Autophagy and the Liver

Autophagy is a cellular process that involves lysosomal degradation and recycling of intracellular organelles and proteins to maintain energy homeostasis during times of cellular stress [1]. It also serves to remove damaged cellular components such as mitochondria and long-lived proteins. Autophagy is catabolic mechanism and although hepatic autophagy performs the standard functions of degrading damaged organelles/aggregated proteins and regulating cell death it also regulates lipid accumulation within the liver. Autophagy can be divided into three distinct sub-groups that are discussed below. This chapter focuses upon the role of autophagy in a variety of liver diseases including hepatocellular carcinoma (HCC) and viral hepatitis. The increased understanding of the cellular machinery regulating autophagy within the liver may foster the development of therapeutic strategies that will ultimately help treat liver disease

Autophagy: A cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes

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

C4b Binding Protein Binds to CD154 Preventing CD40 Mediated Cholangiocyte Apoptosis: A Novel Link between Complement and Epithelial Cell Survival

Activation of CD40 on hepatocytes and cholangiocytes is critical for amplifying Fas-mediated apoptosis in the human liver. C4b-Binding Protein (C4BP) has been reported to act as a potential surrogate ligand for CD40, suggesting that it could be involved in modulating liver epithelial cell survival. Using surface plasmon resonance (BiaCore) analysis supported by gel filtration we have shown that C4BP does not bind CD40, but it forms stable high molecular weight complexes with soluble CD40 ligand (sCD154). These C4BP/sCD154 complexes bound efficiently to immobilised CD40, but when applied to cholangiocytes they failed to induce apoptosis or proliferation or to activate NFkB, AP-1 or STAT 3, which are activated by sCD154 alone. Thus C4BP can modulate CD40/sCD154 interactions by presenting a high molecular weight multimeric sCD154/C4BP complex that suppresses critical intracellular signalling pathways, permitting cell survival without inducing proliferation. Immunohistochemistry demonstrated co-localisation and enhanced expression of C4BP and CD40 in human liver cancers. These findings suggest a novel pathway whereby components of the complement system and TNF ligands and receptors might be involved in modulating epithelial cell survival in chronic inflammation and malignant disease

Upgrade of the MARI spectrometer at ISIS

The MARI direct geometry time-of-flight neutron spectrometer at ISIS has been upgraded with an $m=3$ supermirror guide and new detector electronics. This has resulted in a flux gain of ${\approx}6{\times}$ at $\lambda=1.8$ {\AA}, and improvements on discriminating electrical noise, allowing MARI to continue to deliver a high quality science program well into its fourth decade of life

Interaction of TWEAK with Fn14 leads to the progression of fibrotic liver disease by directly modulating hepatic stellate cell proliferation

Tumour necrosis factor‐like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor‐inducible 14 (Fn14) have been associated with liver regeneration in vivo. To further investigate the role of this pathway we examined their expression in human fibrotic liver disease and the effect of pathway deficiency in a murine model of liver fibrosis. The expression of Fn14 and TWEAK in normal and diseased human and mouse liver tissue and primary human hepatic stellate cells (HSCs) were investigated by qPCR, western blotting and immunohistochemistry. In addition, the levels of Fn14 in HSCs following pro‐fibrogenic and pro‐inflammatory stimuli were assessed and the effects of exogenous TWEAK on HSCs proliferation and activation were studied in vitro. Carbon tetrachloride (CCl(4)) was used to induce acute and chronic liver injury in TWEAK KO mice. Elevated expression of both Fn14 and TWEAK were detected in acute and chronic human liver injury, and co‐localized with markers of activated HSCs. Fn14 levels were low in quiescent HSCs but were significantly induced in activated HSCs, which could be further enhanced with the profibrogenic cytokine TGFβ in vitro. Stimulation with recombinant TWEAK induced proliferation but not further HSCs activation. Fn14 gene expression was also significantly up‐regulated in CCl(4) models of hepatic injury whereas TWEAK KO mice showed reduced levels of liver fibrosis following chronic CCl(4) injury. In conclusion, TWEAK/Fn14 interaction leads to the progression of fibrotic liver disease via direct modulation of HSCs proliferation, making it a potential therapeutic target for liver fibrosis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Development of Clinical Criteria for Functional Assessment to Predict Primary Nonfunction of High-Risk Livers Using Normothermic Machine Perfusion

Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow &gt;150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers.</p

Discarded livers tested by normothermic machine perfusion in the VITTAL trial:Secondary end points and 5-year outcomes

Normothermic machine perfusion (NMP) enables pretransplant assessment of high-risk donor livers. The VITTAL trial demonstrated that 71% of the currently discarded organs could be transplanted with 100% 90-day patient and graft survivals. Here, we report secondary end points and 5-year outcomes of this prospective, open-label, phase 2 adaptive single-arm study. The patient and graft survivals at 60 months were 82% and 72%, respectively. Four patients lost their graft due to nonanastomotic biliary strictures, one caused by hepatic artery thrombosis in a liver donated following brain death, and 3 in elderly livers donated after circulatory death (DCD), which all clinically manifested within 6 months after transplantation. There were no late graft losses for other reasons. All the 4 patients who died during the study follow-up had functioning grafts. Nonanastomotic biliary strictures developed in donated after circulatory death livers that failed to produce bile with pH &gt;7.65 and bicarbonate levels &gt;25 mmol/L. Histological assessment in these livers revealed high bile duct injury scores characterized by arterial medial necrosis. The quality of life at 6 months significantly improved in all but 4 patients suffering from nonanastomotic biliary strictures. This first report of long-term outcomes of high-risk livers assessed by normothermic machine perfusion demonstrated excellent 5-year survival without adverse effects in all organs functioning beyond 1 year (ClinicalTrials.gov number NCT02740608).</p

Isolation of Primary Human Hepatocytes from Normal and Diseased Liver Tissue: A One Hundred Liver Experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples

Human intrahepatic tregs are functional, require IL-2 from effector cells for survival and are susceptible to fas ligand mediated apoptosis

Regulatory T cells (T(reg)) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of T(reg) in the liver, suggesting that the local hepatic microenvironment might affect T(reg) stability, survival, and function. We hypothesized that interactions between T(reg) and endothelial cells during recruitment and then with epithelial cells within the liver affect T(reg) stability, survival, and function. To model this, we explored the function of T(reg) after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated T(reg) [PEM T(reg)]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of T(reg). Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the T(reg) survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect T(reg) stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM T(reg) phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating T(reg) reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of T(reg) compared with CD8 effector cells. T(reg) from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair T(reg) stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of T(reg) to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain T(reg) dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction with T(reg) therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138–150