Prominent role of gut dysbiosis in the pathogenesis of cystic fibrosis-related liver disease in mice.

BACKGROUND & AIMS: Cystic fibrosis-related liver disease (CFLD) is a chronic cholangiopathy that increases morbidity and mortality in patients with CF. Current treatments are unsatisfactory, and incomplete understanding of CFLD pathogenesis hampers therapeutic development. We have previously shown that mouse CF cholangiocytes respond to lipopolysaccharide with excessive inflammation. Thus, we investigated the role of the gut-liver axis in the pathogenesis of CFLD. METHODS: Wild-type (WT), whole-body Cftr knockout (CFTR-KO) and gut-corrected (CFTR-KO-GC) mice were studied. Liver changes were assessed by immunohistochemistry and single-cell transcriptomics (single-cell RNA sequencing), inflammatory mediators were analysed by proteome array, faecal microbiota by 16S ribosomal RNA sequencing and gut permeability by FITC-dextran assay. RESULTS: The livers of CFTR-KO mice showed ductular proliferation and periportal inflammation, whereas livers of CFTR-KO-GC mice had no evident pathology. Single-cell RNA sequencing analysis of periportal cells showed increased presence of neutrophils, macrophages and T cells, and activation of pro-inflammatory and pathogen-mediated immune pathways in CFTR-KO livers, consistent with a response to gut-derived stimuli. CFTR-KO mice exhibited gut dysbiosis with enrichment of Enterobacteriaceae and Enterococcus spp., which was associated with increased intestinal permeability and mucosal inflammation, whereas gut dysbiosis and inflammation were absent in CFTR-KO-GC mice. Treatment with nonabsorbable antibiotics ameliorated intestinal permeability and liver inflammation in CFTR-KO mice. Faecal microbiota transfer from CFTR-KO to germ-free WT mice did not result in dysbiosis nor liver pathology, indicating that defective intestinal CFTR is required to maintain dysbiosis. CONCLUSION: Defective CFTR in the gut sustains a pathogenic microbiota, creates an inflammatory milieu, and alters intestinal permeability. These changes are necessary for the development of cholangiopathy. Restoring CFTR in the intestine or modulating the microbiota could be a promising strategy to prevent or attenuate liver disease. IMPACT AND IMPLICATIONS: Severe cystic fibrosis-related liver disease (CFLD) affects 10% of patients with cystic fibrosis (CF) and contributes to increased morbidity and mortality. Treatment options remain limited due to a lack of understanding of disease pathophysiology. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates Cl- and HCO3- secretion in the biliary epithelium and its defective function is thought to cause cholestasis and excessive inflammatory responses in CF. However, our study in Cftr-knockout mice demonstrates that microbial dysbiosis, combined with increased intestinal permeability caused by defective CFTR in the intestinal mucosa, acts as a necessary co-factor for the development of CFLD-like liver pathology in mice. These findings uncover a major role for the gut microbiota in CFLD pathogenesis and call for further investigation and clinical validation to develop targeted therapeutic strategies acting on the gut-liver axis in CF.

Dysregulation of the Scribble/YAP/ß-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 PKHD1 gene, leading to defective fibrocystin (FPC), changes in planar cell polarity (PCP) and increased ß-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 ß-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 ß-catenin nuclear expression, and CTGF, integrin ß6, CXCL1, and CXCL10 mRNA levels, whereas inhibition of ß-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 ß-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 ß-catenin with gain of a fibroinflammatory phenotype. The Scribble/YAP/ß-catenin axis is a critical factor in the sequence of events linking the genetic defect to fibrocystic trait of cholangiocytes in CHF.

IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Recent Advances in Practical Methods for Liver Cell Biology: A Short Overview.

Molecular and cellular research modalities for the study of liver pathologies have been tremendously improved over the recent decades. Advanced technologies offer novel opportunities to establish cell isolation techniques with excellent purity, paving the path for 2D and 3D microscopy and high-throughput assays (e.g., bulk or single-cell RNA sequencing). The use of stem cell and organoid research will help to decipher the pathophysiology of liver diseases and the interaction between various parenchymal and non-parenchymal liver cells. Furthermore, sophisticated animal models of liver disease allow for the in vivo assessment of fibrogenesis, portal hypertension and hepatocellular carcinoma (HCC) and for the preclinical testing of therapeutic strategies. The purpose of this review is to portray in detail novel in vitro and in vivo methods for the study of liver cell biology that had been presented at the workshop of the 8th meeting of the European Club for Liver Cell Biology (ECLCB-8) in October of 2018 in Bonn, Germany.

Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.

BACKGROUND & AIMS: In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. METHODS: Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. RESULTS: In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRß inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. CONCLUSION: PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. LAY SUMMARY: Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves cystic fibrosis transmembrane conductance regulator correctors efficacy.

Cystic fibrosis transmembrane conductance regulator (CFTR), the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e., cholangiocytes) of the liver. Progressive clinical liver disease (CF-associated liver disease; CFLD) occurs in around 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD; however, the lack of human-derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC-cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. Protein kinase A/cAMP-mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX-770 and VX-809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and Toll-like receptor 4 and proinflammatory changes, including increased nuclear factor kappa-light-chain-enhancer of activated B cells activation, secretion of proinflammatory chemokines (i.e., monocyte chemotactic protein 1 and interleukin-8), as well as alterations of the F-actin cytoskeleton. Treatment with Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src, along with administration of VX-770 and VX-809, successfully restored fluid secretion to normal levels. CONCLUSION: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. (Hepatology 2018;67:972-988).

ß-Catenin and interleukin-1ß-dependent chemokine (C-X-C motif) ligand 10 production drives progression of disease in a mouse model of congenital hepatic fibrosis.

Congenital hepatic fibrosis (CHF), a genetic disease caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, encoding for the protein fibrocystin/polyductin complex, is characterized by biliary dysgenesis, progressive portal fibrosis, and a protein kinase A-mediated activating phosphorylation of ß-catenin at Ser675. Biliary structures of Pkhd1del4/del4 mice, a mouse model of CHF, secrete chemokine (C-X-C motif) ligand 10 (CXCL10), a chemokine able to recruit macrophages. The aim of this study was to clarify whether CXCL10 plays a pathogenetic role in disease progression in CHF/Caroli disease and to understand the mechanisms leading to increased CXCL10 secretion. We demonstrate that treatment of Pkhd1del4/del4 mice for 3 months with AMG-487, an inhibitor of CXC chemokine receptor family 3, the cognate receptor of CXCL10, reduces the peribiliary recruitment of alternative activated macrophages (cluster of differentiation 45+ F4/80+ cells), spleen size, liver fibrosis (sirius red), and cyst growth (cytokeratin 19-positive area), consistent with a pathogenetic role of CXCL10. Furthermore, we show that in fibrocystin/polyductin complex-defective cholangiocytes, isolated from Pkhd1del4/del4 mice, CXCL10 production is mediated by Janus kinase/signal transducer and activator of transcription 3 in response to interleukin 1beta (IL-1ß) and ß-catenin. Specifically, IL-1ß promotes signal transducer and activator of transcription 3 phosphorylation, whereas ß-catenin promotes its nuclear translocation. Increased pro-IL-1ß was regulated by nuclear factor kappa-light-chain-enhancer of activated B cells, and increased secretion of active IL-1ß was mediated by the activation of Nod-like receptors, pyrin domain containing 3 inflammasome (increased expression of caspase 1 and Nod-like receptors, pyrin domain containing 3). CONCLUSION: In fibrocystin/polyductin complex-defective cholangiocytes, ß-catenin and IL-1ß are responsible for signal transducer and activator of transcription 3-dependent secretion of CXCL10; in vivo experiments show that the CXCL10/CXC chemokine receptor family 3 axis prevents the recruitment of macrophages, reduces inflammation, and halts the progression of the disease; the increased production of IL-1ß highlights the autoinflammatory nature of CHF and may open novel therapeutic avenues. (Hepatology 2018;67:1903-1919).

Pathophysiologic implications of innate immunity and autoinflammation in the biliary epithelium.

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.

The deleterious interplay between tumor epithelia and stroma in cholangiocarcinoma.

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.

Adenylyl cyclase 5 links changes in calcium homeostasis to cAMP-dependent cyst growth in polycystic liver disease.

BACKGROUND & AIMS: Genetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca2+]ER depletion is increased, while store-operated Ca2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca2+]ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca2+, are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca2+]ER depletion. METHODS: PC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis. RESULTS: When comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca2+]ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca2+]ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca2+ channel that mediates SOCE. CONCLUSION: [Ca2+]ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target. LAY SUMMARY: Polycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease.

Emerging concepts in biliary repair and fibrosis.

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.

The cystic fibrosis transmembrane conductance regulator controls biliary epithelial inflammation and permeability by regulating Src tyrosine kinase activity.

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).

Macrophage recruitment by fibrocystin-defective biliary epithelial cells promotes portal fibrosis in congenital hepatic fibrosis.

UNLABELLED: Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a ß-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvß6 integrin, an activator of latent local transforming growth factor-ß1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. CONCLUSION: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time.

Posttranslational regulation of polycystin-2 protein expression as a novel mechanism of cholangiocyte reaction and repair from biliary damage.

UNLABELLED: Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)-mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2(-/-) knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2(-/-) mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. CONCLUSION: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases.

Stimulation of nuclear receptor peroxisome proliferator-activated receptor-γ limits NF-κB-dependent inflammation in mouse cystic fibrosis biliary epithelium.

UNLABELLED: Cystic fibrosis-associated liver disease is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of toll-like receptor 4/nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB)-dependent immune mechanisms plays a major role in the pathogenesis of cystic fibrosis-associated liver disease and may represent a therapeutic target. Nuclear receptors are transcription factors that regulate several intracellular functions. Some nuclear receptors, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counterregulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in cystic fibrosis transmembrane conductance regulator (Cftr) knockout mice exposed to dextran sodium sulfate and in vitro in primary cholangiocytes isolated from wild-type and from Cftr-knockout mice exposed to lipopolysaccharide. We found that in CFTR-defective biliary epithelium expression of PPAR-γ is increased but that this does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) up-regulates PPAR-γ-dependent genes, while inhibiting the activation of NF-κB and the secretion of proinflammatory cytokines (lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-2, granulocyte colony-stimulating factor, keratinocyte chemoattractant) in response to lipopolysaccharide. PPAR-γ agonists modulate NF-κB-dependent inflammation by up-regulating nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, a negative regulator of NF-κB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-knockout mice exposed to a dextran sodium sulfate-induced portal endotoxemia. CONCLUSION: These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes.

Protein kinase A-dependent pSer(675) -ß-catenin, a novel signaling defect in a mouse model of congenital hepatic fibrosis.

UNLABELLED: Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and ß-catenin. In cholangiocytes isolated and cultured from Pkhd1(del4/del4) mice, stimulation of cAMP/PKA signaling (forskolin 10 µM) stimulated Ser(675) -phosphorylation of ß-catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down-regulation of E-cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 µM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1(del4/del4) cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer(675) -ß-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1(del4/del4) and was inhibited by: (1) PKI, (2) silencing ß-catenin (siRNA), and (3) the Rac-1 inhibitor NSC 23766. CONCLUSION: These data show that in fibrocystin-defective cholangiocytes, cAMP/PKA signaling stimulates pSer(675) -phosphorylation of ß-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer(675) -ß-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1(del4/del4) cholangiocytes. ß-Catenin-dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target.

Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)ß. PDGF-D, a PDGFRß agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRß blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. CONCLUSION: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRß and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.

Notch signaling regulates tubular morphogenesis during repair from biliary damage in mice.

BACKGROUND & AIMS: Repair from biliary damages requires the biliary specification of hepatic progenitor cells and the remodeling of ductular reactive structures into branching biliary tubules. We hypothesized that the morphogenetic role of Notch signaling is maintained during the repair process and have addressed this hypothesis using pharmacologic and genetic models of defective Notch signaling. METHODS: Treatment with DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) or ANIT (alpha-naphthyl-isothiocyanate) was used to induce biliary damage in wild type mice and in mice with a liver specific defect in the Notch-2 receptor (Notch-2-cKO) or in RPB-Jk. Hepatic progenitor cells, ductular reaction, and mature ductules were quantified using K19 and SOX-9. RESULTS: In DDC treated wild type mice, pharmacologic Notch inhibition with dibenzazepine decreased the number of both ductular reaction and hepatic progenitor cells. Notch-2-cKO mice treated with DDC or ANIT accumulated hepatic progenitor cells that failed to progress into mature ducts. In RBP-Jk-cKO mice, mature ducts and hepatic progenitor cells were both significantly reduced with respect to similarly treated wild type mice. The mouse progenitor cell line BMOL cultured on matrigel, formed a tubular network allowing the study of tubule formation in vitro; γ-secretase inhibitor treatment and siRNAs silencing of Notch-1, Notch-2 or Jagged-1 significantly reduced both the length and number of tubular branches. CONCLUSIONS: These data demonstrate that Notch signaling plays an essential role in biliary repair. Lack of Notch-2 prevents biliary tubule formation, both in vivo and in vitro. Lack of RBP-Jk inhibits the generation of biliary-committed precursors and tubule formation.