Defining the trajectory of cells during differentiation and disease is key for uncovering the mechanisms driving cell fate and identity. However, trajectories of human cells remain largely unexplored due to the challenges of studying them with human samples. In this study, we investigate the proteome trajectory of iPSCs differentiation to hepatic stellate cells (diHSCs) and identify RORA as a key transcription factor governing the metabolic reprogramming of HSCs necessary for diHSCs' commitment, identity, and activation. Using RORA deficient iPSCs and pharmacologic interventions, we show that RORA is required for early differentiation and prevents diHSCs activation by reducing the high energetic state of the cells. While RORA knockout mice have enhanced fibrosis, RORA agonists rescue multi-organ fibrosis in in vivo models. Notably, RORA expression correlates negatively with liver fibrosis and HSCs activation markers in patients with liver disease. This study reveals that RORA regulates cell metabolic plasticity, important for mesoderm differentiation, pericyte quiescence, and fibrosis, influencing cell commitment and disease.

Background & Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of chronic liver disease worldwide. Autoantibodies (Ab), such as antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA), are frequently detected in MASLD, but their role in disease progression remains controversial. This study aimed to evaluate the prevalence of positive Ab and the histological features of autoimmune hepatitis (AIH) in MASLD and their association with liver-related outcomes. Methods: We conducted a multicenter, retrospective, longitudinal study of patients with biopsy-proven MASLD from the HEPAmet Registry. Data on ANA (≥1/80), ASMA (≥1/40), and AIH histological features (portal inflammation, interface hepatitis, and plasma cell infiltration) were analyzed for their association with compensated advanced chronic liver disease (cACLD), liver decompensation, and death. Results: Of the 460 patients (49% women, median age 58 years, median BMI 33 kg/m2, and 45% with advanced fibrosis), 17% and 25% tested positive for ANA and ASMA, respectively. Histological features of AIH included interface hepatitis (19%), moderate/severe portal inflammation (12%), and plasma cell clusters (10%). Possible AIH based on histological criteria was present in 8% of patients. The presence of positive Ab was independently associated with cACLD development (odds ratio 2.890, p <0.030), liver decompensation (hazard ratio 3.969, p = 0.001), and death (hazard ratio 2.546, p = 0.036). In contrast, the presence of isolated histologic autoimmune features was not correlated with serological markers and did not affect the prognosis of MASLD. Conclusions: ANA and ASMA are commonly found in patients with MASLD and are associated with poorer liver-related outcomes and reduced survival, whereas isolated histological autoimmune features provide no additional prognostic value. Impact and implications: Metabolic dysfunction-associated steatotic liver disease (MASLD) can coexist with other liver diseases, including autoimmune hepatitis. The role of autoantibodies and histological autoimmune features in MASLD progression remains controversial. Understanding the relationship between autoimmune characteristics and disease progression in MASLD may help physicians identify high-risk populations, enhance risk stratification, and personalize disease treatment.

Background & Aims: Alcohol-associated liver disease (ALD) is a major cause of liver disease worldwide with scarce therapeutic options. Animal models poorly recapitulate advanced ALD precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool. However, current patient-derived liver organoids fail to recapitulate the epithelial heterogeneity and its generation requires liver surgical resections, thus limiting personalized disease modeling. Here, we report the development of organoids from liver needle biopsies (b-Orgs) from patients with ALD. Methods: b-Orgs were generated from tru-cut biopsies from patients at early (n=28) and advanced (n=34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single cell RNA-sequencing and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis (AH) and evaluate response to prednisolone. Results: Phenotypic and functional analysis of b-Orgs showed hepatocyte-enriched features. Single-cell RNA-sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Moreover, b-Orgs preserved disease-stage features and allowed to identify the association of ELF3 with cell plasticity and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including ROS production, lipid accumulation, inflammation and decreased cell proliferation, which were mitigated in response to prednisolone. Conclusions: Overall, we provide a human-based model that recapitulates epithelial complexity and patient specific features, allowing to identify drivers of cell plasticity and expanding organoid-based liver disease modeling for personalized medicine. ### Competing Interest Statement The authors have declared no competing interest.

Mitochondrial reactive oxygen species (mROS) are central to physiology1,2. Excess mROS production has been associated with several disease states2,3; however, the precise sources, regulation and mechanism of generation in vivo remain unclear, which limits translational efforts. Here we show that in obesity, hepatic coenzyme Q (CoQ) synthesis is impaired, which increases the CoQH2 to CoQ (CoQH2/CoQ) ratio and drives excessive mROS production through reverse electron transport (RET) from site IQ in complex I. Using multiple complementary genetic and pharmacological models in vivo, we demonstrate that RET is crucial for metabolic health. In patients with steatosis, the hepatic CoQ biosynthetic program is also suppressed, and the CoQH2/CoQ ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Background & Aims: Impaired phagocytic capacity due to activation of the PD-1/PD-L1 pathway has been implicated in the development of bacterial infections in patients with cirrhosis. Soluble PD-L1 (sPD-L1) is easily measurable in plasma and has been proposed as a biomarker of sepsis. In the current study, we aim to evaluate the role of sPD-L1 as a biomarker of bacterial infection development in patients with cirrhosis. Methods: Plasma samples from 995 patients with chronic liver disease grouped in three cohorts were analyzed: an initial cohort of 268 hospitalized patients with acute decompensated cirrhosis, 327 out-patients with non-acute decompensated cirrhosis and finally 400 patients with high-risk alcohol consumption, including all stages of liver fibrosis, from mild/no fibrosis to cirrhosis (F0–F4). The main outcomes of the study were development of bacterial infection and mortality. Results: Patients who developed bacterial infections had higher median levels of sPD-L1 than those who did not (160 [IQR 116-221] vs. 136 [IQR 97-193] pg/ml, respectively, p value <0.001; hazard ratio 1.034, 95% CI 1.014-1.055). Levels of sPD-L1 were associated with bacterial infection development after adjustment for confounding factors. During follow-up, patients who died had higher median sPD-L1 levels than survivors, after adjustment for MELD Na (180 [IQR 143-267] vs. 134 [IQR 97-187] pg/ml, respectively; p value <0.001; HR 1.066, 95% CI 1.043-1.089). These findings were observed in all cohorts. Conclusions: Plasma levels of sPD-L1 are associated with the risk of bacterial infection development irrespective of the stage of chronic liver disease. Furthermore, higher sPD-L1 levels are linked to increased mortality. Measurement of sPD-L1 levels may help identify patients at high risk of developing bacterial infections and guide the implementation of new preventive strategies. Impact and implications: This study explores the role of soluble PD-L1 as a biomarker of immune dysfunction and its association with clinical outcomes in patients with chronic liver disease. Our findings demonstrate that soluble PD-L1 levels increase with the progression of liver disease and they are independently associated with an increased risk of bacterial infection development and mortality. These results could help physicians identify high-risk individuals earlier and implement preventive strategies.

Background & Aims: Expression of P21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD); however, the underlying mechanisms remain unknown. In the present study, we investigated the function of CDKN1A in SLD. Methods: CDKN1A expression levels were evaluated in different patient cohorts with SLD, fibrosis, and advanced chronic liver disease (ACLD). Cdkn1a-/- and Cdkn1a+/+ mice were fed with either a Western diet (WD), a Lieber-DeCarli (LdC) diet plus multiple EtOH (ethanol) binges, or a DuAL diet (metabolic dysfunction-associated fatty liver disease and alcohol-related liver). Primary hepatocytes were isolated and functional assays performed. Results: A significant increase in CDKN1A expression was observed in patients with steatohepatitis and fibrosis (with a positive correlation with both NAFLD Activity Score and fibrosis staging scores), cirrhosis and ACLD. Cdkn1a+/+ mice, fed a DuAL diet exhibited liver injury and cell death increased reactive oxygen species (ROS), and markers of senescence (γH2AX, β-GAL, Cdkn1a/p53) contributing to steatosis and inflammation. In contrast, Cdkn1a-/- mutant mice showed a significant decrease in senescence-associated markers as well as in markers of liver injury, hepatic steatosis and an increase in fatty acid oxidation and reduction in free fatty acid uptake as well as de novo lipogenesis. Mechanistically, activation of the AMPK-SIRT3 was observed in Cdkn1a-deleted animals. Conclusions: Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis via the AMPK-SIRT3 axis. CDKN1A expression was found to be directly correlated with increased severity of NAFLD Activity Score and fibrosis in patients with SLD. CDKN1A could be a potential theragnostic target for the treatment of metabolic dysregulation in patients with SLD, with and without alcohol consumption. Impact and implications:: Expression of p21, encoded by the CDKN1A gene, has been associated with fibrosis progression in steatotic liver disease (SLD), but the molecular mechanisms remain elusive. Interestingly, in this study we found that Cdkn1a deletion protected against preclinical SLD by promoting fatty acid oxidation and preventing free fatty acid uptake and de novo lipogenesis, via the AMPK-SIRT3 axis. Translationally, Cdkn1a expression was found to be directly correlated with increased severity of NAFLD Activity Score (NAS) and fibrosis in SLD patients, and therefore, CDKN1A might be used potential theragnostic target for the treatment of metabolically induced SLD, with and without alcohol consumption.

BACKGROUND & AIMS:Animal models poorly recapitulate advanced alcohol-associated liver disease (ALD), precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool; however, current patient-derived liver organoids fail to capture epithelial heterogeneity and require surgical resections, limiting their use in personalized disease modeling. Here, we describe the development of organoids from liver needle biopsies (b-Orgs) obtained from patients with ALD. METHODS:b-Orgs were generated from tru-cut biopsies from patients at early (n = 28) and advanced (n = 34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single-cell RNA sequencing, and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis and evaluate response to prednisolone. RESULTS:Phenotypic and functional analysis of b-Orgs showed hepatocyte-enriched features. Single-cell RNA sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Importantly, b-Orgs preserved disease stage features and revealed an association between ELF3, cell plasticity, and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including reactive oxygen species production, lipid accumulation, inflammation and decreased cell proliferation, which were attenuated by prednisolone. CONCLUSIONS:We describe a human-based model that captures epithelial complexity and patient-specific features of ALD. This approach enables the identification of drivers of cell plasticity and highlights the potential of organoid-based liver disease modeling for personalized medicine. IMPACT AND IMPLICATIONS:While organoids have emerged as a powerful human-based preclinical tool, current patient-derived liver organoids fail to capture epithelial heterogeneity and require surgical resections, limiting their use in personalized disease modeling. Here, we describe the generation of biopsy-derived organoids (b-Orgs) from patients with varying stages of alcohol-related liver disease. b-Orgs capture the liver epithelial cell composition found in patients' liver tissue and are efficiently generated from different stages of the disease, providing a platform for patient-tailored disease modeling and drug testing.

Background and objectives Fibrosis contributes to 45% of deaths in industrialized nations and is characterized by an abnormal accumulation of extracellular matrix (ECM). There are no specific anti-fibrotic treatments for liver fibrosis, and previous unsuccessful attempts at drug development have focused on preventing ECM deposition. Because liver fibrosis is largely acknowledged to be reversible, regulating fibrosis resolution could offer novel therapeutical options. However, little is known about the mechanisms controlling ECM remodeling during resolution. Changes in proteolytic activity are essential for ECM homeostasis and macrophages are an important source of proteases. Herein, in this study we evaluate the role of macrophage-derived cathepsin D (CtsD) during liver fibrosis. Methods CtsD expression and associated pathways were characterized in single-cell RNA sequencing and transcriptomic datasets in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD and hepatocyte-CtsD knock-out mice. Results Analysis of single-cell RNA sequencing datasets demonstrated CtsD was expressed in macrophages and hepatocytes in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD (CtsDΔMyel) and hepatocyte-CtsD knock-out mice. CtsD deletion in macrophages, but not in hepatocytes, resulted in enhanced liver fibrosis. Both inflammatory and matrisome proteomic signatures were enriched in fibrotic CtsDΔMyel livers. Besides, CtsDΔMyel liver macrophages displayed functional, phenotypical and secretomic changes, which resulted in a degradomic phenotypical shift, responsible for the defective proteolytic processing of collagen I in vitro and impaired collagen remodeling during fibrosis resolution in vivo. Finally, CtsD-expressing mononuclear phagocytes of cirrhotic human livers were enriched in lysosomal and ECM degradative signaling pathways. Conclusions Our work describes for the first-time CtsD-driven lysosomal activity as a central hub for restorative macrophage function during fibrosis resolution and opens new avenues to explore their degradome landscape to inform drug development.

BACKGROUND & AIMS: Alcoholic foamy degeneration (AFD) is a condition with similar clinical presentation to alcoholassociated hepatitis (AH), but with a speci fi c histologic pattern. Information regarding the prevalence and prognosis of AFD is scarce and there are no tools for a noninvasive diagnosis. METHODS: A cohort of patients admitted to the Hospital Clinic of Barcelona for clinical suspicion of AH who underwent liver biopsy was included. Patients were classi fi ed as AFD, AH, or other fi ndings, according to histology.Clinical features, histology, and genetic expression of liver biopsy specimens were analyzed. The accuracy of National Institute on Alcohol Abuse and Alcoholism criteria and laboratory parameters for differential diagnosis were investigated. RESULTS: Of 230 patients with a suspicion of AH, 18 (8%) met histologic criteria for AFD, 184 (80%) had de fi nite AH, and 28 (12%) had other fi ndings. In patients with AFD, massive steatosis was more frequent and the fi brosis stage was lower. AFD was characterized by down -regulation of liver fi brosis and in fl ammation genes and up -regulation of lipid metabolism and mitochondrial function genes. Patients with AFD had markedly better long-term survival (100% vs 57% in AFD vs AH; P [ .002) despite not receiving corticosteroid treatment, even in a model for end -stage liver disease - matched sensitivity analysis. Serum triglyceride levels had an area under the receiver operating characteristic of 0.886 (95% CI, 0.807 - 0.964) for the diagnosis of AFD, whereas the National Institute on Alcohol Abuse and Alcoholism criteria performed poorly. A 1 -step algorithm using triglyceride levels of 225 mg/dL (sensitivity, 0.77; speci fi city, 0.90; and Youden index, 0.67) is proposed for differential diagnosis. CONCLUSIONS: AFD in the setting of suspicion of AH is not uncommon. A differential diagnosis is important because prognosis and treatment differ largely. Triglyceride levels successfully identify most patients with AFD and may be helpful in decision making.

Background and Aims:Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocytes markers and showing immature features. However, the mechanisms and the impact of hepatocyte dedifferentiation in liver disease are poorly understood.Methods:HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ALD). Hepatocyte- specific overexpression or deletion of CXCR4, and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity.Results:Here we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients' outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness and cancer gene programs. CXCR4 pathway was highly enriched in HB cells, and correlated with disease severity and hepatocyte dedifferentiation. In vitro , CXCR4 was associated with biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased hepatocyte specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression.Conclusions:This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH.Lay summary:Here we describe that hepatocyte dedifferentiation is associated with disease severity and a reduced synthetic capacity of the liver. Moreover, we identify the CXCR4 pathway as a driver of hepatocyte dedifferentiation and as a therapeutic target in alcohol-related hepatitis.