Biliary Cells Research Articles

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

Prom1+ Biliary Progenitor Cells Display Abnormal Cellular Polarity and Ciliopathy in Murine Biliary Atresia

INTRODUCTION: Although the pathogenesis of the extrahepatic bile duct (EHBD) occlusion in biliary atresia (BA), the leading cause of liver failure, is unclear, BA has been associated with genetic polymorphisms linked to ciliopathies. Prominin-1 (Prom1) promotes intrahepatic fibrosis in BA. PROM1+ biliary progenitor cells (PCs) regenerate the EHBD after injury, whereas Prom1 knockout (KO) mice have impaired EHBD regeneration. Prom1 has been shown to maintain PC ciliary structure. Thus, we hypothesize that Prom1 regulates PC polarity and ciliary structure integral to EHBD regeneration. METHODS: Wild-type (WT) and KO EHBDs 18 days after murine BA rhesus rotavirus (RRV) or saline injection at day of life 3 were collected and sectioned. WT and KO EHBDs were dissociated and grown in Matrigel. All were stained via immunofluorescence and imaged with confocal microscopy. Images were processed with Arivis Vision4D. Paired t-tests were performed for statistical analyses (p < 0.05). RESULTS: EHBDs of WT mice after RRV injury demonstrate normal cell polarity with cytoskeletal protein F-actin localized to the apical membrane, but KO EHBD do not. Further analysis of WT organoids demonstrates apical co-localization of PROM1 with F-actin, phosphorylated EZRIN, and cystic fibrosis transmembrane conductance regulator (CFTR). In contrast, KO organoids exhibit less apical expression of F-actin, pEZRIN, and CFTR compared with WT (F-actin: 12% vs 84%, p = 0.0367). KO also manifest shorter and fewer cilia by α-acetyl-tubulin staining (Figure).CONCLUSION:Prom1 maintains cellular polarity and ciliary structure of biliary PCs that may support EHBD regeneration. Prom1 dysfunction may be associated EHBD obliteration in BA.

A comprehensive transcriptomic comparison of hepatocyte model systems improves selection of models for experimental use

The myriad of available hepatocyte in vitro models provides researchers the possibility to select hepatocyte-like cells (HLCs) for specific research goals. However, direct comparison of hepatocyte models is currently challenging. We systematically searched the literature and compared different HLCs, but reported functions were limited to a small subset of hepatic functions. To enable a more comprehensive comparison, we developed an algorithm to compare transcriptomic data across studies that tested HLCs derived from hepatocytes, biliary cells, fibroblasts, and pluripotent stem cells, alongside primary human hepatocytes (PHHs). This revealed that no HLC covered the complete hepatic transcriptome, highlighting the importance of HLC selection. HLCs derived from hepatocytes had the highest transcriptional resemblance to PHHs regardless of the protocol, whereas the quality of fibroblasts and PSC derived HLCs varied depending on the protocol used. Finally, we developed and validated a web application (HLCompR) enabling comparison for specific pathways and addition of new HLCs. In conclusion, our comprehensive transcriptomic comparison of HLCs allows selection of HLCs for specific research questions and can guide improvements in culturing conditions.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential

Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers. In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. Invitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. Invivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver. A cell population with clonal growth and organoid formation capability, which has liver differentiation potency invitro and invivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver. Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans.

Novel parameter for cancer chemosensitivity to fibroblast growth factor receptor inhibitors.

Fibroblast growth factor receptor inhibitors (FGFRi) were introduced into clinical trials on several cancer types and found to be particularly efficacious on urothelial cancer and cholangiocarcinoma. Although many enrolled patients responded well in clinical trials, there were some patients who did not respond to FGFRi even though their tumors carried the genomic changes that met the enrollment criteria. As already established, fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR) share the downstream signaling pathway of MAPK activation. Accordingly, it is conceivable that targeted inhibition of FGFR alone could leave the MAPK signaling unaffected when the signaling through EGFR is relatively strong. To test this hypothesis, we calculated here the FGFR to EGFR mRNA ratio (F/E for short) of biliary tract and urothelial cancer cell lines utilized in preclinical studies. In six biliary tract cancer cell lines, two responsive lines had an F/E of 9.5 and 9.0, whereas the F/E of four nonresponsive lines was 0.1-1.8. In 22 urothelial cancer cell lines, four of the five responsive lines showed an F/E of 2.8-4.9 (median, 3.6), whereas the F/E range of 17 nonresponsive lines was 0.01-2.7 (median, 0.6) (p=0.004). We further investigated our 47 patient-derived colorectal cancer-stem cell spheroid lines. The 18 responsive lines showed relatively high F/E (median, 16.4), whereas 29 nonresponsive lines had low F/E (median, 9.2) (p=0.0006). These results suggest that F/E is another strong predictor of responses to FGFRi that is as useful as the current genomic criteria based solely on the FGFR genomic changes.

A missense mutation in the proprotein convertase gene furinb causes hepatic cystogenesis during liver development in zebrafish.

Hepatic cysts are fluid‐filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time‐lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole‐genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4‐phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.

Patch grafting of organoids of stem/progenitors into solid organs can correct genetic-based disease states

Patch grafting, a novel strategy for transplantation of stem/progenitor organoids into porcine livers, has been found successful also for organoid transplantation into other normal or diseased solid organs in pigs and mice. Each organoid contained ∼100 cells comprised of biliary tree stem cells (BTSCs), co-hepato/pancreatic stem/progenitors, and partnered with early lineage stage mesenchymal cells (ELSMCs), angioblasts and precursors to endothelia and stellate cells. Patch grafting enabled transplantation into livers or pancreases of ≥108th (pigs) or ≥106th−7th (mice) organoids/patch. Graft conditions fostered expression of multiple matrix-metalloproteinases (MMPs), especially secretory isoforms, resulting in transient loss of the organ's matrix-dictated histological features, including organ capsules, and correlated with rapid integration within a week of organoids throughout the organs and without emboli or ectopic cell distribution. Secondarily, within another week, there was clearance of graft biomaterials, followed by muted expression of MMPs, restoration of matrix-dictated histology, and maturation of donor cells to functional adult fates.The ability of patch grafts of organoids to rescue hosts from genetic-based disease states was demonstrated with grafts of BTSC/ELSMC organoids on livers, able to rescue NRG/FAH-KO mice from type I tyrosinemia, a disease caused by absence of fumaryl acetoacetate hydrolase. With the same grafts, if on pancreas, they were able to rescue NRG/Akita mice from type I diabetes, caused by a mutation in the insulin 2 gene. The potential of patch grafting for cell therapies for solid organs now requires translational studies to enable its adaptation and uses for clinical programs.

Ileal bile acid transporter inhibition in Cyp2c70 KO mice ameliorates cholestatic liver injury

Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.

Metabolic syndrome and cancer risk

Cancer has become a leading cause of death among patients with metabolic syndrome (MetS). The more components of MetS a patient has, the higher his risk of cancer. MetS is causally associated with colorectal, pancreatic, gallbladder, biliary, hepatocellular, gastric, esophageal, thyroid, breast, endometrial and renal cell cancers. MetS increases cancer mortality up to 2,4-fold. Intentional long-term weight loss reduces the excess cancer risk of obese MetS-patients. Both a low-risk lifestyle and cancer screening are effective and decrease the burden of cancer.

Abstract 5334: Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers

Abstract Background: Human epithelial growth factor receptor 2 (HER2) is a well-established therapeutic target in HER2-positive breast and gastric cancer. Recently, novel HER2-targeting agents are being developed after trastuzumab, and HER2-targeted therapies have been attempted in across solid tumor types, including biliary tract cancer. In these aspects, it is essential to understand and elucidate the resistant mechanism of the HER2-targeting strategies. Yes-associated protein (YAP), a transcription factor of the Hippo pathway, function as proto-oncoproteins by inducing target genes involved in cancer cell survival and proliferation (Zhao B, et al. Genes Develop 2008). Also, YAP is emerging as a resistance mechanism of cytotoxic and targeted drugs. In this study, we explore the role of YAP in overcoming resistance to trastuzumab in HER2-positive cancer cells. Methods: Four trastuzumab-resistant (HR) cell lines were established using HER2-postive gastric and biliary tract cancer cell lines (N87, SNU216, SNU2670, and SNU2773) (Jin MH, et al. Mol Cancer Ther. 2017). YAP siRNA and Verteporfin (YAP-TEAD inhibitor) were used to downregulate YAP. MTT assay, cell cycle analysis, western blot and migration assay were performed to analyze the antitumor effects through YAP downregulation. In order to elucidate tumor cell immune-modulation by YAP, human PBMC co-culture was used and immune markers, such as programmed death-ligand 1 (PD-L1), were analyzed in HR cell lines. Results: Increase of receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) expression was observed in the HR cells. Relatively high expression of pYAP, YAP, and transcriptional co-activator with PDZ-binding motif (TAZ) were observed in the HR cells (SNU216HR, SNU2670HR and SNU2773HR) compared to parental cells. Reducing overexpressed YAP in HR cells resulted in tumor cell growth inhibition. In cell cycle analysis, the increase of subG1 phase through YAP downregulation was observed, and cyclinD, B, A and BCL-XL were effectively decreased. Migration was also inhibited by the decrease in YAP expression. By verteporfin treatment, changes in immune markers including increase of CD80 and decrease of PD-L1 was found. In addition, p-STAT3 and IL6, Which regulate PD-L1, were also decreased through the reduction of YAP. In HR cells treated with siYAP, there was a tendency to increase CD8+ T cells upon PBMC co-culture. Conclusion: YAP is upregulated in trastuzumab-resistant (HR) cells and upregulated YAP induce PD-L1 expression. Inhibition of YAP shows anti-tumor effects and modulates immune status. Collectively, our results suggest that inhibition of YAP is one of promising strategies to overcome trastuzumab resistance in HER2-positive cancers. Citation Format: Ah Rong Nam, Jeesun Yoon, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Tae-Yong Kim, Do-Youn Oh. Targeting the hippo transducer YAP overcomes trastuzumab-resistance in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5334.

Abstract 2618: Convergent MAPK pathway alterations mediate acquired resistance to FGFR inhibitors in cholangiocarcinoma with FGFR fusions/rearrangements

Abstract Purpose: Genomic aberrations in fibroblast growth factor receptor (FGFR) are oncogenic drivers in several cancers. FGFR inhibitors (FGFRi) have demonstrated antitumor activity in cholangiocarcinoma (CCA) with FGFR2 fusions/rearrangements, though acquired resistance remains a therapeutic challenge and has been linked to FGFR2 mutations other than fusions/rearrangements. We sought to investigate mechanisms of acquired resistance to FGFRi and approaches to overcome resistance. Methods: Longitudinal plasma samples were collected from patients with FGFR pathway alterations enrolled in the futibatinib phase I trial (NCT02052778) and sequenced using a targeted, 73-gene panel. A separate retrospective analysis was conducted to evaluate possible evolution of genomic aberrations in CCA patients with FGFR2 fusion/rearrangement who had additional tumor and/or plasma next-generation sequencing (NGS) following FGFRi therapy. To assess the efficacy of futibatinib in cells with FGFR2 fusions in vitro, a FGFR2-BICC1 fusion H69 cholangiocyte cell line was developed. MAPK pathway alterations (BRAF_V600E or KRAS_G12D) were introduced to determine the impact of these co-alterations on FGFRi sensitivity, and combinations were tested to determine if efficacy could be enhanced. Cell viability assays, colony formation assays, and western blots were utilized to determine the effects of these agents in engineered cells. Results: A total of 58 plasma samples were collected from 17 patients with FGFR pathway alterations who were enrolled in the futibatinib phase I trial, including 13 (76.5%) of which had CCA. One patient with a FGFR2-CTNNA3 fusion who had NRAS G12D and BRAF A694T at baseline which were undetected during treatment had a dramatic increase in their variant allele frequency (VAF) upon progression (4.2%-0.0%-100% and 2.8%-0.0%-50.9%, respectively). Further, additional MAPK alterations were detected at time of progression, including BRAF V600E, NRAS Q61K, NRAS G12C, NRAS G13D and KRAS G12K mutations. In our separate retrospective series, of 17 patients who underwent repeat tumor and/or plasma NGS following treatment with one or more FGFRi, 10 (58.8%) had newly detectable alterations in MAPK pathway genes, 10 (58.8%) had new FGFR2 alterations, and 7 (41.2%) developed new alterations in both FGFR2 and MAPK pathway genes. In vitro studies demonstrated that in isogenic H69 biliary cell lines, introduction of FGFR2-BICC1 robustly sensitized to FGFRi when compared to a parental cell line, which was blunted by the introduction of secondary KRAS_G12D or BRAF_V600E mutations. Conclusions: Convergent genomic evolution in the MAPK pathway may be a potential mechanism of acquired resistance to FGFRi therapy. Work is ongoing to determine if targeting co-alterations may enhance the efficacy of FGFRi in FGFR2-fusion driven malignancies. Citation Format: TImothy P. DiPeri, Ming Zhao, Tyler Moss, Michael Kahle, Payal Rauli, Sunyoung S. Lee, Abdel Halim, Hiroshi Hirai, Volker Wacheck, Karim Benhadji, Jordi Rodon, Milind Javle, Funda Meric-Bernstam. Convergent MAPK pathway alterations mediate acquired resistance to FGFR inhibitors in cholangiocarcinoma with FGFR fusions/rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2618.

Sorafenib Chemosensitization by Caryophyllane Sesquiterpenes in Liver, Biliary, and Pancreatic Cancer Cells: The Role of STAT3/ABC Transporter Axis.

A combination of anticancer drugs and chemosensitizing agents has been approached as a promising strategy to potentiate chemotherapy and reduce toxicity in aggressive and chemoresistant cancers, like hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and pancreatic ductal adenocarcinoma (PDAC). In the present study, the ability of caryophyllane sesquiterpenes to potentiate sorafenib efficacy was studied in HCC, CCA, and PDAC cell models, focusing on the modulation of STAT3 signaling and ABC transporters; tolerability studies in normal cells were also performed. Results showed that the combination of sorafenib and caryophyllane sesquiterpenes synergized the anticancer drug, especially in pancreatic Bx-PC3 adenocarcinoma cells; a similar trend, although with lower efficacy, was found for the standard ABC transporter inhibitors. Synergistic effects were associated with a modulation of MDR1 (or Pgp) and MRP transporters, both at gene and protein level; moreover, activation of STAT3 cascade and cell migration appeared significantly affected, suggesting that the STAT3/ABC-transporters axis finely regulated efficacy and chemoresistance to sorafenib, thus appearing as a suitable target to overcome drawbacks of sorafenib-based chemotherapy in hepato-biliary-pancreatic cancers. Present findings strengthen the interest in caryophyllane sesquiterpenes as chemosensitizing and chemopreventive agents and contribute to clarifying drug resistance mechanisms in HCC, CCA, and PDAC cancers and to developing possible novel therapeutic strategies.

Benign focal liver lesions: The role of magnetic resonance imaging.

Liver lesions are common findings in radiologists’ daily routine. They are a complex category of pathology that range from solitary benign lesions to primary liver cancer and liver metastases. Benign focal liver lesions can arise from different liver cell types: Epithelial (hepatocytes and biliary cells) and nonepithelial (mesenchymal cells). Liver magnetic resonance imaging (MRI) is a fundamental radiological method in these patients as it allows with its multiparametric approach optimal non-invasive tissue characterization. Furthermore, advanced liver MRI techniques such as diffusion-weighted imaging and hepatobiliary contrast agents have improved the detection of focal liver lesions and can be highly effective in differentiating pseudotumor from tumors, as well as benign from malignant lesions, and can also be used for differential diagnosis. Although histological examination can be useful in making a definitive diagnosis, MRI is an important modality in the diagnosis of liver lesions with a significant impact on patient care. This aim of this review is to provide a comprehensive overview of benign liver lesions on MRI.

Dysregulation of the Scribble/YAP/β-catenin axis sustains the fibroinflammatory response in a PKHD1-/- mouse model of congenital hepatic fibrosis.

Congenital hepatic fibrosis (CHF), a genetic cholangiopathy characterized by fibropolycystic changes in the biliary tree, is caused by mutations in the PKHD1gene, leading to defective fibrocystin (FPC), changes in planar cell polarity (PCP) and increased β-catenin-dependent chemokine secretion. In this study, we aimed at understanding the role of Scribble (a protein involved in PCP), Yes-associated protein (YAP), and β-catenin in the regulation of the fibroinflammatory phenotype of FPC-defective cholangiocytes. Immunohistochemistry showed that compared with wild type (WT) mice, in FPC-defective (Pkhd1del4/del4 ) mice nuclear expression of YAP/TAZ in cystic cholangiocytes, significantly increased and correlated with connective tissue growth factor (CTGF) expression and pericystic fibrosis, while Scribble expression on biliary cyst cells was markedly decreased. Cholangiocytes isolated from WT mice showed intense Scribble immunoreactivity at the membrane, but minimal nuclear expression of YAP, which conversely increased, together with CTGF, after small interfering RNA (siRNA) silencing of Scribble. In FPC-defective cholangiocytes, inhibition of YAP nuclear import reduced β-catenin nuclear expression, and CTGF, integrin β6, CXCL1, and CXCL10mRNA levels, whereas inhibition of β-catenin signaling did not affect nuclear translocation of YAP. Notably, siRNA silencing of Scribble and YAP in WT cholangiocytes mimics the fibroinflammatory changes of FPC-defective cholangiocytes. Conditional deletion of β-catenin in Pkhd1del4/del4 mice reduced cyst growth, inflammation and fibrosis, without affecting YAP nuclear expression. In conclusion, the defective anchor of Scribble to the membrane facilitates the nuclear translocation of YAP and β-catenin with gain of a fibroinflammatory phenotype. The Scribble/YAP/β-catenin axis is a critical factor in the sequence of events linking the genetic defect to fibrocystic trait of cholangiocytes in CHF.

Differential responses of receptor tyrosine kinases C‐Met and EGFR in mice

In previous studies, we have demonstrated the critical roles of EGFR and HGFR (c‐Met) for liver regeneration and for conversion of hepatocytes to cholangiocytes by a “clean” partial hepatectomy model, in which hepatocytes and cholangiocytes grow rapidly and synchronously. In contrast, the vast majority of liver diseases involve a prolonged pathologic duration, therefore multiple types of cells participate and play different roles in the variant stages. However, it is not fully understood how EGFR and Met play their roles in this complicated setting. In this study, we explore the role of MET and EGFR signaling pathways in biliary injury after common bile duct ligation. Mice with inhibition of EGFR signaling alone (EGFRi) showed abolished hepatocyte proliferation and biliary duct cell proliferation (Ki67 immunochemistry staining) at 2, 5, and 7 days after BDL. However, Met knockout mice (KO) showed increased proliferation of cholangiocytes at 2, 5, and 7 days. Met KO mice subjected to BDL surgery had an increase of the CK19‐ marked ductular reaction at 2, 5, and 7 days. In contrast, EGFRi mice showed a reduced ductular reaction at the same time point. There was enhanced immunohistochemistry for Sox9 in EGFRi mice, but absence of its expression in Met KO mice at 7 days post‐BDL. From western blot results of total liver tissue lysate, we found increased EpCAM expression after BDL at 7 days in Met KO but not in EGFRi mice. In addition, we also observed that both p‐Merlin and cleaved p‐Mst1, components of the Hippo pathway, decreased only in Met knockout mice. Mice with combined disruption of Met and EGFR signaling became very ill by 24 hours and died at 1 to 2 days after bile duct ligation. From Kaplan‐Meier analysis, both EGFR inhibition and Met knockout mice had decreased survival rates starting at 11 days, though the survival rate of Met KO mice was overall much better than EGFRi mice by around 40%. We also observed that p‐Ezrin (Thr567) was downregulated in either Met KO or EGFRi mice, while the total Ezrin expression did not change.ConclusionsOur study demonstrates that both c‐Met and EGFR control biliary response to bile duct ligation. The cholangiocyte responses, however, were very different between these two receptors. Their combined disruption led to acute liver failure and early death following bile duct ligation. The receptor tyrosine kinases c‐Met and EGFR may play different roles in the biliary injury‐repair responses, including proliferation, trans‐differentiation, and bi‐potential progenitor cells activation.

β‐catenin Overexpressing Hepatocytes Reduce Bile Stasis by Contributing to Cholangiocyte‐Like Phenotype in Murine Model of Intrahepatic Cholestasis

Study ObjectiveTo investigate the role of β‐catenin overexpression in hepatocytes (HC) in hepatocyte‐to‐biliary reprogramming during intrahepatic cholestasis.HypothesisWe previously reported that mice transgenic for Ser‐45 mutant β‐catenin (TG) show increased hepatocyte‐to‐biliary reprogramming after administration of the porphyrinogenic biliary toxin 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet for 1 month. However, whether these cholangiocyte‐like hepatocytes fully convert into cholangiocytes (CC) or maintain an intermediate phenotype, remains unclear. We hypothesized that TG livers will have more HC‐derived CC than wild type and contribute to functional de novo biliary branches that will alleviate disease severity.MethodsAge matched WT control (Con) and TG, both containing ROSA26‐stopflox/flox, were injected with AAV8‐TBG‐Cre to permanently label hepatocytes with EYFP and then fed 0.1% DDC diet for different time points ‐ 30d, 60d, 90d, 120d, 150d. Before sacrifice, bile flow analysis was measured for 1h to obtain flow rate and bile was collected. Liver histology and serum biochemistry were analyzed for fibrosis and parameters of injury. Porphyrin measurement was assessed in the liver tissues. Relative gene expression for bile transporter genes and immunofluorescence staining were performed to determine the expression of biliary markers.ResultsTG mice showed significantly increased bile flow rate as compared to Con mice after 150d of DDC diet. Liver histology and porphyrin measurement analysis showed significantly less porphyrin accumulation in TG as compared to the Con. Analysis of bile homeostasis and transport genes demonstrated comparable levels of expression in TG and Con mice suggesting a possible mechanism by which HC contribute to bile duct formation and thus, increase bile flow rate observed in TG. HC‐derived ducts positive for both EYFP and Sox9 were observed more frequently in TG mice compared to Con. Nevertheless, serum biochemistry did not show any differences in hepatic and biliary injury in these groups.ConclusionsMice with β‐catenin overexpressing HC showed significantly improved bile flow rate and reduced porphyrin accumulation in response to the biliary toxin DDC. Using HC fate‐tracing, we observed increased biliary cell markers in TG hepatocytes that is suggestive of the mechanism of enhanced bile flow. Further studies would be focused on assessing the functional comparison of these HC‐derived CC to native CC and evaluate if β‐catenin overexpression mediates similar biliary reprogramming in response to short‐term DDC diet.

Deletion of kif3a in CK19 positive cells leads to primary cilia loss, biliary cell proliferation and cystic liver lesions in TAA-treated mice.

Loss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood. Primary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3af/f;CK19CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays. At the end of 20weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro. TAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.

Bile acid-sensitive tuft cells regulate biliary neutrophil influx.

Inflammation and dysfunction of the extrahepatic biliary tree are common causes of human pathology, including gallstones and cholangiocarcinoma. Despite this, we know little about the local regulation of biliary inflammation. Tuft cells, rare sensory epithelial cells, are particularly prevalent in the mucosa of the gallbladder and extrahepatic bile ducts. Here, we show that biliary tuft cells express a core genetic tuft cell program in addition to a tissue-specific gene signature and, in contrast to small intestinal tuft cells, decreased postnatally, coincident with maturation of bile acid production. Manipulation of enterohepatic bile acid recirculation revealed that tuft cell abundance is negatively regulated by bile acids, including in a model of obstructive cholestasis in which inflammatory infiltration of the biliary tree correlated with loss of tuft cells. Unexpectedly, tuft cell-deficient mice spontaneously displayed an increased gallbladder epithelial inflammatory gene signature accompanied by neutrophil infiltration that was modulated by the microbiome. We propose that biliary tuft cells function as bile acid-sensitive negative regulators of inflammation in biliary tissues and serve to limit inflammation under homeostatic conditions.

Cysteinyl leukotrienes and acetylcholine are biliary tuft cell cotransmitters.

The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.

Serum keratin 19 (CYFRA21-1) is a prognostic biomarker in severe alcoholic hepatitis.

Up to 30% of patients with severe alcoholic hepatitis (sAH) die within 3 months of presentation. The degree of ductular reaction, characterized by accumulation of biliary and liver progenitor cells, confers a poor prognosis. Keratin fragments are established serological surrogates of liver injury and keratin 19 (K19) is a histological marker of the ductular reaction. We assessed the relationship between serum K19 levels (viz. CYFRA21-1), histology and outcome in patients with sAH. Serum CYFRA21-1 was quantified in pre-treatment serum samples from 824 patients enrolled in the STOPAH trial. The cohort was randomly divided into two groups to test mortality associations; histological analyses were performed using the 87 cases with suitable samples. CYFRA21-1 levels were elevated in sAH and strongly predicted alcoholic steatohepatitis (ASH) on biopsy (area under the receiver operated characteristic [AUROC] 0.785 [95% Confidence Interval 0.602-0.967]) and 90-day survival (AUROC 0.684/0.693). The predictive ability of CYFRA21-1 was comparable with the model of end-stage liver disease (MELD) score and was independently associated with survival in multivariable analysis. CYFRA21-1 moderately correlated with hepatocellular injury markers M30/M65 but displayed a more robust predictive ability. A combination of MELD and CYFRA21-1 conferred a modest improvement in the AUROC value (0.731/0.743). In sAH serum, CYFRA21-1 levels associate with the presence of ASH on biopsy and independently predict 90-day survival. As a single marker performance is comparable to established scoring systems. Therefore, CYFRA21-1, which is available in many clinical laboratories, may become a useful component of prognostic models.