Hepatic progenitor cell-originated ductular reaction facilitates liver fibrosis through activation of hedgehog signaling.

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-shGli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl4/2-AAF-treated), Μdr2-/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl4-treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro. Enhanced Gli1 expression was identified in KRT19+-reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-shGli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate-stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis.

The crosstalk between cholangiocytes and hepatic stellate cells promotes the progression of epithelial-mesenchymal transition and periductal fibrosis during Clonorchis sinensis infection.

ABSTRACT: BACKGROUND: Clonorchis sinensis infection is one of the risk factors that provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis and even cholangiocarcinoma (CCA). Disrupted or aberrant intercellular communication among liver-constituting cells leads to pathological states that cause various hepatic diseases. This study was designed to investigate the pathological changes caused by C. sinensis excretory-secretory products (ESPs) in non-cancerous human cell lines (cholangiocytes [H69 cell line] and human hepatic stellate cells [LX2 cell line]) and their intercellular crosstalk, as well the pathological changes in infected mouse liver tissues. METHODS: The cells were treated with ESPs, following which transforming growth factor beta 1 (TGF-ß1) and interleukin-6 (IL-6) secretion levels and epithelial-mesenchymal transition (EMT)- and fibrosis-related protein expression were measured. The ESP-mediated cellular motility (migration/invasion) between two cells was assessed using the Transwell and three-dimensional microfluidic assay models. The livers of C. sinensis-infected mice were stained using EMT and fibrotic marker proteins. RESULTS: Treatment of cells with ESPs increased TGF-ß1 and IL-6 secretion and the expression of EMT- and fibrosis-related proteins. The ESP-mediated mutual cell interaction further affected the cytokine secretion and protein expression levels and promoted cellular motility. N-cadherin overexpression and collagen fiber deposition were observed in the livers of C. sinensis-infected mice. CONCLUSIONS: These findings suggest that EMT and biliary fibrosis occur through intercellular communication between cholangiocytes and hepatic stellate cells during C. sinensis infection, promoting malignant transformation and advanced hepatobiliary abnormalities.

Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

Cholangiocyte organoids to study drug-induced injury.

BACKGROUND: Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. METHODS: Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. RESULTS: CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. CONCLUSION: These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.

Modulation of hepatic cellular tight junctions via coculture with cholangiocytes enables non-destructive bile recovery.

Estimation of the biliary clearance of drugs and their metabolites in humans is crucial for characterizing hepatobiliary disposition and potential drug-drug interactions. Sandwich-cultured hepatocytes, while useful for in vitro bile analysis, require cell destruction for bile recovery, limiting long-term or repeated dose drug effect evaluations. To overcome this limitation, we investigated the feasibility of coculturing a human hepatic carcinoma cell line (HepG2-NIAS cells) and a human cholangiocarcinoma cell line (TFK-1 cells) using the collagen vitrigel membrane in a variety of coculture configurations. The coculture configuration with physiological bile flow increased the permeability of fluorescein-labeled bile acids (CLF) across the HepG2-NIAS cell layer by approximately 1.2-fold compared to the HepG2-NIAS monoculture. This enhancement was caused by paracellular leakage due to the loosened tight junctions of HepG2-NIAS, confirmed by the use of an inhibitor for bile acid transporters, the increase of permeability of dextran, and the decrease of the transepithelial electrical resistance (TEER) value. Based on the results of loosening hepatic tight junctions via coculture with TFK-1 in the CLF permeability assay, we next attempted to collect the CLF accumulated in the bile canaliculi of HepG2-NIAS. The recovery of the CLF accumulated in the bile canaliculi was increased 1.4 times without disrupting hepatic tight junctions by the coculture of HepG2-NIAS cells and TFK-1 cells compared to the monoculture of HepG2-NIAS cells. This non-destructive bile recovery has the potential as a tool for estimating the biliary metabolite and provides valuable insights to improve in vitro bile analysis.

Spatially segregated defects and IGF1-responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome.

BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1Ndr/Ndr mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1Ndr/Ndr mice, can improve biliary outcomes. METHODS: Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1+/+ and Jag1Ndr/Ndr livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining. RESULTS: Cell growth assays and transcriptomics demonstrated that Jag1Ndr/Ndr ICOs were less proliferative than Jag1+/+ ICOs. IGF1 specifically rescued survival and growth of Jag1Ndr/Ndr pICOs. Jag1Ndr/Ndr hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1Ndr/Ndr hICOs) and in vivo (Jag1Ndr/Ndr hilar portal tracts) analyses revealed ectopic HNF4a+ hepatocytes. CONCLUSIONS: Hilar and peripheral Jag1Ndr/Ndr ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1Ndr/Ndr pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.

Diagnostic yield and novel candidate genes by next generation sequencing in 166 children with intrahepatic cholestasis.

BACKGROUND AND AIMS: Cholestatic liver disease is a leading referral to pediatric liver transplant centers. Inherited disorders are the second most frequent cause of cholestasis in the first month of life. METHODS: We retrospectively characterized the genotype and phenotype of 166 participants with intrahepatic cholestasis, and re-analyzed phenotype and whole-exome sequencing (WES) data from patients with previously undetermined genetic etiology for newly published genes and novel candidates. Functional validations of selected variants were conducted in cultured cells. RESULTS: Overall, we identified disease-causing variants in 31% (52/166) of our study participants. Of the 52 individuals, 18 (35%) had metabolic liver diseases, 9 (17%) had syndromic cholestasis, 9 (17%) had progressive familial intrahepatic cholestasis, 3 (6%) had bile acid synthesis defects, 3(6%) had infantile liver failure and 10 (19%) had a phenocopy of intrahepatic cholestasis. By reverse phenotyping, we identified a de novo variant c.1883G > A in FAM111B of a case with high glutamyl transpeptidase (GGT) cholestasis. By re-analyzing WES data, two patients were newly solved, who had compound heterozygous variants in recently published genes KIF12 and USP53, respectively. Our additional search for novel candidates in unsolved WES families revealed four potential novel candidate genes (NCOA6, CCDC88B, USP24 and ATP11C), among which the patients with variants in NCOA6 and ATP11C recapitulate the cholestasis phenotype in mice models. CONCLUSIONS: In a single-center pediatric cohort, we identified monogenic variants in 22 known human intrahepatic cholestasis or phenocopy genes, explaining up to 31% of the intrahepatic cholestasis patients. Our findings suggest that re-evaluating existing WES data from well-phenotyped patients on a regular basis can increase the diagnostic yield for cholestatic liver disease in children.

Autophagy impairment in human bile duct carcinoma cells.

Bile duct epithelial cells, named cholangiocytes, may undergo a neoplastic transformation leading to cholangiocarcinoma. The role autophagy plays in cancer is still debated and few information are available in cholangiocarcinoma. We report in vitro data, at least in part validated in vivo,i ndicating that autophagy is impaired in intrahepatic cholangiocarcinoma cells, as compared to healthy cholangiocytes, evaluated through LC3II and p62 Western blot analyses. Autophagy impairment was found to be associated with low expression of TFEB protein and high expression of three proteins i.e., c-FLIP, caspase-10 and cleaved BCLAF-1, as compared to healthy cholangiocytes. We highlight biological effects of autophagy impairment in cholangiocarcinoma showing that autophagy induction, via rapamycin, as well as caspase inhibition, via Q-VD-OPh, are able to reduce proliferation marker PCNA level, colony size and protein content of cultured cholangiocarcinoma cells. The increased protein expression of p62, c-FLIP, caspase-10 observed in vitro in cholangiocarcinoma cells was paralleled by significant increase at gene expression levels in vivo; in fact, significant increase of transcript levels of p62, c-FLIP and caspase-10 was observed in 34 biopsies from human cholangiocarcinoma patients compared to 9 biopsies from 9 healthy controls, as reported in the GEPIA2 public database. The significant increase of p62 level in cholangiocarcinoma was found as a relatively uncommon finding in solid cancers, since it was also found in only 7 cancer types out of 31 cancer types investigated, including melanoma and hepatocarcinoma. In conclusion, we present data suggesting a molecular machinery controlling autophagy in cholangiocytes and autophagy impairment in cholangiocarcinoma.

Inhibiting Wnt Signaling Reduces Cholestatic Injury by Disrupting the Inflammatory Axis.

BACKGROUND & AIMS: ß-Catenin, the effector molecule of the Wnt signaling pathway, has been shown to play a crucial role in bile acid homeostasis through direct inhibition of farnesoid X receptor (FXR), which has pleiotropic effects on bile acid homeostasis. We hypothesize that simultaneous suppression of ß-catenin signaling and activation of FXR in a mouse model of cholestasis will reduce injury and biliary fibrosis through inhibition of bile acid synthesis. METHODS: To induce cholestasis, we performed bile duct ligation (BDL) on wild-type male mice. Eight hours after surgery, mice received FXR agonists obeticholic acid, tropifexor, or GW-4064 or Wnt inhibitor Wnt-C59. Severity of cholestatic liver disease and expression of target genes were evaluated after either 5 days or 12 days of treatment. RESULTS: We found that although the FXR agonists worsened BDL-induced injury and necrosis after 5 days, Wnt-C59 did not. After 12 days of BDL, Wnt-C59 treatment, but not GW-4064 treatment, reduced both the number of infarcts and the number of inflammatory cells in liver. RNA sequencing analysis of whole livers revealed a notable suppression of nuclear factor kappa B signaling when Wnt signaling is inhibited. We then analyzed transcriptomic data to identify a cholangiocyte-specific signature in our model and demonstrated that Wnt-C59-treated livers were enriched for genes expressed in quiescent cholangiocytes, whereas genes expressed in activated cholangiocytes were enriched in BDL alone. A similar decrease in biliary injury and inflammation occurred in Mdr2 KO mice treated with Wnt-C59. CONCLUSIONS: Inhibiting Wnt signaling suppresses cholangiocyte activation and disrupts the nuclear factor kappa B-dependent inflammatory axis, reducing cholestatic-induced injury.

Cellular heterogeneity and plasticity during NAFLD progression.

Nonalcoholic fatty liver disease (NAFLD) is a progressive liver disease that can progress to nonalcoholic steatohepatitis (NASH), NASH-related cirrhosis, and hepatocellular carcinoma (HCC). NAFLD ranges from simple steatosis (or nonalcoholic fatty liver [NAFL]) to NASH as a progressive form of NAFL, which is characterized by steatosis, lobular inflammation, and hepatocellular ballooning with or without fibrosis. Because of the complex pathophysiological mechanism and the heterogeneity of NAFLD, including its wide spectrum of clinical and histological characteristics, no specific therapeutic drugs have been approved for NAFLD. The heterogeneity of NAFLD is closely associated with cellular plasticity, which describes the ability of cells to acquire new identities or change their phenotypes in response to environmental stimuli. The liver consists of parenchymal cells including hepatocytes and cholangiocytes and nonparenchymal cells including Kupffer cells, hepatic stellate cells, and endothelial cells, all of which have specialized functions. This heterogeneous cell population has cellular plasticity to adapt to environmental changes. During NAFLD progression, these cells can exert diverse and complex responses at multiple levels following exposure to a variety of stimuli, including fatty acids, inflammation, and oxidative stress. Therefore, this review provides insights into NAFLD heterogeneity by addressing the cellular plasticity and metabolic adaptation of hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells during NAFLD progression.

Galanin System in the Human Bile Duct and Perihilar Cholangiocarcinoma.

BACKGROUND: Perihilar cholangiocarcinoma (pCCA) is characterised by poor outcomes. Early diagnosis is essential for patient survival. The peptide galanin (GAL) and its receptors GAL1-3 are expressed in various tumours. Detailed characterisation of the GAL system in pCCA is lacking. Our study sought to characterise GAL and GAL1-3 receptor (GAL1-3-R) expression in the healthy human bile duct, in cholestasis and pCCA. METHODS: Immunohistochemical staining was performed in healthy controls (n = 5) and in the peritumoural tissues (with and without cholestasis) (n = 20) and tumour tissues of pCCA patients (n = 33) using validated antibodies. The score values of GAL and GAL1-3-R expression were calculated and statistically evaluated. RESULTS: GAL and GAL1-R were expressed in various bile duct cell types. GAL2-R was only slightly but still expressed in almost all the examined tissues, and GAL3-R specifically in cholangiocytes and capillaries. In a small pCCA patient cohort (n = 18), high GAL expression correlated with good survival, whereas high GAL3-R correlated with poor survival. CONCLUSIONS: Our in-depth characterisation of the GAL system in the healthy human biliary duct and pCCA in a small patient cohort revealed that GAL and GAL3-R expression in tumour cells of pCCA patients could potentially represent suitable biomarkers for survival.

Proteomic Modulation in TGF-ß-Treated Cholangiocytes Induced by Curcumin Nanoparticles.

Curcumin is a natural polyphenol that exhibits a variety of beneficial effects on health, including anti-inflammatory, antioxidant, and hepato-protective properties. Due to its poor water solubility and membrane permeability, in the present study, we prepared and characterized a water-stable, freely dispersible nanoformulation of curcumin. Although the potential of curcumin nanoformulations in the hepatic field has been studied, there are no investigations on their effect in fibrotic pathological conditions involving cholangiocytes. Exploiting an in vitro model of transforming growth factor-ß (TGF-ß)-stimulated cholangiocytes, we applied the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomic approaches to study the proteome modulation induced by curcumin nanoformulation. Our results confirmed the well-documented anti-inflammatory properties of this nutraceutic, highlighting the induction of programmed cell death as a mechanism to counteract the cellular damages induced by TGF-ß. Moreover, curcumin nanoformulation positively influenced the expression of several proteins involved in TGF-ß-mediated fibrosis. Given the crucial importance of deregulated cholangiocyte functions during cholangiopathies, our results provide the basis for a better understanding of the mechanisms associated with this pathology and could represent a rationale for the development of more targeted therapies.

Liver cell therapies: cellular sources and grafting strategies.

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.

Morphological aspects of small-duct cholangiopathies: A minireview.

The biliary system consists of intrahepatic and extrahepatic bile ducts lined by biliary epithelial cells (cholangiocytes). Bile ducts and cholangiocytes are affected by a variety of disorders called cholangiopathies, which differ in aetiology, pathogenesis, and morphology. Classification of cholangiopathies is complex and reflects pathogenic mechanisms (immune-mediated, genetic, drug- and toxin-induced, ischaemic, infectious, neoplastic), predominant morphological patterns of biliary injury (suppurative and non-suppurative cholangitis, cholangiopathy), and specific segments of the biliary tree affected by the disease process. While the involvement of large extrahepatic and intrahepatic bile ducts is typically visualised using radiology imaging, histopathological examination of liver tissue obtained by percutaneous liver biopsy still plays an important role in the diagnosis of cholangiopathies affecting the small intrahepatic bile ducts. To increase the diagnostic yield of a liver biopsy and determine the optimal therapeutic approach, the referring clinician is tasked with interpreting the results of histopathological examination. This requires knowledge and understanding of basic morphological patterns of hepatobiliary injury and an ability to correlate microscopic findings with results obtained by imaging and laboratory methods. This minireview describes the morphological aspects of small-duct cholangiopathies pertaining to the diagnostic process.

MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.

Is liver regeneration key in hepatocellular carcinoma development?

The liver is the largest organ of the mammalian body and has the remarkable ability to fully regenerate in order to maintain tissue homeostasis. The adult liver consists of hexagonal lobules, each with a central vein surrounded by six portal triads localized in the lobule border containing distinct parenchymal and nonparenchymal cells. Because the liver is continuously exposed to diverse stress signals, several sophisticated regenerative processes exist to restore its functional status following impairment. However, these stress signals can affect the liver's capacity to regenerate and may lead to the development of hepatocellular carcinoma (HCC), one of the most aggressive liver cancers. Here, we review the mechanisms of hepatic regeneration and their potential to influence HCC development.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Mesenchymal stem cells: A novel treatment option for primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a progressive liver disease for which there is no effective therapy. Hepatocytes and cholangiocytes from a PSC patient were cocultured with mesenchymal stem cells (MSCs) to assess in vitro change. A single patient with progressive PSC was treated with 150 million MSCs via direct injection into the common bile duct. Coculture of MSCs with cholangiocytes and hepatocytes showed in vitro improvement. Local delivery of MSCs into a single patient with progressive PSC was safe. Radiographic and endoscopic evaluation showed stable distribution of multifocal structuring in the early postoperative period. MSCs may be effective for the treatment of PSC.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Immunobiology of the biliary tract system.

The biliary tract is a complex tubular organ system spanning from the liver to the duodenum. It is the site of numerous acute and chronic disorders, many of unknown origin, that are often associated with cancer development and for which there are limited treatment options. Cholangiocytes with proinflammatory capacities line the lumen and specialised types of immune cells reside in close proximity. Recent technological breakthroughs now permit spatiotemporal assessments of immune cells within distinct niches and have increased our understanding of immune cell tissue residency. In this review, a comprehensive overview of emerging knowledge on the immunobiology of the biliary tract system is provided, with a particular emphasis on the role of distinct immune cells in biliary disorders.