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Hepatic Stem/Progenitor Cell Activation Differs between Primary Sclerosing and Primary Biliary Cholangitis

Open ArchivePublished:December 14, 2017DOI:https://doi.org/10.1016/j.ajpath.2017.11.010
      Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are human primary cholangiopathies characterized by the damage of mature cholangiocytes and by the appearance of ductular reaction (DR) as the results of hepatic progenitor cell activation. This study evaluated the differences in progenitor cell niche activation between these two cholangiopathies. Liver tissue was obtained from healthy liver donors (n = 5) and from patients with PSC (n = 20) or PBC (n = 20). DR, progenitor cell phenotype, and signaling pathways were investigated by IHC analysis and immunofluorescence. Our results indicated that DR was more extended, appeared earlier, and had a higher proliferation index in PBC compared with PSC. In PBC, DR was strongly correlated with clinical prognostic scores. A higher percentage of sex determining region Y–box (SOX)9+ and cytokeratin 19+ cells but fewer features of hepatocyte fate characterized progenitor cell activation in PBC versus PSC. Lower levels of laminin and neurogenic locus notch homolog protein 1 but higher expression of wingless-related integration site (WNT) family pathway components characterize progenitor cell niche in PSC compared with PBC. In conclusion, progenitor cell activation differs between PSC and PBC and is characterized by a divergent fate commitment and different signaling pathway predominance. In PBC, DR represents a relevant histologic prognostic marker.
      Primary sclerosing cholangitis (PSC) is a chronic cholangiopathy characterized by diffuse inflammation and fibrosis of the bile ducts.
      • Lazaridis K.N.
      • LaRusso N.F.
      Primary sclerosing cholangitis.
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      • Adams D.H.
      Primary sclerosing cholangitis.
      The progressive injury primarily involves the extrahepatic and large intrahepatic bile ducts, leading to concentric periductal fibrosis and obliterating strictures.
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      • LaRusso N.F.
      Primary sclerosing cholangitis.
      • Hirschfield G.M.
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      • Adams D.H.
      Primary sclerosing cholangitis.
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      • Rossi M.
      • Karlsen T.H.
      • Alvaro D.
      • Gaudio E.
      Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.
      The pathogenesis of PSC is still largely unknown, and the mechanisms involved in the characteristic bile duct strictures are poorly understood.
      • Tabibian J.H.
      • O'Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
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      • Strazzabosco M.
      • Manns M.
      • Trauner M.
      Characterization of animal models for primary sclerosing cholangitis (PSC).
      Recently, the involvement of biliary tree stem/progenitor cells (BTSCs) was demonstrated as a determinant in the progression of duct lesions in PSC.
      • Carpino G.
      • Cardinale V.
      • Renzi A.
      • Hov J.R.
      • Berloco P.B.
      • Rossi M.
      • Karlsen T.H.
      • Alvaro D.
      • Gaudio E.
      Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.
      BTSCs represent a stem/progenitor cell niche located within the glands of the biliary tree (peribiliary glands).
      • Cardinale V.
      • Wang Y.
      • Gaudio E.
      • Carpino G.
      • Mendel G.
      • Alpini G.
      • Reid L.M.
      • Alvaro D.
      The biliary tree: a reservoir of multipotent stem cells.
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      • Wang Y.
      • Semeraro R.
      • Anceschi M.
      • Brunelli R.
      • Alvaro D.
      • Reid L.M.
      • Gaudio E.
      Biliary tree stem/progenitor cells in glands of extrahepatic and intrahepatic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.
      • Lanzoni G.
      • Cardinale V.
      • Carpino G.
      The hepatic, biliary, and pancreatic network of stem/progenitor cell niches in humans: a new reference frame for disease and regeneration.
      Massive BTSC proliferation and peribiliary gland hyperplasia were revealed in PSC but not in primary biliary cholangitis (PBC). Peribiliary gland activation resulted in obliterative concentric fibrosis through the accumulation of myofibroblasts in the walls of the extrahepatic and large intrahepatic bile ducts in PSC.
      • Carpino G.
      • Cardinale V.
      • Renzi A.
      • Hov J.R.
      • Berloco P.B.
      • Rossi M.
      • Karlsen T.H.
      • Alvaro D.
      • Gaudio E.
      Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.
      • Lanzoni G.
      • Cardinale V.
      • Carpino G.
      The hepatic, biliary, and pancreatic network of stem/progenitor cell niches in humans: a new reference frame for disease and regeneration.
      Moreover, PSC and PBC share the appearance of ductular reaction (DR) within the liver parenchyma. DR is thought to represent the activation of the hepatic stem/progenitor cell (HpSC) niche that resides within the bile ductules.
      • Carpino G.
      • Cardinale V.
      • Renzi A.
      • Hov J.R.
      • Berloco P.B.
      • Rossi M.
      • Karlsen T.H.
      • Alvaro D.
      • Gaudio E.
      Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.
      • Stueck A.E.
      • Wanless I.R.
      Hepatocyte buds derived from progenitor cells repopulate regions of parenchymal extinction in human cirrhosis.
      Interestingly, the contribution of HpSCs in human liver regeneration is variable based on disease pathogenesis.
      • Katoonizadeh A.
      • Nevens F.
      • Verslype C.
      • Pirenne J.
      • Roskams T.
      Liver regeneration in acute severe liver impairment: a clinicopathological correlation study.
      • Spee B.
      • Carpino G.
      • Schotanus B.A.
      • Katoonizadeh A.
      • Vander Borght S.
      • Gaudio E.
      • Roskams T.
      Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
      Different signaling pathways are involved in generating divergent responses due to the specific liver injury.
      • Spee B.
      • Carpino G.
      • Schotanus B.A.
      • Katoonizadeh A.
      • Vander Borght S.
      • Gaudio E.
      • Roskams T.
      Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
      • Boulter L.
      • Govaere O.
      • Bird T.G.
      • Radulescu S.
      • Ramachandran P.
      • Pellicoro A.
      • Ridgway R.A.
      • Seo S.S.
      • Spee B.
      • Van Rooijen N.
      • Sansom O.J.
      • Iredale J.P.
      • Lowell S.
      • Roskams T.
      • Forbes S.J.
      Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease.
      In the HpSC niche, the neurogenic locus notch homolog protein (NOTCH) and wingless-related integration site (WNT) family pathways can be differently modulated based on disease etiology and stage.
      • Spee B.
      • Carpino G.
      • Schotanus B.A.
      • Katoonizadeh A.
      • Vander Borght S.
      • Gaudio E.
      • Roskams T.
      Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
      • Boulter L.
      • Govaere O.
      • Bird T.G.
      • Radulescu S.
      • Ramachandran P.
      • Pellicoro A.
      • Ridgway R.A.
      • Seo S.S.
      • Spee B.
      • Van Rooijen N.
      • Sansom O.J.
      • Iredale J.P.
      • Lowell S.
      • Roskams T.
      • Forbes S.J.
      Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease.
      However, differences in HpSC response and DR phenotype between PSC and PBC have not been yet investigated.
      Therefore, the aims of the present study were to compare PSC and PBC in terms of i) DR extent and HpSC activation in progressive disease stages, ii) DR phenotype and commitment, and iii) expression of signaling pathways by HpSCs.

      Materials and Methods

      Samples

      Formalin-fixed and paraffin-embedded liver tissue from patients with PSC or PBC or from healthy livers was obtained from the Norwegian PSC Research Center, Oslo University Hospital Rikshospitalet (Oslo, Norway), from the Sapienza University of Rome (Rome, Italy), and from the University of Padua (Padua, Italy).
      Specimens included i) livers with normal findings on histologic examination (controls) from liver donors (n = 5); ii) PSC (n = 20), obtained from liver biopsy samples and from explanted livers; and iii) PBC (n = 20), from liver biopsy samples and from explanted livers.
      Informed consent was obtained from each patient, and the study protocol was conformed to the ethics guideline of the 1975 Declaration of Helsinki. The research protocol was reviewed and approved by the ethics committee at Umberto I Policlinico (Rome, Italy), and the regional committees for medical and health research ethics in southeastern Norway. No donor organs were obtained from executed prisoners or other institutionalized individuals.

      Clinical Parameters

      The diagnostic criteria for PSC and PBC followed the Clinical Practice Guidelines from the European Association for the Study of the Liver.
      European Association for the Study of the Liver
      EASL clinical practice guidelines: management of cholestatic liver diseases.
      Clinical and biochemical parameters were collected during biopsy or during transplantation, and, if appropriate, 1 year after ursodeoxycholic acid administration. Clinical parameters were collected to determine prognostic classes according to the Mayo Clinic's Revised Natural History Model for PSC and the Updated Natural History Model for PBC, while the Global PBC
      • Lammers W.J.
      • Hirschfield G.M.
      • Corpechot C.
      • Nevens F.
      • Lindor K.D.
      • Janssen H.L.
      • Floreani A.
      • Ponsioen C.Y.
      • Mayo M.J.
      • Invernizzi P.
      • Battezzati P.M.
      • Pares A.
      • Burroughs A.K.
      • Mason A.L.
      • Kowdley K.V.
      • Kumagi T.
      • Harms M.H.
      • Trivedi P.J.
      • Poupon R.
      • Cheung A.
      • Lleo A.
      • Caballeria L.
      • Hansen B.E.
      • van Buuren H.R.
      Global PBC Study Group
      Development and validation of a scoring system to predict outcomes of patients with primary biliary cirrhosis receiving ursodeoxycholic acid therapy.
      and UK-PBC
      • Carbone M.
      • Sharp S.J.
      • Flack S.
      • Paximadas D.
      • Spiess K.
      • Adgey C.
      • Griffiths L.
      • Lim R.
      • Trembling P.
      • Williamson K.
      • Wareham N.J.
      • Aldersley M.
      • Bathgate A.
      • Burroughs A.K.
      • Heneghan M.A.
      • Neuberger J.M.
      • Thorburn D.
      • Hirschfield G.M.
      • Cordell H.J.
      • Alexander G.J.
      • Jones D.E.
      • Sandford R.N.
      • Mells G.F.
      UK-PBC Consortium
      The UK-PBC risk scores: derivation and validation of a scoring system for long-term prediction of end-stage liver disease in primary biliary cholangitis.
      risk scores were used to stratify the risk for progression in PBC patients. Patients who presented with overlapping syndromes or concomitant liver diseases, comprising alcoholic steatohepatitis or nonalcoholic fatty liver disease, were excluded.

      Light Microscopy, Histopathology, and IHC Analysis

      Specimens were fixed in 10% buffered formalin for 2 to 4 hours and embedded in low-temperature–fusion paraffin (55°C to 57°C), and 3- to 4-μm sections were stained with hematoxylin and eosin and Sirius Red/Fast Green, according to standard protocols. PSC and PBC samples were staged in accordance to the common histologic scoring systems
      • Ludwig J.
      • Dickson E.R.
      • McDonald G.S.
      Staging of chronic nonsuppurative destructive cholangitis (syndrome of primary biliary cirrhosis).
      • de Vries E.M.
      • de Krijger M.
      • Farkkila M.
      • Arola J.
      • Schirmacher P.
      • Gotthardt D.
      • Goeppert B.
      • Trivedi P.J.
      • Hirschfield G.M.
      • Ytting H.
      • Vainer B.
      • Buuren H.R.
      • Biermann K.
      • Harms M.H.
      • Chazouilleres O.
      • Wendum D.
      • Kemgang A.D.
      • Chapman R.W.
      • Wang L.M.
      • Williamson K.D.
      • Gouw A.S.
      • Paradis V.
      • Sempoux C.
      • Beuers U.
      • Hubscher S.G.
      • Verheij J.
      • Ponsioen C.Y.
      Validation of the prognostic value of histologic scoring systems in primary sclerosing cholangitis: an international cohort study.
      and further categorized as early (not cirrhosis) or cirrhosis. The extent of fibrosis was evaluated in Sirius Red/Fast Green stains, as detailed previously.
      • Della Corte C.
      • Carpino G.
      • De Vito R.
      • De Stefanis C.
      • Alisi A.
      • Cianfarani S.
      • Overi D.
      • Mosca A.
      • Stronati L.
      • Cucchiara S.
      • Raponi M.
      • Gaudio E.
      • Byrne C.D.
      • Nobili V.
      Docosahexanoic acid plus vitamin D treatment improves features of NAFLD in children with serum vitamin D deficiency: results from a single centre trial.
      Briefly, Sirius Red/Fast Green–stained slides were scanned by a digital scanner (ScanScope CS System, Aperio Technologies, Inc., Oxford, UK) and processed by ImageScope software version 11.2 (Leica Biosystems, Milan, Italy). An image analysis algorithm was used to quantify the proportion of Sirius Red–stained area. The algorithm was applied on the entire section. The extent of collagen deposition was expressed as the proportion (%) of Sirius Red–stained area with respect to the total biopsy area, providing a quantitative value on a continuous scale.
      For immunohistochemistry analysis, endogenous peroxidase activity was blocked by a 30-minute incubation in methanolic hydrogen peroxide (2.5%). Antigens were retrieved, as indicated by the vendor, by applying proteinase K (catalog number S3020; Dako Cytomation, Glostrup, Denmark) for 10 minutes at room temperature. Sections were then incubated overnight at 4°C with primary antibodies (Table 1). Samples were rinsed twice with phosphate-buffered saline for 5 minutes and incubated for 20 minutes at room temperature with secondary biotinylated antibody and then with streptavidin–horseradish peroxidase (LSAB + System-HRP; catalog number K0690; Dako Cytomation). Diaminobenzidine (Dako Cytomation) was used as a substrate, and sections were counterstained with hematoxylin.
      • Onori P.
      • Alvaro D.
      • Floreani A.R.
      • Mancino M.G.
      • Franchitto A.
      • Guido M.
      • Carpino G.
      • De Santis A.
      • Angelico M.
      • Attili A.F.
      • Gaudio E.
      Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.
      For immunofluorescence, nonspecific protein binding was blocked by 5% normal goat serum. Specimens were incubated with primary antibodies. Specimens were washed and incubated for 1 hour with labeled isotype-specific secondary antibodies (anti-mouse Alexa Fluor 546, anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 488, and anti-goat Alexa Fluor 546; Invitrogen, Life Technologies Ltd, Paisley, UK) and counterstained with DAPI for visualization of cell nuclei.
      • Carpino G.
      • Renzi A.
      • Cardinale V.
      • Franchitto A.
      • Onori P.
      • Overi D.
      • Rossi M.
      • Berloco P.B.
      • Alvaro D.
      • Reid L.M.
      • Gaudio E.
      Progenitor cell niches in the human pancreatic duct system and associated pancreatic duct glands: an anatomical and immunophenotyping study.
      For all immunoreactions, negative controls (the primary antibody was replaced with preimmune serum) were also included. Sections were examined in a coded fashion by DM 4500 B Light and Fluorescence Microscopy (Leica Microsystems, Weltzlar, Germany), equipped with a Prog Res C10 Plus Videocam (Jenoptik, Jena, Germany). Immunofluorescence stains were also analyzed by confocal microscopy (model TCS-SP2; Leica Microsystems). Slides were further processed with Image Analysis System software version 7 (Delta Sistemi, Rome, Italy) and were independently evaluated by two researchers (G.C. and P.O.) in a blinded fashion.
      Table 1List of Antibodies Used in This Study
      NameHostSupplierCatalog no.Dilution
      CD326/EpCAMMouseDako CytomationM35251:100
      Cytokeratin 7MouseDako CytomationM70181:100
      Cytokeratin 19MouseDako CytomationM08881:100
      Hep-Par1MouseDako CytomationM71581:100
      LamininRabbitDako CytomationZ00971:25
      Notch1GoatSanta Cruz Biotechnology, Heidelberg, GermanySc-60141:50
      pβ-CateninMouseCell Signaling Technology, Leiden, the Netherlands#4176S1:100
      PCNAMouseDako CytomationM08791:100
      SOX9RabbitEMD Millipore, Darmstadt, GermanyAB58091:200
      WNT1GoatSanta CruzSc.62801:50
      WNT3aMouseSanta CruzSc-1361631:50
      EpCAM, epithelial cell adhesion molecule; Hep-Par1, hepatocyte paraffin 1; Notch1, neurogenic locus notch homolog protein 1; p-, phosphorylated; PCNA, proliferating cell nuclear antigen; SOX9, sex determining region Y-box (SOX)9; WNT, wingless-related integration site family.
      The extent of DR was evaluated by cytokeratin (CK)-7 immunoreactivity. CK7-stained slides were scanned by a digital scanner (ScanScope CS System; Aperio Digital Pathology, Leica Biosystems) and processed by ImageScope software.
      • Carpino G.
      • Nobili V.
      • Renzi A.
      • De Stefanis C.
      • Stronati L.
      • Franchitto A.
      • Alisi A.
      • Onori P.
      • De Vito R.
      • Alpini G.
      • Gaudio E.
      Macrophage activation in pediatric nonalcoholic fatty liver disease (NAFLD) correlates with Hepatic progenitor cell response via Wnt3a pathway.
      • Nobili V.
      • Carpino G.
      • Alisi A.
      • Franchitto A.
      • Alpini G.
      • De Vito R.
      • Onori P.
      • Alvaro D.
      • Gaudio E.
      Hepatic progenitor cells activation, fibrosis and adipokines production in pediatric nonalcoholic fatty liver disease.
      The area occupied by CK7+ cells was quantified by an image analysis algorithm. The extent of DR was expressed as the percentage of the parenchymal area occupied by reactive ductules.
      • Carpino G.
      • Nobili V.
      • Renzi A.
      • De Stefanis C.
      • Stronati L.
      • Franchitto A.
      • Alisi A.
      • Onori P.
      • De Vito R.
      • Alpini G.
      • Gaudio E.
      Macrophage activation in pediatric nonalcoholic fatty liver disease (NAFLD) correlates with Hepatic progenitor cell response via Wnt3a pathway.
      • Nobili V.
      • Carpino G.
      • Alisi A.
      • Franchitto A.
      • Alpini G.
      • De Vito R.
      • Onori P.
      • Alvaro D.
      • Gaudio E.
      Hepatic progenitor cells activation, fibrosis and adipokines production in pediatric nonalcoholic fatty liver disease.
      Cholangiocytes lining the interlobular bile ducts were excluded from the counts. CK7-stained slides were further counterstained with Sirius Red for better visualization of the spatial relationship between DR and fibrosis.
      With regard to DR phenotype, sex determining region Y–box (SOX)9 and epithelial cell adhesion molecule (EpCAM) were used as stem/progenitor cell markers; the coexpression of CK7 and CK19 was considered indicative of the maintenance of a biliary phenotype, and the expression of the hepatocyte marker hepatocyte paraffin 1 (Hep-Par1) and the loss of the biliary CK19 expression were considered as signs of hepatocyte commitment.
      • Spee B.
      • Carpino G.
      • Schotanus B.A.
      • Katoonizadeh A.
      • Vander Borght S.
      • Gaudio E.
      • Roskams T.
      Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.
      • Turner R.
      • Lozoya O.
      • Wang Y.
      • Cardinale V.
      • Gaudio E.
      • Alpini G.
      • Mendel G.
      • Wauthier E.
      • Barbier C.
      • Alvaro D.
      • Reid L.M.
      Human hepatic stem cell and maturational liver lineage biology.
      The DR proliferation index (PI) was automatically calculated as the percentage of DR cells expressing the proliferating cell nuclear antigen by specific algorithms.
      For all other immunoreactions, the number of positive cells was automatically calculated by an algorithm on the entire section and, then, a semiquantitative scoring system was applied (0 = <1%; 1 = 1% to 5%; 2 = 6% to 30%; 3 = 31% to 50%; and 4 = >50%).
      • Carpino G.
      • Cardinale V.
      • Renzi A.
      • Hov J.R.
      • Berloco P.B.
      • Rossi M.
      • Karlsen T.H.
      • Alvaro D.
      • Gaudio E.
      Activation of biliary tree stem cells within peribiliary glands in primary sclerosing cholangitis.
      Immunofluorescence stains were scanned with a digital scanner (ScanScope FL System; Aperio Technologies) and processed with ImageScope software. An algorithm was used to quantify the proportion of positive pixel area for a single fluorophore or the area with colocalization of two fluorophores.
      • Carpino G.
      • Cardinale V.
      • Gentile R.
      • Onori P.
      • Semeraro R.
      • Franchitto A.
      • Wang Y.
      • Bosco D.
      • Iossa A.
      • Napoletano C.
      • Cantafora A.
      • D'Argenio G.
      • Nuti M.
      • Caporaso N.
      • Berloco P.
      • Venere R.
      • Oikawa T.
      • Reid L.
      • Alvaro D.
      • Gaudio E.
      Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder.

      Intermediate EpCAM+ Hepatocytes and Bud Definition

      Buds were defined as hepatocytes, single or in clusters, immediately adjacent to cholangiocytes and located in fibrous septa (ie, in regions with parenchymal extinction). Buds were classified as levels 0 to 4. Buds of each maturation level (1 to 4) were counted on the CK19 slides.
      • Stueck A.E.
      • Wanless I.R.
      Hepatocyte buds derived from progenitor cells repopulate regions of parenchymal extinction in human cirrhosis.
      To quantitate the presence of apparent bud-derived tissue, we defined the immaturity index (IMI)-EpCAM as the presence of hepatocytes with EpCAM positivity.
      • Stueck A.E.
      • Wanless I.R.
      Hepatocyte buds derived from progenitor cells repopulate regions of parenchymal extinction in human cirrhosis.
      • Yoon S.M.
      • Gerasimidou D.
      • Kuwahara R.
      • Hytiroglou P.
      • Yoo J.E.
      • Park Y.N.
      • Theise N.D.
      Epithelial cell adhesion molecule (EpCAM) marks hepatocytes newly derived from stem/progenitor cells in humans.
      The presence of EpCAM+ hepatocytes was further confirmed by double immunofluorescence for EpCAM and Hep-Par1 and scored as: 0 = no positive cells; 1 (level 1) = single occasional; or 2 (level 2) = clusters of EpCAM+ hepatocytes.
      • Stueck A.E.
      • Wanless I.R.
      Hepatocyte buds derived from progenitor cells repopulate regions of parenchymal extinction in human cirrhosis.
      • Katoonizadeh A.
      • Nevens F.
      • Verslype C.
      • Pirenne J.
      • Roskams T.
      Liver regeneration in acute severe liver impairment: a clinicopathological correlation study.
      Analyses were independently performed by two different researchers (G.C. and P.O.) in a blinded fashion.

      Statistical Analysis

      Data are expressed as means ± SD. The t-test or Mann–Whitney U-test was used to determine the significance of differences between groups for normally or non-normally distributed data, respectively. The Pearson correlation coefficient or the Spearman nonparametric correlation was used. A P value of <0.05 was considered statistically significant. Analyses were performed using SPSS software version 23 (IBM, Milan, Italy).

      Results

      Clinical and Serologic Parameters

      In PSC patients, ALP was similar between early-PSC and cirrhotic-PSC patients (Table 2). In contrast, serum bilirubin, serum albumin, and international normalized ratio were significantly altered in cirrhotic- compared with noncirrhotic-PSC patients. The cirrhotic-PSC patients presented a high Mayo risk score, whereas noncirrhotic-PSC patients showed a low or intermediate score.
      Table 2Biochemical and Prognostic Parameters in Patients with Primary Sclerosing Cholangitis
      ParameterEarly PSC (n = 10)Cirrhotic PSC (n = 10)P value
      Age (y)32.9 ± 5.557.13 ± 4.13<0.001
      M/F, n6/46/4
      Bilirubin (mg/dL)0.95 ± 0.586.43 ± 5.57<0.05
      Albumin (g/L)44.5 ± 3.525.3 ± 4.1<0.001
      AST (× ULN)2.14 ± 0.633.79 ± 2.050.07
      ALP (× ULN)4.23 ± 2.111.99 ± 1.610.14
      GGT (× ULN)3.55 ± 1.992.73 ± 3.080.61
      INR (0.8–1.2)0.92 ± 0.041.9 ± 0.5<0.001
      Mayo PSC riskLow to intermediateHigh
      Unless otherwise indicated, data are expressed as means ± SD. P values <0.05 are in bold.
      F, female; M, male; ALP, alkaline phosphatase; AST, aspartate aminotransferase; GGT, γ-glutamyl transferase; INR, international normalized ratio; PSC, primary sclerosing cholangitis; ULN, upper limit of normal.
      PBC patients were young adult and middle-aged women (Table 3). ALP was significantly increased in cirrhotic-PBC compared with early-PBC patients (P < 0.05), whereas none of the differences were significant in ALT, bilirubin, or albumin level. PBC patients, according to the Mayo Clinic's Updated Natural History Model for PBC, had an estimated probability of survival at 24 months of 99% to 100%. Liver biochemistry was evaluated 1 year after the start of ursodeoxycholic acid treatment for the calculation of the Global PBC and UK-PBC risk scores. Both the UK-PBC and Global PBC risk scores discriminated between early-PBC and cirrhotic-PBC patients (both, P < 0.05).
      Table 3Biochemical and Prognostic Parameters in Patients with Primary Biliary Cholangitis
      ParameterEarly PBC (n = 10)Cirrhotic PBC (n = 10)P value
      Age (y)46.33 ± 11.546.0 ± 8.540.96
      M/F, n0/100/10
      Bilirubin (mg/dL)0.63 ± 0.291.43 ± 1.220.14
      Albumin (g/L)40.3 ± 4.342.4 ± 4.30.5
      AST (× ULN)0.89 ± 0.51.72 ± 2.040.39
      ALP (× ULN)2.11 ± 1.945.62 ± 2.48<0.05
      GGT (× ULN)3.52 ± 3.5414.83 ± 5.98<0.01
      INR (0.8–1.2)0.92 ± 0.041.9 ± 0.5<0.001
      UK risk score, 5 years (%)1.73 ± 1.259.69 ± 9.12<0.05
      UK risk score, 15 years (%)7.05 ± 4.329.47 ± 19.32<0.05
      Global PBC score, 3 years (%)95.93 ± 1.290.9 ± 3.24<0.01
      Global PBC score, 5 years (%)92.85 ± 2.1384.3 ± 5.4<0.01
      PBC score, 10 years (%)82.47 ± 7.7266.87 ± 8.61<0.01
      Mayo PBC risk, 24 months (%)99–10099–100
      Unless otherwise indicated, data are expressed as means ± SD. P values <0.05 are in bold.
      F, female; M, male; ALP, alkaline phosphatase; AST, aspartate aminotransferase; GGT, γ-glutamyl transferase; INR, international normalized ratio; PBC, primary biliary cholangitis; ULN, upper limit of normal.

      Ductular Reaction Extent and HpSC Proliferation Indexes

      In livers with normal findings on histologic examination (controls), resident HpSCs can be recognized as CK7 (or EpCAM) positive cells located in the smallest branches (canals of Hering and bile ductules) of the biliary tree at the interface with portal tracts (Figure 1A ). In the noncirrhotic stages of PBC (stages I to III; early PBC) (Figure 1B), the presence of reactive ductules was revealed, and DR extent was increased (5.05 ± 0.61) compared with that in healthy livers (2.41 ± 1.67; P < 0.01). Cirrhotic-PBC samples (stage IV) (Figure 1, C and D) were characterized by a greater DR extent (11.90 ± 1.40) compared with those in early PBC (P < 0.01) and controls (P < 0.001). DR extent was strongly correlated with PBC stage (r = 0.83, P < 0.001).
      Figure thumbnail gr1
      Figure 1Ductular reaction (DR) in human cholangiopathies. A–C: Immunohistochemistry(IHC) analysis for cytokeratin (CK)-7 in controls, primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). A: In control livers, CK7 immunoreaction individuates canals of Hering and bile ductules. The boxed area in the left panel corresponds with the magnified view of a canal of Hering in the right panel. B: In the early stages of PBC and PSC, the presence of DR is revealed. C: In cirrhotic samples, DR extent is greater compared with that in early stages. D: Differences in DR extent among groups. E: IHC analysis for CK7 was counterstained with Sirius Red for the visualization of the association between DR and fibrosis extent. F: Scatterplot showing correlation between DR and fibrosis in PBC and PSC. Data are expressed as means ± SD (D). P < 0.05 versus control; P < 0.05 versus early PSC; P < 0.05 versus early stages (PBC and PSC); §P < 0.05 versus cirrhotic PSC. Scale bars: 100 μm (A–C); 200 μm (E). Original magnification, ×40 (A, right panel)
      In PSC (Figure 1, B and D), DR extent was slightly, but not significantly, increased in early-PSC biopsy samples (3.16 ± 1.15) compared with livers with normal findings on histologic examination (P = 0.28). Cirrhotic-PSC samples showed a greater DR extent (7.01 ± 1.96) compared with those in early-PSC stages (P < 0.05) and controls (P < 0.05). DR extent was significantly less in PSC compared with PBC samples (P < 0.01).
      In both PBC and PSC samples, DR extent was significantly correlated with fibrosis extent (PBC: r = 0.63, P < 0.01; PSC: r = 0.62, P < 0.01) (Figure 1, E and F).
      When the PI of cells within reactive ductules (DR-PI) was calculated (Figure 2), the DR-PI in cirrhotic PBC (38.4 ± 6.2) was higher in comparison with controls (5.8 ± 2.9; P < 0.01) and early-PBC samples (18.0 ± 2.1; P < 0.01). The DR-PI in early PBC was significantly higher compared with that in control specimens (P < 0.05). The DR-PI in cirrhotic PSC (24.4 ± 3.8) was significantly higher in comparison with those in control (P < 0.001) and early-PSC samples (10.0 ± 3.7; P < 0.001), and in early-PSC samples compared with normal specimens (P < 0.05). The DR-PI was lower in PSC compared with PBC samples (P < 0.001).
      Figure thumbnail gr2
      Figure 2Ductular reaction (DR) proliferation index (PI) in human cholangiopathies. A: Immunohistochemistry (IHC) analysis for proliferating cell nuclear antigen (PCNA) in control livers, primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). PCNA+ cells within DR are more numerous (arrows) in PBC and PSC compared with controls. PCNA expression is higher in PBC compared with PSC samples and in cirrhotic compared with early stages. B: Immunofluorescence for cytokeratin (CK)-7 and PCNA in late stages of PBC and PSC samples. Nuclei are displayed in blue. Immunofluorescence confirms the higher PCNA expression in CK7+ DR in PBC versus PSC samples (arrows). C: Differences in DR-PI among groups. Data are expressed as means ± SD (C). P < 0.05 versus control; P < 0.05 versus early PSC; P < 0.05 versus early stages (PBC and PSC); §P < 0.05 versus cirrhotic PSC. Scale bars: 100 μm (A); 25 μm (B).
      With regard to clinical parameters, DR in PSC samples was correlated with the Mayo PSC risk score (r = 0.827, P < 0.05) (Supplemental Figure S1A) and with bilirubin (r = 0.841, P < 0.05) and was inversely correlated with serum albumin (r = −0.838, P < 0.05). No correlation with ALP serum levels was evident in PSC patients. In PBC samples, DR was correlated with total bilirubin (r = 0.937, P = 0.001); γ-glutamyl transferase (r = 0.736, P = 0.037); and UK-PBC risk scores up to 5 years (r = 0.785, P < 0.001), 10 years (r = 0.820, P < 0.001), and 15 years (r = 0.847, P = 0.001) (Supplemental Figure S1B) and Global PBC score (r = 0.791, P = 0.019) and Global PBC score orthotopic liver transplantation–free survival at 3 years (r = −0.852, P = 0.007), 5 years (r = −0.850, P = 0.008), and 10 years (r = −0.833, P = 0.010) (Supplemental Figure S1B). No correlation was found with the History Model for PBC from the Mayo Clinic.

      Phenotype of Reactive Ductules

      In control specimens with normal findings on histologic examination (Figure 3), double immunofluorescence showed that the majority of CK7+ cells lining the canals of Hering and the bile ductules coexpressed SOX9 (score = 3.6 ± 0.5) (Figure 3A) and CK19 (score = 4 ± 0) (Figure 3B). In cirrhotic PBC, double immunofluorescence showed that reactive ductules were mainly located within fibrous septa and that the majority of CK7+ cells were SOX9+ (score = 3.8 ± 0.4) (Figure 3A) and CK19+ (score = 3.7 ± 0.5) (Figure 3B). There were no or very few cells within the bile and reactive ductules that were Hep-Par1+ (score = 0; ie, <5%) in control and PBC samples (Figure 3C). Conversely, in cirrhotic PSC, reactive ductules were located at the interface with the fibrotic septa, and numerous cells penetrated deep into the cirrhotic nodules; variable percentages of CK7+ DR cells were positive for SOX9 (score = 1.2 ± 0.4) (Figure 3A) and CK19 (score = 1.65 ± 0.5) (Figure 3B), with lower values compared with controls and cirrhotic-PBC samples (both, P < 0.01). Interestingly, reactive ductules contained Hep-Par1+ cells in the cirrhotic-PSC samples (score = 1 ± 0.8) (Figure 3C) but not in the early-PSC samples (data not shown).
      Figure thumbnail gr3
      Figure 3Phenotype of reactive ductules in human cholangiopathies. A: Immunofluorescence for cytokeratin (CK)-7 and transcription factor sex determining region Y-box (SOX)9 in control livers, primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). In cirrhotic PSC, ductular reaction (DR) is composed of a higher percentage of CK7+/SOX9 (blue arrows) compared with controls and cirrhotic PBC. Red arrows indicate CK7+/SOX9+ cells. The graph on the right summarizes differences in SOX9 positivity among groups. B: Immunofluorescence for CK7 and CK19. In cirrhotic PSC, a higher percentage of DR cells are CK19 compared with controls and cirrhotic PBC. Green arrows indicate CK7+/CK19 DR, and yellow arrows indicate CK7+/CK19+ DR. The graph on the right summarizes differences in CK19 positivity among groups. C: IHC analysis for hepatocyte paraffin 1 (Hep-Par1). Reactive ductules contain Hep-Par1+ cells (arrows) in cirrhotic PSC, but not in controls or cirrhotic PBC. Arrowheads indicate negative DR cells. The graph on the right summarizes differences in Hep-Par1 positivity among groups. Data are expressed as means ± SD. P < 0.05 versus other groups. Scale bars = 50 μm.
      Hepatocyte buds (Figure 4A ) were not present in controls and were more numerous in cirrhotic PSC compared with cirrhotic PBC (11.4 ± 3.7 versus 5.4 ± 3.4; P < 0.05). The immaturity index, calculated as the presence of EpCAM+/Hep-Par1+ newly derived hepatocytes (Figure 4, B and C), was higher in cirrhotic-PSC samples compared with cirrhotic-PBC samples (1.8 ± 0.5 versus 0.6 ± 0.6; P < 0.01). No EpCAM+ hepatocytes were present in control livers (Figure 4, B and C). Few EpCAM+ hepatocytes were present in early stages of PSC and PBC (data not shown).
      Figure thumbnail gr4
      Figure 4Features of hepatocyte commitment in hepatic stem/progenitor cell (HpSC) compartment in human cholangiopathies. A: Immunohistochemistry (IHC) analysis for cytokeratin (CK)-19 in control (CTR) livers, primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). The evaluation of hepatocyte buds indicated a higher prevalence in cirrhotic PSC (arrows) compared with controls and cirrhotic PBC. The graph on the right summarizes differences in hepatocyte bud number among groups. B: IHC analysis for epithelial cell adhesion molecule (EpCAM). EpCAM+ hepatocytes are rarely present in normal and cirrhotic-PBC samples, whereas clusters of EpCAM+ hepatocytes are frequently observed in cirrhotic-PSC samples (arrows). The graph on the right summarizes differences in EpCAM+ hepatocytes (Heps) among groups. C: Immunofluorescence for EpCAM and hepatocyte paraffin 1 (Hep-Par1). Nuclei are displayed in blue. Immunofluorescence confirms the presence of clusters of EpCAM+/Hep-Par1+ hepatocytes in PSC (yellow asterisks) but not in PBC (white asterisks). Dashed lines individuate the interface between the portal tract (PT) and the liver parenchyma. Data are expressed as means ± SD (A and B). P < 0.05 versus other groups. Scale bars: 50 μm (A and B); 100 μm (C).

      Signaling Pathways in the HpSC Niche: Expression of Laminin, NOTCH, and WNT Pathways

      The presence of laminin around the reactive ductules and the expression of the NOTCH and WNT pathways by DR cells were evaluated by immunohistochemistry analysis on serial sections and confirmed by double immunofluorescence (Figures 5 and 6).
      Figure thumbnail gr5
      Figure 5Laminin and neurogenic locus notch homolog protein 1 (NOTCH1) expression in human cholangiopathies. A: Immunohistochemistry (IHC) analysis for laminin in controls, primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC). In cirrhotic PBC but not in controls and cirrhotic PSC, ductular reaction (DR) is surrounded by a laminin-rich matrix (arrows). Vessels represent positive controls (arrowheads). B: Immunofluorescence for cytokeratin (CK)-7 and laminin shows laminin (arrowheads) around DR (arrows) in PBC. Nuclei are displayed in blue. C: IHC analysis for NOTCH1. NOTCH1+ DR cells (arrows) are more numerous in cirrhotic PBC compared with controls and cirrhotic PSC. D: Immunofluorescence for NOTCH1 and sex determining region Y-box (SOX)9 confirms high NOTCH1 (green arrows) expression in SOX9+ (red arrows) cells in cirrhotic PBC. Nuclei are displayed in blue. Scale bars: 100 μm (A and C); 50 μm (B and D).
      Figure thumbnail gr6
      Figure 6Wingless-related integration site family (WNT)/β-catenin expression in human cholangiopathies. A: Immunohistochemistry (IHC) analysis for WNT1 in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). WNT1+ (arrows) cells in ductular reaction (DR) are more numerous in cirrhotic PSC compared with cirrhotic PBC. B: Immunofluorescence for WNT1 and cytokeratin (CK)-7 confirms WNT1 (red arrows) expression by CK7+ DR (yellow arrows) in cirrhotic PSC. Nuclei (NU) are displayed in blue. C and D: Immunofluorescence for WNT3a and sex determining region Y-box (SOX)9 shows fewer WNT3a+/SOX9+ DR cells (arrows) in cirrhotic PBC compared with cirrhotic PSC. Nuclei are displayed in blue. E: IHC analysis for phosphorylated (p)β-catenin shows higher expression (arrows) by DR in cirrhotic PSC compared with cirrhotic PBC. F: Immunofluorescence for pβ-catenin and CK7 confirms the cytoplasmic/nuclear pβ-catenin expression by CK7+ DR (arrows) in cirrhotic PSC. Nuclei are displayed in blue. G: The heat map shows differences in IHC analysis expression of laminin, NOTCH1, WNT1, WNT3a, and pβ-catenin by DR. Color range refers to the used semiquantitative scoring system, as follows: 0 = <1%; 1 = 1% to 5%; 2 = 6% to 30%; 3 = 31% to 50%; and 4 = >50%. Data are expressed as means ± SD in each cell (G). P < 0.05 versus control; P < 0.05 versus PSC; P < 0.05 versus PBC. Scale bars: 100 μm (A, C–E); 50 μm (B and F).
      In cirrhotic PBC, the percentage of DR elements surrounded by laminin was higher (score = 2.8 ± 0.5) compared with those in the control (score = 0.5 ± 0.1) and cirrhotic-PSC samples (score = 0.6 ± 0.4) (Figure 5, A and B). NOTCH1 expression by DR was increased in cirrhotic-PBC samples (score = 3.6 ± 0.5) and in cirrhotic-PSC samples (score = 2.8 ± 0.4) compared with controls (0.5 ± 0.1) (P < 0.05) (Figure 5, C and D); cirrhotic-PBC samples showed significantly higher values with respect to those in cirrhotic PSC (P < 0.05). The study of the WNT pathway revealed that the expression of WNT1 (Figure 6, A and B), WNT3a (Figure 6, C and D), and phosphorylated β-catenin (Figure 6, E and F) were higher in cirrhotic-PSC samples (2.8 ± 0.9, 2.0 ± 1.0, and 3.3 ± 1.0, respectively; all, P < 0.05) and in cirrhotic-PBC samples [0.6 ± 0.5 (P < 0.01); 0.2 ± 0.5 (P < 0.01); and 1.5 ± 1.0 (P < 0.05)] compared with controls (0, 0, and 0.5 ± 0.1); cirrhotic-PSC samples showed significantly higher values compared with cirrhotic PBC (all, P < 0.05). Results are visually summarized by the heat map in Figure 6G.

      Discussion

      The main results of the present study indicate that i) DR in PBC is more expansive, appears earlier, and has a higher PI compared with PSC; ii) DR extent correlates with fibrosis and clinical prognostic risk scores both in PSC and PBC patients; iii) the phenotype of reactive ductules differs between PSC and PBC, with a predominant biliary phenotype in PBC and prominent signs of hepatocyte commitment in PSC; and iv) the observed differences in fate commitment were associated with signaling pathways in HpSC niche, as characterized by lower levels of laminin and NOTCH1 and higher WNT/β-catenin pathway expression in PSC compared with PBC.
      DR is composed of anastomosing strands of tortuous ductules, and its presence is thought to be the result of HpSC activation and proliferation.
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      In conclusion, our results indicate a different pattern of HpSC activation in the context of human primary cholangiopathies, which reflects the involvement of different portions of the biliary tree as the primary target of damage. These aspects may have implications in the pathogenesis of different cholangiopathies. Interestingly, the evaluation of DR in biopsy samples may individuate patients with progressive fibrosis and could have prognostic value, especially in PBC. Future studies may reveal the role of the HpSC-activation patterns in furnishing valuable information in the clinical management of cholestatic diseases.

      Acknowledgments

      We thank Melissa Kerr for proofreading the manuscript.
      G.C. conceived, designed, and performed experiments, analyzed data, and wrote the manuscript; V.C. and D.O. analyzed data, performed experiments, and wrote the manuscript; T.H.K., T.F., A.Fr., N.C., P.O., P.B.B., and A.Fl. acquired data and wrote the manuscript; and D.A. and E.G. conceived and designed experiments, interpreted data, provided financial support, and edited the manuscript. G.C. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Correlation between ductular reaction (DR) and prognostic clinical risk score. A: In patients affected by primary sclerosing cholangitis (PSC), the scatterplot shows correlation between DR and Mayo PSC risk score. B: In patients affected by primary biliary cholangitis (PBC), the scatterplot shows correlation between DR and UK-PBC risk (15 years) and Global PBC score (10 years). P < 0.05.

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