Delia Blaya

The biliary epithelium gives rise to liver progenitor cells

Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)‐positive hepatocytes. In order to investigate the role of HNF1β‐expressing cells we used a tamoxifen‐inducible Hnf1βCreER/R26RYfp/LacZ mouse to lineage‐trace Hnf1β+ biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β+ cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β+ cells to hepatocyte was detected. We next assessed the contribution of Hnf1β+‐derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl‐1,4‐dihydro‐collidin (DDC)‐diet and a choline‐deficient ethionine‐supplemented (CDE)‐diet. The contribution of Hnf1β+ cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC‐diet treatment, mice fed with a CDE‐diet showed a small population of hepatocytes derived from Hnf1β+ cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome‐wide expression profile of Hnf1β+‐derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. Conclusion: HNF1β+ biliary duct cells are the origin of LPC. HNF1β+ cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration. (Hepatology 2014;60:1367–1377)

CCL20 mediates lipopolysaccharide induced liver injury and is a potential driver of inflammation and fibrosis in alcoholic hepatitis

Objective Chemokines are known to play an important role in the pathophysiology of alcoholic hepatitis (AH), a form of acute-on-chronic liver injury frequently mediated by gut derived lipopolysaccharide (LPS). In our study, we hypothesise that chemokine CCL20, one of the most upregulated chemokines in patients with AH, is implicated in the pathogenesis of AH by mediating LPS induced liver injury. Design CCL20 gene expression and serum levels and their correlation with disease severity were assessed in patients with AH. Cellular sources of CCL20 and its biological effects were evaluated in vitro and in vivo in chronic, acute and acute-on-chronic experimental models of carbon tetrachloride and LPS induced liver injury. RNA interference technology was used to knockdown CCL20 in vivo. Results CCL20 hepatic and serum levels were increased in patients with AH and correlated with the degree of fibrosis, portal hypertension, endotoxaemia, disease severity scores and short term mortality. Moreover, CCL20 expression was increased in animal models of liver injury and particularly under acute-on-chronic conditions. Macrophages and hepatic stellate cells (HSCs) were identified as the main CCL20 producing cell types. Silencing CCL20 in vivo reduced LPS induced aspartate aminotransferase and lactate dehydrogenase serum levels and hepatic proinflammatory and profibrogenic genes. CCL20 induced proinflammatory and profibrogenic effects in cultured primary HSCs. Conclusions Our results suggest that CCL20 upregulation is strongly associated with LPS and may not only represent a new potential biomarker to predict outcome in patients with AH but also an important mediator linking hepatic inflammation, injury and fibrosis in AH.

Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.

Integrative microRNA profiling in alcoholic hepatitis reveals a role for microRNA-182 in liver injury and inflammation

Objective MicroRNAs (miRNAs) are well-known regulators of disease pathogenesis and have great potential as biomarkers and therapeutic targets. We aimed at profiling miRNAs in alcoholic hepatitis (AH) and identifying miRNAs potentially involved in liver injury. Design MiRNA profiling was performed in liver samples from patients with AH, alcohol liver disease, non-alcoholic steatohepatitis, HCV disease and normal liver tissue. Expression of miRNAs was assessed in liver and serum from patients with AH and animal models. Mimic and decoy miR-182 were used in vitro and in vivo to evaluate miR-182s biological functions. Results MiRNA expression profile in liver was highly altered in AH and distinctive from alcohol-induced cirrhotic livers. Moreover, we identified a set of 18 miRNAs predominantly expressed in AH as compared with other chronic liver conditions. Integrative miRNA-mRNA functional analysis revealed the association of AH-altered miRNAs with nuclear receptors, IGF-1 signalling and cholestasis. Interestingly, miR-182 was the most highly expressed miRNA in AH, which correlated with degree of ductular reaction, disease severity and short-term mortality. MiR-182 mimic induced an upregulation of inflammatory mediators in biliary cells. At experimental level, miR-182 was increased in biliary cells in mice fed with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet but not upregulated by alcohol intake or fibrosis. Inhibition of miR-182 in DDC-fed mice reduced liver damage, bile acid accumulation and inflammatory response. Conclusions AH is characterised by a deregulated miRNA profile, including miR-182, which is associated with disease severity and liver injury. These results highlight the potential of miRNAs as therapeutic targets and biomarkers in AH.

Adipocyte fatty-acid binding protein is overexpressed in cirrhosis and correlates with clinical outcomes

Fatty-acid-binding proteins (FABPs) are small intracellular proteins that coordinate lipid-mediated processes by targeting metabolic and immune response pathways. The aim of the study was to investigate plasma FABPs levels and their relationship with clinical outcomes in cirrhosis. Plasma levels of L-FABP1(liver and kidney), I-FABP2(intestine), and A-FABP4(adipocyte and macrophages) were measured in 274 patients with decompensated cirrhosis. Hepatic gene expression of FABPs was assessed in liver biopsies from patients with decompensated cirrhosis and in liver cell types from mice with cirrhosis. Immunohistochemistry of A-FABP4 in human liver biopsy was also performed. Plasma levels of FABPs were increased in patients with decompensated cirrhosis compared to those of healthy subjects (L-FABP1: 25 (17–39) vs 10 (9–17) ng/mL p = 0.001, I-FABP2: 1.1 (0.5–2.1) vs 0.6 (0.4–1) ng/mL p = 0.04 and A-FABP4: 37 (20–68) vs 16 (11–33) ng/mL p = 0.002), respectively. Increased A-FABP4 levels were associated with complications of cirrhosis, acute-on-chronic liver failure and poor survival. Hepatic A-FABP4 gene expression was upregulated in decompensated cirrhosis. Macrophages were the main liver cell that over-expressed A-FABP4 in experimental cirrhosis and increased A-FABP4 was found in macrophages of human biopsies by immunohistochemistry. A-FABP4 levels are increased in decompensated cirrhosis and correlate with poor outcomes. Liver macrophages appear to be the main source of A-FABP4 in decompensated cirrhosis.

Pentraxin‐3 modulates lipopolysaccharide‐induced inflammatory response and attenuates liver injury

Acute‐on‐chronic liver injury is characterized by an important inflammatory response frequently associated with endotoxemia. In this context, acute‐phase proteins such as Pentraxin‐3 (PTX3) are released; however, little is known about their role in chronic liver disease. The aim of this study was to elucidate the role of PTX3 in liver injury. The role of PTX3 was evaluated in cultured human cells, liver tissue slices, and mice with acute‐on‐chronic liver injury. PTX3 expression was assessed in tissue and serum samples from 54 patients with alcoholic hepatitis. PTX3 expression was up‐regulated in animal models of liver injury and strongly induced by lipopolysaccharide (LPS). Liver cell fractionation showed that macrophages and activated hepatic stellate cells were the main cell types expressing PTX3 in liver injury. Ex vivo and in vivo studies showed that PTX3 treatment attenuated LPS‐induced liver injury, inflammation, and cell recruitment. Mechanistically, PTX3 mediated the hepatic stellate cell wound‐healing response. Moreover, PTX3 modulated LPS‐induced inflammation in human primary liver macrophages and peripheral monocytes by enhancing a TIR domain–containing adapter‐inducing interferon–dependent response and favoring a macrophage interleukin‐10‐like phenotype. Additionally, hepatic and plasma PTX3 levels were increased in patients with alcoholic hepatitis, a prototypic acute‐on‐chronic condition; and its expression correlated with disease severity scores, endotoxemia, infections, and short‐term mortality, thus suggesting that expression of PTX3 found in patients could be a counterregulatory response to injury. Conclusion: Experimental and human evidence suggests that, in addition to being a potential biomarker for alcoholic hepatitis, PTX3 participates in the wound‐healing response and attenuates LPS‐induced liver injury and inflammation; therefore, administration of PTX3 could be a promising therapeutic strategy in acute‐on‐chronic conditions, particularly those associated with endotoxemia. (Hepatology 2017;66:953–968).

Pentraxin‐3 Modulates LPS‐induced Inflammatory Response and Attenuates Liver Injury

Acute‐on‐chronic liver injury is characterized by an important inflammatory response frequently associated with endotoxemia. In this context, acute‐phase proteins such as Pentraxin‐3 (PTX3) are released; however, little is known about their role in chronic liver disease. The aim of this study was to elucidate the role of PTX3 in liver injury. The role of PTX3 was evaluated in cultured human cells, liver tissue slices, and mice with acute‐on‐chronic liver injury. PTX3 expression was assessed in tissue and serum samples from 54 patients with alcoholic hepatitis. PTX3 expression was up‐regulated in animal models of liver injury and strongly induced by lipopolysaccharide (LPS). Liver cell fractionation showed that macrophages and activated hepatic stellate cells were the main cell types expressing PTX3 in liver injury. Ex vivo and in vivo studies showed that PTX3 treatment attenuated LPS‐induced liver injury, inflammation, and cell recruitment. Mechanistically, PTX3 mediated the hepatic stellate cell wound‐healing response. Moreover, PTX3 modulated LPS‐induced inflammation in human primary liver macrophages and peripheral monocytes by enhancing a TIR domain–containing adapter‐inducing interferon–dependent response and favoring a macrophage interleukin‐10‐like phenotype. Additionally, hepatic and plasma PTX3 levels were increased in patients with alcoholic hepatitis, a prototypic acute‐on‐chronic condition; and its expression correlated with disease severity scores, endotoxemia, infections, and short‐term mortality, thus suggesting that expression of PTX3 found in patients could be a counterregulatory response to injury. Conclusion: Experimental and human evidence suggests that, in addition to being a potential biomarker for alcoholic hepatitis, PTX3 participates in the wound‐healing response and attenuates LPS‐induced liver injury and inflammation; therefore, administration of PTX3 could be a promising therapeutic strategy in acute‐on‐chronic conditions, particularly those associated with endotoxemia. (Hepatology 2017;66:953–968).

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 -/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 -/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 -/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6 -/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression

Objective Alcoholic liver disease (ALD) is a leading cause of death among chronic liver diseases. However, its pathogenesis has not been completely established. MicroRNAs (miRNAs) are key contributors to liver diseases progression. This study investigated hepatocyte-abundant miRNAs dysregulated by ALD, its impact on hepatocyte injury and the underlying basis. Design Alcoholic hepatitis (AH) human and animal liver samples and hepatocytes were used to assess miR-148a levels. Pre-miR-148a was delivered specifically to hepatocytes in vivo using lentivirus. Immunoblottings, luciferase reporter assays, chromatin immunoprecipitation and immunofluorescence assays were carried out in cell models. Results The miRNA profile and PCR analyses enabled us to find substantial decrease of miR-148a in the liver of patients with AH. In mice subjected to Lieber-DeCarli alcohol diet or binge alcohol drinking, miR-148a levels were also markedly reduced. In cultured hepatocytes and mouse livers, alcohol exposure inhibited forkhead box protein O1 (FoxO1) expression, which correlated with miR-148a levels and significantly decreased in human AH specimens. FoxO1 was identified as a transcription factor for MIR148A transactivation. MiR-148a directly inhibited thioredoxin-interacting protein (TXNIP) expression. Consequently, treatment of hepatocytes with ethanol resulted in TXNIP overexpression, activating NLRP3 inflammasome and caspase-1-mediated pyroptosis. These events were reversed by miR-148a mimic or TXNIP small-interfering RNA transfection. Hepatocyte-specific delivery of miR-148a to mice abrogated alcohol-induced TXNIP overexpression and inflammasome activation, attenuating liver injury. Conclusion Alcohol decreases miR-148a expression in hepatocytes through FoxO1, facilitating TXNIP overexpression and NLRP3 inflammasome activation, which induces hepatocyte pyroptosis. Our findings provide information on novel targets for reducing incidence and progression of ALD.

Expression of microRNA‐155 in inflammatory cells modulates liver injury

MicroRNA 155 (miR‐155) is involved in immune and inflammatory diseases and is associated with liver fibrosis and steatohepatitis. However, the mechanisms involved in miR‐155 regulation of liver injury are largely unknown. The role of miR‐155 in acute liver injury was assessed in wild‐type (WT), miR‐155−/−, and miR‐155−/− mice transplanted with WT bone marrow. Additionally, miR‐155 expression was evaluated in liver tissue and peripheral blood mononuclear cells of patients with autoimmune hepatitis. Concanavalin A, but not acetaminophen, treatment increased the expression of miR‐155 in liver tissue of WT mice. Concanavalin A induced increases in cell death, liver aminotransferases, and expression of proinflammatory cytokines (chemokine [C‐X‐C motif] ligands 1, 5, 9, 10, and 11; chemokine [C‐C motif] ligands 2 and 20; and intercellular cell adhesion molecule 1) in miR‐155−/− compared to WT mice. Importantly, these animals showed a significant decrease in cluster of differentiation 4–positive/chemokine (C‐X‐C motif) receptor 3–positive and forkhead box p3–positive cell recruitment but no changes in other inflammatory cell populations. Mechanistically, miR‐155‐deficient regulatory T cells showed increased SH2 domain–containing inositol 5‐phosphatase 1 expression, a known target of miR‐155. Inhibition of SH2 domain–containing inositol 5‐phosphatase 1 in miR‐155−/− mice restored forkhead box p3 recruitment and reduced liver cytokine expression. Transplantation of bone marrow from WT animals into miR‐155−/− mice partially reversed the effect of concanavalin A on miR‐155−/− mice as assessed by proinflammatory cytokines and cell death protein expression. Patients with autoimmune hepatitis showed a marked increase in miR‐155 expression in the liver but reduced expression of miR‐155 in peripheral blood mononuclear cells. Conclusion: miR‐155 expression is altered in both liver tissue and circulating inflammatory cells during liver injury, thus regulating inflammatory cell recruitment and liver damage; these results suggest that maintaining miR‐155 expression in inflammatory cells might be a potential strategy to modulate liver injury. (Hepatology 2018).