Caspase-8 in Liver Parenchymal Cells (Lpc), but Not Rip3, is an Essential Orchestrator of Experimental Cholestatic Liver Disease

ORAL PRESENTATIONS Methods: Experiments with 3 GC (dexamethasone, prednisolone and budesonide) commonly used in clinical pharmacology were carried out in mice: C57BL/6 wild-type strain, and KO for Fxr (Fxr-/-), Fgf15 (Fgf15-/-) or intestinal GC receptor (GR-/-). In vitro studies were performed in Alexander hepatoma cells with negligible basal expression of FXR and CYP7A1, which were transfected with FXR/ RXR. Results: In wild-type mice, GC treatment (ip: 0.25–5 mg/kg/day for 7 days) dramatically reduced, in a dose-dependent manner, ileal expression of Fgf15, whereas Asbt was up-regulated, but Fxr was not modified. Using a non-hepatotoxic dose of dexamethasone (ip: 0.5 mg/kg/day for 7 days), as indicated by normal levels of ALT, AST and BA (measured by HPLC-MS/MS), down-regulation of intestinal Fgf15 without up-regulation of liver Cyp7a1, the key enzyme in neutral pathway of bile acid synthesis, was found. The expression in mouse liver of other genes involved in BA homeostasis, such as Bsep, Ntcp, Mrp2 (BA transport) and Baat (BA conjugation) was not modified. In Fxr-/- and GR-/- mice, basal expression of Fgf15 was lowered. Treatment with GC resulted in further reduction of Fgf15 expression in Fxr-/-, but not GR-/- mice. Liver Cyp7a1 expression was not significantly changed in any case. In contrast, in Fgf15-/- mice, GC treatment was able to induce up-regulation of Cyp7a1. When Alexander cells transfected with FXR/RXR were treated with GC, a marked increase in BSEP expression was observed. However, GW4064-induced FGF19 and SHP up-regulation was limited by GC. This inhibition was also observed in Alexander cells transfected with empty plasmids. In contrast, the expression of CYP27A1, the ratelimiting enzyme in the acidic pathway of BA synthesis was stimulated by GC, in an FXR-independent way. Conclusions: GC interfere with the regulation of hepatic BA synthesis by altering FXR-mediated signalling via ileal FGF19 secretion, which may lead to adverse effects accounting for hepatobiliary alterations in chronic therapy with GC. PS090 CD362+ HUMAN MESENCHYMAL STROMAL CELLS REDUCE HEPATIC INFLAMMATION AND INDUCE M2 MACROPHAGE POLARISATION IN MURINE MODELS OF PRIMARY SCLEROSING CHOLANGITIS V. Vigneswara1, M. Alfaifi1, D. Hedegaard1, D. Haldar1, Y. Chen1, M. Gargesha2, A. Owen1, S. Siew1, C. Baan3, A. Viola4, D. Roy2, M. Hoogduijn3, S. Elliman5, G.M. Hirschfield1, P.N. Newsome1. 1 National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, United Kingdom; 2BioInVision, Cleveland, United States; 3Erasmus Medical Centre, University of Rotterdam, Rotterdam, Netherlands; 4UNIPD, Padua, Italy; 5Orbsen Therapeutics, Galway, Ireland E-mail: [email protected] Background and Aims: Primary sclerosing cholangitis (PSC) is an inflammatory condition characterised by bile duct damage. The ability of mesenchymal stromal cells (MSC) to immunomodulate offers therapeutic potential but the inherent heterogeneity of unsorted MSC populations may explain varied/reduced function and poses regulatory challenges. Thus we set out to study the efficacy of purified CD362+ MSC in relevant pre-clinical models of PSC as a prelude to a clinical trial. Methods: MSC were provided by Orbsen Therapeutics. Unsorted human bone marrow (BM) and unsorted and CD362+ sorted umbilical cord (UC) derived MSC were injected intravenously into 6–7 wk old male Mdr2-/- mice (FVB/N background) & Ova-Bil mice (allo-immune liver injury). The extent of liver damage was determined by liver histology, serum analysis and FACS analysis of whole liver cell digest two weeks after cell infusion. A time-course of bio-distribution was determined by whole mouse cryo-imaging of Qdot labelled MSC.

Results: Infusions of unsorted BM/UC MSC reduced liver damage in both models of PSC with reductions in serum ALT and bile acids (BA). In the MDR2-/- mouse infusion of a 250 k dose of CD362+ UC-MSC was the most effective in reducing inflammation as compared to infusion of control PBS (ALT 662 IU to 290 IU; BA 112 μmol/L to 47 μmol/L). There was also a reduction in CD45+ staining on liver sections although there was no change in Picrosirius red staining for fibrosis. FACS analysis of digested livers indicated a reduction in total hepatic lymphocytes in mice treated with CD362+ UC-MSC (38225 cells/g tissue to 27583/g tissue) compared to control PBS infused mice. This was accompanied by a significant reduction in hepatic CD4+ T cells (36% to 23%) whereas the percentage of CD3+CD4+CD25+ regulatory T cells also increased (7.85% to 14.4%) compared to controls. In addition, there was a significant increase in the % of hepatic M2 macrophage (20% to 36%) in CD362+ UC-MSC infused mice compared with other treatment groups. A cryo-imaging sevenday time-course indicated MSC were cleared rapidly although there was a liver-specific increase in MSC 2–3 days after infusion. Conclusions: CD362+ human MSC exert a potent anti-inflammatory action in murine models of PSC. MSC are cleared rapidly after infusion and despite modest hepatic homing induce significant M2 polarisation of hepatic monocytes. PS091 CASPASE-8 IN LIVER PARENCHYMAL CELLS (LPC), BUT NOT RIP3, IS AN ESSENTIAL ORCHESTRATOR OF EXPERIMENTAL CHOLESTATIC LIVER DISEASE F.J. Cubero1, J. Peng1, M. Hatting1, G. Zhao1, M.E. Zoubek1, R. MaciasRodriguez1, A. Ruiz-Margain1, J. Reissing1, H.W. Zimmermann1, N. Gassler2, T. Luedde1, C. Liedtke1, C. Trautwein1. 1Medicine III, University Hospital RWTH Aachen, Aachen; 2Institute of Pathology, Klinikum Braunschweig, Braunschweig, Germany E-mail: [email protected] Background and Aims: Different studies have strongly supported a mechanistic role for death receptor (DR) family members in cholestatic liver disease. The cystein-aspartate protease caspase-8 (CASP8) is the apical initiator caspase in DR-mediated apoptosis. Necroptosis—programmed necrosis depending on the kinases receptor interacting protein 1 (RIP1) and RIP3—represents an alternative programmed cell-death pathway. Here we hypothesize that CASP8 might play a critical for the pathogenesis of cholestatic liver disease. Methods: Liver injury was characterized in a cohort of human sera and biopsies from PBC patients (n = 28). In parallel, liver parenchymal cell (LPC)-specific Casp8 (Casp8 Δhepa), constitutive deletion of Rip3 (Rip3 -/-) and Casp8 Δhepa/Rip3 -/- double knockout and floxed (WT) mice were subjected to surgical ligation of the common bile duct (BDL) for 28 days. Results: Patients with PBC displayed typical symptoms (fatigue and pruritus) and cholestatic pattern in liver function tests (LFT), with high levels of alkaline phosphatase, transaminases, cholesterol as well as elevated titers of anti-mitochondrial antibodies. Overexpression of cleaved caspase-8, RIP3 and JNK was characteristic of liver explants from PBC patients compared with healthy controls, accompanied by elevated protein levels of TNF and IL-6. Casp8 Δhepa after 28 days BDL displayed decreased number and size of necrotic foci compared with Casp8f/f mice, associated with significantly decreased serum transaminases, TUNEL staining, and cleaved Caspase-3 and cytochrome C protein overexpression were found in Casp8 Δhepa mice 28 days after BDL, along with diminished compensatory proliferation (Ki-67, PCNA) and ductular reaction (CK19). These results were translated into a decreased inflammatory profile elicited by lower IL-6, TNF-α protein levels and reduced immune infiltration. Overall, liver fibrogenesis (Sirius red, Collagen IA1, ASMA) was significantly ameliorated in Casp8 Δhepa. Lower activation of JNK, RIP1 and RIP3 correlated with the protection shown after chronic BDL. Moreover, no differences were found in

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ORAL PRESENTATIONS markers of liver injury Rip3 -/- compared with WT mice, 28 days after BDL. Interestingly, Casp8 Δhepa/Rip3 -/- displayed improvement of liver function after BDL. Conclusions: CASP8 in LPC, but not RIP3, is an essential mediator of cholestatic liver injury, thus, it might be an interesting pharmacologic target that can be used in the clinic as treatment for cholestatic liver diseases. PS092 PROTEOMIC ANALYSIS OF LIVER FIBROTIC SEPTA AND PERIPORTAL SPACES IN PRIMARY SCLEROSING CHOLANGITIS, PRIMARY BILIARY CHOLANGITIS AND ALCOHOLIC LIVER DISEASE G. Mazza1, A. Telese1, E. Schrumpf2–4, J. Gilbertson5, N. Rendell5, B. Lombardi6, J. Godovac-Zimmermann6, G.W. Taylor5, D. Thorburn1, T.H. Karlsen2–4,7, M. Pinzani1. 1UCL Institute for Liver and Digestive Health, Division of Medicine, University College London & Sheila Sherlock Liver Centre, Royal Free London NHS Foundation Trust, London, United Kingdom; 2Norwegian PSC Research Center, Department of Transplantation Medicine, Division of Cancer Medicine, Surgery and Transplantation, Oslo University Hospital Rikshospitalet, Oslo, Norway; 3 K.G. Jebsen Inflammation Research Centre, Institute of Clinical Medicine, University of Oslo, Oslo, Norway; 4Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; 5Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, Royal Free Hospital. University College London, London, United Kingdom; 6Proteomics and Molecular Cell Dynamics, Center for Nephrology, School of Life and Medical Sciences, University College London, Royal Free Campus, Rowland Hill Street, London, United Kingdom; 7Section of Gastroenterology, Department of Transplantation Medicine, Oslo University Hospital, Oslo, Norway E-mail: [email protected] Background and Aims: Primary Sclerosing Cholangitis (PSC) is a chronic, cholestatic liver disease of unknown etiology characterized by periductular inflammation, fibrosis and strictures of bile ducts. There is no medical treatment available for PSC and identification of prognostic biomarkers are crucial for patient counseling and liver transplantation. The aim of this study was to evaluate differences in term of protein composition within fibrotic septa between PSC and other types of liver fibrosis. Methods: Formalin fixed paraffin embedded liver samples were collected from 21 explanted livers (Male 71%; Age 51.5 ± 12.3; MELD 15.3 ± 11.2), including primary sclerosing cholangitis (PSC;11), primary biliary cirrhosis (PBC;5) and alcoholic liver diseases (ALD;5). The slices have been laser microdissected (LMD) by employing a Leica LMD system. Specific fibrotic area (e.g fibrotic bile ducts, portal-to-portal fibrotic septa) were dissected and analyzed with liquid chromatography electrospray tandem mass spectrometry. Furthermore, sub analysis was performed within the PSC patients that were stratified in accordance to the MELD score (cut off value >12). Results: A total of 215 proteins were detected of which 124 were identified within the three groups, including main collagens such as: Collagen α-1(I) chain, Collagen α-1(VI) chain, Collagen α-1(XIV) chain, Collagen α-2(I) chain, Collagen α-2(VI) chain, Collagen α-3(VI) chain. Interestingly, the PSC-PBC combined group showed the peculiar presence of proteoglycans such as: Biglycan, Lumican, Mimecan. Notably, exclusive proteins were detected for each disease (PSC 14, PBC 10, ALD 18). The PSC group was characterized by the expression of: Transforming growth factor-β-induced protein ig-h3, Fibronectin and Tryptase peptides. However, Collagen IV-α and Pterin 4α were only detected into the ALC group Moreover, the analysis of the PSC group in accordance with the MELD score identified proteins that were exclusively (Betaine-homocysteine S-methyltransferase, Peroxiredoxin-2, Plastin-2) or preferentially (Retinal dehydrogenase 1, Heat shock 70 kDa protein-6 peptides) expressed in patients presenting MELD score <12.

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Conclusions: This is the first pilot study evaluating fibrotic septa protein composition by employing LMD-proteomic. Quantitative analyses of tissue and serum samples are required in order to validate these findings. However, this study opens new opportunities for the discovery of disease-specific biomarkers. PS093 GENOME-WIDE ASSOCIATION STUDY (GWAS) OF LIVER FIBROSIS PHENOTYPES IN PATIENTS WITH PRIMARY SCLEROSING CHOLANGITIS (PSC) REVEALS COMMON GENETIC VARIATION INFLUENCING SERUM LEVELS OF LYSYL OXIDASE-LIKE-2 (LOXL2) P.R. Shea1, B. Eksteen2, G.M. Hirschfield3, M.L. Shiffman4, H.L. A. Janssen5, A.J. Montano-Loza6, Z. Goodman7, S.E. Kleinstein1, R.P. Myers8, G.M. Subramanian8, J.G. McHutchison8, C.L. Bowlus9, M. Manns10, R. Chapman11, C. Levy12, A.J. Muir13, D.B. Goldstein1. 1 Institute for Genomic Medicine, Columbia University, New York, United States; 2University of Calgary, Calgary, Canada; 3Center for Liver Research, NIHR Biomedical Research Unit, University of Birmingham, Birmingham, United Kingdom; 4Liver Institute of Virginia, Richmond, United States; 5University of Toronto, Toronto; 6University of Alberta, Edmonton, Canada; 7Inova Fairfax Hospital, Falls Church; 8Gilead Sciences, Inc., Foster City; 9University of California Davis, Sacramento, United States; 10Hannover Medical School, Hannover, Germany; 11 University of Oxford, Oxford, United Kingdom; 12University of Miami, Miami; 13Duke Clinical Research Institute, Durham, United States E-mail: [email protected] Background and Aims: LOXL2 plays a central role in fibrosis by catalyzing collagen cross-linkage and its serum levels (sLOXL2) correlate with fibrosis in PSC patients. In order to better understand the genetic factors that may influence PSC-related fibrosis, we performed a GWAS of common genetic variation among affected patients. Methods: We genotyped PSC patients enrolled in a phase 2b trial of simtuzumab, a monoclonal antibody directed against LOXL2. Genotyping was performed at over 5 million single nucleotide polymorphism (SNP) loci using the Illumina Omni5 high density BeadArray chip. GWAS were conducted using linear or logistic regression of fibrosis-related phenotypes; specifically, advanced fibrosis (Ishak stage 3–6), hepatic collagen content assessed by computer-assisted morphometry on picrosirius red-stained biopsies, and sLOXL2 (VIDAS®, BioMérieux, Marcy L’Etoile, France). All analyses were Bonferroni adjusted and included age, gender, race, and BMI. Table: Top Results from GWAS for Fibrosis-Related Endpoints in PSC Patients Endpoint sLOXL2

Hepatic collagen Advanced fibrosis

Top SNPs rs17636009 rs75294187 rs7941097 rs28820314 rs74567273 rs10145540 rs17035257 rs35612858

Chromosome 13q32 11q14 11q14 14q24 15q14 14q24 4q32 1p32

Nearest Gene

p-value

GPC5 TYR TENM4 ISM2 C15orf53 ISM2 PDGFC C1orf87

3.416 × 10−11 6.732 × 10−09 1.216 × 10−08 1.841 × 10−08 1.945 × 10−08 2.122 × 10−08 9.828 × 10−08* 2.469 × 10−06*

*p-values do not exceed the threshold for significance.

Results: 200 of 234 randomized patients (85%) consented to genetic analysis. The median age was 45 years, 64% were male, 86% were Caucasian, and 47% had ulcerative colitis. 53% of the patients had advanced fibrosis and the median (IQR) hepatic collagen and sLOXL2 levels were 4.5% (2.7–7.0%) and 102 pg/mL (70–145), respectively. We identified four genomic regions with associations with sLOXL2 that exceeded the genome-wide threshold for significance (Table). The strongest association ( p = 3.42 × 10−11) was with a region on chromosome 13q31 near the glypican 5 (GPC5) gene. SNPs near the

Journal of Hepatology 2016 vol. 64 | S159–S182