (n = 10). Targeted, multiplex polymerase chain reaction ultra-deep sequencing was performed using a liver cancer-specific primer panel for genes ARID1A, ARID2, AXIN1, ATM, CTNNB1, HNF1A and TP53. Concordance of mutations in plasma ctDNA and HCC tissue DNA was determined, and associations with clinical outcomes were analysed. RESULTS: Plasma cell-free DNA was detected in all samples. Lower plasma cell-free DNA levels were seen in Barcelona Clinic Liver Cancer (BCLC A compared with BCLC stage B/C/D (median concentration 122.89 ng/mL versus 168.21 ng/mL, p = 0.041). 29 mutations in the eight genes (21 unique mutations) were detected in 18/51 patients (35%), median 1.5 mutations per patient (interquartile range 1-2). Mutations were most frequently detected in ARID1A (11.7%), followed by CTNNB1 (7.8%) and TP53 (7.8%). In patients with matched tissue DNA, all mutations detected in plasma ctDNA detected were confirmed in HCC DNA; however, 71% of patients had mutations identified in HCC tissue DNA that were not detected in matched ctDNA. CONCLUSION: ctDNA is quantifiable across all HCC stages and allows detection of mutations in key driver genes of hepatic carcinogenesis. This study demonstrates high specificity but low sensitivity of plasma ctDNA for detecting mutations in matched HCC tissue.

The Inflammation-Based Index Can Predict Response and Improve Patient Selection in NETs Treated With PRRT: A Pilot Study.

Background: Peptide receptor radionuclide therapy (PRRT) is an effective treatment of certain patients with metastatic neuroendocrine tumors (NETs). Tumor response is highly variable; no biomarker in clinical practice has been demonstrated to reliably predict outcome. The inflammation-based index (IBI), derived from serum C-reactive protein and albumin levels, predicts survival and response to treatment in patients in several cancer types and was therefore explored in this setting. Materials and Methods: Clinico-pathological data from patients undergoing PRRT for metastatic NETs were collected at baseline and during treatment. The primary endpoint was progression-free survival (PFS) with a secondary endpoint of overall survival (OS). Cox regression analysis tested associations between baseline variables and their dynamic changes and PFS and OS. Decision curve analysis (DCA) was used to determine the net benefit associated with a treatment strategy determined by the baseline IBI and nonresponse to PRRT. Results: Fifty-five patients were recruited. Baseline IBI > 0 was associated with inferior PFS (hazard ratio, 14.2; 95% CI, 5.25 to 38.5; P < 0.001) and OS (P < 0.001). Multivariate analysis confirmed an independent association between IBI and PFS (P = 0.001). DCA indicated a net clinical benefit at risk thresholds between 0.03 and 0.58. Conclusion: Baseline IBI score and its dynamic change through treatment are associated with both PFS and OS. At a risk threshold equivalent to the currently accepted rate of nonresponse to therapy, implementation of this easily derived score could avoid a substantial number of futile treatments. These findings should be validated in additional independent cohorts.

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.

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.