Browsing by Author "Carpino, Guido"
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Item The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling(MDPI, 2022-05-09) Mancinelli, Romina; Ceci, Ludovica; Kennedy, Lindsey; Francis, Heather; Meadows, Vik; Chen, Lixian; Carpino, Guido; Kyritsi, Konstantina; Wu, Nan; Zhou, Tianhao; Sato, Keisaku; Pannarale, Luigi; Glaser, Shannon; Chakraborty, Sanjukta; Alpini, Gianfranco; Gaudio, Eugenio; Onori, Paolo; Franchitto, Antonio; Medicine, School of MedicineBackground & aims: Cholangiocytes are the target cells of liver diseases that are characterized by biliary senescence (evidenced by enhanced levels of senescence-associated secretory phenotype, SASP, e.g., TGF-β1), and liver inflammation and fibrosis accompanied by altered bile acid (BA) homeostasis. Taurocholic acid (TC) stimulates biliary hyperplasia by activation of 3',5'-cyclic cyclic adenosine monophosphate (cAMP) signaling, thereby preventing biliary damage (caused by cholinergic/adrenergic denervation) through enhanced liver angiogenesis. Also: (i) α-calcitonin gene-related peptide (α-CGRP, which activates the calcitonin receptor-like receptor, CRLR), stimulates biliary proliferation/senescence and liver fibrosis by enhanced biliary secretion of SASPs; and (ii) knock-out of α-CGRP reduces these phenotypes by decreased cAMP levels in cholestatic models. We aimed to demonstrate that TC effects on liver phenotypes are dependent on changes in the α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-β1/VEGF axis. Methods: Wild-type and α-CGRP-/- mice were fed with a control (BAC) or TC diet for 1 or 2 wk. We measured: (i) CGRP levels by both ELISA kits in serum and by qPCR in isolated cholangiocytes (CALCA gene for α-CGRP); (ii) CALCRL immunoreactivity by immunohistochemistry (IHC) in liver sections; (iii) liver histology, intrahepatic biliary mass, biliary senescence (by β-GAL staining and double immunofluorescence (IF) for p16/CK19), and liver fibrosis (by Red Sirius staining and double IF for collagen/CK19 in liver sections), as well as by qPCR for senescence markers in isolated cholangiocytes; and (iv) phosphorylation of PKA/ERK1/2, immunoreactivity of TGF-β1/TGF- βRI and angiogenic factors by IHC/immunofluorescence in liver sections and qPCR in isolated cholangiocytes. We measured changes in BA composition in total liver by liquid chromatography/mass spectrometry. Results: TC feeding increased CALCA expression, biliary damage, and liver inflammation and fibrosis, as well as phenotypes that were associated with enhanced immunoreactivity of the PKA/ERK1/2/TGF-β1/TGF-βRI/VEGF axis compared to BAC-fed mice and phenotypes that were reversed in α-CGRP-/- mice fed TC coupled with changes in hepatic BA composition. Conclusion: Modulation of the TC/ α-CGRP/CALCRL/PKA/ERK1/2/TGF-β1/VEGF axis may be important in the management of cholangiopathies characterized by BA accumulation.Item Endothelin Receptor-A Inhibition Decreases Ductular Reaction, Liver Fibrosis, and Angiogenesis in a Model of Cholangitis(Elsevier, 2023) Owen, Travis; Carpino, Guido; Chen, Lixian; Kundu, Debjyoti; Wills, Payton; Ekser, Burcin; Onori, Paolo; Gaudio, Eugenio; Alpini, Gianfranco; Francis, Heather; Kennedy, Lindsey; Medicine, School of MedicineBackground & aims: Primary sclerosing cholangitis (PSC) leads to ductular reaction and fibrosis and is complicated by vascular dysfunction. Cholangiocyte and endothelial cell crosstalk modulates their proliferation in cholestatic models. Endothelin (ET)-1 and ET-2 bind to their receptor, ET-A, and cholangiocytes are a key source of ET-1 after bile duct ligation. We aimed to evaluate the therapeutic potential of ET-A inhibition in PSC and biliary-endothelial crosstalk mediated by this pathway. Methods: Wild-type and multidrug resistance 2 knockout (Mdr2-/-) mice at 12 weeks of age were treated with vehicle or Ambrisentan (ET-A antagonist) for 1 week by daily intraperitoneal injections. Human control and PSC samples were used. Results: Mdr2-/- mice at 4, 8, and 12 weeks displayed angiogenesis that peaked at 12 weeks. Mdr2-/- mice at 12 weeks had enhanced biliary ET-1/ET-2/ET-A expression and secretion, whereas human PSC had enhanced ET-1/ET-A expression and secretion. Ambrisentan reduced biliary damage, immune cell infiltration, and fibrosis in Mdr2-/- mice. Mdr2-/- mice had squamous cholangiocytes with blunted microvilli and dilated arterioles lacking cilia; however, Ambrisentan reversed these alterations. Ambrisentan decreased cholangiocyte expression of pro-angiogenic factors, specifically midkine, through the regulation of cFOS. In vitro, ET-1/ET-A caused cholangiocyte senescence, endothelial cell angiogenesis, and macrophage inflammation. In vitro, human PSC cholangiocyte supernatants increased endothelial cell migration, which was blocked with Ambrisentan treatment. Conclusions: ET-A inhibition reduced biliary and liver damage in Mdr2-/- mice. ET-A promotes biliary angiocrine signaling that may, in turn, enhance angiogenesis. Targeting ET-A may prove therapeutic for PSC, specifically patients displaying vascular dysfunction.Item FGF1 Signaling Modulates Biliary Injury and Liver Fibrosis in the Mdr2-/- Mouse Model of Primary Sclerosing Cholangitis(Wolters Kluwer, 2022) O’Brien, April; Zhou, Tianhao; White, Tori; Medford, Abigail; Chen, Lixian; Kyritsi, Konstantina; Wu, Nan; Childs, Jonathan; Stiles, Danaleigh; Ceci, Ludovica; Chakraborty, Sanjukta; Ekser, Burcin; Baiocchi, Leonardo; Carpino, Guido; Gaudio, Eugenio; Wu, Chaodong; Kennedy, Lindsey; Francis, Heather; Alpini, Gianfranco; Glaser, Shannon; Medicine, School of MedicineFibroblast growth factor 1 (FGF1) belongs to a family of growth factors involved in cellular growth and division. MicroRNA 16 (miR-16) is a regulator of gene expression, which is dysregulated during liver injury and insult. However, the role of FGF1 in the progression of biliary proliferation, senescence, fibrosis, inflammation, angiogenesis, and its potential interaction with miR-16, are unknown. In vivo studies were performed in male bile duct-ligated (BDL, 12-week-old) mice, multidrug resistance 2 knockout (Mdr2-/-) mice (10-week-old), and their corresponding controls, treated with recombinant human FGF1 (rhFGF1), fibroblast growth factor receptor (FGFR) antagonist (AZD4547), or anti-FGF1 monoclonal antibody (mAb). In vitro, the human cholangiocyte cell line (H69) and human hepatic stellate cells (HSCs) were used to determine the expression of proliferation, fibrosis, angiogenesis, and inflammatory genes following rhFGF1 treatment. PSC patient and control livers were used to evaluate FGF1 and miR-16 expression. Intrahepatic bile duct mass (IBDM), along with hepatic fibrosis and inflammation, increased in BDL mice treated with rhFGF1, with a corresponding decrease in miR-16, while treatment with AZD4547 or anti-FGF1 mAb decreased hepatic fibrosis, IBDM, and inflammation in BDL and Mdr2-/- mice. In vitro, H69 and HSCs treated with rhFGF1 had increased expression of proliferation, fibrosis, and inflammatory markers. PSC samples also showed increased FGF1 and FGFRs with corresponding decreases in miR-16 compared with healthy controls. Conclusion: Our study demonstrates that suppression of FGF1 and miR-16 signaling decreases the presence of hepatic fibrosis, biliary proliferation, inflammation, senescence, and angiogenesis. Targeting the FGF1 and miR-16 axis may provide therapeutic options in treating cholangiopathies such as PSC.Item Knockout of secretin ameliorates biliary and liver phenotypes during alcohol-induced hepatotoxicity(BMC, 2023-01-09) Kyritsi, Konstantina; Wu, Nan; Zhou, Tianhao; Carpino, Guido; Baiocchi, Leonardo; Kennedy, Lindsey; Chen, Lixian; Ceci, Ludovica; Meyer, Alison Ann; Barupala, Nipuni; Franchitto, Antonio; Onori, Paolo; Ekser, Burcin; Gaudio, Eugenio; Wu, Chaodong; Marakovits, Corinn; Chakraborty, Sanjukta; Francis, Heather; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineBackground: Alcohol-related liver disease (ALD) is characterized by ductular reaction (DR), liver inflammation, steatosis, fibrosis, and cirrhosis. The secretin (Sct)/secretin receptor (SR) axis (expressed only by cholangiocytes) regulates liver phenotypes in cholestasis. We evaluated the role of Sct signaling on ALD phenotypes. Methods: We used male wild-type and Sct-/- mice fed a control diet (CD) or ethanol (EtOH) for 8 wk. Changes in liver phenotypes were measured in mice, female/male healthy controls, and patients with alcoholic cirrhosis. Since Cyp4a10 and Cyp4a11/22 regulate EtOH liver metabolism, we measured their expression in mouse/human liver. We evaluated: (i) the immunoreactivity of the lipogenesis enzyme elongation of very-long-chain fatty acids 1 (Elovl, mainly expressed by hepatocytes) in mouse/human liver sections by immunostaining; (ii) the expression of miR-125b (that is downregulated in cholestasis by Sct) in mouse liver by qPCR; and (iii) total bile acid (BA) levels in mouse liver by enzymatic assay, and the mRNA expression of genes regulating BA synthesis (cholesterol 7a-hydroxylase, Cyp27a1, 12a-hydroxylase, Cyp8b1, and oxysterol 7a-hydroxylase, Cyp7b11) and transport (bile salt export pump, Bsep, Na+-taurocholate cotransporting polypeptide, NTCP, and the organic solute transporter alpha (OSTa) in mouse liver by qPCR. Results: In EtOH-fed WT mice there was increased biliary and liver damage compared to control mice, but decreased miR-125b expression, phenotypes that were blunted in EtOH-fed Sct-/- mice. The expression of Cyp4a10 increased in cholangiocytes and hepatocytes from EtOH-fed WT compared to control mice but decreased in EtOH-fed Sct-/- mice. There was increased immunoreactivity of Cyp4a11/22 in patients with alcoholic cirrhosis compared to controls. The expression of miR-125b decreased in EtOH-fed WT mice but returned at normal values in EtOH-fed Sct-/- mice. Elovl1 immunoreactivity increased in patients with alcoholic cirrhosis compared to controls. There was no difference in BA levels between WT mice fed CD or EtOH; BA levels decreased in EtOH-fed Sct-/- compared to EtOH-fed WT mice. There was increased expression of Cyp27a1, Cyp8b1, Cyp7b1, Bsep, NTCP and Osta in total liver from EtOH-fed WT compared to control mice, which decreased in EtOH-fed Sct-/- compared to EtOH-fed WT mice. Conclusions: Targeting Sct/SR signaling may be important for modulating ALD phenotypes.Item Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis(American Physiological Society, 2023) Meadows, Vik; Marakovits, Corinn; Ekser, Burcin; Kundu, Debjyoti; Zhou, Tianhao; Kyritsi, Konstantina; Pham, Linh; Chen, Lixian; Kennedy, Lindsey; Ceci, Ludovica; Wu, Nan; Carpino, Guido; Zhang, Wenjun; Isidan, Abdulkadir; Meyer, Alison; Owen, Travis; Gaudio, Eugenio; Onori, Paolo; Alpini, Gianfranco; Francis, Heather; Medicine, School of MedicinePrimary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC. NEW & NOTEWORTHY: We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.Item Mast cells selectively target large cholangiocytes during biliary injury via H2HR-mediated cAMP/pERK1/2 signaling(Wolters Kluwer, 2022) Zhou, Tianhao; Meadows, Vik; Kundu, Debjyoti; Kyritsi, Konstantina; Owen, Travis; Ceci, Ludovica; Carpino, Guido; Onori, Paolo; Gaudio, Eugenio; Wu, Nan; Glaser, Shannon; Ekser, Burcin; Alpini, Gianfranco; Kennedy, Lindsey; Francis, Heather; Medicine, School of MedicineBile ducts are heterogenous in structure and function, and primary sclerosing cholangitis (PSC) damages specific bile ducts leading to ductular reaction (DR), mast cell (MC) infiltration, increased histamine release, inflammation, and fibrosis. Bile duct ligation (BDL) induces large duct damage via cyclic adenosine monophosphate (cAMP)/extracellular signal-related protein kinase (ERK) signaling, and large cholangiocytes express H2 histamine receptor (H2HR). We evaluated how MCs interact with large cholangiocytes during cholestasis. Male wild-type (WT) and MC-deficient (KitW-sh ) mice 10-12 weeks of age were subjected to BDL for 7 days. Select KitW-sh mice were injected with MCs pretreated with control or H2HR antagonist (ranitidine, 25 μm, 48 h) via tail vein injection. In vitro, MC migration toward small mouse cholangiocytes (SMCCs) and large mouse cholangiocytes (LMCCs) treated with lipopolysaccharide or histamine (±ranitidine) was measured. LMCCs were stimulated with MC supernatants pretreated with control, α-methyl-dl-histidine (to block histamine release), or ranitidine. Liver damage, large duct DR/senescence, inflammation, fibrosis, and cAMP/ERK immunoreactivity increased in BDL WT and KitW-sh +MC mice but decreased in BDL KitW-sh and KitW-sh +MC-H2HR mice. In vitro, MCs migrate toward damaged LMCCs (but not SMCCs) blocked by inhibition of H2HR. Loss of MC histamine or MC-H2HR decreases LMCC proliferation, senescence, H2HR, and cAMP/ERK levels. Human PSC livers have increased MC number found near DR, senescent ducts, and H2HR-positive ducts. Conclusion: Infiltrating MCs preferentially interact with large ducts via H2HR signaling promoting biliary and liver damage. Mediation of MCs may be a therapeutic strategy for PSC.Item Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury(Wiley, 2021) Wu, Nan; Carpino, Guido; Ceci, Ludovica; Baiocchi, Leonardo; Francis, Heather; Kennedy, Lindsey; Zhou, Tianhao; Chen, Lixian; Sato, Keisaku; Kyritsi, Konstantina; Meadows, Vik; Ekser, Burcin; Franchitto, Antonio; Mancinelli, Romina; Onori, Onori; Gaudio, Eugenio; Glaser, Shannon; Alpini, Gianfranco; Medicine, School of MedicineBackground and Aims Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein–coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFβ receptor type I (TGFβRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. Approach and Results Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2−/−) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2−/− mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin’s interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFβR1 signaling, which was reduced by loss of MT1. Conclusions Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFβR1 activation. Blocking GPR50/TGFβR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.Item p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling(Wolters Kluwer, 2023) Kundu, Debjyoti; Kennedy, Lindsey; Zhou, Tianhao; Ekser, Burcin; Meadows, Vik; Sybenga, Amelia; Kyritsi, Konstantina; Chen, Lixian; Ceci, Ludovica; Wu, Nan; Wu, Chaodong; Glaser, Shannon; Carpino, Guido; Onori, Paolo; Gaudio, Eugenio; Alpini, Gianfranco; Francis, Heather; Medicine, School of MedicineBackground and aims: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. Approach and results: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. Conclusion: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.Item Prolonged Administration of Melatonin Ameliorates Liver Phenotypes in Cholestatic Murine Model(Elsevier, 2022) Ceci, Ludovica; Chen, Lixian; Baiocchi, Leonardo; Wu, Nan; Kennedy, Lindsey; Carpino, Guido; Kyritsi, Konstantina; Zhou, Tianhao; Owen, Travis; Kundu, Debjyoti; Sybenga, Amelia; Isidan, Abdulkadir; Ekser, Burcin; Franchitto, Antonio; Onori, Paolo; Gaudio, Eugenio; Mancinelli, Romina; Francis, Heather; Alpini, Gianfranco; Glaser, Shannon; Medicine, School of MedicineBackground & aims: Primary sclerosing cholangitis (PSC) is characterized by biliary senescence and hepatic fibrosis. Melatonin exerts its effects by interacting with Melatonin receptor 1 and 2 (MT1/MT2) melatonin receptors. Short-term (1 wk) melatonin treatment reduces a ductular reaction and liver fibrosis in bile duct-ligated rats by down-regulation of MT1 and clock genes, and in multidrug resistance gene 2 knockout (Mdr2-/-) mice by decreased miR200b-dependent angiogenesis. We aimed to evaluate the long-term effects of melatonin on liver phenotype that may be mediated by changes in MT1/clock genes/miR200b/maspin/glutathione-S transferase (GST) signaling. Methods: Male wild-type and Mdr2-/- mice had access to drinking water with/without melatonin for 3 months. Liver damage, biliary proliferation/senescence, liver fibrosis, peribiliary inflammation, and angiogenesis were measured by staining in liver sections, and by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay in liver samples. We confirmed a link between MT1/clock genes/miR200b/maspin/GST/angiogenesis signaling by Ingenuity Pathway Analysis software and measured liver phenotypes and the aforementioned signaling pathway in liver samples from the mouse groups, healthy controls, and PSC patients and immortalized human PSC cholangiocytes. Results: Chronic administration of melatonin to Mdr2-/- mice ameliorates liver phenotypes, which were associated with decreased MT1 and clock gene expression. Conclusions: Melatonin improves liver histology and restores the circadian rhythm by interaction with MT1 through decreased angiogenesis and increased maspin/GST activity.Item Role of ductular reaction and ductular-canalicular junctions in identifying severe primary biliary cholangitis(Elsevier, 2022-08-19) Overi, Diletta; Carpino, Guido; Cristoferi, Laura; Onori, Paolo; Kennedy, Lindsey; Francis, Heather; Zucchini, Nicola; Rigamonti, Cristina; Viganò, Mauro; Floreani, Annarosa; D’Amato, Daphne; Gerussi, Alessio; Venere, Rosanna; Alpini, Gianfranco; Glaser, Shannon; Alvaro, Domenico; Invernizzi, Pietro; Gaudio, Eugenio; Cardinale, Vincenzo; Carbone, Marco; Medicine, School of MedicineBackground & aims: Primary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC. Methods: Liver biopsies were collected from naïve people with PBC (N = 87). Clinical-serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2 -/- mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence. Results: Ductular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals' estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular-canalicular junctions. Conclusions: Extensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis. Lay summary: In people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at diagnosis correlates with the lack of response to first-line therapy with ursodeoxycholic acid and serves to restore ductular-canalicular junctions in people with PBC. Assessing ductular reaction extension at diagnosis may add valuable information for clinicians.