Acquisition of epithelial plasticity in human chronic liver disease.

For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.

Spleen Size Does Not Correlate With Histological Stage of Liver Disease in People With Nonalcoholic Fatty Liver Disease.

Splenomegaly in the context of liver disease is classically associated with advanced cirrhosis and portal hypertension.1 More recently, we observed an increasing number of patients with splenomegaly and nonalcoholic fatty liver disease (NAFLD), but in whom intensive work-up revealed no evidence of advanced liver disease or portal hypertension. In this study, we found no correlation between spleen size and histological stage of NAFLD, and a strong correlation between body weight, height and serum high density lipoprotein (HDL) levels.

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

ABCB4 Mutations in Adults Cause a Spectrum Cholestatic Disorder Histologically Distinct from Other Biliary Disease.

BACKGROUND: Mutations in the ABCB4 gene are associated with failure of bile acid emulsification leading to cholestatic liver disease. Presentations range from progressive familial intrahepatic cholestasis type 3 (PFIC3) in childhood, to milder forms seen in adulthood. AIMS: We sought to characterize adult disease with particular reference to histology which has been hitherto poorly defined. METHODS: Four unrelated adults (three female, mean age 39 years) and three sisters presenting with cholestatic liver disease in adulthood, associated with variants in the ABCB4 gene, were identified. Clinical review and detailed blinded histopathological analysis were performed. RESULTS: Two novel pathogenic ABCB4 variants were identified: c.620 T > G, p.(Ile207Arg) and c.2301dupT, p.(Thr768TyrfsTer26). Sub-phenotypes observed included low-phospholipid-associated cholelithiasis syndrome (LPAC), intrahepatic cholestasis of pregnancy (ICP), drug-induced cholestasis, idiopathic adulthood ductopenia, and adult PFIC3. Of note, 5/7 had presented with gallstone complications (4 meeting LPAC definition) and 4/6 females had a history of ICP. Considerable overlap was observed phenotypically and liver transplantation was required in 3/7 of patients. Histologically, cases generally demonstrated ductopenia of the smaller tracts, mild non-ductocentric portal inflammation, bilirubinostasis, significant copper-associated protein deposition, and varying degrees of fibrosis. CONCLUSIONS: Adults with ABCB4 mutations may harbor a spectrum of cholestatic disease phenotypes and can progress to liver transplantation. We observed a distinct histological pattern which differs from classical biliary disease and describe two novel pathogenic ABCB4 variants. ABCB4 sequencing should be considered in patients with relevant cholestatic phenotypes and/or suggestive histology; accurate diagnosis can guide potential interventions to delay progression and inform family screening.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

Suppression of insulin-induced gene 1 (INSIG1) function promotes hepatic lipid remodelling and restrains NASH progression.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a silent pandemic associated with obesity and the metabolic syndrome, and also increases cardiovascular- and cirrhosis-related morbidity and mortality. A complete understanding of adaptive compensatory metabolic programmes that modulate non-alcoholic steatohepatitis (NASH) progression is lacking. METHODS AND RESULTS: Transcriptomic analysis of liver biopsies in patients with NASH revealed that NASH progression is associated with rewiring of metabolic pathways, including upregulation of de novo lipid/cholesterol synthesis and fatty acid remodelling. The modulation of these metabolic programmes was achieved by activating sterol regulatory element-binding protein (SREBP) transcriptional networks; however, it is still debated whether, in the context of NASH, activation of SREBPs acts as a pathogenic driver of lipotoxicity, or rather promotes the biosynthesis of protective lipids that buffer excessive lipid accumulation, preventing inflammation and fibrosis. To elucidate the pathophysiological role of SCAP/SREBP in NASH and wound-healing response, we used an Insig1 deficient (with hyper-efficient SREBPs) murine model challenged with a NASH-inducing diet. Despite enhanced lipid and cholesterol biosynthesis, Insig1 KO mice had similar systemic metabolism and insulin sensitivity to Het/WT littermates. Moreover, activating SREBPs resulted in remodelling the lipidome, decreased hepatocellular damage, and improved wound-healing responses. CONCLUSIONS: Our study provides actionable knowledge about the pathways and mechanisms involved in NAFLD pathogenesis, which may prove useful for developing new therapeutic strategies. Our results also suggest that the SCAP/SREBP/INSIG1 trio governs transcriptional programmes aimed at protecting the liver from lipotoxic insults in NASH.

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Pilot study of a noninvasive real-time optical backscatter probe in liver transplantation.

Transplantation of severely steatotic donor livers is associated with early allograft dysfunction and poorer graft survival. Histology remains the gold standard diagnostic of donor steatosis despite the lack of consensus definition and its subjective nature. In this prospective observational study of liver transplant patients, we demonstrate the feasibility of using a handheld optical backscatter probe to assess the degree of hepatic steatosis and correlate the backscatter readings with clinical outcomes. The probe is placed on the surface of the liver and emits red and near infrared light from the tip of the device and measures the amount of backscatter of light from liver tissue via two photodiodes. Measurement of optical backscatter (Mantel-Cox P < 0.0001) and histopathological scoring of macrovesicular steatosis (Mantel-Cox P = 0.046) were predictive of 5-year graft survival. Recipients with early allograft dysfunction defined according to both Olthoff (P = 0.0067) and MEAF score (P = 0.0097) had significantly higher backscatter levels from the donor organ. Backscatter was predictive of graft loss (AUC 0.75, P = 0.0045). This study demonstrates the feasibility of real-time measurement of optical backscatter in donor livers. Early results indicate readings correlate with steatosis and may give insight to graft outcomes such as early allograft dysfunction and graft loss.

The Alpha-1 Antitrypsin Polymer Load Correlates With Hepatocyte Senescence, Fibrosis Stage and Liver-Related Mortality.

BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is an important, inherited cause of chronic liver disease. Marked variation in fibrosis stages in patients with homozygous deficiency and those factors that determine whether heterozygous carriers develop liver fibrosis, remain unexplained. Murine studies implicate polymerized alpha-1 antitrypsin (AAT) within hepatocytes as pathogenic. AIMS AND METHODS: The relationship between the quantity of polymerized AAT within hepatocytes (polymer load), stage of hepatic fibrosis and liver-related clinical outcomes (death, evolution to hepatocellular carcinoma, or need for liver transplantation) were investigated using liver tissue from 92 patients at first presentation with either homozygous or heterozygous AATD. Further tissue-based studies were undertaken to determine if polymerized AAT was associated with failure of cell cycle progression, accelerated aging or hepatocyte senescence by immunohistochemical analysis. RESULTS: The AAT polymer load correlated closely with hepatic fibrosis stage and long-term clinical outcome, independent of homozygous or heterozygous status. AAT polymers within hepatocytes correlated closely with failure of cell cycle progression assessed using cell cycle phase markers, accelerated aging manifest as shortened telomeres and other markers consistent with hepatocyte senescence manifest as the presence of nuclear p21 expression and enlarged nuclei. The proportion of p21 positive hepatocytes or hepatocytes with enlarged nuclei correlated with hepatic fibrosis stage and the long-term clinical outcome. CONCLUSION: These data suggest that accumulation of AAT polymers within hepatocytes drives senescence. Quantitation of both the AAT polymer load or hepatocyte senescence markers correlated with hepatic fibrosis stage and the long-term clinical outcome. Either or both could be considered markers of disease severity and treatment response in clinical trials.

Pervasive lesion segregation shapes cancer genome evolution.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

OTULIN protects the liver against cell death, inflammation, fibrosis, and cancer.

Methionine-1 (M1)-linked polyubiquitin chains conjugated by the linear ubiquitin chain assembly complex (LUBAC) control NF-κB activation, immune homoeostasis, and prevents tumour necrosis factor (TNF)-induced cell death. The deubiquitinase OTULIN negatively regulates M1-linked polyubiquitin signalling by removing the chains conjugated by LUBAC, and OTULIN deficiency causes OTULIN-related autoinflammatory syndrome (ORAS) in humans. However, the cellular pathways and physiological functions controlled by OTULIN remain poorly understood. Here, we show that OTULIN prevents development of liver disease in mice and humans. In an ORAS patient, OTULIN deficiency caused spontaneous and progressive steatotic liver disease at 10-13 months of age. Similarly, liver-specific deletion of OTULIN in mice leads to neonatally onset steatosis and hepatitis, akin to the ORAS patient. OTULIN deficiency triggers metabolic alterations, apoptosis, and inflammation in the liver. In mice, steatosis progresses to steatohepatitis, fibrosis and pre-malignant tumour formation by 8 weeks of age, and by the age of 7-12 months the phenotype has advanced to malignant hepatocellular carcinoma. Surprisingly, the pathology in OTULIN-deficient livers is independent of TNFR1 signalling. Instead, we find that steatohepatitis in OTULIN-deficient livers is associated with aberrant mTOR activation, and inhibition of mTOR by rapamycin administration significantly reduces the liver pathology. Collectively, our results reveal that OTULIN is critical for maintaining liver homoeostasis and suggest that M1-linked polyubiquitin chains may play a role in regulation of mTOR signalling and metabolism in the liver.

Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.

Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Severe steroid-resistant anti-PD1 T-cell checkpoint inhibitor-induced hepatotoxicity driven by biliary injury.

INTRODUCTION: Hepatotoxicity from T-cell checkpoint blockade is an increasingly common immune-related adverse event, but remains poorly characterised and can be challenging to manage. Such toxicity is generally considered to resemble autoimmune hepatitis, although this assumption is extrapolated from limited clinicopathological reports of anti-cytotoxic T-lymphocyte-associated protein 4-induced hepatotoxicity. METHODS: Here we report, with full clinicopathological correlation, three cases of T-cell checkpoint inhibitor-induced hepatotoxicity associated with anti-programmed cell death protein 1 agents. RESULTS: We find that a major feature of these cases is biliary injury, including a unique case of vanishing bile duct syndrome, and that such toxicity was poorly responsive to long-term immunosuppression (corticosteroids and mycophenolate mofetil). Any potential benefits of long-term immunosuppression in these cases were outweighed by therapy-related complications. DISCUSSION: We discuss potential aetiologies and risk factors for immune-mediated biliary toxicity in the context of the limited literature in this field, and provide guidance for the investigation and supportive management of affected patients.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Lipid zonation and phospholipid remodeling in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) can progress from simple steatosis (i.e., nonalcoholic fatty liver [NAFL]) to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Currently, the driver for this progression is not fully understood; in particular, it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. In this study, we used dietary and genetic mouse models of NAFL and NASH and translated the results to humans by correlating the spatial distribution of lipids in liver tissue with disease progression using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples and observed partial to complete loss of lipid zonation in NASH. In addition, we found increased hepatic expression of genes associated with remodeling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species that promote inflammation and cell injury. The results of our immunohistochemistry analyses suggest that the zonal location of remodeling enzyme LPCAT2 plays a role in the change in spatial distribution for AA-containing lipids. This results in a cycle of AA-enrichment in pericentral hepatocytes, membrane release of AA, and generation of proinflammatory eicosanoids and may account for increased oxidative damage in pericentral regions in NASH. CONCLUSION: NAFLD is associated not only with lipid enrichment, but also with zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD. (Hepatology 2017;65:1165-1180).

Any value in a specialist review of liver biopsies? Conclusions of a 4-year review.

AIMS: Liver pathology is a challenging subspeciality, with histopathologists frequently seeking specialist opinions. This study aims to determine the impact of specialist reviews on the final diagnosis and patient management. METHODS AND RESULTS: Agreement with the initial reporting centre in the histopathological diagnosis of 1265 liver biopsies was determined. The nature of differences was explored in more depth for 103 discrepant cases. Differences in the histopathological interpretation were present in 749 of 1265 (59%) biopsies, of which 505 of 749 (67%) were predicted at the time of reporting to impact upon patient management. Agreement was good in cases with chronic viral hepatitis, fatty liver disease, malignancy and minimal pathological changes, while diagnostic differences occurred in more than 70% with biliary disease, autoimmune hepatitis or vascular/architectural changes. A clinical review of a subset of reports with histopathological differences predicted changes in patient management in 63 of 103 (61%). CONCLUSIONS: Clinically significant differences in liver biopsy interpretation between local pathologists and subspecialists are common. Diagnoses with frequent discrepancies, such as biliary disease, may benefit from a specialist review as standard when diagnosed initially, while cases requiring specialist advice from disease subgroups where discrepancies are less common, such as chronic viral hepatitis, could be selected during the clinicopathological conference process.

Endoplasmic Reticulum Stress Is Implicated in Intestinal Failure-Associated Liver Disease.

BACKGROUND: Intestinal failure-associated liver disease (IFALD) is the most serious consequence of long-term parenteral nutrition for intestinal failure. Little is known about the pathogenesis of IFALD, although many of the risk factors are also linked to endoplasmic reticulum stress (ERS). We propose that ERS may have a role in the development of IFALD. METHODS: Archived liver tissue from patients with early and late IFALD, as well as from normal controls, was used for RNA extraction and immunohistochemistry to demonstrate the presence of ERS markers. RESULTS: Mean relative RNA levels of glucose regulatory protein 78 in normal liver (n = 3), early IFALD (n = 15), and late IFALD (n = 5) were 0.5, 37.86, and 212.11, respectively. Mean relative expression of ERDj4 (ER DnaJ homologue 4, a downstream ERS effector) in normal liver, early IFALD, and late IFALD was 5.51, 216.68, and 213.22, respectively. The degree of splicing of X-box binding protein 1 in IFALD compared with normal liver was significantly higher (mean, 0.0779 normal, 0.102 early IFALD, 0.2063 late IFALD). CONCLUSIONS: This is the first description of ERS in IFALD. This information may open up new therapeutic possibilities in the form of chemical chaperones known to ameliorate ERS.