BACKGROUND AIMS: The common characteristics for alcohol-associated liver injury (ALI) include abnormal liver function, infiltration of inflammatory cells, and generation of oxidative stress. The gastrin-releasing peptide receptor (GRPR) is activated by its neuropeptide ligand, gastrin-releasing peptide (GRP). GRP/GRPR appears to induce the production of cytokines in immune cells and promotes neutrophil migration. However, the effects of GRP/GRPR in ALI are unknown. APPROACH RESULTS: We found high GRPR expression in the liver of patients with alcoholic steatohepatitis and increased pro-GRP levels in peripheral blood mononuclear cells of these patients compared to that of the control. Increased expression of GRP may be associated with histone H3 lysine 27 acetylation induced by alcohol, which promotes the expression of GRP and then GRPR binding. Grpr-/- and Grprflox/floxLysMCre mice alleviated ethanol-induced liver injury with relieved steatosis, lower serum alanine aminotransferase, aspartate aminotransferase, triglycerides, malondialdehyde, and superoxide dismutase levels, reduced neutrophil influx, and decreased expression and release of inflammatory cytokines and chemokines. Conversely, overexpression of GRPR showed opposite effects. The pro-inflammatory and oxidative stress roles of GRPR might be dependent on IRF1-mediated Caspase-1 inflammasome and NOX2-dependent reactive oxygen species pathway, respectively. In addition, we verified the therapeutic and preventive effects of RH-1402, a novel GRPR antagonist, for ALI. CONCLUSIONS: A knockout or antagonist of GRPR during excess alcohol intake could have anti-inflammatory and antioxidative roles, as well as provide a platform for histone modification-based therapy for ALI.

BACKGROUND: The value of hepatocellular carcinoma (HCC) surveillance is determined by the balance between benefits and harms; however, no studies have enumerated psychological harms. METHODS: We fielded surveys measuring psychological harms to patients with cirrhosis in a multi-center randomized trial of HCC surveillance outreach. All patients with positive or indeterminate surveillance results and matched patients with negative results were invited to complete surveys measuring: 1)depression via PHQ-9, 2)anxiety via State-Trait Anxiety Inventory, 3)HCC-specific worry via Psychological Consequences Questionnaire, and 4)decisional regret. Patients were classified into four groups: true positive (TP), false positive (FP), indeterminate, and true negative (TN). Multivariable longitudinal regression analysis using generalized estimating equation method were performed to compare means of measures across groups. We conducted 89 semi-structured interviews in a subset of patients, stratified by health system and test result. RESULTS: Of 2872 patients in the trial, 311 completed 1 + follow-up survey (63 FP, 77 indeterminate, 38 TP, and 133 TN). Moderate depression decreased in TN patients, increased in TP, and had intermittent but mild increases in those with FP and indeterminate results. High anxiety temporarily increased in TP patients but resolved over time and was stable in those with FP and indeterminate results. Decisional regret was low and did not differ across groups. In semi-structured interviews, patients reported apprehension, anxiety, emotional distress, and coping related to HCC surveillance. CONCLUSION: Psychological harms of HCC surveillance appear mild but differ by test result. Future research should determine the impact of psychological harms on value of HCC surveillance programs. CLINICAL TRIALS NUMBER: NCT02582918 and NCT03756051.

The indolent and aggressive behaviors of hepatocellular carcinoma (HCC) might have a role in clinical trial (CT) results; however, the indolent HCC is less analyzed compared to others cancer. Indolent profile could be characterized as follow: (a) patients with low risk of progression itself due to the HCC molecular profile and/or due to the interaction between cancer cell their microenvironment; (b) patients who achieve objective response or present spontaneous regression and (c) patients who develop radiological progression with no consequence on either the liver function or general status, and without trigger a change in the tumor stage. Patients with ' indolent HCC' generally never develop cancer-related symptoms neither die for HCC-related causes. Thus, we hypothesize that the imbalance in the proportion of 'indolent' versus 'aggressive HCC' between arms or the under-/over-estimation of HCC behavior at baseline in single arm CT could be associated to CT failure or under- overestimation of trial results. The ' indolent progression' may also explain the discrepancy between radiological progression-based end-points and survival. Moreover, we discuss the related-causes that explain the indolent profile of HCC and propose (a) refining the progression-related end-point by the pattern of progression to minimize the limitations of the current end-points; (b) considering alternative statistical tools for survival analysis such as Milestone Survival, or Restricted Mean Survival Time to capture the value of indolent HCC. According to these considerations, we propose incorporating novel end-points into the single arm of phase I/II CT as exploratory analysis or as a secondary end-point in phase III CT.

BACKGROUND AND AIMS: Normal alkaline phosphatase (ALP) levels in ursodeoxycholic acid (UDCA)-treated patients with primary biliary cholangitis (PBC) are associated with better long-term outcome. However, second-line therapies are currently recommended only when ALP levels remain above 1.5 times the upper limit of normal (xULN) after 12-month UDCA. We assessed whether, in patients considered good responders to UDCA, normal ALP levels were associated with significant survival gains. APPROACH AND RESULTS: We performed a retrospective cohort study of 1,047 patients with PBC who attained an adequate response to UDCA according to Paris-2 criteria. Time to liver-related complications, liver transplantation or death was assessed using adjusted restricted mean survival time analysis. The overall incidence rate of events was 17.0 (95%CI 13.7 - 21.1) per 1,000 out of 4,763.2 patient-years. On the whole population, normal serum ALP values (but not normal GGT, ALT, or AST; or total bilirubin < 0.6 xULN) were associated with an overall absolute complication-free survival gain at 10 years of 7.6 months (95%CI 2.7 - 12.6, p = 0.003). In subgroup analysis, this association was significant in patients with a liver stiffness measurement ≥ 10 kPa and/or age ≤ 62 years, with a 10-year absolute complication-free survival gain of 52.8 months (95%CI 45.7 - 59.9, p < 0.001) when these two conditions were met. CONCLUSIONS: PBC patients with an adequate response to UDCA and persistent ALP elevation between 1.1 and 1.5 xULN, particularly those with advanced fibrosis and/or who are sufficiently young, remain at risk of poor outcome. Further therapeutic efforts should be considered for these patients.

Cholangiocarcinoma (CCA) is a highly aggressive and lethal cancer that originates from biliary epithelium. Systemic treatment options for CCA are currently limited, and the first targeted drug of CCA, pemigatinib, emerged till 2020 for CCA treatment by inhibiting FGFR2 phosphorylation. However, the regulatory mechanism of FGFR2 phosphorylation is not fully elucidated. Here we screened the FGFR2-interacting proteins and showed that protein tyrosine phosphatase N9 (PTPN9) interacts with FGFR2 and negatively regulates FGFR2pY656/657. Using phosphatase activity assays and modeling the FGFR2-PTPN9 complex structure, we identified FGFR2pY656/657 as a substrate of PTPN9, and found that sec. 14p domain of PTPN9 interacts with FGFR2 via ACAP1 mediation. Co-expression of PTPN9 and ACAP1 indicates a favorable prognosis for CCA. Additionally, we identified key amino acids and motifs involved in sec. 14p-APCP1-FGFR2 interaction, including the "YRETRRKE" motif of sec. 14p, Y471 of PTPN9, as well as the PH and Arf-GAP domain of ACAP1. Moreover, we discovered that the FGFR2I654V substitution can decrease PTPN9-FGFR2 interaction and thereby reduce the effectiveness of pemigatinib treatment. Using a series of in vitro and in vivo experiments including PDX, we showed that PTPN9 synergistically enhances pemigatinib effectiveness and suppresses CCA proliferation, migration, and invasion by inhibiting FGFR2pY656/657. In conclusion, our study identifies PTPN9 as a negative regulator of FGFR2 phosphorylation and a synergistic factor for pemigatinib treatment. The molecular mechanism, oncogenic function and clinical significance of PTPN9-ACAP1-FGFR2 complex are revealed, providing more evidence for CCA precision treatment.

BACKGROUND AIMS: Primary sclerosing cholangitis (PSC) is a chronic progressive liver disease characterized by infiltration of intrahepatic tissue-resident memory CD8+ T cells (TRM). Itaconate has demonstrated therapeutic potential in modulating inflammation. An unmet need for PSC is the reduction of biliary inflammation, and we hypothesized that itaconate may directly modulate pathogenic TRM. METHODS: The numbers of intrahepatic CD103+ TRM were evaluated by immunofluorescence in PSC (n = 32) and serum levels of itaconate in PSC (n = 64), primary biliary cholangitis (PBC) (n = 60), autoimmune hepatitis (AIH) (n = 49), and healthy controls (n = 109) were determined by LC-MS/MS. Additionally, the frequencies and immunophenotypes of intrahepatic TRM using explants from PSC (n = 5) and healthy donors (n = 6) were quantitated by flow cytometry. The immunomodulatory properties of 4-octyl itaconate (4-OI, a cell-permeable itaconate derivative) on CD103+ TRM were studied in vitro. Finally, the therapeutic potential of itaconate was studied by administration of 4-OI and deficiency of immune-responsive gene 1(Irg1, encodes the aconitate decarboxylase producing itaconate) in murine models of PSC. RESULTS: Intrahepatic CD103+ TRM was significantly expanded in PSC and was positively correlated with disease severity. Serum itaconate levels decreased in PSC. Importantly, 4-OI inhibited the induction and effector functions of CD103+ TRM in vitro. Mechanistically, 4-OI blocked DNA demethylation of RUNX3 in CD8+T cells. Moreover, 4-OI reduced intrahepatic CD103+ TRM and ameliorated liver injury in murine models of PSC. CONCLUSIONS: Itaconate exerted immunomodulatory activity on CD103+ TRM in both in vitro and murine PSC models. Our study suggests that targeting pathogenic CD103+ TRM with itaconate has therapeutic potential in PSC.

Nonalcoholic steatohepatitis (NASH) represents a severe stage of fatty liver disease characterized by hepatocyte injury, inflammation, and liver fibrosis. Myeloid-derived innate immune cells, such as macrophages and dendritic cells, play an important role in host defense and disease pathogenesis. Despite this, the nature of transcriptomic reprogramming of myeloid cells in NASH liver and its contribution to disease progression remain incompletely defined. In this study, we performed bulk and single-cell RNA sequencing analysis to delineate the landscape of macrophage and dendritic cell transcriptomes in healthy and NASH liver. Our analysis uncovered cell type-specific patterns of transcriptomic reprogramming upon diet-induced NASH. We identified Brain abundant membrane attached signal protein 1 (Basp1) as a myeloid-enriched gene that is markedly induced in mouse and human NASH liver. Myeloid-specific inactivation of Basp1 attenuates the severity of diet-induced NASH pathologies as shown by reduced hepatocyte injury and liver fibrosis in mice. Mechanistically, cultured macrophages lacking Basp1 exhibited diminished response to pro-inflammatory stimuli, impaired NLRP3 inflammasome activation, and reduced cytokine secretion. Together, these findings uncover Basp1 as a critical regulator of myeloid inflammatory signaling that underlies NASH pathogenesis.

In advanced, liver-only intrahepatic cholangiocarcinoma (iCCA), Selective Internal Radiation Therapy (SIRT) has been suggested as promising in non-randomized studies. We aimed to compare data from patients with advanced, liver-only iCCA treated in first line in clinical trials with either chemotherapy alone or the combination with SIRT. We collected individual patients' data from the ABC-01, ABC-02, ABC-03, BINGO, AMEBICA and MISPHEC prospective trials. Data from patients with liver-only iCCA treated in chemotherapy-only arms of the first five trials were compared with data from patients treated with SIRT and chemotherapy in MISPHEC. Emulated target trial paradigm and Inverse Probability of Treatment Weighting (IPTW methods) using the propensity score were used to minimize biases. We compared 41 patients treated with the combination with 73 patients treated with chemotherapy alone, the main analysis being in 43 patients treated with cisplatin-gemcitabine or gemcitabine-oxaliplatin. After weighting, overall survival was significantly higher in patients treated with SIRT: median 21.7 months [95% Confidence Interval (CI): 14.1; not reached] vs 15.9 months [95%CI: 9.8; 18.9], Hazard Ratio = 0.59 [95%CI: 0.34; 0.99], p = 0.049. Progression-free survival was significantly improved: median 14.3 months [95%CI: 7.8; not reached] vs 8.4 months [95%CI: 5.9; 12.1], Hazard Ratio = 0.52 [95% CI: 0.31; 0.89], p < 0.001. Results were confirmed in most sensitivity analyses. Conclusion: This analysis derived from prospective clinical trials suggests that SIRT combined with chemotherapy might improve outcomes over chemotherapy alone in patients with advanced, liver-only iCCA. Randomized controlled evidence is needed to confirm these findings.

BACKGROUND AIMS: Hepatocellular carcinoma (HCC) can increase the risk of non-neoplastic portal vein thrombosis (PVT) in cirrhosis. However, natural history of PVT and its prognostic role in HCC patients are unknown. APPROACH RESULTS: Consecutive cirrhotic HCC patients undergoing laparoscopic ablation were retrospectively evaluated and followed-up to 36 months. HCC and PVT characteristics and evolution were reviewed. PVT was categorized according to lumen occupancy (≤ 50%, > 50% < 100% and = 100%) and extension to other veins. Evolution of thrombosis was considered at 1 year from diagnosis. Variables associated with presence of PVT and evolution patterns were analyzed, as well as their impact on survival. Seven-hundreds-fifty patients were included, 88 with PVT. On multivariate analysis, the occurrence of PVT at HCC diagnosis was associated with pre-treatment total tumor volume (p < 0.001) and clinically-significant portal hypertension (p = 0.005). During follow-up, 46 de novo PVT occurred, 27/46 (58.7%) in presence of viable tumor. Among 115 PVT diagnosed in presence of HCC, 83 had available radiological follow-up, and 22 were anticoagulated. The 'complete/progressive' evolution pattern was associated with non-response to HCC treatment in non-anticoagulated patients. The presence of PVT was independently associated with lower overall survival, particularly when progressive or occlusive (p < 0.001). A higher competing risk of death emerged for 'complete and progressive' PVT, both for HCC-related (p < 0.001) and non-HCC-related (p = 0.002) death. CONCLUSIONS: HCC represents an independent risk factor for the occurrence and progression of PVT in cirrhosis. Since progressive and occlusive PVT seems to be an independent factor associated with mortality, screening and prompt treatment of this complication should be considered.

BACKGROUND: Early identification of those with NAFLD activity score ≥ 4 and significant fibrosis (≥F2) or "at-risk MASH" is a priority as these patients are at increased risk for disease progression and may benefit from therapies. We developed and validated a highly specific metabolomics-driven score to identify at-risk MASH. METHODS: We included derivation (n = 790) and validation (n = 565) cohorts from international tertiary centers. Patients underwent laboratory assessment and liver biopsy for MASLD. Based on 12 lipids, body mass index, aspartate aminotransferase, and alanine aminotransferase, the MASEF score was developed to identify at-risk MASH and compared to the FibroScan-AST (FAST) score. We further compared the performance of a FIB-4 + MASEF algorithm to that of FIB-4 + liver stiffness measurements (LSM) by transient elastography (VCTE). RESULTS: The diagnostic performance of the MASEF score showed an area under the receiver-operating characteristic curve, sensitivity, specificity, positive and negative predictive values of 0.76 (95% CI 0.72-0.79), 0.69, 0.74, 0.53, and 0.85 in the derivation cohort, and 0.79 (95% CI 0.75-0.83), 0.78, 0.65, 0.48, and 0.88 in the validation cohort, while FAST performance in the validation cohort was 0.74 (95% CI 0.68-0.79; p = 0.064), 0.58, 0.79, 0.67, and 0.73, respectively. FIB-4 + MASEF showed similar overall performance compared to FIB-4 + LSM by VCTE (p = 0.69) to identify at-risk MASH. CONCLUSION: MASEF is a promising diagnostic tool for the assessment of at-risk MASH. It could be used alternatively to LSM by VCTE in the algorithm that is currently recommended by several guidance publications.

BACKGROUND AIMS: Hepatoblastoma (HB) is the most common liver cancer in children, posing a serious threat to children's health. Chemoresistance is the leading cause of mortality in HB patients. A more explicit definition of the features of chemotherapy resistance in HB represents a fundamental urgent need. APPROACH RESULTS: We performed an integrative analysis including single-cell RNA sequencing, whole-exome sequencing, and bulk RNA sequencing in 180 HB samples, to reveal genomic features, transcriptomic profiles, and the immune microenvironment of HB. Multi-color immunohistochemistry staining and in vitro experiments were performed for validation. Here, we reported four HB transcriptional subtypes primarily defined by differential expression of transcription factors (TFs). Among them, the S2A subtype, characterized by strong expression of progenitor (MYCN, MIXL1) and mesenchymal TFs (TWIST1, TBX5), was defined as a new chemo-resistant subtype. The S2A subtype showed increased transforming growth factor beta (TGF-β) cancer-associated fibroblast and an immunosuppressive microenvironment induced by the upregulated TGF-β of HB. Interestingly, the S2A subtype enriched SBS24 signature and significantly higher serum aflatoxin B1-albumin (AFB1-ALB) level in comparison with other subtypes. Functional assays indicated that aflatoxin promotes hepatoblastoma to upregulate TGF-β. Furthermore, Clinical prognostic analysis showed that serum AFB1-ALB is a potential indicator of HB chemoresistance and prognosis. CONCLUSIONS: Our studies offer new insights into the relationship between aflatoxin and HB chemoresistance and provide important implications for its diagnosis and treatment.

BACKGROUNDS: Prolyl-4-hydroxylases (P4Hs) are key enzymes in collagen synthesis. The P4HA subunit (P4HA1, P4HA2, and P4HA3) contains a substrate binding and catalyzation domain. We postulated that P4HA2 would play a key role in the cholangiocyte pathology of cholestatic liver diseases. METHODS: We studied humans with primary biliary cholangitis (PBC) and Primary sclerosing cholangitis (PSC), P4HA2 -/- mice injured by DDC, and P4HA2 -/- /MDR2 -/- double knockout mice. A parallel study was performed in patients with PBC, PSC, and controls using immunohistochemistry and immunofluorescence. In the murine model, the level of ductular reaction and biliary fibrosis were monitored by histology, qPCR, immunohistochemistry, and Western blotting. Expression of Yes1 Associated Transcriptional Regulator (YAP) phosphorylation was measured in isolated mouse cholangiocytes. The mechanism of P4HA2 was explored in RBE and 293T cell lines by using qPCR, Western blot, immunofluorescence, and co-immunoprecipitation. RESULTS: The hepatic expression level of P4HA2 was highly elevated in patients with PBC or PSC. Ductular reactive cholangiocytes predominantly expressed P4HA2. Cholestatic patients with more severe liver injury correlated with levels of P4HA2 in the liver. In P4HA2 -/- mice, there was a significantly reduced level of ductular reaction and fibrosis compared with controls in the DDC-induced chronic cholestasis. Decreased liver fibrosis and ductular reaction were observed in P4HA2 -/- /MDR2 -/- mice compared with MDR2 -/- mice. Cholangiocytes isolated from P4HA2 -/- /MDR2 -/- mice displayed a higher level of YAP phosphorylation, resulting in cholangiocytes proliferation inhibition. In vitro studies showed that P4HA2 promotes RBE cell proliferation by inducing SAV1 degradation, eventually resulting in the activation of YAP. CONCLUSIONS: P4HA2 promotes hepatic ductular reaction and biliary fibrosis by regulating the SAV1-mediated Hippo signaling pathway. P4HA2 is a potential therapeutic target for PBC and PSC.

BACKGROUND AND AIMS: We evaluated the diagnostic accuracy of simple, noninvasive tests (NITs) in NAFLD patients with type 2 diabetes (T2D). METHODS AND RESULTS: This was an individual patient data meta-analysis of 1780 patients with biopsy-proven NAFLD and T2D. The index tests of interest were FIB-4, NAFLD Fibrosis Score (NFS), aspartate aminotransferase-to-platelet ratio index, liver stiffness measurement (LSM) by vibration-controlled transient elastography, and AGILE 3+. The target conditions were advanced fibrosis, NASH, and fibrotic NASH(NASH plus F2-F4 fibrosis). The diagnostic performance of noninvasive tests. individually or in sequential combination, was assessed by area under the receiver operating characteristic curve and by decision curve analysis. Comparison with 2278 NAFLD patients without T2D was also made. In NAFLD with T2D LSM and AGILE 3+ outperformed, both NFS and FIB-4 for advanced fibrosis (area under the receiver operating characteristic curve:LSM 0.82, AGILE 3+ 0.82, NFS 0.72, FIB-4 0.75, aspartate aminotransferase-to-platelet ratio index 0.68; p < 0.001 of LSM-based versus simple serum tests), with an uncertainty area of 12%-20%. The combination of serum-based with LSM-based tests for advanced fibrosis led to a reduction of 40%-60% in necessary LSM tests. Decision curve analysis showed that all scores had a modest net benefit for ruling out advanced fibrosis at the risk threshold of 5%-10% of missing advanced fibrosis. LSM and AGILE 3+ outperformed both NFS and FIB-4 for fibrotic NASH (area under the receiver operating characteristic curve:LSM 0.79, AGILE 3+ 0.77, NFS 0.71, FIB-4 0.71; p < 0.001 of LSM-based versus simple serum tests). All noninvasive scores were suboptimal for diagnosing NASH. CONCLUSIONS: LSM and AGILE 3+ individually or in low availability settings in sequential combination after FIB-4 or NFS have a similar good diagnostic accuracy for advanced fibrosis and an acceptable diagnostic accuracy for fibrotic NASH in NAFLD patients with T2D.

BACKGROUND AND AIMS: Gut microbiota are recognized to be important for anticancer therapy, yet the underlying mechanism is not clear. Here, through the analysis of clinical samples, we identify the mechanism by which the gut microbial metabolite butyrate inhibits HCC and then explore new strategies for HCC treatment. APPROACH AND RESULTS: In our study, we demonstrate that gut microbial metabolite butyrate improves anticancer therapy efficacy by regulating intracellular calcium homeostasis. Using liquid chromatography-mass spectrometry analysis, we found that butyrate metabolism is activated in HCC patients compared with healthy individuals. Butyrate levels are lower in the plasma of HCC patients by gas chromatography-mass spectrometry (GC-MS) analysis. Butyrate supplementation or depletion of short-chain Acyl-CoA dehydrogenase (SCAD) gene (ACADS), encoding a key enzyme for butyrate metabolism, significantly inhibits HCC proliferation and metastasis. The profiling analysis of genes upregulated by butyrate supplementation or ACADS knockdown reveals that calcium signaling pathway is activated, leading to dysregulation of intracellular calcium homeostasis and production of reactive oxygen species. Butyrate supplementation improves the therapy efficacy of a tyrosine kinase inhibitor sorafenib. On the basis of these findings, we developed butyrate and sorafenib coencapsulated mPEG-PLGA-PLL nanoparticles coated with anti-GPC3 antibody (BS@PEAL-GPC3) to prolong the retention time of drugs and enhance drug targeting, leading to high anticancer efficacy. BS@PEAL-GPC3 nanoparticles significantly reduce HCC progression. In addition, BS@PEAL-GPC3 nanoparticles display excellent HCC targeting with excellent safety. CONCLUSIONS: In conclusion, our findings provide new insight into the mechanism by which the gut microbial metabolites inhibit HCC progression, suggesting a translatable therapeutics approach to enhance the clinical targeted therapeutic efficacy.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Comment in Hepatology. 2023 Jul 1;78(1):E3.

Comment in Hepatology. 2023 Jul 1;78(1):E12-E13.

Comment in Hepatology. 2023 Jul 1;78(1):E6-E7.

BACKGROUND AND AIMS: Patients with severe alcohol-associated hepatitis have high morbidity and mortality. Novel therapeutic approaches are urgently needed. The aims of our study were to confirm the predictive value of cytolysin-positive Enterococcus faecalis ( E. faecalis ) for mortality in patients with alcohol-associated hepatitis and to assess the protective effect of specific chicken immunoglobulin Y (IgY) antibodies against cytolysin in vitro and in a microbiota-humanized mouse model of ethanol-induced liver disease. APPROACH AND RESULTS: We investigated a multicenter cohort of 26 subjects with alcohol-associated hepatitis and confirmed our previous findings that the presence of fecal cytolysin-positive E. faecalis predicted 180-day mortality in those patients. After combining this smaller cohort with our previously published multicenter cohort, the presence of fecal cytolysin has a better diagnostic area under the curve, better other accuracy measures, and a higher odds ratio to predict death in patients with alcohol-associated hepatitis than other commonly used liver disease models. In a precision medicine approach, we generated IgY antibodies against cytolysin from hyperimmunized chickens. Neutralizing IgY antibodies against cytolysin reduced cytolysin-induced cell death in primary mouse hepatocytes. The oral administration of IgY antibodies against cytolysin decreased ethanol-induced liver disease in gnotobiotic mice colonized with stool from cytolysin-positive patients with alcohol-associated hepatitis. CONCLUSIONS: E. faecalis cytolysin is an important mortality predictor in alcohol-associated hepatitis patients, and its targeted neutralization through specific antibodies improves ethanol-induced liver disease in microbiota-humanized mice.

Comment in Hepatology. 2023 Jul 1;78(1):E16-E17.

BACKGROUND AIMS: Prolonged systemic inflammation contributes to poor clinical outcomes in severe alcohol-associated hepatitis (AH) even after the cessation of alcohol use. However, mechanisms leading to this persistent inflammation remain to be understood. APPROACH RESULTS: We show that while chronic alcohol induces nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation in the liver, alcohol binge results not only in NLRP3 inflammasome activation but also in increased circulating extracellular apoptosis-associated speck-like protein containing a caspase recruitment domain (ex-ASC) specks and hepatic ASC aggregates both in patients with AH and in mouse models of AH. These ex-ASC specks persist in circulation even after the cessation of alcohol use. Administration of alcohol-induced-ex-ASC specks in vivo in alcohol-naive mice results in sustained inflammation in the liver and circulation and causes liver damage. Consistent with the key role of ex-ASC specks in mediating liver injury and inflammation, alcohol binge failed to induce liver damage or IL-1β release in ASC-deficient mice. Our data show that alcohol induces ex-ASC specks in liver macrophages and hepatocytes, and these ex-ASC specks can trigger IL-1β release in alcohol-naive monocytes, a process that can be prevented by the NLRP3 inhibitor, MCC950. In vivo administration of MCC950 reduced hepatic and ex-ASC specks, caspase-1 activation, IL-1β production, and steatohepatitis in a murine model of AH. CONCLUSIONS: Our study demonstrates the central role of NLRP3 and ASC in alcohol-induced liver inflammation and unravels the critical role of ex-ASC specks in the propagation of systemic and liver inflammation in AH. Our data also identify NLRP3 as a potential therapeutic target in AH.