E Humphreys

Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell–mediated liver damage

Hepatitis B virus (HBV) causes chronic infection in more than 350 million people worldwide. It replicates in hepatocytes but is non-cytopathic; liver damage is thought to be immune mediated. Here, we investigated the role of innate immune responses in mediating liver damage in patients with chronic HBV infection. Longitudinal analysis revealed a temporal correlation between flares of liver inflammation and fluctuations in interleukin (IL)-8, interferon (IFN)-α, and natural killer (NK) cell expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) directly ex vivo. A cross-sectional study confirmed these findings in patients with HBV-related liver inflammation compared with healthy carriers. Activated, TRAIL-expressing NK cells were further enriched in the liver of patients with chronic HBV infection, while their hepatocytes expressed increased levels of a TRAIL death–inducing receptor. IFN-α concentrations found in patients were capable of activating NK cells to induce TRAIL-mediated hepatocyte apoptosis in vitro. The pathogenic potential of this pathway could be further enhanced by the ability of the IFN-α/IL-8 combination to dysregulate the balance of death-inducing and regulatory TRAIL receptors expressed on hepatocytes. We conclude that NK cells may contribute to liver inflammation by TRAIL-mediated death of hepatocytes and demonstrate that this non-antigen–specific mechanism can be switched on by cytokines produced during active HBV infection.

CXCR3-dependent recruitment and CCR6-mediated positioning of Th-17 cells in the inflamed liver

Background & Aims IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown. Methods The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy. Results IL-17+ T cells comprised 1–3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17+ cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts. Conclusions CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand.

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.

Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth

Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.

Quantitative assessment of the cell surface proteome to identify novel therapeutic targets in cholangiocarcinoma

BACKGROUND Cholangiocarcinoma has a high mortality and morbidity. Median survival is less than 6 months. Surgical resection is appropriate in certain circumstances. Because distal cholangiocarcinoma is difficult to distinguish from pancreatic cancers, patients might not receive optimum therapy. Proteomics is the study of complex cellular proteins using mass spectrometry. The aim of this study was to determine the constituent proteins on the cell surface of a model of cholangiocarcinoma. METHODS A sample preparation technique to enrich for cell surface proteins of the intrahepatic cholangiocarcinoma cell line CC-SW-1 was developed by modifying a NeutrAvidin-biotin system. After isolation, trypsin digestion, and purification, peptides were fractionated for tandem mass spectrometry before being analysed with the NCBInr database and the Mascot search algorithm. Results were confirmed by immunohistochemistry using a peroxidase detection technique on paraffin-embedded sections from resected specimens. FINDINGS Peptide enrichment was confirmed by electrophoresis. 862 proteins were consistently expressed between samples (n=3). 271 of these proteins were attributed only to the cell surface. They included proteins used clinically for staging disease (cytokeratin 19 [CK19]), identifying cancer stem cells (epithelial cell adhesion molecule [EpCAM], neural cell adhesion molecule [NCAM], epithelial growth factor receptor [EGFR]), and indicating potential for differentiation (Frozzled receptor, Notch pathway). Novel markers from the tumour necrosis factor (TNF) receptor superfamily were also identified. Immunohistochemistry confirmed these findings. INTERPRETATION The results from this surface proteomic profiling could help to identify novel therapeutic targets in cholangiocarcinoma. Further development of this technique could be translated to distinguish between distal cholangiocarcinoma and pancreatic cancers. FUNDING UK Medical Research Council.

OC-033 The Tweak And Fn14 Pathway As Potential Mediator Of Liver Fibrosis

Introduction The TNF superfamily ligand TWEAK and its cognate receptor Fn14 have been implicated in the pathogenesis of liver disease and have been predominantly associated with liver progenitor cell proliferation and ductular reaction. We hypothesised that TWEAK and Fn14 may also be involved in the establishment and progression of fibrosis via a direct effect on hepatic stellate cell (HSC) function. Methods TWEAK and Fn14 expression was studied by qPCR, western blot and immunostaining in tissue and stromal cells from explanted human liver specimens and normal donor livers surplus to surgical requirements, or as a byproduct of surgical resection. The responses of HSCs to TWEAK were investigated by western blot, live cell imaging and proliferation assays. TWEAK was measured in HSC supernatant by ELISA. Results Confocal microscopy revealed localisation of Fn14 to cells expressing stromal markers in normal human livers, with significant upregulation in diseased livers. Fn14 expression was confirmed in both primary human HSCs and myofibroblasts in vitro . Stimulation with recombinant TWEAK led to an upregulation of NF-kB signalling and induced proliferation in cultured HSCs. TWEAK immunostaining localised the protein to the fibrotic areas of ALD and NASH liver sections suggestive of an autocrine regulation of Fn14 signalling. We confirmed that HSCs express TWEAK and release it into their environment by qPCR and ELISA, and demonstrated that function-blocking TWEAK antibodies reduced the proliferative capacity of HSC. Conclusion Our study suggests that TWEAK/Fn14 promotes liver fibrosis via enhanced proliferation of HSC, possibly through an autocrine mechanism driven by HSC production of TWEAK. Disclosure of Interest None Declared.

PMO-118 The effects of th17 cytokines on liver parenchymal cells shape the microenvironment for local generation of TH17/TC17 in inflammatory liver disease

Introduction IL-17 secreting T cells have been implicated in autoimmunity, inflammatory disease and provide a link between the innate and adaptive immune responses. High numbers of IL-17-producing T cells which also secrete IL-21 and IL-22 are found in close proximity to bile ducts in several liver diseases. Th17 related cytokines have multiple effects and may be involved in both effector responses and repair and regeneration. Methods Primary human parenchymal cells were assessed for cytokine receptor expression by western blotting. The effects of stimulation with recombinant IL-17, IL-21, IL-22, TNFa or IFN-g alone or in combination were compared for apoptosis using annexin staining, proliferation was measured by in situ Ki67 staining and adhesion molecule expression was assessed by flow cytometry. Secretion of IL-1b, IL-6, IL-23 and TGF-b1 was assessed by ELISA. Results All parenchymal cells expressed IL-17R, IL-21R and IL-22R. Th17 related cytokines did not cause apoptosis but led to parenchymal cell proliferation. Cholangiocytes and hepatocytes responded best to IL-17, whereas sinusoidal endothelial cells were responsive to IL-22. Endothelial cells upregulated adhesion molecules in response to Th17 related cytokines. Cholangiocytes responded to Th17 cytokines by secreting high levels of IL-1b, IL-6, IL-23 and TGF-b1 all cytokines that support the survival of Th17 and Tc17 cells. Conclusion Liver parenchymal cells express IL-17, IL-21 and IL-22 receptors and proliferate in response to Th17 cytokines. Upregulation of adhesion molecules by sinusoidal endothelial cells promotes lymphocyte recruitment and retention. Cholangiocytes also respond by secreting Th17/Tc17 polarising cytokines. Therefore Th17 related cytokines secreted by infiltrating lymphocytes may activate the epitheliome to generate a local environment characterised by cholangiocyte proliferation and Th17/Tc17 cell survival, thus contributing to bile duct proliferation and persistent chronic inflammation that characterises many liver diseases. Competing interests None declared.

Fn14 is expressed on neoplastic cholangiocytes in intrahepatic cholangiocarcinoma and promotes necrosis after interaction with TWEAK

Abstract Background Cholangiocarcinoma is the second most common primary hepatic malignancy worldwide. The incidence of intrahepatic cholangiocarcinoma in the UK has steadily increased over the past 40 years. The main treatment is chemotherapy, and 5-year survival after radical surgery is only 25%. The carcinogenic mechanisms involved in cholangiocarcinoma remain elusive. The fibroblast growth factor-inducible 14 (Fn14)/tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor-ligand system has been shown to be of importance in cellular proliferation and tumour angiogenesis in hepatocellular carcinoma. The aim of this study was to demonstrate the expression of Fn14 and TWEAK in cholangiocarcinoma and determine the functional significance of Fn14/TWEAK interaction on neoplastic cholangiocytes. Methods Human liver samples were obtained with consent from the Queen Elizabeth Hospital liver transplant programme. Sections were stained for Fn14 with immunohistochemical techniques. Expression of Fn14 on a cholangiocarcinoma cell line (CC-LP-1) stimulated with TNFα, interferon γ and fibroblast growth factor (FGF) basic was established quantitatively with flow cytometry. Stimulated CC-LP-1 were exposed to different concentrations of TWEAK for 24h. Apoptosis, necrosis, autophagy, and reactive oxygen species production at 24 h were determined by flow cytometry using annexin, 7AAD, dansylcadaverine, and dichlorofluorescein assays, respectively. Proliferation was determined with Ki69 nuclear staining. Findings Immunohistochemistry revealed Fn14 on the intrahepatic malignant small bile ducts in cholangiocarcinoma. Exposure of neoplastic cholangiocytes to TWEAK for 24h induced necrosis and reduced apoptosis in FGF-activated neoplastic cholangiocytes in a dose-dependent manner. Interpretation Fn14 is expressed on neoplastic cholangiocytes in intrahepatic cholangiocarcinoma. Activation of the Fn14/TWEAK receptor-ligand system induces necrosis. The role of Fn14/TWEAK in cholangiocarcinoma needs further investigation to ascertain the mechanisms involved and outcome on overall tumour viability. Funding UK Medical Research Council.

PMO-113 FN14 is expressed on cholangiocytes and promotes biliary ductular remodelling via apoptosis and reactive oxygen species after interaction with tweak

Introduction The mortality from chronic liver disease in the UK has increased by 50%.1 The prevalence of cholangiopathies, diseases of the bile ducts, has increased fourfold.2 These include primary biliary cirrhosis, primary sclerosing cholangitis and allograft rejection after transplantation.3 4 It is increasingly observed in livers donated for transplantation after cardiac death, a source of organs on which the NHS is becoming more reliant.5 6 It is characterised by inflammation and destruction of intrahepatic bile ducts.7 When sustained it may drive portal fibrosis to end-stage liver disease when the only therapeutic option for patients is liver transplantation.8 The novel TNF superfamily member TNF-like weak inducer of apoptosis (TWEAK) and its cognate receptor FGF-inducible protein 14 (Fn14) are implicated in hepatic inflammation and remodelling.9 10 TWEAK is mainly secreted as a soluble cytokine by myelomonocytic cells.11 Fn14-TWEAK interaction in other systems promotes cell growth, apoptosis, autophagy and transdifferentiation via activation of TRAF and NF-kB pathways.12 Aim To demonstrate the expression of Fn14 and TWEAK on cholangiocytes and the functional significance of Fn14/TWEAK interaction on biliary ductular remodelling. Methods Human liver samples were obtained with consent from the Queen Elizabeth Hospital liver transplant programme. Sections were stained for Fn14 and TWEAK using immunohistochemical techniques. Expression of Fn14 and TWEAK on cholangiocytes stimulated with TNF-α, IFN-γ and FGF was established quantitatively using flow cytometry. Cholangiocytes stimulated with FGF were exposed to TWEAK for 48 h. Apoptosis and reactive oxygen species production at this time point were determined by flow cytometry using annexin and dichlorofluorescein assays respectively. Results Immunohistochemistry reveals Fn14 on the intra-hepatic small bile ducts of inflamed livers, especially around the Canals of Hering. Fn14 expression is increased on cholangiocytes in vitro by 26% after stimulation with FGF. Exposure of cholangiocytes to TWEAK for 48 h induces apoptosis and upregulation of reactive oxygen species in FGF-activated cholangiocytes. Conclusion Fn14 is expressed on cholangiocytes in inflamed human livers. Activation of the Fn14/TWEAK receptor-ligand system induces apoptosis using a novel mechanism partly dependent on the generation of reactive oxygen species. Competing interests None declared. References 1. Williams JG, Roberts SE, Ali MF, et al. Gastroenterology services in the UK. The burden of disease, and the organisation and delivery of services for gastrointestinal and liver disorders: a review of the evidence. Gut 2007;56(Suppl 1):1–113. 2. James OF, Bhopal R, Howel D, et al. Primary biliary cirrhosis once rare, now common in the United Kingdom? Hepatology 1999;30:390–4. 3. Burt AD. Primary biliary cirrhosis and other ductopenic diseases. Clin Liver Dis 2002;6:363–80, vi. 4. Polson J, Lee WM; American Association for the Study of Liver Disease. AASLD position paper: the management of acute liver failure. Hepatology 2005;41:1179–97. 5. Foley DP, Fernandez LA, Leverson G, et al. Biliary complications after liver transplantation from donation after cardiac death donors: an analysis of risk factors and long-term outcomes from a single center. Ann Surg 2011;253:817–25. 6. NHSBT Monthly Statistics. 2011. 7. Desmet VJ. Histopathology of chronic cholestasis and adult ductopenic syndrome. J Clin Liver Dis 1998;2:249–64, viii. 8. Eksteen B, Afford SC, Wigmore SJ, Immune-mediated liver injury. Semin Liver Dis 2007;27:351–66. 9. Jakubowski A, Ambrose C, Parr M, et al. TWEAK induces liver progenitor cell proliferation. J Clin Invest 2005;115:2330–40. 10. Tirnitz-Parker JE, Viebahn CS, Jakubowski A, et al. Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells. Hepatology. 2010;52:291–302. 11. Kaduka Y, Takeda K, Nakayama M. TWEAK mediates anti-tumor effect of tumor-infiltrating macrophage. Biochem Biophys Res Commun. 2005;331:384–90. 12. Roos C, Wicovsky A, Müller N, et al. Soluble and transmembrane TNF-like weak inducer of apoptosis differentially activate the classical and noncanonical NF-kappa B pathway. J Immunol 2010;185:1593–605.

PMO-131 FGF inducible protein 14 is upregulated in neovessels during chronic inflammatory liver disease and promotes intrahepatic endothelial cell angiogenesis in vitro following stimulation via tweak

Introduction TWEAK and Fn14 members of the TNF superfamily of ligands and receptors collectively regulate a diverse range of immune, inflammatory and regenerative responses. Recent studies indicate a potential role of TWEAK and Fn14 in tissue repair following liver injury where they may promote angiogenesis and neovessel growth. TWEAK/Fn14 could therefore facilitate inflammatory cell recruitment and promote portal associated lymphoid tissue development during inflammation. Aims (1). To investigate TWEAK/Fn14 expression in human liver tissue during chronic liver disease. (2) To study Fn14 expression in isolated intra-hepatic endothelial cells (IHEC). (3) To determine angiogenic responses of IHEC to TWEAK. Methods Tissue sections from explanted human livers at the time of hepatobiliary surgery, including normal donor; normal tissue adjacent to malignant lesions; HCV; ALD; NASH; Chronic Allograft Rejection; PSC and PBC, were subjected to immunohistochemistry or dual immunofluorescence using antibodies to TWEAK, Fn14 and phenotypic markers CD31 and CD68. Isolated IHEC were cultured with combinations of TNFα, IFNg, FGF, and assessed for TWEAK/Fn14 expression using flow cytometry. In addition IHEC were incubated with recombinant TWEAK in presence ± TNFα and assessed using a matrigel angiogenesis assay for vessel formation and branching. Results Fn14 expression was low in normal tissue in portal vessels and sinusoids, whereas in disease portal neovessels (CD31 +ve) were highly positive for Fn14. TWEAK expression was low in normal tissue but highly expressed in CD68+ve monocytic cells surrounding areas of neovascularisation and inflammatory cell aggregation. Fn14 expression significantly up-regulated on isolated IHEC when stimulated with the TNFα. Confocal imaging showed that the expression of Fn14 was predominantly cytoplasmic unless stimulated with TNF which enhanced cell surface expression. IHEC were consistently negative for TWEAK and TWEAK stimulation increased IHEC angiogenesis, where a change in cell morphology and enhanced branching of capillary like structures was observed. Pre-incubation with Fn14 antagonistic mAb completely abrogated vessel formation and branching. Conclusion These new data show that Fn14 activation stimulates neovessel branching in IHEC and that TNF-α promotes mobilisation of cytoplasmic Fn14 to the cell surface suggesting an important regulatory role for TWEAK/Fn14 in neovascularisation and portal lymphoid aggregation during hepatic inflammation. Competing interests None declared.