S Erschfeld

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 growth factor/c-Met signalling is important for the selection of transplanted hepatocytes.

Background At present hepatocyte transplantation is a promising option for cellular therapy of end-stage liver diseases. However, the underlying molecular mechanisms need to be better defined in order to translate this technique into clinical use. This study investigated the cursiv relevance of hepatocyte growth factor (HGF)/c-Met signalling for hepatocyte repopulation after transplantion. Methods Wild-type mice (c-MetloxP/loxP) and hepatocyte-specific conditional c-Met (HGF receptor) knockout (c-MetΔhepa) mice were used as donors and recipients for hepatocyte transplantation. Results Transplantation experiments revealed two major findings. First it was demonstrated that c-Met is indispensable in donor cells, as c-MetΔhepa cells did not repopulate recipient livers after transplantation. Second, genetic deletion of c-Met in recipient hepatocytes resulted in enhanced expansion of unmodified donor cells in host livers (up to 250-fold after 12 weeks). The relevant mechanisms for this observation in c-MetΔhepa host hepatocytes could be defined. c-MetΔhepa hepatocytes showed enhanced apoptosis, reduced cellular proliferation and a lack of AKT-kinase and STAT3 activation. In addition, tissue remodelling was changed in c-MetΔhepa recipient livers. Therefore, the lack of pro-proliferative transcription factors, increased apoptosis and changes in matrix-remodelling inhibit host cell proliferation in c-MetΔhepa recipient livers and thus favour repopulation of transplanted hepatocytes. Therapeutically liver repopulation could be increased through adenoviral expression of NK-4—an inhibitor of HGF signalling—in host hepatocytes. Conclusion HGF/c-Met plays a crucial role in host and donor cells of the liver for the cursiv selection of transplanted hepatocytes. Modulating HGF-dependent signalling seems a promising therapeutic option to favour expansion of transplanted hepatocytes.

Lack of hepatic c-Met and gp130 expression is associated with an impaired antibacterial response and higher lethality after bile duct ligation.

The prognosis of liver failure is often determined by infectious and cholestatic complications. As HGF/c-Met and interleukin (IL)-6/gp130 control hepatic cytoprotective pathways, we here investigated their cooperative role during the onset of cholestatic liver injury. Conditional hepatocyte-specific (Δhepa) c-Met, gp130 and c-Met/gp130 knockout mice (Cre-loxP system) were subjected to bile duct ligation (BDL) and lipopolysaccharide (LPS) stimulation. gp130Δhepa and c-Met/gp130Δhepa mice displayed increased lethality associated with severe bacteraemia early after BDL, whereas c-MetΔhepa and wild-type mice showed normal survival. Analysis of the innate immune response and the regulation of hepatic antibacterial pathways showed that the LPS-triggered hepatocellular response via the Toll-like receptor-4 pathway was regulated differentially by HGF/c-Met and IL-6/gp130. Activation of p38MAPK, c-Jun N-terminal kinase and signalling transducer and activator of transcription-3 was impaired in gp130Δ and c-MetΔhepa livers. In addition, the acute-phase response (APR) was reduced in c-MetΔhepa livers, whereas gp130Δhepa displayed a completely abolished APR. In contrast, TNF-α-dependent NF-κB activation was enhanced in gp130Δhepa and c-MetΔhepa mice and it was associated with a higher rate of apoptosis and inflammation. Moreover, expression of the neutrophil produced and secreted cathelin-related antimicrobial peptide and of genes related to the inflammasome complex correlated with the strength of the bacterial infection and with TNF-α expression. In conclusion, Gp130 and c-Met are involved in the hepatic antibacterial and innate immune response, control the APR and thus prevent sepsis and liver injury during cholestatic conditions.

Glycoprotein 130–dependent pathways in host hepatocytes are important for liver repopulation in mice

Hepatocyte transplantation (HT) is still restricted by the limited amount of transplantable cells. Therefore, a better understanding of the mechanisms involved in cellular engraftment, proliferation, and in vivo selection is important. Here we aimed to evaluate the role of the interleukin 6 (IL‐6)/glycoprotein 130 (gp130) system for liver repopulation. Mice carrying a conditional hepatocyte‐specific deletion of the common IL‐6 signal transducer gp130 (gp130Δhepa) were used for HT. First, we compared bone marrow transplantation (BMT), partial hepatectomy (PH), and retrorsine treatment of recipient mice to optimize the in vivo selection of transplanted hepatocytes. BMT combined with PH was sufficient to induce a 30‐fold increase in the number of transplanted donor hepatocytes, whereas additional retrorsine pretreatment led to an up to 40‐fold increase. Next, the influence of gp130 signaling in hepatocytes on cell selection was evaluated. Wild‐type (WT) hepatocytes repopulated WT recipients at the same rate as gp130Δhepa cells. In contrast, liver repopulation by transplanted cells was enhanced in gp130Δhepa recipient mice. This was associated with higher proliferation of donor hepatocytes and enhanced apoptosis in gp130Δhepa recipient livers. Additionally, the acute phase response was strongly induced after HT in WT recipients but blunted in gp130Δhepa recipients. As a result, significantly more liver remodeling, evidenced by stronger hepatic stellate cell activation and collagen accumulation, was found in gp130Δhepa mice after HT. In conclusion, the HT model established here can be efficiently applied to investigate cell‐specific mechanisms in liver repopulation. Moreover, we have shown that gp130‐dependent pathways in host hepatocytes are important for controlling liver repopulation. Liver Transpl 16:23–32, 2010.

The Anti-Microbial Peptide LL-37/CRAMP Is Elevated in Patients with Liver Diseases and Acts as a Protective Factor during Mouse Liver Injury

Background: Antimicrobial peptides (AMP) are an important defense mechanism of the innate immune system and can modulate the course of various diseases. However, their significance during liver pathogenesis is currently not well defined. Methods: Patients with liver diseases were analyzed for LL-37/CRAMP, human beta-defensin-2 (hBD2), and complement 5a (C5a) serum levels. Mice deficient in CRAMP (Cathelicidin-related Antimicrobial Peptide), the mouse homolog for human LL-37, were fed with a methionine- and choline-deficient diet (MCD) and underwent bile-duct ligation (BDL). Results: First, serum samples from patients with chronic liver diseases were investigated. Therefore, significantly enhanced levels for LL-37, hBD2, and complement C5a were detected, all of which comprise antimicrobial properties. Next, CRAMP-knockout (CRAMP-KO) mice were investigated, to better define a functional role of LL-37/CRAMP in animal models of liver diseases. MCD feeding and bile-duct ligation of CRAMP-KO mice resulted in an enhanced degree of liver injury during the early treatment phase. MCD feeding in CRAMP-KO mice led to stronger intrahepatic fat accumulation and significantly enhanced matrix remodeling, whereas BDL caused more extensive liver necrosis. At the late 28 days time point, MCD-fed CRAMP-KO mice displayed a higher intrahepatic fat load. Long-term changes in bile-duct-ligated mice included higher collagen content as a sign of enhanced fibrosis progression if CRAMP was absent. Conclusion: The study shows a clear correlation of antimicrobial peptide serum levels in patients with chronic liver diseases. Furthermore, we were able to demonstrate protective functions of LL-37/CRAMP in two independent mouse models of chronic liver injury.

Hepatic Tissue Environment in NEMO-Deficient Mice Critically Regulates Positive Selection of Donor Cells after Hepatocyte Transplantation

Background Hepatocyte transplantation (HT) is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF)-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. Methods Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT) and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa). Results Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. Conclusion This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and opens a new door for the applicability of this technique to combat liver disease in the human clinic.

Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH

We have recently shown that hepatocyte-specific c-met deficiency accelerates the progression of nonalcoholic steatohepatitis in experimental murine models resulting in augmented production of reactive oxygen species and accelerated development of fibrosis. The aim of this study focuses on the elucidation of the underlying cellular mechanisms driven by Nrf2 overactivation in hepatocytes lacking c-met receptor characterized by a severe unbalance between pro-oxidant and antioxidant functions. Control mice (c-metfx/fx), single c-met knockouts (c-metΔhepa), and double c-met/Keap1 knockouts (met/Keap1Δhepa) were then fed a chow or a methionine-choline-deficient (MCD) diet, respectively, for 4 weeks to reproduce the features of nonalcoholic steatohepatitis. Upon MCD feeding, met/Keap1Δhepa mice displayed increased liver mass albeit decreased triglyceride accumulation. The marked increase of oxidative stress observed in c-metΔhepa was restored in the double mutants as assessed by 4-HNE immunostaining and by the expression of genes responsible for the generation of free radicals. Moreover, double knockout mice presented a reduced amount of liver-infiltrating cells and the exacerbation of fibrosis progression observed in c-metΔhepa livers was significantly inhibited in met/Keap1Δhepa. Therefore, genetic activation of the antioxidant transcription factor Nrf2 improves liver damage and repair in hepatocyte-specific c-met-deficient mice mainly through restoring a balance in the cellular redox homeostasis.

Contents Vol. 91, 2015

Founded as ‘Archiv für Verdauungskrankheiten’ 1895 by I. Boas Continued as ‘Gastroenterologia’ 1939–1967 Former Editors: P. Morawitz (1934–1936), R. Staehelin (1937–1943), A. Hurst (1940–1945), W. Löffler (1943–1961), T.C. Hunt (1947–1967), N. Henning (1953–1962), B. Ihre (1953–1967), H. Bartelheimer (1963–1967), M. Demole (1963–1971), H. Kapp (1968–1970), R. Lambert (1972–1978), W. Creutzfeldt (1979–1992), R. Arnold (1993–2003), C. Beglinger (2004–2011), B. Göke (2004–2014)

Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation

Introduction Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Methods Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130ΔMx), or to selectively disrupt gp130-dependent Ras (gp130ΔMxRas) or STAT signalling (gp130ΔMxSTAT) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. Results BM derived from gp130 deficient donor mice (gp130ΔMx) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130ΔMxRas and gp130ΔMxSTAT donor BM. BMT of gp130ΔMxSTAT cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130ΔMxRas BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Conclusion Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.