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Cholestasis is a frequent clinical condition initiating or complicating chronic liver diseases, particularly cholangiopathies, where the biliary epithelium is the primary target of the pathogenetic sequence. Until a few decades ago, understanding of cholestasis relied mostly on the experimental model of bile duct ligation in rodents. However, a simple model of biliary obstruction cannot reproduce the complex mechanisms and networks leading to cholestasis in cholangiopathies. These networks are underpinned by an intricate dysregulation of pro-inflammatory and pro-fibrotic signals involving besides cholangiocytes, multiple cell elements of both innate and adaptive immunity. Therefore, in the last years, a wide range of animal models of biliary injury have been developed, mostly in mice, following three main approaches, chemical induction, immunization and genetic manipulation. In this review, we will give an update of the animal models of the two main cholangiopathies, primary sclerosing cholangitis and primary biliary cholangitis, which have provided us with the most relevant insights into the pathogenesis of these still controversial diseases.

Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases. Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential.

Despite recent advances, pathogenesis of cholangiocarcinoma, a highly lethal cancer, remains enigmatic. Furthermore, treatment options are still limited and often disappointing. For this reason, in the last few years there has been a mounting interest towards the generation of experimental models able to reproduce the main features associated with this aggressive behavior. Toxic and infestation-induced, genetically engineered and cell implantation rodent models have been generated, contributing to a deeper understanding of the complex cell biology of the tumor, sustained by multiple cell interactions and driven by a huge variety of molecular perturbations. Herein, we will overview the most relevant animal models of biliary carcinogenesis, highlighting the methodological strategy, the molecular, histological and clinical phenotypes consistent with the human condition, their particular strengths and weaknesses and the novel therapeutic approaches that have been developed.

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype.

Prognosis of cholangiocarcinoma, a devastating liver epithelial malignancy characterized by early invasiveness, remains very dismal, though its incidence has been steadily increasing. Evidence is mounting that in cholangiocarcinoma, tumor epithelial cells establish an intricate web of mutual interactions with multiple stromal components, largely determining the pervasive behavior of the tumor. The main cellular components of the tumor microenvironment (i.e. myofibroblasts, macrophages, lymphatic endothelial cells), which has been recently termed as 'tumor reactive stroma', are recruited and activated by neoplastic cells, and in turn, deleteriously mold tumor behavior by releasing a huge variety of paracrine signals, including cyto/chemokines, growth factors, morphogens and proteinases. An abnormally remodeled and stiff extracellular matrix favors and supports these cell interactions. Although the mechanisms responsible for the generation of tumor reactive stroma are largely uncertain, hypoxia presumably plays a central role. In this review, we will dissect the intimate relationship among the different cell elements cooperating within this complex 'ecosystem', with the ultimate goal to pave the way for a deeper understanding of the mechanisms underlying cholangiocarcinoma aggressiveness, and possibly, to foster the development of innovative, combinatorial therapies aimed at halting tumor progression. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen

Chronic diseases of the biliary tree (cholangiopathies) represent one of the major unmet needs in clinical hepatology and a significant knowledge gap in liver pathophysiology. The common theme in cholangiopathies is that the target of the disease is the biliary tree. After damage to the biliary epithelium, inflammatory changes stimulate a reparative response with proliferation of cholangiocytes and restoration of the biliary architecture, owing to the reactivation of a variety of morphogenetic signals. Chronic damage and inflammation will ultimately result in pathological repair with generation of biliary fibrosis and clinical progression of the disease. The hallmark of pathological biliary repair is the appearance of reactive ductular cells, a population of cholangiocyte-like epithelial cells of unclear and likely mixed origin that are able to orchestrate a complex process that involves a number of different cell types, under joint control of inflammatory and morphogenetic signals. Several questions remain open concerning the histogenesis of reactive ductular cells, their role in liver repair, their mechanism of activation, and the signals exchanged with the other cellular elements cooperating in the reparative process. This review contributes to the current debate by highlighting a number of new concepts derived from the study of the pathophysiology of chronic cholangiopathies, such as congenital hepatic fibrosis, biliary atresia, and Alagille syndrome.

Resistance to conventional chemotherapeutic agents, a typical feature of cholangiocarcinoma, prevents the efficacy of the therapeutic arsenal usually used to combat malignancy in humans. Mechanisms of chemoresistance by neoplastic cholangiocytes include evasion of drug-induced apoptosis mediated by autocrine and paracrine cues released in the tumor microenvironment. Here, recent evidence regarding molecular mechanisms of chemoresistance is reviewed, as well as associations between well-developed chemoresistance and activation of the cancer stem cell compartment. It is concluded that improved understanding of the complex interplay between apoptosis signaling and the promotion of cell survival represent potentially productive areas for active investigation, with the ultimate aim of encouraging future studies to unveil new, effective strategies able to overcome current limitations on treatment.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubulestabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma.

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF-related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll-like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates toll-like receptor 4, resulting in activation of nuclear factor kappa-light-chain-enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa-light-chain-enhancer of activated B cells-dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr knockout mice). Conclusion: These findings reveal a novel function of CFTR as a regulator of toll-like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF-related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118-2134).

The diseases of the intrahepatic biliary tree are a large group of potentially evolutive congenital and acquired liver disorders affecting both the adult and pediatric populations. They represent a relevant cause of liver-related morbidity and mortality and an important indication for liver transplantation, particularly in children. While the practical approach to patients affected by biliary tree diseases has not significantly changed yet, the conceptual approach to the pathophysiology of cholangiopathies has witnessed important advances that will be discussed. The primary cell target of the pathogenetic sequence of these disorders is the biliary epithelium. Cholangiocytes have multifaceted functions, not limited to bile production. Their capability to secrete a range of different pro-inflammatory mediators, cytokines, and chemokines indicates a major role of cholangiocytes in the inflammatory reaction. Furthermore, paracrine secretion of growth factors and peptides mediates an extensive cross-talk with other liver cell types, including hepatocytes, stellate, and endothelial and inflammatory cells. Cholangiopathies share a number of pathogenetic mechanisms, including inflammation, cholestasis, fibrosis, apoptosis, altered development, and neoplastic transformation. These basic disease mechanisms will be discussed in detail, along with the distinct features of a number of cholangiopathies. Furthermore, an increase in the biliary cell compartment is a common response to many forms of liver injury, from cholangiopathies to viral and fulminant hepatitis. Elucidation of these pathophysiologic mechanisms will likely provide clues for future therapeutic strategies. Furthermore, understanding the role of cholangiocytes in liver regeneration/repair and the mechanisms of cholangiocyte activation and their relationship with liver progenitor cell will be of further interest.