Invasive Ductular Reaction Operates Hepatobiliary Junctions upon Hepatocellular Injury in Rodents and Humans
2019; Elsevier BV; Volume: 189; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2019.04.011
ISSN1525-2191
AutoresLaure‐Alix Clerbaux, Rita Manco, Noémi Van Hul, Caroline Bouzin, Amedeo Sciarra, Christine Sempoux, Neil D. Theise, Isabelle Leclercq,
Tópico(s)Liver Disease and Transplantation
ResumoDuctular reaction (DR) is observed in virtually all liver diseases in both humans and rodents. Depending on the injury, DR is confined within the periportal area or invades the parenchyma. On severe hepatocellular injury, invasive DR has been proposed to arise for supplying the liver with new hepatocytes. However, experimental data evidenced that DR contribution to hepatocyte repopulation is at the most modest, unless replicative capacity of hepatocytes is abrogated. Herein, we proposed that invasive DR could contribute to operating hepatobiliary junctions on hepatocellular injury. The choline-deficient ethionine-supplemented mouse model of hepatocellular injury and human liver samples were used to evaluate the hepatobiliary junctional role of the invasive form of DR. Choline-deficient ethionine-supplemented–induced DR expanded as biliary epithelium into the lobule and established new junctions with the canaliculi. By contrast, no new ductular-canalicular junctions were observed in mouse models of biliary obstructive injury exhibiting noninvasive DR. Similarly, in humans, an increased number of hepatobiliary junctions were observed in hepatocellular diseases (viral, drug induced, or metabolic) in which DR invaded the lobule but not in biliary diseases (obstruction or cholangitis) in which DR was contained within the portal mesenchyme. In conclusion, our data in rodents and humans support that invasive DR plays a hepatobiliary junctional role to maintain structural continuity between hepatocytes and ducts in disorders affecting hepatocytes. Ductular reaction (DR) is observed in virtually all liver diseases in both humans and rodents. Depending on the injury, DR is confined within the periportal area or invades the parenchyma. On severe hepatocellular injury, invasive DR has been proposed to arise for supplying the liver with new hepatocytes. However, experimental data evidenced that DR contribution to hepatocyte repopulation is at the most modest, unless replicative capacity of hepatocytes is abrogated. Herein, we proposed that invasive DR could contribute to operating hepatobiliary junctions on hepatocellular injury. The choline-deficient ethionine-supplemented mouse model of hepatocellular injury and human liver samples were used to evaluate the hepatobiliary junctional role of the invasive form of DR. Choline-deficient ethionine-supplemented–induced DR expanded as biliary epithelium into the lobule and established new junctions with the canaliculi. By contrast, no new ductular-canalicular junctions were observed in mouse models of biliary obstructive injury exhibiting noninvasive DR. Similarly, in humans, an increased number of hepatobiliary junctions were observed in hepatocellular diseases (viral, drug induced, or metabolic) in which DR invaded the lobule but not in biliary diseases (obstruction or cholangitis) in which DR was contained within the portal mesenchyme. In conclusion, our data in rodents and humans support that invasive DR plays a hepatobiliary junctional role to maintain structural continuity between hepatocytes and ducts in disorders affecting hepatocytes. The biliary tree is an arborizing network of conduits that drains bile secreted by hepatocytes to the gut. Bile secretion is an active and tightly regulated process resulting in extrusion of biliary components at the apical pole of hepatocytes into a space sealed by tight junctions between adjacent hepatocytes, the canaliculus. Coordinated contractions of the pericanalicular microfilaments drain bile downstream to bile ductules delineated by cholangiocytes enclosed in the portal mesenchyme. The canal of Hering (CoH), a transitional structure formed by the apical poles of hepatocytes in the periportal region and by cholangiocytes of the most proximal extremities of the bile ductules, represents the anatomic interface between the canaliculi and the ducts.1Saxena R. Theise N.D. Canals of Hering: recent insights and current knowledge.Semin Liver Dis. 2004; 24: 43-48Crossref PubMed Scopus (116) Google Scholar Small ductules converge to form larger ducts, then carry the bile to the gallbladder and the gut.2Roskams T.A. Theise N.D. Balabaud C. Bhagat G. Bhathal P.S. Bioulac-sage P. Brunt E.M. Crawford J.M. Crosby H.A. Desmet V. Finegold M.J. Geller S.A. Gouw A.S.H. Hytiroglou P. Knisely A.S. Kojiro M. Lefkowitch J.H. Nakanuma Y. Olynyk J.K. Park Y.N. Portmann B. Saxena R. Scheuer P.J. Nomenclature of the finer branches of the biliary.Hepatology. 2004; 39: 1739-1745Crossref PubMed Scopus (559) Google Scholar The morphology and functional properties of cholangiocytes vary gradually along the proximal to distal axis.3Maroni L. Haibo B. Ray D. Zhou T. Wan Y. Meng F. Marzioni M. Alpini G. Functional and structural features of cholangiocytes in health and disease.Cell Mol Gastroenterol Hepatol. 2015; 1: 368-380Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 4Han Y. Glaser S. Meng F. Francis H. Marzioni M. McDaniel K. Alvaro D. Venter J. Carpino G. Onori P. Gaudio E. Alpini G. Franchitto A. Recent advances in the morphological and functional heterogeneity of the biliary epithelium.Exp Biol Med. 2013; 238: 549-565Crossref PubMed Scopus (54) Google Scholar, 5Tabibian J.H. Masyuk A.I. Masyuk T.V. O'Hara S.P. LaRusso N.F. 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Schaap F.G. Hampe J. Hengstler J.G. The ascending pathophysiology of cholestatic liver disease.Hepatology. 2017; 65: 722-738Crossref PubMed Scopus (174) Google Scholar However, a hallmark of chronic liver diseases, including cholestatic disorders, is the appearance of ductular reaction (DR).9Gouw A.S.H. Clouston A.D. Theise N.D. Ductular reactions in human liver: diversity at the interface.Hepatology. 2011; 54: 1853-1863Crossref PubMed Scopus (188) Google Scholar DR morphology may range from structures formed by cuboid cells, delineating a clear lumen and constrained within the portal mesenchyme, to elongated cells with a migratory phenotype, invading the parenchyma. This diversity of DR pattern, observed in both humans and rodent models, has been related to the nature and cell compartment being injured.9Gouw A.S.H. Clouston A.D. Theise N.D. Ductular reactions in human liver: diversity at the interface.Hepatology. 2011; 54: 1853-1863Crossref PubMed Scopus (188) Google Scholar, 10Kaneko K. Kamimoto K. Miyajima A. Itoh T. Adaptive remodeling of the biliary architecture underlies liver homeostasis.Hepatology. 2015; 61: 2056-2066Crossref PubMed Scopus (77) Google Scholar, 11Köhn-Gaone J. Dwyer B.J. Grzelak C.A. Miller G. Shackel N.A. Ramm G.A. McCaughan G.W. Elsegood C.L. Olynyk J.K. Tirnitz-Parker J.E.E. Divergent inflammatory, fibrogenic, and liver progenitor cell dynamics in two common mouse models of chronic liver injury.Am J Pathol. 2016; 186: 1762-1774Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 12Clerbaux L.-A. Van Hul N. Gouw A.S.H. Manco R. Español-Suñer R. Leclercq I.A. Relevance of the CDE and DDC mouse models to study ductular reaction in chronic human liver diseases. Animal Models for Human Diseases. Edited by Ibeh B.InTech Open. 2018; Google Scholar, 13Alison M. Golding M. Lalani E.-N. Sarraf C. Wound healing in the liver with particular reference to stem cells.Philos Trans R Soc London. 1998; 353: 877-894Crossref PubMed Scopus (72) Google Scholar Proliferation of pseudoducts is typically seen on cholangiocellular diseases, such as in primary biliary cholangitis or primary sclerosing cholangitis. In experimental animals, bile duct ligation (BDL) or diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet cause a type of DR that has been demonstrated to represent the two-dimensional proliferative rearrangement of the biliary epithelium.10Kaneko K. Kamimoto K. Miyajima A. Itoh T. Adaptive remodeling of the biliary architecture underlies liver homeostasis.Hepatology. 2015; 61: 2056-2066Crossref PubMed Scopus (77) Google Scholar, 14Vartak N. Damle-Vartak A. Richter B. Dirsch O. Dahmen U. Hammad S. Hengstler J.G. Cholestasis-induced adaptive remodeling of interlobular bile ducts.Hepatology. 2016; 63: 951-964Crossref PubMed Scopus (86) Google Scholar In hepatocellular diseases, DR manifests as the invasion of the parenchyma by elongated cells expressing biliary markers. This is seen in viral hepatitis C or autoimmune disease in humans or in the choline-deficient ethionine-supplemented (CDE) model in rodents.12Clerbaux L.-A. Van Hul N. Gouw A.S.H. Manco R. Español-Suñer R. Leclercq I.A. Relevance of the CDE and DDC mouse models to study ductular reaction in chronic human liver diseases. Animal Models for Human Diseases. Edited by Ibeh B.InTech Open. 2018; Google Scholar Whether DR is ever responsible for parenchymal reconstitution remains controversial. Parenchymal reconstitution from DR has been suggested in severe acute injury in human livers15Theise N.D. Saxena R. Portmann B.C. Thung S.N. Yee H. Chiriboga L. Kumar A. Crawford J.M. The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (613) Google Scholar and in advanced-stage chronic human disease.16Roskams T. Progenitor cell involvement in cirrhotic human liver diseases: from controversy to consensus.J Hepatol. 2003; 39: 431-434Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Roskams T.A. Libbrecht L. Desmet V.J. Progenitor cells in diseased human liver.Semin Liver Dis. 2003; 23: 385-396Crossref PubMed Scopus (274) Google Scholar Some studies in rodents18Lu W.-Y. Bird T.G. Boulter L. Tsuchiya A. Cole A.M. Hay T. Guest R.V. Wojtacha D. Man T.Y. Mackinnon A. Ridgway R.A. Kendall T. Williams M.J. Jamieson T. Raven A. Hay D.C. Iredale J.P. Clarke A.R. Sansom O.J. Forbes S.J. Hepatic progenitor cells of biliary origin with liver repopulation capacity.Nat Cell Biol. 2015; 17: 971-983Crossref PubMed Scopus (303) Google Scholar, 19Raven A. Lu W.-Y. Man T.Y. Ferreira-Gonzalez S. O'Duibhir E. 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Hepatocytes could accomplish regeneration in the unlined regions of parenchyma, whereas the DR compartment could be required to preserve or repair a canalicular-ductal morphologic link.29Theise N.D. Dollé L. Kuwahara R. Low hepatocyte repopulation from stem cells: a matter of hepatobiliary linkage not massive production.Gastroenterology. 2013; 145: 253-254Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 30Desmet V.J. Ductal plates in hepatic ductular reactions: hypothesis and implications, I: types of ductular reaction reconsidered.Virchows Arch. 2011; 458: 251-259Crossref PubMed Scopus (96) Google Scholar Herein, we show that CDE-induced DR expands into the parenchyma as biliary epithelium, which establishes de novo junctions with canaliculi. Reduction of CDE-induced DR extent significantly decreased the number of hepatobiliary junctions. By contrast, new ductular-canalicular junctions were not observed in the BDL or DDC models of biliary obstructive injury. In a similar manner, an increased number of hepatobiliary junctions in viral, drug-induced, or metabolic hepatocellular diseases in which DR invades the lobule, but not in biliary diseases (obstruction, primary biliary cholangitis, or primary sclerosing cholangitis) in which DR is contained within the portal mesenchyme was observed in humans. These findings support that the invasive form of DR connects with the canalicular system and operates hepatobiliary junctions after disorders affecting predominantly hepatocytes. All animal experiments were performed with approval of the University Animal Welfare Committee (2012UCLMD026; 2016UCLMD003). Five-week–old male C57Bl/6J mice (weight, 80 μm from the border of the portal mesenchyma, the associated DR was classified as invasive; when junctions were observed between 20 and 80 μm, DR was categorized as minimally invasive; and when junctions were seen <20 μm or within the portal mesenchyma, DR was classified as noninvasive. Mouse liver sections were incubated with primary antibodies against CK19 (dilution 1:10; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), then with a horseradish peroxidase–conjugated secondary antibody and binding revealed with 3,3’-diaminobenzidine. Diaminobenzidine-stained sections were digitalized with a SCN400 slide scanner (Leica, Wetzlar, Germany). On CK19-stained slides, the stained area was measured using Tissue IA software version 2.0.04 (Leica Biosystems, Dublin, Ireland). For immunofluorescence labeling, liver sections were exposed to antibodies directed against CK19 (dilution 1:10; Developmental Studies Hybridoma Bank), mucin-1 (dilution 1:200; MUC-1 Ab-5; NeoMarkers, Fremont, CA), laminin (dilution 1:50; ab11575; Abcam, Cambridge, UK), acetylated α-tubulin (dilution 1:1000; T6793; Sigma), carcinoembryonic antigen–related cell adhesion molecule (ceacam)-1 (dilution 1:500; LS-C106710; LifeSpan Biosciences, Seattle, WA), and YFP (dilution 1:250; ab6673; Abcam). Secondary antibodies were anti-rat IgG, anti-goat IgG, or anti-rabbit IgG, combined to AlexaFluor 594, AlexaFluor 488, or AlexaFluor 647 (dilution 1:1000; Invitrogen, Merelbeke, Belgium), as appropriate. For mucin-1 immunodetection, sections were incubated with anti-hamster IgG (dilution 1:250; 127-065-160; Jackson ImmunoResearch, Ely, UK) and then with AlexaFluor 488 (1:1000; Invitrogen). Hoechst (dilution 1:10,000) was used to reveal the nuclei. After double immunofluorescence of ceacam-1 and mucin-1, optical sections were generated by structured illumination using an AxioImager microscope (Carl Zeiss Company, Oberkochen, Germany) and then analyzed using the image analysis tool Author version 6.0.0 (Visiopharm, Hørsholm, Denmark). Portal fields were delineated manually; then, a concentric area of 170 μm was automatically extended by the Visiopharm software. The junctions between mucin-1 (green) and ceacam-1 (red) were assessed manually, and the shortest distance between each junction and the portal vein was measured by the Visiopharm software. For Z-stack imaging, liver slides (vibratome; 100 μm thick) were exposed under agitation for 2 days at 4°C first to primary antibody against mucin-1 and then for 2 days at 4°C to primary antibody against ceacam-1, followed by 2 days at 4°C with secondary antibody anti-hamster IgG, and finally with a mixture of AlexaFluor 488, secondary antibody anti-mouse IgG combined to AlexaFluor 594, and Hoechst. Liver sections were examined with a Zeiss LSM510 confocal microscope. Total RNA was extracted using TRIzol (Invitrogen). Quantitative real-time PCR was performed by AB StepOne Plus (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (Applied Biosystems). Expression of 36B4 was used as an internal standard. Results are expressed as fold expression relative to expression in the control (value set at 1) using the ΔΔCt method. All data are presented as means ± SD or means ± SEM when indicated, and were compared using the unpaired two-tailed t-test or one-way analysis of variance. CDE-fed mice display a DR that progressively invades the hepatic lobule.33Van Hul N. Abarca-Quinones J. Sempoux C. Horsmans Y. Leclercq I.A. Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.Hepatology. 2009; 49: 1625-1635Crossref PubMed Scopus (132) Google Scholar In livers of mice controls or fed with CDE diet for 3 days, staining of the well-established biliary/DR marker CK19 was restricted to cholangiocytes of the bile ducts and single isolated cells around the PTs, corresponding to CoH (Figure 1). After 9 days of CDE, CK19+ DR expands outside the portal area; and after 21 days, DR number significantly increased as they invaded the hepatic lobule (Figure 1A). Mucin-1 is a glycoprotein produced by and lining the apical surface of cholangiocytes facing bile duct lumen in normal livers (Figure 1B). While on the basal side, cholangiocytes (but not hepatocytes) lie on a laminin basement membrane (Figure 1C).22Español-Suñer R. Carpentier R. Van Hul N. Legry V. Achouri Y. Cordi S. Jacquemin P. Lemaigre F. Leclercq I.A. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.Gastroenterology. 2012; 143: 1564-1575Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar Like for cholangiocytes, mucin-1 staining was polarized at the apical pole of DR cells (Figure 1B), whereas laminin was located at their basal pole (Figure 1C). Cholangiocytes carry a primary cilium, a sensory organelle that protrudes from the apical pole into the duct lumen and detects changes in bile flow and composition. As in bile ducts, CDE-induced DR exhibited acetylated α-tubulin–positive staining, aligning longitudinally along the lumen (Figure 1D). Cholangiocytes release bicarbonate,3Maroni L. Haibo B. Ray D. Zhou T. Wan Y. Meng F. Marzioni M. Alpini G. Functional and structural features of cholangiocytes in health and disease.Cell Mol Gastroenterol Hepatol. 2015; 1: 368-380Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar through activation of the secretin receptor (SR), the cystic fibrosis transmembrane conductance regulator (CFTR), and the chloride/bicarbonate anion exchanger 2 (AE2). Hepatic mRNA expression levels of SR and CFTR (but not of AE2; data not shown) were significantly increased in 9- and 21-day CDE livers, when infiltrative DR was seen and positively correlated with CK19 mRNA hepatic expression (Figure 1, E and F). Altogether, these data support that DRs expand as polarized biliary epithelium expressing the machinery needed to sense and modify bile (flow). DR and bile ducts constitute together a continuous network in CDE livers.10Kaneko K. Kamimoto K. Miyajima A. Itoh T. Adaptive remodeling of the biliary architecture underlies liver homeostasis.Hepatology. 2015; 61: 2056-2066Crossref PubMed Scopus (77) Google Scholar, 34Lenzi R. Liu M. Tarsetti F. Slott P. Alpini G. Zhai W. Paronetto F. Lenzen R. Tavolini N. Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis: evidence for a biliary epithelial nature of oval cells.Lab Invest. 1992; 66: 390-402PubMed Google Scholar Whether these ductular ramifications emerging in the continuity of the biliary tree connect, on the other side, to the canalicular system has never been explored. To visualize the ductular-canalicular junctions, double staining was performed with mucin-1 to label the apical pole of biliary/DR cells; and ceacam-1 was used for the hepatocyte canaliculi.22Español-Suñer R. Carpentier R. Van Hul N. Legry V. Achouri Y. Cordi S. Jacquemin P. 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Desdouets C. Leclercq I. Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury.J Hepatol. 2019; 70: 1180-1191Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar The mucin-1–ceacam-1 junctions appeared as either continuous on the same plane or overlapping, depending on the angle of the two-dimensional (2D) analysis (Figure 2B). Between zero and two ductular-canalicular junctions were identified per PT in control livers (Figure 2B). As confirmed using tamoxifen-injected osteopontin-iCreERT2;Rosa26-YFP mice, in which biliary cells are readily identified by their YFP tag,22Español-Suñer R. Carpentier R. Van Hul N. Legry V. Achouri Y. Cordi S. Jacquemin P. Lemaigre F. Leclercq I.A. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.Gastroenterology. 2012; 143: 1564-1575Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar mucin-1–expressing cells engaged in these junctions were isola
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