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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.

"Orthoplastics" is a relatively new approach to lower limb reconstruction, where an integration of both plastic and orthopedic expertise is required, together with the availability of well-equipped facilities. Acute shortening and long-term frames for lengthening are generally considered alternatives to length preservation and soft tissue microsurgical reconstruction, but an integration of external fixation and reconstructive microsurgery is gaining an increasing role with refinements of joint approaches.Data on sixteen patients who underwent microsurgical lower limb reconstruction and external fixation with an orthoplastic approach, following acute or chronic tibial injury, were retrospectively reviewed. All patients presented a post traumatic soft tissue defect associated with a Gustilo III tibial fracture or a tibial septic pseudarthrosis. Data on type and timing of bone and soft tissue reconstruction, outcomes, complications and need for re-operation were extrapolated and compared to an historic group of patients treated with an orthopedic-based approach.In the orthoplastic group, soft tissues were reconstructed with an ALT flap in most cases; a muscle-sparing VL or ALT-VL chimeric flap was necessary in cases with a very extensive defect. In the orthopedic group, soft tissues were left to heal by second intention or patients were lately referred to plastic surgeons. Statistical comparison between the two groups has showed significant differences on the following data: time for soft tissue healing, time to bone union, number of reinterventions, post-operative deep infection rate, time to return to work.The orthoplastic approach to complex leg defects yields shorter treatment time and better functional results compared to the orthopedic-based approach. External fixation and microsurgical reconstruction are not necessarily alternative procedures but can integrate in an orthoplastic path to address at best both soft tissue and bone reconstruction.Die „Orthoplastie“ ist ein relativ neuer Ansatz zur Rekonstruktion der unteren Extremitäten, der eine Integration von plastisch-chirurgischer und orthopädischer Fachkompetenz sowie die Verfügbarkeit gut ausgestatteter Einrichtungen erfordert. Die akute Verkürzung mit längerfristig angelegtem Rahmen zur Verlängerung gelten generell als Alternativen zum Längenerhalt mit mikrochirurgischer Weichteilrekonstruktion, doch gewinnt die integrierte Behandlung mit Fixateur externe und rekonstruktiver Mikrochirurgie zunehmend an Bedeutung und die gemeinsamen Ansätze werden weiter verfeinert.Es erfolgte eine retrospektive Analyse der Daten von sechzehn Patienten, die sich nach akuter oder chronischer Tibiaverletzung einem orthoplastischen Verfahren mit mikrochirurgischer Rekonstruktion der unteren Extremität und Fixateur externe unterzogen. Bei allen Patienten fand sich ein posttraumatischer Weichteildefekt in Verbindung mit einer Tibiafraktur vom Grad III nach Gustilo oder einer septischen Pseudarthrose der Tibia. Die Daten zu Art und Zeitpunkt der Knochen- und Weichteilrekonstruktion, Ergebnissen, Komplikationen und Notwendigkeit einer Reoperation wurden extrapoliert und mit denen einer historischen Patientengruppe verglichen, die orthopädisch behandelt worden war.In der orthoplastischen Gruppe wurden die Weichteile in den meisten Fällen mit einem anterolateralen Oberschenkellappen (ALT-Flap) rekonstruiert. Bei Patienten mit großflächigem Defekt war eine muskelschonende Vastus-lateralis-Lappenplastik (VL) oder eine chimäre ALT-VL-Lappenplastik erforderlich. In der orthopädischen Gruppe wurden die Weichteile einer Sekundärheilung überlassen oder die Patienten wurden später an einen plastischen Chirurgen überwiesen. Ein statistischer Vergleich zwischen den beiden Gruppen ergab signifikante Unterschiede hinsichtlich folgender Daten: Zeit bis zur Heilung der Weichteile, Zeit bis zur knöchernen Durchbauung, Anzahl der Reinterventionen, Rate tiefer postoperativer Infektionen und Zeit bis zur Wiederaufnahme der Berufstätigkeit.Der orthoplastische Ansatz bei komplexen Beindefekten bietet im Vergleich zu orthopädischen Verfahren kürzere Behandlungszeiten und liefert bessere funktionelle Ergebnisse. Die Behandlung mit Fixateur externe und die mikrochirurgische Rekonstruktion müssen sich nicht gegenseitig ausschließen, sondern können in einem orthoplastischen Behandlungspfad miteinander kombiniert werden, um eine bestmögliche Rekonstruktion sowohl der Weichteile als auch des Knochens zu erzielen.

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).

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 β-catenin-dependent secretion of a range of chemokines, including CXCL1, CXCL10 and CXCL12, 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 αvβ6 integrin, an activator of latent local TGFβ1. 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. our results show that 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. This article is protected by copyright. All rights reserved.

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. (Hepatology 2015;62:1551-1562)

Cystic fibrosis-associated liver disease (CFLD) is a chronic cholangiopathy that negatively impacts the quality of life and survival of patients with Cystic Fibrosis (CF). CF is a disease of secretory epithelia caused by genetically-determined defective function of CFTR (cystic fibrosis conductance regulator), a cAMP-activated Cl- channel that, in the liver, is uniquely expressed in the biliary epithelium. The pathogenesis of CFLD is thought to be related to the ductal cholestasis caused by the defective channel function of CFTR. However, a number of evidence indicate that, in association with the genetic defect, other pathogenetic factors concur to determine the liver phenotype. During the first year of my doctorate we have demonstrated that in CFTR-defective biliary epithelium, exposed to bacterial-derived endotoxins, TLR4/NF-kB-dependent innate immune responses are increased, indicating a loss of “endotoxin tolerance”. The current standard of care for CFLD is limited to the administration of choleretic agents. Based on our novel interpretation of the pathogenesis of CFLD, a rational therapeutic approach to the management of CFLD should be to target the exaggerated inflammatory response of the biliary epithelium. Nuclear receptors (NRs), ligand-activated transcription factors that regulate several intracellular metabolic functions by controlling the expression of specific target genes, are now emerging as important regulators of inflammation in several conditions. In this work we sought to investigate the ability of NRs to modulate the TLR4/NF-kB-dependent innate immune responses in CF biliary epithelia. Our data indicate that cholangiocytes express several subtypes of NRs, including Peroxisome Proliferator Activated Receptor (PPAR) subgroup α, γ and β/δ, Liver X Receptors (LXR) subgroup β, Farnesoid X Receptor (FXR) and Vitamin D Receptor (VDR). Among these NRs, PPAR-γ was barely expressed in wild type (WT) cholangiocytes , but its expression, both at the gene and protein levels, was strongly and significantly more expressed in CF cells. In spite of its increased expression in the nucleus, PPAR-γ was not transcriptionally active in CF cholangiocytes; in fact the basal level of expression of PPAR-γ target genes (Acaa1b, Angptl4 and Hmgcs2) was similar to WT cholangiocytes. On the other hand, treatment of CF cholangiocytes with the PPAR-γ agonist pioglitazone significantly increased the expression of its target genes, indicating that the transcriptional activity of nuclear PPAR-γ could be readily activated in the presence of proper agonists. Consistent with the higher expression level of the receptor, the increase in PPAR-γ target genes stimulated by pioglitazone was higher in CF than in WT cholangiocytes. The observation that PPAR-γ, while inactive at baseline levels, was able to respond to exogenous stimulation, indicates that a defect in the availability of endogenous PPAR-γ activators might impair PPAR-γ function in CF and increase the receptor expression as counter-regulatory mechanism. Thus, to study the availability of endogenous PPAR-γ activators in CF, we performed lipidomic analysis of extracts from cultured cholangiocytes, and quantified the major ω-3 and ω-6 polyunsaturated fatty acids (PUFAs), i.e. the precursors of the active form of PPAR-γ ligands. We detected an increased ratio between arachidonic acid and docosahexaenoic acid (AA/DHA) and between arachidonic acid and linoleic acid (AA/LA) in CF cells compared to their controls. LA, the precursor of AA, is the main source of pro-inflammatory mediators, while DHA is an important precursor of anti-inflammatory eicosanoids that are able to activate PPAR-γ. This finding might explain, at least in part, the increased receptor expression in CF biliary cells. Having shown that CF cells express higher amounts of PPAR-γ, that can be activated by exogenous ligands, but it is not transcriptionally active because of the lack of endogenous ligands, we next tested if exogenous activation of PPAR-γ by synthetic ligands, might decrease the TLR4/NF-kB-driven inflammation in CFTR-defective biliary epithelium. We found that in Cftr-KO cells, in the presence of pioglitazone, nuclear translocation of p65/NF-kB, as well as its transcriptional activity, were significantly inhibited, both before and after stimulation with bacterial LPS. Moreover, the gene expression and protein secretion of several NF-kB-dependent pro-inflammatory cytokines, such as LIX (LPS-induced CXC chemokine), MCP-1 (monocyte chemotactic protein-1), MIP-2 (macrophage inflammatory protein 2), G-CSF (granulocyte colony-stimulating factor) and KC (keratinocyte chemo-attractant) were significantly inhibited both at baseline and after LPS stimulation. The anti-inflammatory effect of pioglitazone was the result of a direct activation of PPAR-γ receptor, as shown by reversal of pioglitazone-induced protection in the presence of the PPAR-γ antagonist GW9662. Finally, to understand the mechanism by which ligand-activated PPAR-γ blocks inflammation in CF cholangiocytes we analyzed the effect of pioglitazone on NF-kB activation pathway steps, and found that pioglitazone up-regulated IkB-α, a negative regulator of NF-kB. In conclusion, our results indicate that a decreased PPAR-γ function caused by a decreased availability of PPAR-γ endogenous agonists might be responsible for the chronic inflammatory state of CFTR-defective cholangiocytes. Stimulation of PPAR-γ signaling by the agonist pioglitazone is able to decrease NF-kB-dependent inflammatory responses by increasing IkB-α, thus may represent a novel strategy to limit inflammation in CFLD. Studies in mice models of liver damage in CFLD have been planned and may represent a proof of concept for anti-inflammatory treatment in CF.

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.

BACKGROUND AND AIM Congenital Hepatic Fibrosis (CHF) is a genetic liver disease caused by mutations in PKHD1, a gene encoding for Fibrocystin, a ciliary protein expressed by cholangiocytes. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis and portal hypertension. In CHF mechanisms of portal fibrosis are unknown. We have recently reported that Pkhd1-/- mice present: 1)activation of b-catenin signaling in cystic cholangiocytes; 2)increased pericystic infiltrate of CD45+/Collagen type1 (Col1)+ cells, reminiscent of fibrocytes. Fibrocytes are monocyte-derived cells, involved in several fibrosing conditions, but their role in liver scarring is controversial. b-catenin is emerging as a regulator of inflammation, therefore we hypothesized that a b-catenin-dependent chemokine production by cholangiocytes drives recruitment of fibrocytes in Pkhd1-/- mice. METHODS In Pkhd1-/- mice we investigated: a)a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); b)the effects of two different b-catenin inhibitors (ICG-001, 25uM, or quercetin, 50uM) on the expression of CXCL1 and CXCL10 (RT-PCR); c)the immunohistochemical expression of CD45/Col1 (fibrocyte markers) and aSMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with portal fibrosis (Sirius-red) in liver samples at 1-12 months; d)the effects of cholangiocyte conditioned media (CM) and CXCL1+CXCL10 on WEHI265.1-monocyte chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT mice served as controls. RESULTS Pkhd1-/- cholangiocytes secreted significantly higher basolateral levels of CXCL1 and CXCL10 as compared to WT. Expression of CXCL1 and CXCL10 was significantly inhibited in cells treated with ICG-001 and quercetin. Pkhd1-/- mice showed progressive fibrosis, but portal accumulation of aSMA+ cells was evident only after 9 months. In contrast, we observed an early peribiliary recruitment of CD45+/Col1+ cells, whose number strongly correlated with the Sirius-red area (r=0.89,p

Liver Cirrhosis (LC) is responsible for high morbidity, mortality and raising costs. Current guidelines set the standard of care for management of cirrhosis in clinical practice, but explicit outcome indicators (OIs) are lacking. If available, OIs could guide clinical care and decision making, so that efforts and resources can be properly allocated. Aim of our study was to generate and test a set of health care OIs for compensated (CC) and decompensated (DC) LC. This study is part of a larger effort (the V.B.M.H. study) to generate OIs for several liver diseases. An expert panel of hepatologists identified a set of OIs for LC according to experience and scientific evidence (as of 2010), and used a modified Delphi method to rate them through a RAND 9-point agreement scale. A final list of 7 indicators with median rating ≥7 and with disagreement index ≤1 was selected. Three OIs were designed for CC and 4 for DC. In the second phase of the study, the selected OIs were tested in clinical practice through a prospective multicenter observational study, involving three tertiary centers in Lombardy, Italy. A web-based EMR was used to collect data. 1732 LC patients were enrolled in 18 months: 984 CC (57%) and 748 DC (43%). 91% of these LC patients had at least two consultations in a 13 months median follow-up. The annual rate of decompensation in CC (OI#1) was 12%, decompensation being more frequent in HCV-related CC. The annual incidence of 1st variceal bleeding (VB) for low-risk varices was 2% and was null for high risk varices, indicating effective primary profilaxis (OI#2). Annual incidence of HCC was 4%, while the percentage of CC patients diagnosed at early stage HCC (BCLC 0/A) was 74%, reflecting the accuracy of oncologic surveillance (OI#3). In DC patients, survival at 1 year after the first episode of decompensation (ascites in 78% of cases) was 99% for CPT A, 87% for CPT B and 73% for CPT C (OI#4). 3% of DC patients had an episode of VB, with survival of 88% after 6 weeks from the VB episode (OI#5) and with recurrence rate of 27% (OI#6). 19 out of 748 DC patients (3%) had spontaneous bacterial peritonitis, with 79% survival after 6 weeks from the episode (OI#7). In conclusion, the selected OIs performed well in monitoring the rate of decompensation in CC and the accuracy of surveillance for HCC; in DC, OIs were able to capture survival and the efficacy of management of major complications. This study represents the first attempt to identify and test a set of value-based OIs for LC, and provides a reference tool for healthcare policy makers to improve quality of care in patients affected by LC

Background & Aims: Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in the biliary epithelium reduces bile flow and alkalinization in patients with cystic fibrosis (CF). Liver damage is believed to result from ductal cholestasis, but only 30% of patients with CF develop liver defects, indicating that another factor is involved. We studied the effects of CFTR deficiency on Toll-like receptor 4 (TLR4)-mediated responses of the biliary epithelium to endotoxins. Methods: Dextran sodium sulfate (DSS) was used to induce colitis in C57BL/6J-Cftrtm1Unc (Cftr-KO) mice and their wild-type littermates. Ductular reaction and portal inflammation were quantified by keratin-19 and CD45 immunolabeling. Cholangiocytes isolated from wild-type and Cftr-KO mice were challenged with lipopolysaccharide (LPS); cytokine secretion was quantified. Activation of nuclear factor κB (NF-κB), phosphorylation of TLR4, and activity of Src were determined. HEK-293 that expressed the secreted alkaline phosphatase reporter and human TLR4 were transfected with CFTR complementary DNAs. Results: DSS-induced colitis caused biliary damage and portal inflammation only in Cftr-KO mice. Biliary damage and inflammation were not attenuated by restoring biliary secretion with 24-nor-ursodeoxycholic acid but were significantly reduced by oral neomycin and polymyxin B, indicating a pathogenetic role of gut-derived bacterial products. Cftr-KO cholangiocytes incubated with LPS secreted significantly higher levels of cytokines regulated by TLR4 and NF-κB. LPS-mediated activation of NF-κB was blocked by the TLR4 inhibitor TAK-242. TLR4 phosphorylation by Src was significantly increased in Cftr-KO cholangiocytes. Expression of wild-type CFTR in the HEK293 cells stimulated with LPS reduced activation of NF-κB. Conclusions: CFTR deficiency alters the innate immunity of the biliary epithelium and reduces its tolerance to endotoxin, resulting in an Src-dependent inflammatory response mediated by TLR4 and NF-κB. These findings might be used to develop therapies for CF-associated cholangiopathy. © 2011 AGA Institute.