Daniela C. Kroy

Lack of interleukin‐6/glycoprotein 130/signal transducers and activators of transcription‐3 signaling in hepatocytes predisposes to liver steatosis and injury in mice

A deregulated cytokine balance is involved in triggering the sequence from steatosis to nonalcoholic steatohepatitis, ultimately leading to liver fibrosis and cancer. To better define the role of proinflammatory interleukin‐6 (IL‐6)‐type cytokines in hepatocytes we investigated the role of IL‐6 and its shared receptor, glycoprotein 130 (gp130), in a mouse model of steatohepatitis. IL‐6−/− mice were fed a choline‐deficient, ethionine‐supplemented (CDE) diet. Conditional gp130 knockout and knockin mice were used to achieve hepatocyte‐specific deletion of gp130 (gp130Δhepa), gp130‐dependent rat sarcoma (Ras)‐(gp130ΔhepaRas), and signal transducers and activators of transcription (STAT)‐(gp130ΔhepaSTAT) activation. CDE‐treated IL‐6−/− mice showed a significant hepatic steatosis at 2 weeks after feeding. The mice rapidly developed elevated fasting blood glucose, insulin serum levels, and transaminases. To better define IL‐6‐dependent intracellular pathways, specifically in hepatocytes, we next treated gp130Δhepa mice with a CDE diet. These animals also developed a marked steatosis with hyperglycemia and displayed elevated insulin serum levels. Additionally, gp130Δhepa animals showed an imbalanced inflammatory response with increased hepatic tumor necrosis factor‐alpha and decreased adiponectin messenger RNA levels. Dissecting the hepatocyte‐specific gp130‐dependent pathways revealed a similar disease phenotype in gp130ΔhepaSTAT mice, whereas gp130ΔhepaRas animals were protected. In CDE‐treated mice lack of gp130‐STAT3 signaling was associated with immune‐cell‐infiltration, jun kinase‐activation, a blunted acute‐phase‐response, and elevated transaminases. Furthermore, gp130Δhepa and gp130ΔhepaSTAT mice showed beginning signs of liver fibrosis compared to gp130ΔhepaRas mice and controls. Conclusion:During CDE treatment mice lacking IL‐6 and gp130‐STAT signaling in hepatocytes are prone to hepatic metabolic changes and inflammation. This ultimately leads to progressive steatohepatitis with signs of liver remodeling. Thus, the presented model allows one to further dissect the role of IL‐6/gp130‐type signaling in hepatocytes during fatty liver degeneration to define new therapeutic targets in metabolic liver diseases. (HEPATOLOGY 2010.)

Lack of Glycoprotein 130/Signal Transducer and Activator of Transcription 3-Mediated Signaling in Hepatocytes Enhances Chronic Liver Injury and Fibrosis Progression in a Model of Sclerosing Cholangitis

The 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) model leads to chronic cholestatic liver injury and therefore resembles human diseases such as sclerosing cholangitis and forms of metabolic liver diseases. The role of the interleukin-6/glycoprotein 130 (gp130) system in this context is still undefined. Therefore, conditional gp130 knockout and knockin mice were used to achieve hepatocyte-specific deletions of gp130 (gp130(Deltahepa)), gp130-dependent ras (gp130(DeltahepaRas)), and signal transducer and activator of transcription (STAT) (gp130(DeltahepaSTAT)) activation. These mice were treated with a DDC-containing diet and analyzed over time. Mice deficient in hepatic gp130 and STAT signaling showed increased and earlier mortality than wild-type and gp130(DeltahepaRas) animals. Over time, significantly more apoptosis and cholestasis became evident in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice. These mice also displayed increased tumor necrosis factor-alpha expression, a diminished acute-phase response (lack of STAT3 and serum amyloid A activation), and enhanced immune cell infiltration in the liver. These were associated with stronger periportal oval cell activation. In addition, DDC treatment in gp130(Deltahepa) and gp130(DeltahepaSTAT) mice resulted in significantly stronger hepatic stellate cell activation. Long-term analysis revealed the development of severe liver fibrosis in gp130(Deltahepa) and gp130(DeltahepaSTAT) animals, as evidenced by increased collagen accumulation. Here we demonstrate that gp130/STAT signaling in hepatocytes provides protection in a cholestatic hepatitis mouse model. STAT3-dependent signaling pathways in hepatocytes protect from apoptosis and tissue injury, which subsequently reduce oval cell activation and prevent fibrosis progression.

Hepatocyte specific deletion of c-Met leads to the development of severe non-alcoholic steatohepatitis in mice

BACKGROUND & AIMSnNon-alcoholic-fatty-liver disease (NAFLD) is part of the metabolic syndrome. The spectrum of NAFLD includes NASH (non-alcoholic steatohepatitis), which is characterised by progressive inflammation associated with oxidative stress and apoptosis, finally triggering liver cirrhosis and hepatocellular carcinoma. HGF (hepatocyte growth factor)/mesenchymal-epithelial transition factor (c-Met) receptor signalling is known to activate distinct intracellular pathways mediating among others anti-apoptotic properties to hepatocytes. Therefore, the aim was to characterise the role of c-Met during NASH development.nnnMETHODSnHepatocyte specific c-Met knockout mice (c-MetΔ(hepa)) using the cre-loxP system and wild type controls (c-Met(loxP/loxP)) were fed a methionine-choline deficient (MCD) diet.nnnRESULTSnMCD feeding triggered massive steatosis, decreased survival and higher transaminases in c-MetΔ(hepa) livers compared to c-Met(loxP/loxP). Gene array analysis demonstrated that genes involved in fatty acid metabolism were strongly upregulated in c-MetΔ(hepa) livers correlating with higher amounts of hepatic free fatty acids. Consequently, c-MetΔ(hepa) mice showed significantly more TUNEL positive cells and more superoxide anion production than c-Met(loxPloxP) animals. Additionally, c-MetΔ(hepa) livers showed significantly larger fractions of infiltrating neutrophils, macrophages, and cytotoxic T cells. These changes correlated with an enhanced progression of liver fibrosis as evidenced by higher collagen deposition in c-MetΔ(hepa) livers. As increased apoptosis was a prominent feature in c-MetΔ(hepa) livers, we generated c-Met/Casp8Δ(hepa) double knockout mice. In these animals compared to c-MetΔ(hepa) animals the increase in apoptosis could be reverted.nnnCONCLUSIONSnc-Met deletion in hepatocytes triggers NASH progression. A prominent mechanism is higher fatty acid accumulation and increased apoptosis, which in part can be reverted by blocking caspase 8.

Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development

Generation of reactive oxygen species (ROS) in response to fatty acids accumulation has been classically proposed as a possible second hit triggering progression from simple steatosis to non-alcoholic steatohepatitis (NASH). In this study we challenged hepatocyte-specific Keap1 knockout mice (Keap1(Δhepa)) and littermate Cre- controls (Keap1(fx/fx)) with two different diet models of NASH in order to evaluate the effects of the anti-oxidant transcription factor Nrf2 over-activation on hepatic metabolism and disease progression. After 4 weeks of MCD diet the liver/body weight ratio of Keap1(Δhepa) mice was significantly higher compared to littermate controls with no differences in total body weight. Strikingly, liver histology revealed a dramatic reduction of lipid droplets confirmed by a decreased content of intra-hepatic triglycerides in Keap1(Δhepa) compared to controls. In parallel to reduced expression of genes involved in lipid droplet formation, protein expression of Liver X Receptor (LXRα/β) and Peroxisome proliferator-activated receptor α (PPARα) was significantly decreased. In contrast, genes involved in mitochondrial lipid catabolism were markedly up-regulated in Keap1(Δhepa) livers. A similar phenotype characterized by inhibition of lipogenesis in favor of increased mitochondrial catabolic activity was also observed after 13 weeks of western diet administration. MCD-induced apoptosis was significantly dampened in Keap1(Δhepa) compared to Keap1(fx/fx) as detected by TUNEL, cleaved caspase-3 and Bcl-2 protein expression analyses. However, no differences in inflammatory F4/80- and CD11b-positive cells and pro-fibrogenic genes were detected between the two groups. Although hepatic lack of Keap1 did not ameliorate inflammation, the resulting constitutive Nrf2 over-activation in hepatocytes strongly reduced hepatic steatosis via enhanced lipid catabolism and repressed de novo lipogenesis during murine NASH development.

Liver transplantation in Germany

Liver transplantation (LT) is a well‐accepted procedure for end‐stage liver disease in Germany. In 2015, 1489 patients were admitted to the waiting list (including 1308 new admissions), with the leading etiologies being fibrosis and cirrhosis (n = 349), alcoholic liver disease (n = 302), and hepatobiliary malignancies (n = 220). Organ allocation in Germany is regulated within the Eurotransplant system based on urgency as expressed by the Model for End‐Stage Liver Disease score. In 2015, only 894 LTs (n = 48 from living donors) were performed at 23 German transplant centers, reflecting a shortage of organs. Several factors may contribute to the low number of organ donations. The German transplant legislation only accepts donation after brain death (not cardiac death), whereas advances in neurosurgery and a more frequently requested “palliative care” approach render fewer patients suitable as potential donors. The legislation further requires the active consent of the donor or first‐degree relatives before donation. Ongoing debates within the German transplant field address the optimal management of patients with alcoholic liver cirrhosis, hepatocellular carcinoma (HCC), and cholangiocarcinoma and measures to increase living donor transplantations. As a result of irregularities at mainly 4 German transplant centers that were exposed in 2012, guiding principles updated by the German authorities have since implemented strict rules (including internal and external auditing, the 8‐eyes principle, mandatory repeated testing for alcohol consumption) to prohibit any manipulations in organ allocation. In conclusion, we will summarize important aspects on the management of LT in Germany, discuss legal and organizational aspects, and highlight challenges mainly related to the relative lack of organ donations, increasing numbers of extended criteria donors, and the peculiarities of the recipient patients. Liver Transplantation 22 1136–1142 2016 AASLD

Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis

Objective Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. Methods Experimental liver fibrosis in mice induced by bile duct ligation or CCl4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. Results In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. Conclusions In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis.

An Unusual Insertion in Jak2 Is Crucial for Kinase Activity and Differentially Affects Cytokine Responses

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-γ and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.

Immunoregulation by lipids during the development of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) represents the most common liver disorder in western countries and it is commonly associated with obesity and progression of the metabolic syndrome. Comprehending a wide spectrum of pathologic features, it is currently well recognized that a key point for the integrity of hepatocyte functionality in NAFLD is the progression from simple steatosis to non-alcoholic steatohepatitis (NASH). Indeed, activation of the innate immune system in response to hepatic metabolic stresses represents a central process that determines the evolution and the reversibility of liver damage. Despite of the burden of studies published in recent years, it is still intriguingly unclear how accumulation of lipids in hepatocytes triggers the activation of the inflammatory response leading to the recruitment of infiltrating cells of extra-hepatic origins. In this review we offer a general view on recent advances pointing out how different classes of lipids are able to specifically affect hepatocytes functionality and survival, thus differently influencing the organization of the hepatic immune response. On the other hand, we gathered recent studies intending to illustrate the basic mechanisms through which several non-parenchymal hepatic and extra-hepatic cell populations get activated in response to lipids. Finally, we indicate latter findings proposing how the immune system majorly contributes to the progression of NASH.

Chemokine (C-X-C motif) ligand 11 levels predict survival in cirrhotic patients with transjugular intrahepatic portosystemic shunt.

Chemokines, such as CXCR3‐ligands, have been identified to play an important role during hepatic injury, inflammation and fibrosis. While CXCL9 is associated with survival in patients receiving transjugular intrahepatic portosystemic shunt (TIPS), the role of CXCL11 in severe portal hypertension remains unknown.

Doppler Ultrasound and Transient Elastography in Liver Transplant Patients for Noninvasive Evaluation of Liver Fibrosis in Comparison with Histology: A Prospective Observational Study.

Background and AimAccurate quantification of progressive liver disease is essential for therapeutic decisions and follow-up for patients who underwent liver transplantation. To evaluate the quality of noninvasive assessment of liver fibrosis in these patients, we compared Doppler ultrasound of the hepatic blood vessels as well as transient elastography (TE, FibroScan®) with liver biopsy following transplantation.MethodsWe performed Doppler ultrasound of the hepatic veins, hepatic artery, and portal vein as well as a TE in 48 patients who underwent liver transplantation 12xa0months ago. Hepatic venous flow was evaluated by determination of the resistance index (HVRI) of the right hepatic vein. Doppler and TE results were compared with histopathologic workup of a 12-month protocol liver biopsy after transplantation.ResultsHVRI showed a high reliability in predicting liver fibrosis stage FII or higher (AUROC of 0.99xa0±xa00.001 for FII or higher, the HVRIxa0<xa01.05 with a sensitivity and specificity of 100 and 91.43xa0%) compared to histopathologic workup (Desmet’s score) and was comparable to TE analysis. Both HVRI and TE differed significantly in no or minimal fibrosis versus FII or higher (pxa0<xa00.001). In contrast, portal vein and hepatic artery did not show significant changes in blood flow in our study population.ConclusionsHepatic vein flow resistance index is a valuable tool in noninvasive evaluation of liver fibrosis in liver transplantation follow-up predicting FII or higher and might help reducing the number of protocol biopsies needed.