Journal of Hepatology

Background & AimsClonal hematopoiesis of indeterminate potential (CHIP) has been linked to chronic liver disease progression, yet its role across the full spectrum of metabolic dysfunction-associated steatotic liver disease (MASLD), from its initial development to end-stage complications, remains unclear. We aimed to comprehensively investigate the association of CHIP and its major subtypes with both the incidence and progression of MASLD. MethodsWe conducted a prospective cohort study of 353,218 UK Biobank participants, stratified into a healthy cohort free of MASLD at baseline (Cohort 1; n=230,270) and a prevalent MASLD cohort (Cohort 2; n=122,948). CHIP was ascertained from whole-exome sequencing data. We used multivariable Cox regression, competing risk models, and mediation analyses to assess the associations of CHIP (overall, by driver gene, and by clone size) with incident MASLD, cirrhosis, hepatocellular carcinoma (HCC), and liver-related death. ResultsIn Cohort 1, CHIP was associated with an increased risk of incident MASLD (HR 1.25, 95% CI 1.08-1.44) and cirrhosis (HR 1.57, 95% CI 1.10-2.25). These associations were driven by non-DNMT3A mutations, particularly TET2, and showed a linear dose-response relationship with clone size. In Cohort 2, non-DNMT3A CHIP was associated with progression to cirrhosis (HR 1.82, 95% CI 1.28-2.58). The associations were more pronounced in males and in individuals without obesity or diabetes. C-reactive protein partially mediated the CHIP-MASLD association. ConclusionCHIP, driven predominantly by non-DNMT3A mutations (particularly TET2) is an independent risk factor for both the development and progression of MASLD. These findings position CHIP as a novel player in the pathophysiology of MASLD and suggest potential avenues for risk stratification and targeted anti-inflammatory intervention. Impact and ImplicationsThis large-scale, prospective study establishes clonal hematopoiesis of indeterminate potential (CHIP) as a novel and independent risk factor for the entire spectrum of metabolic dysfunction-associated steatotic liver disease (MASLD), from its initial development to its progression to cirrhosis and liver-related death. For hepatologists and hematologists, these findings identify a genetically defined, high-risk subpopulation, particularly individuals with non-DNMT3A mutations, who may benefit from enhanced liver surveillance. The identification of systemic inflammation as a partial mediator of the CHIP-MASLD association suggests that anti-inflammatory therapies currently under development for liver disease could represent a targeted treatment strategy for this growing patient population.

Microbial bile salt hydrolase (BSH) plays a central role in shaping bile acid composition and gut-liver metabolic signaling, yet its therapeutic potential in metabolic dysfunction-associated steatohepatitis (MASH) remains incompletely defined. Here, we evaluated the efficacy of the non-absorbable BSH inhibitor GR-7 in a diet induced mouse model of steatohepatitis using early and late intervention strategies with different dosing regimens. GR-7 reduced food intake and exerted stage- and dose-dependent therapeutic effects, with early intervention robustly suppressing hepatic fibrosis even at low dose, whereas late-stage administration of high-dose GR-7 markedly reduced hepatic steatosis and inflammation, as evidenced by decreased liver weight, hepatic triglyceride and cholesterol levels, and plasma ALT. Although late intervention did not result in statistically significant histological reversal of fibrosis, a trend toward improvement was observed, together with suppression of fibrogenic gene expression, suggesting that prolonged treatment may further enhance antifibrotic efficacy. Mechanistically, GR-7 effectively inhibited microbial BSH activity in vivo, leading to reduced cecal unconjugated primary and secondary bile acids--including deoxycholic acid and lithocholic acid, which was associated with improved gut barrier integrity and reduced hepatic inflammation. In parallel, BSH inhibition reprogrammed hepatic bile acid metabolism toward activation of the alternative CYP27A1-mediated synthesis pathway, accompanied by reduced food intake, thereby contributing to improved hepatic lipid accumulation. Furthermore, late-stage high-dose treatment selectively remodeled the hepatic immune landscape rather than fully restoring homeostasis, highlighting immune recalibration as a key component of therapeutic response. Together, these findings identify microbial BSH inhibition as a promising microbiome-targeted therapeutic strategy for MASH. HighlightsO_LIThe non-absorbable BSH inhibitor GR-7 improves steatosis, inflammation, and fibrosis in of Western diet-induced steatohepatitis model in mice in a dose-dependent manner. C_LIO_LIGR-7 reduces food intake and body weight gain. C_LIO_LIGR-7 reduces cytotoxic secondary bile acids, including DCA and LCA. C_LIO_LIGR-7 reprograms hepatic bile acid metabolism and immune responses. C_LI

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer therapy by restoring anti-tumor immunity. However, persistent antigen exposure drives T cell exhaustion, limiting the effectiveness of ICIs. Ignorant T cells are antigen-specific T cells that maintain a naive state by regaining stem-like properties, allowing them to remain fully responsive to subsequent immunization. Virus-related hepatocellular carcinoma (HCC) demonstrates superior responses to ICIs compared to non-viral HCC, prompting us to investigate whether immunologically ignorant T cells exist in HBV-associated HCC and represent a promising target for improving immunotherapy outcomes. MethodsSingle-cell RNA sequencing (scRNA-seq) was performed on tumor tissues from patients with HBV-associated HCC. For validation, immunostaining was conducted on the discovery cohort and an independent cohort of 16 non-B non-C HCC and 22 HBV HCC. The enrichment of TIGIT and NECTIN3 in the proposed ignorant T cell was further validated using the TCGA database. ResultsscRNA-seq identified distinct HBV-infected HCC populations and revealed NECTIN3 upregulation in HBV-enriched subsets. CellChat analysis uncovered a novel NECTIN3-TIGIT tumor-immune interaction in HBV-enriched subsets, which shifted toward TIGIT-NECTIN2 as viral transcription declines. Trajectory analysis revealed the emergence of ignorant CD8 T cells following T cell exhaustion. TIGIT-NECTIN2/3 interactions deliver a weak exhaustion signal. This allows T cells to survive and regain naive-like properties as ignorant cells. Integration of bulk RNA-seq data identified CD24, STMN1, and EZH2 as potential biomarkers of ignorant CD8 T cells. ConclusionsTIGIT-NECTIN2/3 interactions present a promising axis for preserving immunologically ignorant T cells and sustaining ICI responsiveness in HBV-associated HCC.

Cirrhosis, the end stage of chronic liver disease marked by fibrosis and impaired liver function, is associated with cirrhosis-associated immune dysfunction, a condition in which systemic inflammation coexists with impaired host defense and increased susceptibility to infections. However, intestinal intraepithelial lymphocytes (IELs), key mediators of epithelial immune defense, remain poorly characterized in this context. Using high-dimensional profiling of paired duodenal biopsies and peripheral blood across disease stages, we define IEL alterations in cirrhosis. Contrary to prior reports of immune exhaustion, lymphocyte effector function was preserved, while disease progression was marked by systemic inflammatory remodeling and increased tumor necrosis factor (TNF) production by circulating T cells. The IEL compartment was markedly altered, with loss of CD8{beta} IELs, expansion of natural killer (NK) IELs, and reduced CCR9CD8{beta} IELs, suggesting altered gut homing. These findings refine cirrhosis-associated immune dysfunction as inflammatory immune reprogramming coupled to impaired epithelial immune surveillance. HighlightsPeripheral lymphocytes from cirrhosis patients retain effector capacity with enhanced inflammatory activity Cirrhosis reshapes the duodenal intraepithelial lymphocyte landscape Reduced frequency of CCR9+CD8{beta} IELs indicates altered gut-homing in cirrhosis

Metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly becoming the leading cause of chronic liver disease and confers substantial cardiometabolic burden. Diet quality and gut microbiota composition have been implicated in MASLD development; however, the interplay among diet, gut microbiota, and hepatic health remains insufficiently characterized. Here, in 9,616 deeply phenotyped middle-aged participants (mean age 52 years) from the Human Phenotype Project, we investigated how five dietary quality indices capturing complementary dimensions of healthy eating, including plant-based (hPDI), Mediterranean-style (AMED), anti-inflammatory (rDII), anti-hyperinsulinemic (rEDIH), and overall quality (AHEI), relate to gut microbial composition and liver steatosis. Dietary pattern scores were derived from two-week continuous diet logs, gut microbiota was characterized by shotgun metagenomic sequencing, and hepatic health was assessed by both ultrasound-derived metrics and prevalent MASLD status. Adherence to each of the five healthy dietary patterns was inversely associated with MASLD prevalence and positively associated with liver speed of sound (SoS), an ultrasound-derived metric that correlates inversely with hepatic fat content. Across all five dietary patterns, greater adherence was consistently associated with 138 gut microbial species, including inverse associations with Flavonifractor plautii, Dysosmobacter welbionis, Ruthenibacterium lactatiformans, Bilophila wadsworthia, and Phocea massiliensis. These five species were also associated with lower liver SoS and higher odds of prevalent MASLD, emerging as potential mediators of the diet-liver relationship in cross-sectional mediation analyses after adjustment for body mass index (BMI). This study identifies candidate microbial targets for future interventional studies investigating dietary strategies for MASLD prevention.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most prevalent chronic liver disease and strongly linked to obesity and insulin resistance. We previously reported that the common nuclear envelope variant rs6461378 (g.842031C>T; SUN1 H118Y) associated with MASLD and related traits including insulin resistance. To gain insight into how wild-type (WT) and H118Y SUN1 might differentially impact insulin signaling, we performed affinity purification-mass spectrometry (AP-MS) in human liver-derived cells stably expressing WT or H118Y SUN1. Unbiased AP-MS revealed a novel SUN1-CUL3 interaction, with comparative analysis showing that WT SUN1 interacted robustly with CUL3, while CUL3 interaction was markedly diminished with H118Y SUN1. Cells in which SUN1 was silenced via siRNA, or in which H118Y SUN1 was ectopically expressed, showed increased CUL3 neddylation, which is required for cullin RING ligase (CRL)-mediated ubiquitination of insulin receptor substrate (IRS) proteins. Inhibition of neddylation restored IRS-1 levels and insulin signaling in H118Y SUN1-expressing cells. Together, our findings provide a potential mechanism of H118Y SUN1-driven insulin resistance and a viable therapeutic approach for its reversal.

Chronic hepatitis C (CHC) remains a leading cause of cirrhosis, hepatocellular carcinoma (HCC), and premature mortality despite effective antiviral therapy, underscoring the need for individualized risk stratification beyond fibrosis stage alone. Using harmonized data from the All of Us Research Program, we developed and internally validated an interpretable multimodal survival framework to predict incident cirrhosis, HCC, and all-cause mortality, explicitly accounting for competing death. Baseline predictors within a {+/-}180-day window around CHC diagnosis included demographics, comorbidities, medications, laboratory biomarkers, socioeconomic context, and selected germline variants. Penalized Cox, ensemble, gradient-boosted, and neural survival models were compared under a consistent training and held-out testing strategy. Best-performing models achieved test C-indices of 0.67 for cirrhosis (Coxnet-LASSO), 0.71 for HCC, and 0.75 for mortality (Random Survival Forest), with stable time-dependent AUROC up to 0.81. Substantial feature compression preserved discrimination: restricting to the top 50% or 25% of predictors resulted in minimal absolute change in test performance (3.5%). Reduced models were anchored in clinically interpretable domains, including age, liver injury markers, hepatic reserve, cardiometabolic burden, deprivation index, and chromosome 19/22 loci. Feature importance reinforces existing known clinical and biological risk factors for liver complications: liver injury markers were most influential for cirrhosis and HCC, whereas hepatic reserve and cardiometabolic burden were more predictive of mortality, with age serving as a central baseline determinant across outcomes. Together, these results support a scalable and parsimonious framework for individualized CHC risk stratification that integrates multimodal determinants.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease for which the mechanisms linking lipid dysregulation to fibrosis remain poorly defined. Hepatic phosphatidylcholine (PC) content is reduced in MASH, but how this alteration drives disease progression is unclear. Here, we identify a role for copper (Cu) homeostasis as a downstream effector of impaired PC biosynthesis. Using single-nucleus RNA sequencing in complementary genetic and dietary mouse models, we found that reduced hepatic PC is associated with marked depletion of hepatic Cu and a concomitant increase in circulating Cu, indicating disrupted Cu distribution. Mechanistically, PC depletion impaired plasma membrane localization of the high-affinity Cu transporter CTR1 (SLC31A1) in hepatocytes, limiting Cu uptake. In human hepatic stellate cells, Cu promoted fibrogenic activation, whereas suppression of Cu import or pharmacologic inhibition of MAPK signaling attenuated fibronectin deposition. In vivo, liver-directed Cu supplementation restored hepatic Cu levels and reduced steatosis but failed to improve fibrosis. In contrast, pharmacologic Cu chelation with bathocuproinedisulfonic acid (BCS) reduced fibrosis without affecting inflammation. Together, these findings identify Cu redistribution as a consequence of impaired PC biosynthesis and implicate Cu-dependent signaling in stellate cell activation, fibrogenesis and MASH pathogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/723926v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@144d748org.highwire.dtl.DTLVardef@91dd8corg.highwire.dtl.DTLVardef@683686org.highwire.dtl.DTLVardef@1d3a0da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Metabolic dysfunction-associated steatotic liver disease (MASLD) afflicts more than one-third of adults globally, contributing significantly to an increased cardiovascular disease risk. Further, patients with severe liver disease experience muscle weakness (sarcopenic obesity) and fatigue. Hypoxia-inducible factor 2 (HIF2) accumulates in the livers of MASLD patients and has been implicated in disease progression. Here we sought to understand the role of hepatic HIF2 in mediating hepatic and extra-hepatic features of MASLD. Using a well-validated obese mouse model of MASLD, we investigated the impact of hepatocyte-specific HIF2 deletion (hHIF2-/-) on hepatic, cardiac and skeletal muscle metabolism, and cardiac function. Over 28 weeks, mice were exposed to a high-fat, high-fructose, high-cholesterol (GAN) diet, which induced obesity alongside hepatic steatosis, fibrosis and inflammation. In contrast to observations in lean mouse models of liver disease, hHIF2-/- did not protect against MASLD, despite greater hepatic NADH-supported mitochondrial respiration and higher intracellular sphingomyelin levels. Instead, in the hearts of GAN-fed mice, hHIF2-/- caused diacylglycerol accumulation independent of diet, accumulation of long-chain acyl-carnitines and exacerbation of ceramide accumulation. Langendorff-perfused hearts from hHIF2-/- mice showed systolic and diastolic dysfunction, including 24% lower left ventricular developed pressure and 34% lower maximal rate of relaxation (dP/dtmin). However, isolated hearts from hHIF2-/- mice were protected against MASLD-associated sympathetic dominance, determined using autonomic receptor agonist stimulation. Both GAN-feeding and hHIF2-/- were associated with lower lean mass (14% and 5.4% lower than respective controls), whilst hHIF2-/- enhanced OXPHOS-associated protein levels in gastrocnemius muscle. Overall, hHIF2-/- resulted in detrimental extra-hepatic effects, including myocardial lipid accumulation, impaired cardiac function, and loss of whole-body lean mass, with no apparent protection against MASLD disease progression.

BackgroundSingle-cell RNA sequencing has provided new insights into hepatocellular carcinoma (HCC); however, a unified understanding of epithelial heterogeneity and immune evasion strategies in HCC remains lacking. MethodsWe re-analyzed publicly available single-cell datasets using conventional bioinformatics pipelines. Cell-type annotation of epithelial and T cell populations was further validated across multiple independent datasets to ensure robustness. ResultsWe systematically examined epithelial, immune, myeloid, and stromal lineages. In addition to recapitulating previously reported findings, we identified several novel observations. Notably, we uncovered a three-step dedifferentiation trajectory in epithelial cells and confirmed a bidirectional differentiation pattern within CD8{square} T cells. We also identified a subset of GZMK{square} CD4{square} T cells, whose transcriptional features resemble but are distinct from T follicular helper (Tfh) cells. Importantly, transcriptional drift within myeloid populations appeared to be closely associated with immune responsiveness. Furthermore, ligand-receptor analysis highlighted a potential cooperative role of LAMP3{square} dendritic cells and Tfh cells in promoting lymphoid follicle formation. ConclusionsIn the era of rapidly evolving single-cell sequencing technologies, we provide a framework for understanding cellular heterogeneity in HCC, which awaits further validation in future studies.

Background & AimsBulevirtide, a viral entry inhibitor used to treat chronic hepatitis delta virus (HDV) infection, targets the hepatic bile salt transporter Na+-Taurocholate Co-transporting Polypeptide (NTCP). As Bulevirtide displays preclinical potential to mitigate cholestatic liver injury, NTCP inhibition is currently explored as treatment for primary sclerosing cholangitis (PSC), a condition frequently associated with colitis. Here, we investigated the immunomodulatory effects of Bulevirtide in lipopolysaccharide (LPS)-induced inflammation and dextran sodium sulfate (DSS)-induced colitis in mice. MethodsThe immunomodulatory properties of the bile salt taurochenodeoxycholic acid (TCDC) were investigated in LPS-challenged mouse bone marrow-derived macrophages (BMDM) and human BLaER1 macrophages. The therapeutic efficacy of Bulevirtide against LPS-induced inflammation and DSS-induced colitis was evaluated in Slco1a/1b-/- FVB and C57BL/6J mice, which recapitulate human bile salt dynamics. ResultsIn BMDMs, TCDC reduced pro-inflammatory tumor necrosis factor alpha (TNF), increased anti-inflammatory interleukin (IL)-10, and suppressed inflammasome activation, as evidenced by reduced IL-1{beta}, IL-18 and cleaved-IL-1{beta} levels. Consistently, TCDC also reduced TNF and IL1B expression in human BLaER1 macrophages. In both FVB and C57BL/6J Slco1a/1b-/- mice, Bulevirtide increased plasma bile salt levels at least 30-fold. This systemic elevation of bile salts reduced plasma TNF and increased IL-10 in LPS-treated mice. Moreover, Bulevirtide attenuated DSS-induced colitis, evidenced by reduced disease scores and reduced intestinal Tnf expression. ConclusionThese findings highlight the anti-inflammatory effects of bile salts in preclinical models of colitis and support NTCP inhibition as a future therapeutic strategy to ameliorate both cholestasis and colitis in PSC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/719641v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@38efbeorg.highwire.dtl.DTLVardef@3e09borg.highwire.dtl.DTLVardef@8f1262org.highwire.dtl.DTLVardef@100179c_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisInhibition of the Na+-Taurocholate Co-transporting Polypeptide using Bulevirtide induces systemic bile salt elevation and mitigates acute inflammation and colitis in mice. These findings support clinical evaluation of Bulevirtide in primary sclerosing cholangitis with protective effects against cholestasis and colitis.

Abstract textO_ST_ABSBackgroundC_ST_ABSAs hepatitis A (HA) incidence declines in Europe and infections occur later in life, clinical presentations may worsen, particularly during outbreaks involving adults. AimWe analysed temporal trends and factors associated with severe disease and mortality among patients hospitalised for HA in France between 2013 and 2024. MethodsICD-10 codes B150 or B159 as primary discharge diagnosis were used to identify HA cases from the National Discharge Data Set. Severity (hepatic and/or extrahepatic organ failure within 12 weeks post-admission) and mortality were analysed using adjusted odds ratios in original and propensity-matched samples. Trends were assessed across five periods covering the 2017 epidemic and COVID-19, with 2013-2016 as reference. ResultsAmong 7,928 cases (60.6% male; median age 30) 29.1% developed severe HA, and 1.43% died. Risk of severe HA increased with age (+17% of risk per decade, p < 0.001), male sex (+39%, p < 0.001), smoking (+25%, p=0.024), liver risk factors (+32%, p=0.026), and cirrhosis (+48%, p = 0.024). Risk of death increased with cirrhosis (3.55-fold, p < 0.001) and high Charlson Comorbidity Index (CCI) (9.95-fold, p < 0.001), but not with advanced age. Compared with 2013-2016, severe HA increased by 60% (p<0.001) and case fatality increased 2.22-fold (p=0.003) in 2021-2024. ConclusionsHA severity and mortality have increased in France over the last decade, with advanced age and male sex increasing severity but not mortality, and high CCI limiting access to organ support, thereby increasing mortality in frail patients. Our findings highlight the need for targeted prevention and optimized care strategies for high-risk groups.

MAIT are a highly versatile population of innate-like T cells that have been implicated in promoting tissue repair-associated process in a variety of tissue and diseases settings in the last years. While certain specific effector molecules responsible for MAIT-cell mediated have been identified, the mechanisms by which MAIT cells exert repair functions remain incompletely understood. Here, we show that hepatic MAIT cells express VEGFA, VEGFB and vimentin, an alternative ligand for the VEGFA-receptor VEGFR2 in both, regenerating and heathy tissue. Expression and secretion of these factors were induced in vitro by combined T cell receptor and cytokine stimulation. Supernatants of activated MAIT cells were able to promote proliferation of different epithelial and endothelial cells, including a liver sinusoidal endothelial-derived cell line in an VEGFR2-dependent manner. Together, our findings expand our understanding of MAIT cell function, especially in the liver and open new opens avenues for exploring MAIT therapeutic potential in modulating tissue repair.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by excessive hepatic lipid accumulation driven by increased de novo lipogenesis (DNL) and impaired lipid oxidation. p107, a member of the retinoblastoma (Rb) family, extensively studied in the context of cell cycle regulation and adipocyte differentiation recently has been identified as a metabolic regulator controlling thermogenic activity. However, its role in hepatic lipid homeostasis remains poorly understood. Here, we identify the cell cycle regulator p107 as a key modulator of hepatic lipid metabolism. p107 expression is increased in patients with MASLD and correlates with disease severity. In mouse models, global and liver-specific p107 deficiency protect against high-fat diet-induced steatosis without affecting body weight. This is associated with reduced expression of lipogenic enzymes including fatty acid synthase (FASN), and enhanced mitochondrial oxidative pathways. Conversely, hepatic restoration of p107 reversed these effects and promoted lipid accumulation and endoplasmic reticulum stress. Consistent with this in human hepatocytes, p107 silencing reduces lipid accumulation, decreases DNL and enhances mitochondrial respiration, whereas p107 overexpression induces the opposite phenotype. Notably, FASN knockdown attenuates the pro-steatotic effects of p107, indicating that it is a critical downstream mediator of p107. Together, these findings establish p107 as a physiological regulator of hepatic lipid metabolism, with its dysregulation contributing to the development of MASLD.

Vascular plasticity is a crucial biological asset enabling our bodies to rapidly adapt to infections and acute inflammation. However, repeated insult during chronic disease can result in these vascular adaptations becoming irreversible, thereby driving disease progression and fibrosis. This study aimed to understand if phenotypic changes in endothelial cell (EC) identity could be indicative of progressive fibrosis and thereby offer new diagnostic and therapeutic opportunities for patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Previous research has documented that a significant shift in EC transcriptomic signature occurs during liver fibrosis in both pre-clinical models and patients. However, the protein expression profile, phenotype and functional role of these new EC subpopulations that are induced during fibrogenesis is unclear. In this study, we integrate high-resolution imaging, proteomic and transcriptomic analysis which collectively highlight a central role for endothelial-to-mesenchymal transition (EndMT)-induced EC plasticity in the derivation of fibrosis-associated EC (FAEC). We demonstrate that: 1) full spectrum flow cytometry can provide new opportunities to categorize and phenotype EC subpopulations, 2) two distinct EndMT-derived FAEC subpopulations expand during fibrogenesis; THY1.2+ICAM1+ and TAGLN+MCAM+ EC that display unique immunomodulatory and metabolic phenotypes, 3) TAGLN+ FAEC are a conserved, pro-fibrotic cell type arising at early stages of MASLD, and 4) increased hepatic expression of TAGLN is significantly associated with detrimental patient outcomes at all stages of liver disease. This study will pave the way for the development of FAEC-specific diagnostic and therapeutic approaches to tackle progressive fibrotic disease.

Chronic co-infections by HBV and its satellite virus HDV are associated with a high risk of progression to cirrhosis and liver cancer, and therapeutic options for achieving a cure are still unsatisfactory. HBs is the main surface glycoprotein of both viruses, and is also massively secreted by infected hepatocytes in the form of empty subviral particles which suppress the host immune responses. This makes HBs an attractive target to develop therapeutic strategies. Here, we took advantage of the known interaction between the Large form HDV antigen (HDAg-L) and the small form of HBs (S-HBs) to develop a non-infectious, minimalistic reporter assay for the assembly and secretion of HDV particles. By screening the existing pharmacopeia for drugs that could interfere with S-HBs and HDAg-L co-secretion, we found that ritonavir and other Cytochrome P450 inhibitors affect the biogenesis of HBs and impair the production of infectious HDV virions. Mechanistically, we established that these drugs induce oxidative stress which dysregulates disulfide bond formation in the endoplasmic reticulum. As a consequence, the production of HBs, which depends on a dense network of disulfide bonds, is markedly affected as evidenced by an abnormal glycosylation profile, altered antigenic properties, and a poor expression of the largest form of HBs (L-HBs) which is essential to virus entry into target cells. This is associated with induction of the unfolded protein response, with the upregulation of CHOP/DDIT3 and key enzymes involved in the synthesis of the reducing metabolite glutathione (PHGDH, SHMT2, MTHFD2). Overall, our results indicate that alterations in redox homeostasis significantly impact HBs biogenesis, and reveal a druggable pathway that could be exploited to eliminate HDV in chronically infected patients. IMPACT AND IMPLICATIONSMore effective therapies are still needed to achieve a functional cure in patients chronically co-infected by HBV and HDV. In this study, we discovered that ritonavir, along with other cytochrome P450 inhibitors, can affect the production of infectious HDV particles in human hepatocyte cultures. Mechanistically, ritonavir induces oxidative stress and the unfolded protein response in the endoplasmic reticulum, thereby altering the biogenesis of HBs, the surface glycoprotein of both viruses. This work highlights the potential benefit and mechanism of action of ritonavir and related molecules in the treatment of co-infected patients.

Chronic hepatitis B persists due to the stability of nuclear covalently closed circular DNA (cccDNA), which maintains viral transcription despite prolonged antiviral therapy, highlighting the need for strategies that suppress cccDNA via host-targeted mechanisms. Here, we identify Spiperone, a clinically approved compound, as a repurposed anti-HBV candidate with strong translational potential. Spiperone robustly reduced HBsAg, HBeAg, viral DNA, and pgRNA across HepG2.2.15, HBV-infected HepG2-NTCP-C4 and HepaRG cells, and multiple in vivo models, including HBV transgenic, hydrodynamic injection, and AAV- HBV1.04x models. Notably, intrahepatic cccDNA was significantly diminished. In combination, Spiperone potentiated tenofovir activity, exhibiting synergistic effects, while both intraperitoneal and oral administration reduced antigenemia and viremia. Mechanistically, Spiperone activated the PERK-eIF2-ATF4 arm of the ER stress response, coupled with mitochondrial perturbation and cytosolic release of oxidized mitochondrial DNA, leading to activation of IFI16-STING-IRF3 signaling. This cascade induced type I interferon (IFN-I) and interferon-stimulated genes. ChIP-qPCR further demonstrated reduced enrichment of activating histone marks on cccDNA, consistent with transcriptional repression. Collectively, these findings position Spiperone as a host-directed antiviral that converges ER stress-linked innate immunity and epigenetic repression to suppress cccDNA, supporting its advancement in combination strategies toward a functional cure for chronic HBV infection.

Semaglutide has shown benefit in metabolic dysfunction-associated steatohepatitis (MASH), but real-world evidence across longitudinal liver phenotypes remains limited, particularly regarding how liver remodeling relates to weight loss and dose exposure. Using a de-identified federated electronic health record network spanning more than 29 million patients in the United States, including 489,785 semaglutide-treated adults, we analyzed 6,734 patients with baseline liver disease burden. We find that higher attained pre-landmark (0-2 years) semaglutide dose was associated with lower post-landmark (2-4 years) risk of steatohepatitis, alcoholic liver disease, and all-cause mortality, whereas greater pre-landmark weight loss was associated with lower post-landmark risk of steatohepatitis, steatotic liver disease, and hepatorenal syndrome, indicating distinct dose- and weight-linked patterns of long-term liver benefits. These associations were notable because semaglutide prescribing was generally lower during the post-landmark period, raising the possibility of durable benefit beyond peak exposure. Towards better understanding mechanistic bases for liver protection, we performed a complementary longitudinal study of 326 adults with paired noninvasive liver elastography measurements before and after treatment initiation. Median liver stiffness decreased from 4.85 [3.02 - 7.20] to 3.9 [2.6 - 5.8] kPa after semaglutide initiation (median change = -0.38 kPa; p<0.001), with 194 of 326 patients (59.5%) showing lower follow-up stiffness. A clinically meaningful reduction of at least 20% was observed in 133 of 326 patients (40.8%), and 69 of 326 (21.2%) shifted to a lower fibrosis stage by prespecified elastography thresholds. Larger improvements were also seen in patients with higher baseline stiffness (p<0.001); notably 80% of patients with cirrhosis-range baseline stiffness ([&ge;]12.5 kPa) achieved [&ge;]20% improvement versus 29.5% with minimal baseline disease (p <0.001). The proportion achieving at least 20% stiffness improvement was similar across weight-loss strata, including patients with no weight loss or weight gain and those with at least 10% weight loss (38.0% in each group), and liver stiffness change showed negligible correlation with changes in weight, BMI, HBA1c, alanine aminotransferase, or aspartate aminotransferase. To provide biological context, single cell RNA analyses demonstrated sparse overall hepatic GLP1R expression (0.0239%), with enrichment in non-parenchymal niches including cholangiocytes, intrahepatic cholangiocytes, liver sinusoidal endothelial cells, and hepatic stellate cells implicated in fibrogenesis and vascular remodeling. Together, this real-world evidence suggests diverse liver benefits for semaglutide beyond weight-loss with intricate dose response relationships.

Hepatocytes undergo extensive proliferation to facilitate liver repair after injury, yet early adaptive changes prior to proliferation remain unclear. Here, we report that during early acetaminophen (APAP)-induced liver injury, hepatocytes exhibit transient proliferation suppression, most pronounced in mid-zone hepatocytes due to zonal APAP metabolism. Using spatial transcriptomics (ST), immunohistochemistry, and functional studies, we identified a unique mid-zone stress-response program. Central to this adaptation is the Atf4-Chop axis, which actively suppresses proliferation via the cell cycle inhibitor Btg2, prioritizing cytoprotection over cell division. This transient arrest is a critical survival strategy: halting energy-intensive proliferation during peak injury allows mid-zone hepatocytes to redirect resources towards protection, enhancing their survival in early APAP-induced liver injury. Thus, Atf4-Chop-mediated quiescence preserves a hepatocyte reservoir necessary for subsequent regenerative proliferation and effective repair. Our findings reveal a key adaptive trade-off in mid-zone hepatocytes where transient proliferation arrest promotes early survival to enable repair.

Background and AimFecal microbiota transplantation (FMT) in Alcohol-related liver disease (ALD) has shown therapeutic potential, with variable efficacy and unclear mechanism. Because dietary protein influences gut microbiota composition, we hypothesized that donor dietary preconditioning could enhance FMT efficacy. We therefore examined in a murine ALD model if high-protein donor diet improves FMT outcome. MethodsALD was induced in C57BL/6N mice using a Lieber-DeCarli ethanol diet combined with thioacetamide administration for 12 weeks. FMT was performed using stool from diet-modulated donors, and recovery was assessed on day7 post-FMT. Multi-omics analysis using 16s rRNA and mass spectroscopy was performed for Gut microbiota composition, plasma- and stool-metabolome, and hepatic proteomes. Multi-omics outcomes were validated in ALD animal and Huh7 hepatocytes. ResultsBoth protein-based FMTs improved ALD recovery; Veg-FMT demonstrated superior efficacy, significantly reducing hepatic injury (AST 1.2-fold, p=0.002; bilirubin 1.2-fold, p=0.03; steatosis 1.7-fold,p=0.01) and restoring gut barrier integrity (occludin 1.5-fold,p=0.04; mucin 2 2.2-fold, p=002; and plasma endotoxin 1.7-fold, p=0.02). A significant 2-fold increase was observed in Lachnospiraceae NK4A136, Coriobacteriaceae UCG-002, and short-chain fatty acids, particularly caproic acid. Functional validation confirmed that caproic acid promoted hepatic fatty acid {beta}-oxidation through PPAR-dependent mechanisms, reducing triglyceride accumulation and lipogenesis in both cellular and animal models. ConclusionDonor preconditioning with a plant-protein enriched diet enhances FMT efficacy in ALD by gut microbiota modulation with increased metabolites like caproic acid. These findings highlight a microbiota-metabolite-host axis through which diet-modulated FMT improves hepatic lipid metabolism and injury, and identifies a pathway via which FMT imparts its effect. SignificanceThis study identifies a mechanistic basis for improving fecal microbiota transplantation (FMT) efficacy in alcohol-related liver disease (ALD) by demonstrating that dietary preconditioning of donor microbiota improves therapeutic outcomes. We show that plant protein-modulated donor microbiota supplements abstinence-associated recovery through increased production of the microbial metabolite caproic acid, which promotes hepatic fatty acid {beta}-oxidation via PPAR signaling. These findings highlight donor dietary conditioning and microbiota-derived metabolites, rather than microbial composition alone, as important determinants of FMT efficacy. The results suggest that microbial metabolites such as caproic acid may represent potential therapeutic targets or biomarkers to enhance and standardize microbiota-based interventions in ALD. Although the current work is based on a murine model, the identified microbiota-metabolite-host metabolic axis provides a framework for future translational studies aimed at optimizing FMT strategies in liver disease.