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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: Current therapies for the treatment of alcohol-related liver disease (ALD) have proven largely ineffective. Patients relapse and the disease progresses even after liver transplantation. Altered epigenetic mechanisms are characteristic of alcohol metabolism given excessive acetate and NAD depletion and play an important role in liver injury. In this regard, novel therapeutic approaches based on epigenetic modulators are increasingly proposed. MicroRNAs, epigenetic modulators acting at the post-transcriptional level, appear to be promising new targets for the treatment of ALD. Methods: MiR-873-5p levels were measured in 23 liver tissue from Patients with ALD, and GNMT levels during ALD were confirmed using expression databases (transcriptome n = 62, proteome n = 68). High-resolution proteomics and metabolomics in mice following the Gao-binge model were used to investigate miR-873-5p expression in ALD. Hepatocytes exposed to 50 mM alcohol for 12 h were used to study toxicity. The effect of anti-miR-873-5p in the treatment outcomes of ALD was investigated. Results: The analysis of human and preclinical ALD samples revealed increased expression of miR-873-5p in the liver. Interestingly, there was an inverse correlation with NNMT, suggesting a novel mechanism for NAD depletion and aberrant acetylation during ALD progression. High-resolution proteomics and metabolomics identified miR-873-5p as a key regulator of NAD metabolism and SIRT1 deacetylase activity. Anti-miR-873-5p reduced NNMT activity, fuelled the NAD salvage pathway, restored the acetylome, and modulated the levels of NF-κB and FXR, two known SIRT1 substrates, thereby protecting the liver from apoptotic and inflammatory processes, and improving bile acid homeostasis. Conclusions: These data indicate that targeting miR-873-5p, a repressor of GNMT previously associated with NAFLD and acetaminophen-induced liver failure. is a novel and attractive approach to treating alcohol-induced hepatoxicity. Impact and implications: The role of miR-873-5p has not been explicitly examined in the progression of ALD, a pathology with no therapeutic options. In this study, inhibiting miR-873-5p exerted hepatoprotective effects against ALD through rescued SIRT1 activity and consequently restored bile acid homeostasis and attenuated the inflammatory response. Targeting hepatic miR-873-5p may represent a novel therapeutic approach for the treatment of ALD.

Background & Aims: : The accumulation of adipose tissue macrophages (ATMs) in obesity has been associated with hepatic injury. However, the contribution of ATMs to hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD) remains to be elucidated. Herein, we investigate the relationship between ATMs and liver fibrosis in patients with patients with NAFLD and evaluate the impact of modulation of ATMs over hepatic fibrosis in an experimental non-alcoholic steatohepatitis (NASH) model. Methods: Adipose tissue and liver biopsies from 42 patients with NAFLD with different fibrosis stages were collected. ATMs were characterised by immunohistochemistry and flow cytometry and the correlation between ATMs and liver fibrosis stages was assessed. Selective modulation of the ATM phenotype was achieved by i.p. administration of dextran coupled with dexamethasone in diet-induced obesity and NASH murine models. Chronic administration effects were evaluated by histology and gene expression analysis in adipose tissue and liver samples. In vitro crosstalk between human ATMs and hepatic stellate cells (HSCs) and liver spheroids was performed. Results: Patients with NAFLD presented an increased accumulation of pro-inflammatory ATMs that correlated with hepatic fibrosis. Long-term modulation of ATMs significantly reduced pro-inflammatory phenotype and ameliorated adipose tissue inflammation. Moreover, ATMs modulation was associated with an improvement in steatosis and hepatic inflammation and significantly reduced fibrosis progression in an experimental NASH model. In vitro, the reduction of the pro-inflammatory phenotype of human ATMs with dextran–dexamethasone treatment reduced the secretion of inflammatory chemokines and directly attenuated the pro-fibrogenic response in HSCs and liver spheroids. Conclusions: Pro-inflammatory ATMs increase in parallel with fibrosis degree in patients with NAFLD and their modulation in an experimental NASH model improves liver fibrosis, uncovering the potential of ATMs as a therapeutic target to mitigate liver fibrosis in NAFLD. Impact and implications: We report that human adipose tissue pro-inflammatory macrophages correlate with hepatic fibrosis in non-alcoholic fatty liver disease (NAFLD). Furthermore, the modulation of adipose tissue macrophages (ATMs) by dextran-nanocarrier conjugated with dexamethasone shifts the pro-inflammatory phenotype of ATMs to an anti-inflammatory phenotype in an experimental murine model of non-alcoholic steatohepatitis. This shift ameliorates adipose tissue inflammation, hepatic inflammation, and fibrosis. Our results highlight the relevance of adipose tissue in NAFLD pathophysiology and unveil ATMs as a potential target for NAFLD.

Background & Aims: The molecular mechanisms driving the progression from early-chronic liver disease (CLD) to cirrhosis and, finally, acute-on-chronic liver failure (ACLF) are largely unknown. Our aim was to develop a protein network-based approach to investigate molecular pathways driving progression from early-CLD to ACLF. Methods: Transcriptome analysis was performed on liver biopsies from patients at different liver disease stages, including fibrosis, compensated cirrhosis, decompensated cirrhosis and ACLF, and control healthy livers. We created 9 liver-specific disease-related protein-protein interaction networks capturing key pathophysiological processes potentially related to CLD. We used these networks as a framework and performed gene set-enrichment analysis (GSEA) to identify dynamic gene profiles of disease progression. Results: Principal component analyses revealed that samples clustered according to the disease stage. GSEA of the defined processes showed an upregulation of inflammation, fibrosis and apoptosis networks throughout disease progression. Interestingly, we did not find significant gene expression differences between compensated and decompensated cirrhosis, while ACLF showed acute expression changes in all the defined liver disease-related networks. The analyses of disease progression patterns identified ascending and descending expression profiles associated with ACLF onset. Functional analyses showed that ascending profiles were associated with inflammation, fibrosis, apoptosis, senescence and carcinogenesis networks, while descending profiles were mainly related to oxidative stress and genetic factors. We confirmed by qPCR the upregulation of genes of the ascending profile and validated our findings in an independent patient cohort. Conclusion: ACLF is characterized by a specific hepatic gene expression pattern related to inflammation, fibrosis, apoptosis, senescence and carcinogenesis. Moreover, the observed profile is significantly different from that of compensated and decompensated cirrhosis, supporting the hypothesis that ACLF should be considered a distinct entity. Lay summary: By using transjugular biopsies obtained from patients at different stages of chronic liver disease, we unveil the molecular pathogenic mechanisms implicated in the progression of chronic liver disease to cirrhosis and acute-on-chronic liver failure. The most relevant finding in this study is that patients with acute-on-chronic liver failure present a specific hepatic gene expression pattern distinct from that of patients at earlier disease stages. This gene expression pattern is mostly related to inflammation, fibrosis, angiogenesis, and senescence and apoptosis pathways in the liver.

Background: Chronic hepatic inflammation leads to liver fibrosis, which may progress to cirrhosis, a condition with high morbidity. Our aim was to assess the as yet unknown role of innate immunity protein CD5L in liver fibrosis. Methods: CD5L was measured by ELISA in plasma samples from cirrhotic (n = 63) and hepatitis (n = 39) patients, and healthy controls (n = 7), by immunohistochemistry in cirrhotic tissue (n = 12), and by quantitative RT-PCR in mouse liver cell subsets isolated by cell sorting. Recombinant CD5L (rCD5L) was administered into a murine model of CCl4-induced fibrosis, and damage, fibrosis and hepatic immune cell infiltration, including the LyC6hi (pro-fibrotic)-LyC6low (pro-resolutive) monocyte ratio were determined. Moreover, rCD5L was added into primary human hepatic stellate cells to study transforming growth factor β (TGFβ) activation responses. Findings: Cirrhotic patients showed elevated plasma CD5L concentrations as compared to patients with hepatitis and healthy controls (Mann-Whitney test p

Introduction and aims. Alcoholic hepatitis is the most severe manifestation of alcoholic liver disease. Unfortunately, there are still some unresolved issues in the diagnosis and management of this disease, such as the need of histological diagnosis, an accurate prognostic stratification, and the development of novel targeted therapies. The present study aimed at addressing these issues by means of metabolomics, a novel high-throughput approach useful in other liver diseases.Material and methods. 64 patients with biopsy-proven alcoholic hepatitis were included and compared with 26 patients with decompensated alcoholic cirrhosis without superimposed alcoholic hepatitis, which was ruled out by liver biopsy.Results. The comparison of the metabolic profiles of patients with alcoholic hepatitis and decompensated cirrhosis showed marked differences between both groups. Importantly, metabolic differences were found among alcoholic hepatitis patients when subjects were stratified according to 90-day survival. Based on these findings, two non-invasive signatures were developed. The first one allowed an accurate non-invasive diagnosis of alcoholic hepatitis (AUROC 0.932; 95% CI 0.901-0.963). The second signature showed a good performance in the prognostic stratification of patients with alcoholic hepatitis (AUROC 0.963; 95% CI 0.895-1.000).Conclusions. Signatures based on metabolomics allowed an accurate non-invasive diagnosis and prognostic stratification of alcoholic hepatitis. The differences observed in the metabolic profile of the patients according to the presence and severity of alcoholic hepatitis are related with different mechanisms involved in the pathophysiology of alcoholic hepatitis such as peroxisomal activity, synthesis of inflammatory mediators or oxidation. This information could be useful for the development of novel targeted therapies.

Background & Aims: MicroRNAs (miRNAs) circulate in several body fluids and can be useful biomarkers. The aim of this study was to identify blood-circulating miRNAs associated with cirrhosis progression and acute-on-chronic liver failure (ACLF). Methods: Using high-throughput screening of 754 miRNAs, serum samples from 45 patients with compensated cirrhosis, decompensated cirrhosis, or ACLF were compared with those from healthy individuals (n = 15). miRNA levels were correlated with clinical parameters, organ failure, and disease progression and outcome. Dysregulated miRNAs were evaluated in portal and hepatic vein samples (n = 33), liver tissues (n = 17), and peripheral blood mononuclear cells (PBMCs) (n = 16). Results: miRNA screening analysis revealed that circulating miRNAs are dysregulated in cirrhosis progression, with 51 miRNAs being differentially expressed among all groups of patients. Unsupervised clustering and principal component analysis indicated that the main differences in miRNA expression occurred at decompensation, showing similar levels in patients with decompensated cirrhosis and those with ACLF. Of 43 selected miRNAs examined for differences among groups, 10 were differentially expressed according to disease progression. Moreover, 20 circulating miRNAs were correlated with model for end-stage liver disease and Child-Pugh scores. Notably, 11 dysregulated miRNAs were associated with kidney or liver failure, encephalopathy, bacterial infection, and poor outcomes. The most severely dysregulated miRNAs (i.e. miR-146a-5p, miR-26a-5p, and miR-191-5p) were further evaluated in portal and hepatic vein blood and liver tissue, but showed no differences. However, PBMCs from patients with cirrhosis showed significant downregulation of miR-26 and miR-146a, suggesting a extrahepatic origin of some circulating miRNAs. Conclusions: This study is a repository of circulating miRNA data following cirrhosis progression and ACLF. Circulating miRNAs were profoundly dysregulated during the progression of chronic liver disease, were associated with failure of several organs and could have prognostic utility. Lay summary: Circulating miRNAs are small molecules in the blood that can be used to identify or predict a clinical condition. Our study aimed to identify miRNAs for use as biomarkers in patients with cirrhosis or acute-on-chronic liver failure. Several miRNAs were found to be dysregulated during the progression of disease, and some were also related to organ failure and disease-related outcomes.