Sortilin regulates ductular reaction and bile duct morphogenesis in cholestasis-induced liver injury via IL-6 and LIF

POSTER PRESENTATIONS ≥2 mm). The following markers of disease activity/severity were used: serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), carbohydrate antigen 19–9 (CA19-9), mAPSC score, BC findings. Results: Overall, 57 ERC and MRI-MRCP were performed in 48 PSC patients (male:31, median age ± interquartile range at PSC diagnosis 31; 21–41, median age ± interquartile range at ERC time 36; 28–44). We found a significant correlation between MRI pBH and cytology findings (IHBD: Spearman’s rho = 0.322, SE:0.095, p = 0.022 and EHBD: Spearman’s rho = 0.322, SE:0.113, p = 0.025); no correlation with other markers of disease activity/severity (i.e., ALT, ALP, CA19-9, biliary neutrophils, intra-epithelial lymphocytes) was seen. Moreover, we found a significant difference between MRCP mAPSC score and serum ALP and CA19-9 level ( p = 0.016 and p < 0.001, respectively) for IHBD and CA 19-9 level ( p = 0.021) for EHBD; a similar difference was seen between ERC mAPSC score and ALP and CA19-9 for IHBD ( p = 0.018 and p = 0.030, respectively). No differences with BC findings was seen. Conclusions: The grade of biliary changes in MRCP and peri-biliary enhancement in MRI may potentially be used as surrogate markers for disease severity and activity in PSC. FRI-399 Expression of the oncofetal marker Nope in the regenerating adult murine liver after disruption of interhepatocytic gap junctions with impaired polarisation via bile duct ligation A. Bowe1, S. Zweerink1, V. Mueck1, T. Goeser1, D. Nierhoff1. 1University of Cologne, Gastroenterology, Cologne, Germany E-mail: [email protected] Background and Aims: Neighbor of Punc E11 (Nope) is strongly expressed in fetal and adult hepatic stem/progenitor cells and in hepatocellular carcinoma but not in terminally differentiated and normally polarized hepatocytes. We here investigated the expression pattern of the oncofetal marker Nope in adult mice after disruption of interhepatocytic gap junctions through liver perfusion or bile duct ligation. Methods: Liver tissue was extracted from adult C57Bl6 mice 24 hours up to 4 weeks after bile duct ligation (BDL). For in-vitro studies, primary hepatocytes were isolated and cultured either on collagencoated dishes or in a matrigel sandwich-culture. Samples were tested for expression levels of Nope via quantitative RT-PCR. Liver tissue was analyzed for Nope and the gap junction protein Connexin 26 via Western blotting and immunohistochemistry. Costainings were performed in combination with CK19 (biliary), E-cadherin (epithelial) or the canalicular marker dipeptidylpeptidase (DPP) IV. Results: BDL leads to a significantly increasing expression level of Nope (after 1 week 87-fold vs. adult liver, p < 0.0001, after 4 weeks 676-fold vs. adult liver, p < 0.001). This high expression level of Nope was confirmed by Western blot, while the expression level of Connexin 26 was decreasing from adult liver until 4 weeks after BDL. In immunohistochemistry, hepatocytes in zone 2 start to express Nope and after 4 weeks of BDL, almost all hepatocytes stain positive for Nope. Costainings with E-cadherin and DPPIV demonstrate a regular sinusoidal expression pattern of Nope on hepatocytes, but no expression on cholangiocytes. Costainings with Connexin 26, that is equally distributed in normal adult liver, reveal a substantial overlap with Nope-positive hepatocytes in zone 2 already in early stages after BDL. While isolated hepatocytes start to express Nope and show an increasing expression over time on collagen-coated dishes, isolated hepatocytes in matrigel sandwich-culture reduce the expression level of Nope back to adult liver within 3 days. Conclusions: We here report the expression of the oncofetal marker Nope on adult hepatocytes after disruption of interhepatocytic gap junctions through liver perfusion or BDL. Since this expression is reversed through sandwich-culture, we postulate that the induction of the membrane protein Nope might be a compensatory mechanism of “disorientated” hepatocytes to regain hepatocellular polarization within the regenerating liver parenchyma. S366

FRI-400 ACE2 therapy using a liver-specific AAV vector inhibits chronic biliary fibrosis in mice I.G. Rajapaksha1, K.Y. Mak1, P.W. Angus2, C.B. Herath1. 1Department of Medicine, The University of Melbourne; 2Department of Gastroenterology and Hepatology, Austin Health, Melbourne, Australia E-mail: [email protected] Background and Aims: There is a major need to develop new therapies to treat chronic biliary diseases such as primary sclerosing cholangitis (PSC). Angiotensin converting enzyme 2 (ACE2) of the alternate renin angiotensin system degrades the potent profibrotic peptide angiotensin II (Ang II) to angiotensin-(1–7) (Ang-(1–7)), a peptide which inhibits fibrosis. We recently showed that a liverspecific adeno-associated viral (AAV) vector carrying ACE2 markedly reduced biliary fibrosis. We therefore investigated the long-term effects of ACE2 gene therapy in the Mdr2-KO murine model of PSC. Methods: A single i.p. injection of either ACE2-AAV or control vector (human serum albumin, HSA-AAV) was administered to 3-month-old Mdr2-KO mice with evolving biliary fibrosis and 7-month-old Mdr2-KO mice which had advanced biliary fibrosis. Hepatic ACE2 gene expression and activity, gene expression of MCP1, COL1 and alpha-SMA, hydroxyproline content, picrosirius staining and hepatic Ang II and Ang-(1–7) peptide levels were measured 3-month post therapy. Additionally, Ang(1–7) was infused into 3-month-old Mdr2-KO mice for 1 month and hepatic fibrosis measured. Moreover, human cholangiocyte cell line, LX2, was used to measure the effect of Ang-(1–7) on the expression of COL1 and epithelial and mesenchymal marker genes. Results: At both time points, ACE2-AAV increased ACE2 gene expression (>60-fold, p < 0.01) and ACE2 protein activity (>2-fold, p < 0.05), resulting in a major decrease in hepatic levels of Ang II with a concomitant increase in Ang-(1–7) levels, compared to controls. ACE2 therapy significantly ( p < 0.05) decreased MCP1, COL1 and alpha-SMA gene expressions compared with control mice livers. These changes were accompanied by ∼50% reduction in hepatic fibrosis ( p < 0.01) and a reduction in ductular proliferation in ACE2 treated mice compared to controls. Ang-(1–7) infusion also significantly ( p < 0.05) reduced liver fibrosis by ∼50%, suggesting that increased Ang-(1–7) production contributes to the beneficial effects of ACE2. Ang-(1–7) treatment of LX2 cells inhibited TGFbeta1induced upregulation of COL1 and S100A4, and dowregulation of CK19 gene expression; all of which was blocked by Ang-(1–7) antagonist A779. Conclusions: These findings suggest that ACE2 improves early and advanced biliary fibrosis in this model by changing the relative hepatic levels of Ang II and Ang-(1–7). We therefore conclude that hepatic ACE2 upregulation has potential as a therapy for patients with severe biliary fibrosis. FRI-401 Sortilin regulates ductular reaction and bile duct morphogenesis in cholestasis-induced liver injury via IL-6 and LIF I. Zvibel1, E. Hubel1, R. Avraham1, O. Shibolet1. 1Tel Aviv Sourasky Medical Center, Tel Aviv, Israel E-mail: [email protected] Background and Aims: Sortilin, a member of the Vps10 domain receptor family, acts both as a trafficking molecule and as a coreceptor. Among the sortilin- trafficked molecules to secretory vesicles is the cytokine IL-6. In its role as co-receptor, sortilin enhances leukemia inhibitory factor (LIF) phosphorylation of STAT3, the downstream signal of the gp130 ligands, a signal that may regulate cholangiocyte proliferation, formation of reactive cholangiocytes and bile duct morphogenesis. We have investigated the role of played by sortilin in the induction of reactive cholangiocytes and in bile duct morphogenesis in cholestatic-induced liver injury, and whether it is mediated via IL-6 levels or via LIF signaling. Methods: Cholestatic injury was induced in wild type (WT) and sortilin null mice (Sort1-/-) by bile duct ligation (BDL).

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POSTER PRESENTATIONS Results: Following BDL, Sort1-/- displayed strikingly impaired ductular reaction, demonstrated by reduced cholangiocyte proliferation, attenuated formation of reactive cholangiocytes and impaired ductular morphogenesis. Sort1-/- and WT displayed similar numbers of CK19-positive cells, however Sort1-/- mice had less CK19-positive cells forming ducts both at early and at later stages following BDL. Mechanistically, the impaired bile duct morphogenesis was accompanied by attenuated expression pSTAT3. In addition, Sort1-/- displayed reduced expression of morphogenesis morphogenesis transcription factor Hes1, a target of Notch2-Jagged1 signaling. Sort1-/- mice had reduced serum IL-6 at all points after BDL. In order to further determine whether sortilin effects were due to reduced serum IL-6, we blocked IL-6 signaling with an IL-6-neutralizing antibody in bile duct- ligated WT mice and observed reduced formation of reactive cholangiocytes, with no effect on bile duct morphogenesis. In order to compensate for reduced LIFR/GP130 signaling Sort1-/- mice, we treated these bile ductligated mice with LIF and found that this treatment corrected the impaired bile duct morphogenesis 14d after BDL, but did not affect formation of reactive cholangiocytes 3d after BDL. Conclusions: Sortilin mediates the ductular reaction and ductular morphogenesis in the setting of biliary fibrosis by regulation of IL-6 levels, which is responsible for reactive cholangiocyte formation and by regulation of LIF signaling, which is involved in bile duct morphogenesis. FRI-402 Pediatric autoimmune hepatitis shows a strong increase of intrahepatic Tregs and an IL-2 associated Treg decline under therapy J. Diestelhorst1,2, N. Junge2, J. Schlue3, C.S. Falk4, M.P. Manns1, U. Baumann2, E. Jaeckel1, R. Taubert1. 1Gastroenterology, Hepatology and Endocrinology; 2Pediatric Gastroenterology and Hepatology; 3 Institute for Pathology; 4Institute for Transplantation Immunology and Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany E-mail: [email protected] Background and Aims: An autoimmune hepatitis (AIH) can manifest in all age groups. A functional and numerical regulatory T cell (Treg) defect has been suggested as a key driver especially in pediatric AIH ( pAIH). Furthermore, a recent in-vitro study showed a reduced IL-2 production in inflamed explanted liver tissue. However, we and others rather found an intrahepatic accumulation of Treg in untreated adult AIH (aAIH) with a disproportionate decline under therapy, especially when remission was not reached. Methods: Liver infiltrating CD4+FOXP3+ Treg, CD4+, CD8+ T- and CD79a+ B cells were quantified before (n = 40) and under (n = 13) therapy in pAIH. In parallel serum cytokines were quantified before (n = 43) and under therapy (n = 28) as well as in pediatric controls without liver or autoimmune diseases (n = 34). Results: Treg accumulated in livers with untreated pAIH. Although portal Treg densities were similar in pAIH and aAIH, the ratios of Treg to T and B cells were higher in pAIH because of less dense portal T and B cell densities. Under therapy portal densities of Treg as well as total T and B cells declined significantly under mostly steroid and azathioprine based therapies in pAIH. However, portal Treg densities decreased disproportionately leading to decreasing ratios of Treg to T and B cells under therapy. Portal Treg densities were similar in treated aAIH and pAIH but with higher Treg ratios in pAIH. Eleven of 28 measured serum cytokines significantly declined under therapy in pAIH. Thereby, IL-2, which is essential for Tregs, showed the strongest decrease, about 10 fold decline of the median with the contraction of the intrahepatic T cell counts under therapy. A similar trend was seen in aAIH as well. However, serum IL-2 levels were similar in untreated pAIH and age and gender matched controls without liver and autoimmune diseases. None of the baseline T- and B cell infiltration parameters were associated with the treatment response in pAIH. Additionally the

intrahepatic B cell/T cell ratio was correlated with the serum immunoglobulin G levels suggesting an autochthonous immunoglobulin production as in aAIH. Conclusions: Intrahepatic Tregs are rather enriched in untreated pAIH without evidence for reduced baseline IL-2 levels. The disproportional decrease of Tregs under therapy may be caused by a decline of the Treg survival factor IL-2. This suggests approaches with Treg facilitating therapies like mTOR inhibitors or low dose IL2 administrations in AIH. FRI-403 Circulating fibroblast growth factor 19 closely correlates with bile Acid synthesis and cholestasis in patients with primary biliary cirrhosis Z.Y. Li1, B. Lin1, Y. Wu1, G. Lin1, Y. Jie1, X. Li1, J. Luo2, Y. Chong1. 1Infectious Disease, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China; 2NGM Biopharmaceuticals, Inc, San Francisco, United States E-mail: [email protected] Background and Aims: Bile acid (BA) synthesis in the liver is regulated by Fibroblast Growth Factor 19 (FGF19) secreted from the ileum as a component of an enterohepatic feedback mechanism. Although FGF19 mRNA is absent in normal liver, hepatic FGF19 gene expression was reported to increase in response to both extrahepatic and intrahepatic cholestasis. The impact of upregulated FGF19 expression on BA synthesis is unclear and investigation the overall role of circulating FGF19 and BA synthesis under cholestatic conditions will further an understanding of PBC pathophysiology and may support the therapeutic potential of blocking hepatic BA synthesis. Methods: Forty-four patients with PBC and 10 healthy subjects were enrolled and BA synthesis was directly quantified by measuring serum concentrations of 7alpha-hydroxycholest-4-en-3-one (C4), along with serum FGF19 and other relevant parameters. The correlations between circulating FGF19, BA synthesis, cholestasis and prognosis were analyzed. Results: Serum levels of C4 were substantially lower, while those of FGF19 were higher, in cirrhotic PBC patients, as compared to those of either healthy subjects or non-cirrhotic PBC patients. Analyses of the relationships between circulating FGF19, BA synthesis and cholestasis revealed that circulating FGF19 was strongly correlated with BA synthesis (r = −0.735, p < 0.0001) and the severity of cholestasis (r = 0.590, p < 0.001). Moreover, BA synthesis was also found to be strongly correlated with the degree of cholestasis (r = 0.522, p = 0.0005). Conclusions: These findings demonstrate that the regulation of BA synthesis in response to cholestasis is primarily controlled by circulating FGF19 and that under cholestatic conditions, the FGF19BA synthesis feedback mechanism remains intact. Administering FGF19, or suitable mimetic, as a pharmacological intervention to increase circulating levels of FGF19 and suppress BA synthesis by inhibiting CYP7A1 gene expression is likely to provide therapeutic benefits for many PBC patients. FRI-404 Exhaustive blood immune phenotype of patients with autoimmune hepatitis A.P. Renand1,2,3, J.-P. Judor1,2,3, H. Aublé4, I. Archambeaud5, J.-F. Mosnier6, J. Gournay5, S. Brouard1,2,3, S. Conchon1,2,3. 1CRTI U1064, INSERM; 2UMR1064, Université de Nantes; 3ITUN; 4CIC Gastro-Nutrition; 5Hépato-Gastro-Entérologie; 6IRCNA, CHU Nantes, Nantes, France E-mail: [email protected] Background and Aims: Autoimmune hepatitis is a rare disease characterized by an immune attack of the liver parenchyma, leading to high levels of aminotransferases, hypergammaglobulinemia, production of autoantibodies and interface hepatitis. Patients with AIH receive corticosteroid therapy, which is not always effective. Until now identification of factors predicting the response to the treatment

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