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.

Background and aimsTo examine the associations between Steatotic liver disease (SLD), its subtypes (MASLD, MetALD, ALD), fibrosis stages and mortality from a broad spectrum of diseases. MethodsWe analysed 486156 UK Biobank participants. We summarised the causes of death during follow-up, by SLD status and subtypes. Multivariable Cox models estimated associations between SLD, SLD subtypes, FIB4 score, and cause-specific mortality outcomes across ICD10 disease categories. ResultsAmong 178336 participants with SLD, 20766 died over a median follow up of 13.8 years. The leading causes of deaths were neoplasm (46.4%), circulatory disease (24.2%), respiratory disease (7.0%), and digestive disease (4.8%). Compared to participants without SLD (n=307820), those with SLD had significantly higher mortality from neoplasm (HR 1.24 (1.20, 1.28)), circulatory (1.57 (1.50, 1.64)), and digestive disease (1.85 (1.67, 2.05)), as well as from metabolic, and genitourinary diseases, across all FIB4 score levels. Elevated risks were also observed for mortality from diabetes (3.40 (2.59, 4.46))), Covid-19 (1.97 (1.76, 2.21)), myocardial infarction (1.69 (1.53, 1.86)), stroke (1.24 (1.04, 1.48)), and several cancers, such as breast (1.50 (1.35, 1.68)), colorectal (1.22 (1.11, 1.34)), and prostate cancer (1.22 (1.09, 1.37)). SLD subtypes exhibited distinct patterns of cause-specific mortality. ConclusionSLD is associated with increased mortality across a wide range of disease categories, with extrahepatic cancers and circulatory diseases being the primary contributors. These findings underscore the importance of multidisciplinary management strategies targeting liver fibrosis, cardiometabolic burden, and cancer prevention in individuals with SLD. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/25342903v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1ee89c0org.highwire.dtl.DTLVardef@195a679org.highwire.dtl.DTLVardef@1e45af3org.highwire.dtl.DTLVardef@10f4503_HPS_FORMAT_FIGEXP M_FIG C_FIG

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/.

Background and AimsChronic hepatitis C (CHC) can progress to cirrhosis and hepatocellular carcinoma (HCC). This study aimed to identify genetic determinants and host-viral interactions that drive this progression to inform risk stratification and personalised treatment strategies. MethodsWe performed a genome-wide association study (GWAS) of cirrhosis (2,829 cases and 1,515 CHC controls), followed by a GWAS of HCC (706 cases and 2,152 cirrhosis controls). We performed cis-eQTL mapping and deconvolution in liver tissue of HCV-infected (136 CHC and 54 cirrhosis) patients to investigate gene expression regulation and cellular heterogeneity. Additionally, ten polygenic risk scores (PRS) for non-viral liver diseases were tested in 3,406 infected individuals. ResultsWe identified the missense risk variant rs738409 in PNPLA3 and a protective variant (rs4386418) in XKR3 in genotype 1-infected patients that were significantly associated with cirrhosis but not HCC progression. HLA fine-mapping identified two amino acids in HLA-DQB1*03:01 and HLA-DRB1*13:01 associated with cirrhosis risk. No genome-wide significant association was observed for HCC, and loci previously linked to non-viral HCC did not replicate. The eQTL analysis revealed 2,060 genes under cis-regulatory control and 129 whose effects were modified by cirrhosis. An intronic eQTL lowered PNPLA3 expression, but was not linked to cirrhosis risk. Deconvolution revealed expansion of plasma cells and macrophages and depletion of hepatocytes in CHC, with further immune-stromal remodelling in cirrhosis. All PRS showed a significant association with cirrhosis risk but not HCC progression. ConclusionCirrhosis in CHC shares genetic architecture with non-viral liver diseases but also displays virus-specific risk variants. Cirrhosis risk involves genetic factors that differ from those underlying progression to HCC.

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.

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.

Background & AimsHepatocellular carcinoma (HCC) is the third top cause of cancer death globally, often arising on a background of cirrhosis. Here, we aimed to establish novel genetic drivers of HCC across ancestries at a population level through a large meta-analysis of all-cause HCC. Approach & ResultsWe included 15 cohorts comprising 17,329 HCC cases and 2,424,298 controls in this meta-analysis. We found 15 genome-wide significant (P < 5x10-8) germline loci, 6 novel in/near GCKR, MTTP, ADH5, MYC, MAP3K9, and GABPB2. MAP3K9, TERT, and GABPB2 variants act independently of cirrhosis on both co-localisation analysis and sensitivity analyses. There was significant ancestral heterogeneity in 6 loci including variants in the HLA locus that had divergent effects on HCC risk between East Asian and European ancestries. Fine-mapping identified 11 potentially causal coding variants, including p.Leu446Pro in GCKR and p.Asp418Glu in MEN1. MEN1, MYC, and TERT are all involved in the beta-catenin pathway transactivation complex. Transcriptome-wide analysis identified enrichment of germline-encoded DHRS1 in HCC. Regulome-wide analysis replicated the germline signal for EPHA2 and found a novel chromatin accessible region containing genes ZNF367 and HABP4. Finally, we demonstrated that population-level genetic architecture for HCC overlaps with steatotic and viral liver disease, and individuals with genetic risk for lower BMI have higher risk of HCC. ConclusionsGenetic risk for HCC is determined by germline susceptibility to beta-catenin pathway activation and cirrhosis. HCC is driven by both heterogenous and homogenous genetic factors across ancestries, which require further ancestral diversity in liver GWAS to fully dissect.

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

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

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.

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.

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is among the most prevalent liver disorders in Western countries. While steatosis is prevalent, molecular mechanisms associated with steatosis grades and how nutrition modulates related metabolic disturbances remain poorly understood. MethodsWe analyzed data from 327 participants of the CHRIS NAFLD study with Type 2 diabetes (T2D) and matched controls, integrating 148 plasma proteins with 175 serum metabolites and lipids to identify circulating biomarkers associated with hepatic steatosis severity. Associations between dietary intake and steatosis were investigated, and a potential influence of diet on the identified biomarkers was evaluated through a sensitivity analysis. Participants were stratified by steatosis grade based on ultrasound-derived measurements. ResultsSteatosis grade was strongly positively correlated with BMI and T2D. Several proteins (e.g., GC, AFM, AZGP1, SERPINC1, VTN, LRG1) and metabolites (e.g., glutamate, tyrosine, valine, lysoPCs) were associated with the highest steatosis grade, mainly reflecting a broader metabolically unhealthy phenotype. Adherence to a plant-based diet was associated with lower steatosis grades independent of circulating proteins, metabolites, and lipids. ConclusionResults suggest that circulating metabolic signatures of steatosis reflect broader metabolic dysregulation and are independent of dietary intake. These data support the use of blood-based multi-omics profiling to distinguish between metabolically healthy and unhealthy individuals, and may thus improve MASLD risk stratification. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/25341334v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@3e7799org.highwire.dtl.DTLVardef@11e03e3org.highwire.dtl.DTLVardef@191529corg.highwire.dtl.DTLVardef@1c68ff4_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMulti-omics profiling reveals biomarkers associated with hepatic steatosis severity C_LIO_LISubstantial overlap between steatosis biomarkers and metabolic health indicators C_LIO_LISteatosis-associated profiles reflect broad metabolic dysfunction C_LIO_LIPlant-based diet is associated with lower steatosis grade C_LIO_LIDietary habits did not affect the identified steatosis blood biomarkers C_LI

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

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: MASLD has become the most prevalent chronic liver disease globally. Although MVPA and plasma fatty acids have been individually studied in relation to metabolic health, their independent and combined associations with MASLD incidence remain unclear. We aimed to investigate these associations. Methods: This study included 51,717 UK Biobank participants free of liver disease at baseline, with MVPA measured using wrist-worn accelerometers and plasma fatty acids quantified via NMR. Multivariable-adjusted Cox models and restricted cubic splines were used. Results: Over a median follow-up of 7.8 years, 472 incident cases were identified. In fully adjusted models, meeting recommended MVPA levels together with higher n-6 PUFA concentrations was associated with a 71% lower risk (HR 0.29, 95% CI 0.18-0.45). The MVPA-MASLD association was nonlinear, with risk reduction plateauing at approximately 189 minutes per week. Higher n-6 PUFA was associated with reduced risk, whereas n-3 PUFA showed no significant association. Conclusions: These findings suggest that behavioral and metabolic factors may jointly influence MASLD risk. Further studies in diverse populations are needed to confirm these associations.

Autoimmune hepatitis (AIH) is a chronic progressive liver disease that despite suggestive serum autoantibodies or plasma cell enrichment, remains functionally a diagnosis of exclusion. Whether the broader cellular composition of the liver might enable improved specificity of diagnosis has not been systematically tested. We prospectively recruited patients undergoing a clinically-indicated liver biopsy for suspected AIH and performed single-nucleus RNA sequencing (snRNA-seq) on biopsy tissue to map the cellular landscape of AIH and its diagnostic mimics. Unsupervised clustering on cell-type abundances alone largely separated AIH from non-AIH samples. Among individual populations, a subset of CD8 T-cells marked by high TOX and PD1 expression was the most discriminating feature: its enrichment perfectly distinguished AIH by both snRNA-seq and in situ density (AUC = 1.00), outperforming plasma cell abundance (AUC = 0.83). CD8TOX T-cell enrichment may therefore be the histologic lesion that marks the diagnosis of AIH.

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.