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.

Liver transcriptomic data from patients with metabolic dysfunction-associated steatotic liver disease (MASLD) offers valuable resource for deciphering pathogenic molecular drivers. Here, we performed a Mega-analysis of MASLD Liver Transcriptomes (MegaMASLD) which reanalysed raw RNAseq data of over 800 livers in a standardized and integrative manner, aiming to unravel druggable molecular events in MASLD. Our analysis revealed a progressive transcriptomic shift predominantly associated with immunopathologies during MASLD progression. The differential transcriptomes produced a MASLD gene signature useful for quantitative assessment of MASLD severity but failed to faithfully recapitulate the exact histological staging. Instead, a histologic-independent unsupervised clustering analysis predicted a high-risk group prone to develop metabolic dysfunction-associated steatohepatitis (MASH), characterized by aberrant changes in humoral immune response and antibody repertoires. These findings were supported by another histologic-independent pseudotime analysis, which also identified several potentially targetable molecular switches, including FGFR, PDGFR, PAK, PRKG1 and CAMK kinase families, activated at various transitory phases of MASLD. The robust analysis has enabled risk stratification and deepened our understanding of the dynamic molecular events driving MASLD, thereby offering new options to enhance precision medicine of MASLD. An online web tool featuring MegaMASLD is available at https://bioanalytics-hs.shinyapps.io/MegaMASLD/.

Liver fibrosis is a strong predictor of clinical outcomes in metabolic dysfunction-associated steatohepatitis (MASH). Fibrosis is a consequence of persistent liver cell injury and inflammation in which Toll-like receptors (TLRs) play a key initiating role. Here we test the hypothesis that TLR5 is involved in the development of MASH and fibrosis using a combination of clinical data from multiple independent patient cohorts, single cell liver transcriptomics and human in vitro and ex vivo models. Hepatic TLR5 expression, but not TLR2 or TLR4, is associated with liver fibrosis and mortality. Plasma levels of TLR5s cognate ligand flagellin are increased in MASH with advanced fibrosis and fall with liver disease improvement. Mechanistically, we identify two parallel TLR5-mediated routes to hepatocyte injury: one elicited by flagellin and the other indirectly by lipid injury. Furthermore, hepatocyte TLR5 inhibition abrogates paracrine activation of hepatic stellate cells to suppress collagen production. This is also seen ex vivo in patient-derived precision-cut liver slices where TLR5 inhibition significantly reduces lipid-induced collagen deposition. These findings reveal a new role for TLR5 signalling, specifically in the development of advanced MASH fibrosis and may offer a novel disease-specific therapeutic approach.

BackgroundLymphangiogenesis plays a critical role in various liver diseases, yet its function in liver fibrosis remains controversial. This study aimed to explore the role of lymphangiogenesis in liver fibrogenesis and its underlying regulatory mechanisms. MethodsLiver fibrotic mice were established by carbon tetrachloride (CCl4) or Thioacetamide (TAA)-induced injection or bile duct ligation. Lymphatic vessels were marked by podoplain (Pdpn) staining in mice and D2-40 staining in clinical samples. Lymphatic vessels area and density were measured to indicate lymphangiogenesis. Multiplexing immunohistochemistry was used to detect co-localization of proteins. ResultsIn the present study, we first verified increased lymphangiogenesis in human and murine fibrotic livers. Afterwards, we identified VEGFC rather than VEGFD as the primary driver of lymphangiogenesis in liver fibrosis. Furthermore, we demonstrated that M1 macrophages serve as the major source of VEGFC. Founctional studies revealed that VEGFC-mediated lymphangiogenesis exacerbates hepatic fibrosis, while its inhibition alleviated fibrosis. Bioinformatic analysis uncovered Midkine (MDK) as a key downstream of lymphangiogenesis. Both in vivo and in vitro studies confirmed that exogenous MDK promotes liver fibrosis via activating hepatic stellate cells (HSCs), whereas MDK inhibition counteracts the profibrotic effects of VEGFC-induced lymphangiogenesis. Importantly, we discovered that MDK activates HSCs through the Hippo/YAP signaling pathway. ConclusionsM1 macrophage-mediated lymphangiogenesis aggravates liver fibrosis via MDK secretion, which activates HSCs. These findings provide novel insights into coordinated crosstalk between macrophages, lymphatic endothelial cells and HSCs in liver fibrosis and suggest lymphangiogenesis and MDK as potential therapeutic targets for fibrotic liver diseases.

Background and AimsThere is an urgent need for new high-quality markers in the early detection of hepatocellular carcinoma (HCC). [A]strom et al. suggested that S2-bound 1-acid glycoprotein (AGP) might be a promising marker. Consequently, we evaluated S2-bound AGP for a predictive advantage in the early detection of HCC. MethodsIn a retrospective case-control study of patients chronically infected with hepatitis C virus (HCV) and treated with direct-acting antiviral agents (n=93), we measured S2-bound AGP using the HepaCheC(R) ELISA kit (Glycobond AB, Linkoping, SE) at treatment start, end of treatment and follow-up (maximum: 78 months). Patients were retrospectively propensity score matched (1:2). 31 patients chronically infected with HCV developed HCC after sustained virological response while 62 did not. In addition, samples of HBV, MASLD and HCC from different etiologies patients were measured. ResultsS2-bound AGP elevation in HCC patients was confirmed. However, we did not observe a predictive advantage of S2-bound AGP in early detection of HCC during treatment and follow-up. Interestingly, S2-bound AGP levels correlated with aspartate aminotransferase ({rho}=0.56, p=9.5x10-15) and liver elastography ({rho}=0.67, p=2.2x10-16). Of note, S2-bound AGP decreased in patients chronically infected with HCV after treatment-induced clearance of HCV. ConclusionFibrosis and liver inflammation are key regulators in the fucosylation of AGP. The potential role of S2-bound AGP as a novel tumor marker requires further investigation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/23298443v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@5440c2org.highwire.dtl.DTLVardef@167a9c1org.highwire.dtl.DTLVardef@b9e494org.highwire.dtl.DTLVardef@4db1f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and AimsChronic hepatitis C virus (HCV) infection can lead to cirrhosis, development of hepatocellular carcinoma (HCC) and several extrahepatic manifestations. A sustained virological response (SVR) is achieved with direct-acting antivirals (DAA) in over 95% of the patients, but sequelae do not improve in all patients, suggesting permanent biological alterations induced by HCV infection. Therefore, we investigated the influence of chronic HCV infection, viral elimination and cirrhosis on inflammatory immune mediators. Approach and ResultsIn 102 chronic HCV patients, 46 with and 56 without cirrhosis, 92 soluble immune mediators (SIM) were measured in plasma samples at therapy start, end of treatment and long-term follow-up (median 96 weeks). 39 HBsAg positive persons with HBeAg negative infection served as controls. At baseline, 42 SIM were altered in chronic HCV patients (adj.p <0.05). Notably, patients with cirrhosis displayed a higher frequency and severity of alterations. At long-term follow-up, the SIM profile of the non-cirrhotic patients recovered to the level of the control group, while 41 SIM remained altered in cirrhotic patients. 33 of these SIM correlated with elastography, among them SIM linked to carcinogenesis as e.g. HGF, IL8 and IL6 (KEGG Pathways hsa05202, hsa05200). ConclusionsHCV-related changes in the inflammatory milieu can persist even after HCV elimination, specifically in cirrhotic patients. These changes are closely associated with liver damage and carcinogenesis. Our findings underscore the need for HCV elimination before extensive liver injury occurs and suggest further investigation of the relationship between persistent inflammatory milieu changes and long-term sequelae after HCV elimination. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/23297828v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@3a885borg.highwire.dtl.DTLVardef@ce3eb1org.highwire.dtl.DTLVardef@7601f6org.highwire.dtl.DTLVardef@17ad655_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background & Aims: Immune-mediated liver injury from immune checkpoint inhibitors (ILICI) is a major immune-related adverse event that limits cancer immunotherapy, yet its tissue-level immunobiology is poorly defined and its management is largely extrapolated from autoimmune hepatitis (AIH). We previously identified a tri-cellular CD8+ T cell-macrophage-hepatocyte injury niche in a murine model of ILICI; here, we tested whether this niche is recapitulated in human disease. Methods: We applied imaging mass cytometry with a 32-marker panel to liver biopsies from patients with ILICI (n = 12), AIH as a disease comparator (n = 14), and healthy controls (n = 2), profiling approximately 297,000 single cells across 144 regions of interest with spatially resolved detection of apoptosis (cleaved caspase-3, cC3) and pyroptosis (cleaved gasdermin D, cGSDMD). Results: We detected histiocyte-rich granulomas in ILICI consisting of macrophages and CD8+ T cells, including activated memory-effector subsets. Permutation-based spatial analysis identified CD8+ T cell-macrophage co-localization as the most frequent significant interaction in ILICI, organizing into integrated innate-adaptive cellular neighborhoods that concentrated cC3- and cGSDMD-positive cells. Descriptively, this contrasted with AIH, in which immune cells and stroma were more spatially compartmentalized. CD8+ T-cell and macrophage densities correlated with Ishak necroinflammation scores, jaundice, and granuloma formation. Conclusions: These findings provide the first single-cell spatial proteomic characterization of human ILICI in situ; they recapitulate the tri-cellular CD8-macrophage-hepatocyte niche we previously defined in a murine model and characterize ILICI as a spatially organized innate-adaptive inflammatory process, nominating myeloid signaling and CD8-macrophage interactions as candidate liver-directed targets to uncouple hepatotoxicity from anti-tumor immunity.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common liver disease, affecting up to 25% of people worldwide, featuring excessive fat accumulation in hepatocytes. Its advanced form, metabolic dysfunction-associated steatohepatitis (MASH), is a serious disease with hepatic inflammation and fibrosis, increasing the need for liver transplants. However, the pathogenic mechanism of MASLD and MASH is not fully understood. We reported that BRUCE (BIRC6) is a liver cancer suppressor and is downregulated in MASLD/MASH patient liver specimens, though the functional role of BRUCE in MASLD/MASH remains to be elucidated. To this end, we generated liver-specific double KO (DKO) mice of BRUCE and PTEN, a major tumor suppressor and MASLD/MASH suppressor. By comparing liver histopathology among 2-3-month-old mice, there were no signs of MASLD or MASH in BRUCE liver-KO mice and only onset of steatosis in PTEN liver-KO mice. Interestingly, DKO mice had developed robust hepatic steatosis with inflammation and fibrosis. Further analysis of mitochondrial function with primary hepatocytes found moderate reduction of mitochondrial respiration, ATP production and fatty acid oxidation in BRUCE KO and the greatest reduction in DKO hepatocytes. Moreover, aberrant activation of pro-fibrotic STAT3 signaling was found in hepatic stellate cells (HSCs) in DKO mice which was prevented by administered STAT3-specific inhibitor (TTI-101). Collectively, the data demonstrates by maintaining mitochondrial metabolism BRUCE works in concert with PTEN to suppress the pro-fibrogenic STAT3 activation in HSCs and consequentially prevent MASLD/MASH. The findings highlight BRUCE being a new co-suppressor of MASLD/MASH.

Background and AimsRecent studies have highlighted the beneficial effect of resolvin D1 (RvD1), a DHA-derived specialized pro-resolving mediator, on metabolic dysfunction-associated steatohepatitis (MASH), but the underlying mechanisms are not well understood. Our study aims to determine the mechanism by which RvD1 protects against MASH progression. MethodsRvD1 was administered to mice with experimental MASH, followed by bulk and single-cell RNA sequencing analysis. Primary cells including bone marrow-derived macrophages (BMDMs), Kupffer cells, T cells, and primary hepatocytes were isolated to elucidate the effect of RvD1 on inflammation, cell death, and fibrosis regression genes. ResultsHepatic tissue levels of RvD1 were decreased in murine and human MASH, likely due to an expansion of pro-inflammatory M1-like macrophages with diminished ability to produce RvD1. Administering RvD1 reduced inflammation, cell death, and liver fibrosis. Mechanistically, RvD1 reduced inflammation by suppressing the Stat1-Cxcl10 signaling pathway in macrophages and prevented hepatocyte death by alleviating ER stress-mediated apoptosis. Moreover, RvD1 induced Mmp2 and decreased Acta2 expression in hepatic stellate cells (HSCs), and promoted Mmp9 and Mmp12 expression in macrophages, leading to fibrosis regression in MASH. ConclusionsRvD1 reduces Stat1-mediated inflammation, mitigates ER stress-induced apoptosis, and promotes MMP-mediated fibrosis regression in MASH. This study highlights the therapeutic potential of RvD1 to treat MASH. Impact and implicationsMetabolic dysfunction-associated steatohepatitis (MASH) is an increasing healthcare burden worldwide. Current treatments for MASH and its sequelae are very limited. Recent studies highlighted the therapeutic benefit of specialized pro-resolving mediators (SPMs), including resolvin D1 (RvD1), in liver diseases. However, the mechanisms underlying these beneficial effects are not well understood. Based on unbiased transcriptomic analyses using bulk and single-cell RNA sequencing in RvD1-treated MASH livers, we show that RvD1 suppresses Stat1-mediated inflammatory responses and ER stress-induced apoptosis, and induces gene expression related to fibrosis regression. Our study provides new mechanistic insight into the role of RvD1 in MASH and highlights its therapeutic potential to treat MASH. HighlightsO_LILiver RvD1 levels are decreased in MASH patients and MASH mice C_LIO_LIRvD1 administration suppresses Stat1-mediated inflammatory response C_LIO_LIRvD1 administration alleviates ER stress-induced apoptosis C_LIO_LIRvD1 administration induces fibrosis regression gene expression C_LI

BackgroundChina has the highest liver cancer burden in the world. Prediction and comparison of the future trends of liver cancer in China and some representative areas may guide further control action. MethodsUsing data from the Global Burden of Disease Study, we assessed incidence, mortality, and disability-adjusted life-years of liver cancer in Mainland China, with reference to representative East Asia areas (Taiwan China, Japan, and Korea) and Western areas (the United Kingdom and the United States). The burden of liver cancer was evaluated and predicted using NOREPRED model from 1990 to 2044. ResultsOverall, the liver cancer incidence (28.1 to 10.6 per 100,000) and mortality (27.5 to 9.7 per 100,000) decreased from 1990 to 2015 in Mainland China, which were consistent with the trends of Eastern Asia areas. However, the disease burden in Mainland China were then plateaued and started to increase during 2015-2044 (10.6 to 14.8 per 100,000 for incidence; 9.7 to 14.02 per 100,000 for mortality), including the hepatitis-related liver cancer incidence (increase from 8.6 to 11.7 per 100,000). While the changing patterns of alcohol- and nonalcoholic steatohepatitis (NASH)- related liver cancer incidence were found similar among Mainland China (0.93 to 1.51 per 100,000 for alcohol; 0.5 to 0.73 per 100,000 for NASH) and Western countries in our projection. ConclusionThe liver cancer burden in Mainland China is unexpectedly predicted to increase again after decades of decline. Future efforts must be made to resolve both the remaining hepatitis-related cancer burden and the changing etiologies.

Accumulation of activated immune cells results in non-specific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. We enrolled 15 CHB patients with active liver damage to receive antiviral therapy, and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8 T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8 T cells, resulting in non-specific Fas ligand-mediated killing of target cells. These results define a CD8 T cell population in the human liver that can drive pathogenesis, and a key pathway involved in their function in CHB patients.

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide despite recent therapeutic advances, driven in part by its marked etiological and molecular heterogeneity and the lack of broadly effective therapeutic targets. Identifying conserved tumor dependencies shared across distinct etiological backgrounds may provide new opportunities for targeted therapy. Here, we developed an integrative computational framework to systematically integrate transcriptomic, functional genomics, and clinical datasets for the identification and prioritization of candidate tumor dependency genes in liver cancer. We reanalyzed transcriptomic data from murine models of liver cancer driven by genotoxic (DEN), oncogenic (c-Myc), and inflammatory (lymphotoxin) stimuli, identifying more than 380 genes consistently upregulated across all tumor models. Functional enrichment analysis revealed a strong overrepresentation of cell cycle-related pathways and liver cancer signatures. Integration with DepMap dependency datasets identified 26 genes with strong dependency scores. Candidate genes were further prioritized by comparing their expression across models of liver regeneration, chronic liver injury, and liver cancer. Analysis of the TCGA-LIHC cohort confirmed significant overexpression of all 26 genes in human HCC, with high expression associated with poor patient survival. Together, these findings establish an integrative framework for identifying conserved tumor dependencies, providing a prioritized set of proliferation-associated genes for functional evaluation as therapeutic targets in HCC.

Nuclear bile acid (BA) signaling plays a central role in liver homeostasis and represents a major therapeutic axis in fibrotic liver diseases. The farnesoid X receptor (FXR), a master nuclear effector of BA signaling, is expressed in several liver-resident cell types, suggesting that it may regulate distinct biological programs beyond the hepatocyte (HC) compartment. Using complementary pharmacological, genetic, and computational approaches across in vitro, ex vivo, and in vivo models of mouse and human origin, we investigated the role of hepatic stellate cell (HSC) FXR (FXRHSC) in both unchallenged and injured livers, which has remained controversial. FXR is robustly expressed in both HCs and HSCs with distinct isoform distributions, and these isoforms exhibited differential capacities to activate gene expression in an HSC context. We found that the potent selective FXR agonist tropifexor triggers a transcriptional program reminiscent of that observed after partial hepatectomy and associated with HC proliferation. This cell cycle-related response was also observed in HSCs and did not require intestinal FXR expression. An HSC-specific response to tropifexor was observed for several genes, including members of the glutathione-S-transferase (GST) family or Scube1. FXRHSC was sufficient to observe the anti-fibrotic effects of tropifexor in precision-cut liver slices, an ex-vivo model of fibrosis. Finally, we identified the regulation of the chemerin-encoding gene Rarres2 as a relevant example of FXRHSC-dependent control of hepatic intercellular communication. Together, these findings identify FXRHSC as an important contributor to hepatic adaptation and therapeutic response to BA analogs and confirmed HSCs as a significant site of nuclear bile acid signaling in liver biology.

BackgroundAside from liver related complications, non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ArLD) are associated with an increased risk of cardiovascular disease (CVD). Liver fibrosis, determined via histology and non-invasive serum fibrosis markers, is associated with cardiovascular events. The association between liver fibrosis and heart failure, and the relationship between PNPLA3 rs738409 and TM6SF2 rs58542926 and heart failure is of particular interest, given the known associations of these genetic polymorphisms with increased risk of liver fibrosis and decreased risk of coronary artery disease. MethodsUsing data from the UK Biobank (UKBB), we examined the relationship between liver fibrosis, determined using non-invasive markers (NAFLD fibrosis score, Fibrosis-4 (FIB-4) and AST to platelet ratio index (APRI score)) and hospitalization or death from heart failure in 413,860 people. Participants were followed up prospectively via electronic linkage to hospital and death records. Cox-regression estimated the hazard ratios (HR) for death or admission with heart failure. The effects of PNPLA3 and TM6SF2 on the association between liver fibrosis and incident heart failure were estimated in an analysis stratified by genotype and by testing for an interaction between genotype and liver fibrosis using a likelihood ratio test. Results12,527 incident cases of heart failure occurred over a median of 10.7 years. Liver fibrosis, determined by single or combination non-invasive tests, was associated with an increased risk of hospitalization or death from heart failure; multivariable adjusted high risk NFS score HR 1.59 [1.45-1.76], p<0.0001, FIB-4 HR 1.69 [1.55-1.84], p<0.0001, APRI HR 1.85 [1.56-2.19], p<0.0001, combined fibrosis scores HR 1.90 [1.44-2.49], p<0.0001). These associations persisted for people with NAFLD or harmful alcohol consumption. Polymorphisms linked to liver fibrosis (PNPLA3 rs738409 GG and TM6SF2 rs58542926 TT) further amplified the positive association between non-invasive liver fibrosis markers and heart failure. A statistically significant interaction was found between PNPLA3 rs738409, FIB-4, APRI score and heart failure. ConclusionLiver fibrosis, determined via non-invasive tests, is associated with an increased risk of hospitalization/death from heart failure in a general population cohort with mixed etiologies of chronic liver disease, including individuals with NAFLD and harmful alcohol consumption. Genetic polymorphisms associated with increased risk of liver fibrosis further increased the risk of heart failure. These findings have important mechanistic, clinical, and public health implications.

Background and aims. The first direct-acting antivirals (DAAs) to treat the viral hepatitis C (HCV) became available in 2011. Despite numerous clinical studies of patient outcomes after treatment, few have evaluated changes in the liver microenvironment. Despite achieving sustained virologic response (SVR), patients may still experience adverse outcomes like cirrhosis and hepatocellular carcinoma. By comparing gene and protein expression in liver biopsies collected before and after treatment, we sought to determine whether specific signatures correlated with disease progression and adverse clinical outcomes. Methods. Biopsies were collected from 22 patients before and after DAA treatment. We measured [~]770 genes and used multispectral imaging with custom machine learning algorithms to analyze phenotypes of intrahepatic macrophages (CD68, CD14, CD16, MAC387, CD163) and T cells (CD3, CD4, CD8, CD45, FoxP3). Results. Before DAA treatment, patients showed two distinct gene expression patterns: one with high pro-inflammatory and antiviral gene expression and another with weaker expression. Patients with adverse outcomes exhibited significantly (p<0.05) more inflammatory activity and had more advanced fibrosis stages in their baseline biopsies than those with liver disease resolution. Patients who achieved SVR had significantly decreased liver enzymes, reduced inflammatory scores, and restored type 1 interferon pathways similar to controls. However, after DAA treatment, patients with persistently high gene expression (67%, pre-hot) still had significantly worse outcomes (p<0.049) despite achieving SVR. A persistent lymphocytic infiltrate was observed in a subset of these patients (76.5%). After therapy, anti-inflammatory macrophages (CD16+, CD16+CD163+, CD16+CD68+) increased, and T cell heterogeneity was more pronounced, showing a predominance of helper and memory T cells (CD3+CD45RO+, CD4+CD45RO+, CD3+CD4+CD45RO+). Conclusions. Patients who have more inflamed livers and more advanced fibrosis before DAA treatment should be closely followed for the development of adverse outcomes, even after achieving SVR. We can enhance patient risk stratification by integrating gene and protein expression profiles with clinical data. This could identify those who may benefit from more intensive monitoring or alternative therapeutic approaches, inspiring a new era of personalized patient care. Lay SummaryDirect-acting antiviral (DAA) therapy has dramatically improved the treatment of chronic HCV, making it curable for most people. This study determined gene and protein expression differences in the liver before and after treatment of HCV. These results will lead to a deeper understanding of the changes in the hepatic immune microenvironment with and without the virus present in the liver in hopes of improving patient surveillance, prognosis, and outcome in the future.

The impact of chronic hepatic infection on antigen non-specific immune cells in circulation is not well understood and may influence long term health. We reported lasting global hyperfunction of circulating CD8 T cells in HCV-infected individuals with cirrhosis. Whether gene expression patterns in bulk CD8 T cells are associated with the severity of liver fibrosis in HCV infection is not known. RNA sequencing of blood CD8 T cells from treatment-naive, HCV-infected individuals with minimal (Metavir F0-1 [&le;] 7.0 kPa) or advanced fibrosis or cirrhosis (F4 [&ge;] 12.5 kPa), before and after direct-acting antiviral therapy, was performed. Principal component analyses determined robust differences in over 350 genes expressed by CD8 T cells from HCV-infected individuals with minimal or advanced fibrosis and data suggests this remains relatively stable after viral clearance. Gene ontology analyses identified disaggregated gene expression related to cellular metabolism, including upregulated phospholipase, phosphatidyl-choline/inositol activity and second-messenger-mediated signaling, while genes in pathways associated with nuclear processes, RNA transport and cytoskeletal dynamics were reduced. Gene Set Enrichment Analysis identified decreased expression of genes regulated by the cMyc and E2f transcription factors in cirrhotics, compared to the minimal fibrosis group, as well as reduced expression of genes linked to oxidative phosphorylation, mTOR signaling, and more. Upregulated gene sets in cirrhotics included IFN-, -{gamma}, TGF-{beta} response genes, apoptosis and apical surface pathways, among others. The hedgehog (Hh) signaling pathway was the top featured gene set upregulated in cirrhotics. Inhibition of Hh signaling with cyclopamine ablated CD8 T cell IFN-{gamma} production, suggesting its involvement in hyperfunction. This is the first analysis of bulk CD8 T cell gene expression profiles in HCV infection in the context of liver fibrosis severity, and suggests cirrhosis significantly reprograms the CD8 T cell pool. The novel finding of increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.

Liver sinusoidal endothelial cells (LSECs) are increasingly recognized as a critical yet underexplored cell type in anti-fibrotic drug development. This study presents a computational drug screening platform integrating LSEC-specific transcriptomic analysis across simple steatosis, fibrotic nonalcoholic steatohepatitis (NASH), and cirrhosis, with tiered gene signature selection combining machine learning, large language model-assisted curation, gene safety assessment, and Connectivity Map-based screening using human endothelial perturbational profiles. The platform identifies 6 clinical-stage and 8 preclinical candidates with LSEC-protective potential. Among these, vorinostat (SAHA), a clinically approved histone deacetylase (HDAC) inhibitor, is selected for experimental validation. In hepatocyte-specific Asah1-deficient mice fed a Paigen diet, SAHA attenuates hepatic inflammation, fibrosis, LSEC dysfunction, and portal hemodynamic abnormalities, with effects confirmed in a hepatotoxin (CCl4)-induced fibrosis model. High mobility group box 1 (HMGB1) is identified as a key hepatocyte-derived paracrine mediator of LSEC injury through Transwell co-culture and glycyrrhizin rescue. Vorinostat dose-dependently reverses HMGB1-induced LSEC dysfunction across inflammation, capillarization, fibrogenesis, and vasoconstriction, associated with endothelial transcription factor reprogramming including KLF2 upregulation, validated in primary LSECs and in vivo. SAHA also protected LSECs from TNF--induced inflammation and reduced monocyte adhesion. These findings establish an LSEC-focused drug repurposing framework and identify candidates for LSEC-protective anti-fibrotic therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/727430v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@a71eb3org.highwire.dtl.DTLVardef@1d090acorg.highwire.dtl.DTLVardef@1ca1450org.highwire.dtl.DTLVardef@1fbb3fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundWe set out to characterise chronic Hepatitis B (CHB) in the primary care population in England and investigate risk factors for progression to hepatocellular carcinoma (HCC). MethodsWe identified 8039 individuals with CHB in individuals aged [&ge;]18 years between 1999-2019 in the English primary care database QResearch. HCC risk factors were investigated using Cox proportional hazards modelling. FindingsMost of those living with CHB were males (60%) of non-White ethnicity (>70%), and a high proportion were in the most deprived Townsend deprivation quintile (44%). Among 7029 individuals with longitudinal data, 161 HCC cases occurred. Increased HCC hazards significantly associated with male sex (adjusted hazards ratio (aHR) 3.44, 95% Confidence Interval (95CI) 2.07-5.73), older age (for age groups 56-55 and [&ge;]66 years of age, compared to 26-35 years, aHRs 7.52 (95CI 4.14-13.67) and11.89 (95CI 6.26-22.60) respectively), socioeconomic deprivation (aHR for fifth Townsend deprivation quintile 1.69, 95CI 1.01-2.84, compared to third), Caribbean ethnicity (aHR 3.32, 95CI 1.43-7.71, compared to White ethnicity), ascites (aHR 1.85, 95CI 1.02-3.36), cirrhosis (aHR 6.52, 95CI 4.54-9.37) and peptic ulcer disease (aHR 2.20, 95CI 1.39-3.49). Reduced HCC hazards were associated with statin use (aHR 0.47, 95CI 0.22-0.99). InterpretationTargeting resources at vulnerable groups, and addressing modifiable risk factors is essential to improve CHB outcomes, and to support progress towards international goals for the elimination of hepatitis infection as a public health threat. FundingWellcome (grant ref 110110/Z/15/Z), UCLH NIHR Biomedical Research Centre, Nuffield Department of Medicine, University of Oxford, GlaxoSmithKline, NIHR Health Informatics Collaborative, Cancer Research UK. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTHE CHB population in England has not been well described. Hepatitis B virus (HBV) reports from the UK Health Security Agency (UHKSA) have not previously reported chronic HBV (CHB) prevalence stratified by relevant subgroups, including ethnicity and socioeconomic status. The burdens of comorbid diseases in this population have also not been characterised. Furthermore, risk factors for the progression of CHB to hepatocellular carcinoma (HCC) have previously been identified largely in homogenous patient samples which may not be widely generalisable. Therefore, risk factors identified in previously published studies require validation in diverse multi-ethnic cohorts. Characterisation of CHB and investigation of novel risk factors for HCC is warranted in a large data source which contains parameters for a large percentage of the population which are collected in a systematic and wide-scale manner in order to improve generalisation of findings. Added value of this studyWe have characterised the largest cohort of CHB individuals in the UK to date, using the QResearch primary care electronic health record database, and describing the demographics and burdens of comorbid disease in the population. This is novel and has not previously been done in a large socioeconomically and ethnically diverse patient sample. We have also analysed risk factors for HCC in the cohort, both validating previously reported factors and investigating novel factors. Implications of all the available evidenceThe findings of this study have important implications for CHB prevention, clinical management, and resource planning. Our detailed description of the demographics and disease profile of the CHB population in the UK may facilitate the targeting of health and prevention resources. Findings concerning HCC risk factors have implications for the clinical management of CHB in order to reduce the risk of progression to HCC.

BackgroundSince liver disease is often clinically unapparent, non-invasive biomarkers predicting future major adverse liver outcomes (MALO) are urgently needed. Therefore, we assessed the usefulness of a novel, proximity extension assay (PEA)-based high-throughput targeted proteomics method to predict MALOs. MethodsPEA plasma proteomic data (>2900 proteins) and clinical information were accessed from the population-based UK Biobank (UKB) cohort, including >53000 individuals with a median follow-up of >10 years and its subcohorts of obese (>12900) and diabetic (>1600) participants. The validation cohorts comprised 287 subjects with severe alpha1-antitrypsin deficiency (AATD), Pi*ZZ genotype, and 960 people living with HIV (PLHIV), who underwent liver stiffness measurement (LSM) via transient elastography. Selected PEA parameters were compared to routine measurements. Bayes-moderated linear models (age and sex as covariates) assessed the differential abundance. Logistic regression was used to identify and validate a novel prognostic score. ResultsRoutine gamma-glutamyltransferase (GGT) and aspartate aminotransferase (AST) levels strongly correlated with PEA-based measurements (r=0.91 and r=0.68, respectively). Similarly, PEA-based thrombospondin-2 levels strongly correlated with immunoassay-based values (r=0.85). Twenty proteins were consistently associated with future MALOs/increased LSM in all cohorts. UKB cohort was used to develop a novel five-component PEA score that demonstrated superior predictive power (AUROC=0.84) compared to established indices/scores, including AST-to-platelet-ratio index (APRI, AUROC=0.73) and Fibrosis-4 index (FIB4, AUROC=0.72) and its attractive predictive power was sustained in diabetic/obese subcohorts. In PLHIV and AATD validation cohorts, all five components showed gradual increases across fibrosis stages, and PEA score numerically outperformed APRI/FIB4 in predicting significant liver disease. ConclusionOur study identifies a new PEA score consisting of epithelial/hepatic stellate cell markers that demonstrates an attractive discriminative ability in several independent cohorts and different liver disease aetiologies.

Cirrhosis and hepatocellular carcinoma (HCC) are long-term complications of chronic liver disease (CLD). In this large multi-ancestry genome-wide association study of all-cause cirrhosis (35,481 cases, 2.36M controls) and HCC (6,680 cases, 1.76M controls), we identified 27 loci associated with cirrhosis (10 novel) and 11 with HCC (three novel). Three novel cirrhosis loci were replicated in independent cohorts (e.g. FGF21, RPTOR, and IFNL3/4). Fifteen cirrhosis loci exhibited differential effects on cirrhosis risk via underlying etiologies, and six HCC loci influenced HCC risk indirectly via cirrhosis. In a gene-burden analysis of rare variants from whole-genome sequencing data in the VA Million Veteran Program (n=102,677), we identified GSTA5 as a novel cirrhosis-associated gene, while APOB and ATP9B were associated with and replicated for HCC. A high genetic risk score for cirrhosis was associated with a nearly doubled risk of CLD progressing to cirrhosis (HR=1.94, P=2x10-68) and of cirrhosis progressing to HCC (HR=1.65, P=7x10-08). Finally, among individuals with chronic hepatitis C who underwent antiviral therapy, cirrhosis risk was modified by variants in PNPLA3, IFNL3/4, and CD81 following pegylated interferon- therapy, and by APOE lead variant following direct-acting antiviral therapy. These findings provide new insights into the complex genetic architecture of CLD progression with potential clinical and therapeutic implications.