M Hatting

Loss of Caspase-8 Protects Mice Against Inflammation-Related Hepatocarcinogenesis but Induces Non-Apoptotic Liver Injury

BACKGROUND & AIMS Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.

Hepatocyte caspase‐8 is an essential modulator of steatohepatitis in rodents

In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death‐receptor‐mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte‐specific Casp8 knockout (Casp8Δhep) mice. Animals were fed with a methionine‐choline‐deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent‐activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8Δhep animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD‐dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8Δhep livers. Conclusion: Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8‐directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189–2201)

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

BACKGROUND & AIMS Non-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. METHODS Hepatocyte 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. RESULTS MCD 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. CONCLUSIONS c-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.

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis

Objective The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. Design Jnk1f/f wildtype (WT), Jnk1−/− and Jnk1Δhepa (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. Results Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1−/− compared with WT and Jnk1Δhepa mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1−/− compared with WT and Jnk1Δhepa livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow–derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1−/− livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. Conclusions Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model

Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130Δhepa) and control animals (gp130f/f) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0–144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130Δhepa animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130Δhepa mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130f/f and gp130Δhepa animals. However, gp130Δhepa livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-β (TGFβ), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.

Haematopoietic cell-derived Jnk1 is crucial for chronic inflammation and carcinogenesis in an experimental model of liver injury

BACKGROUND & AIMS Chronic liver injury triggers complications such as liver fibrosis and hepatocellular carcinoma (HCC), which are associated with alterations in distinct signalling pathways. Of particular interest is the interaction between mechanisms controlled by IKKγ/NEMO, the regulatory IKK subunit, and Jnk activation for directing cell death and survival. In the present study, we aimed to define the relevance of Jnk in hepatocyte-specific NEMO knockout mice (NEMO(Δhepa)), a genetic model of chronic inflammatory liver injury. METHODS We generated Jnk1(-/-)/NEMO(Δhepa) and Jnk2(-/-)/NEMO(Δhepa) mice by crossing NEMO(Δhepa) mice with Jnk1 and Jnk2 global deficient animals, respectively, and examined the progression of chronic liver disease. Moreover, we investigated the expression of Jnk during acute liver injury, evaluated the role of Jnk1 in bone marrow-derived cells, and analysed the expression of NEMO and p-JNK in human diseased-livers. RESULTS Deletion of Jnk1 significantly aggravated the progression of liver disease, exacerbating apoptosis, compensatory proliferation and carcinogenesis in NEMO(Δhepa) mice. Conversely, Jnk2(-/-)/NEMO(Δhepa) displayed hepatic inflammation. By using bone marrow transfer, we observed that Jnk1 in haematopoietic cells had an impact on the progression of chronic liver disease in NEMO(Δhepa) livers. These findings are of clinical relevance since NEMO expression was downregulated in hepatocytes of patients with HCC whereas NEMO and p-JNK were expressed in a large amount of infiltrating cells. CONCLUSIONS A synergistic function of Jnk1 in haematopoietic cells and hepatocytes might be relevant for the development of chronic liver injury. These results elucidate the complex function of Jnk in chronic inflammatory liver disease.

TAL deficiency, all roads lead to oxidative stress?†

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1–/– and Taldo1 /– mice spontaneously developed HCC, and Taldo1–/– mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1–/– livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ ADP ratio. Reduced -catenin phosphorylation and enhanced c-Jun expression in Taldo1–/– livers reflected adaptation to oxidative stress. Taldo1–/– hepatocytes were resistant to CD95/ Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1–/– mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency. (HEPATOLOGY 2009; 50:.)

Inactivation of caspase 8 in liver parenchymal cells confers protection against murine obstructive cholestasis

BACKGROUND & AIMS Caspase 8 (CASP8) is the apical initiator caspase in death receptor-mediated apoptosis. Strong evidence for a link between death receptor signaling pathways and cholestasis has recently emerged. Herein, we investigated the role of CASP8-dependent and independent pathways during experimental cholestasis. METHODS Liver injury was characterized in a cohort of human sera (n = 28) and biopsies from patients with stage IV primary biliary cholangitis. In parallel, mice with either specific deletion of Casp8 in liver parenchymal cells (Casp8Δhepa) or hepatocytes (Casp8Δhep), and mice with constitutive Ripk3 (Ripk3-/-) deletion, were subjected to surgical ligation of the common bile duct (BDL) from 2 to 28 days. Floxed (Casp8fl/fl) and Ripk3+/+ mice were used as controls. Moreover, the pan-caspase inhibitor IDN-7314 was used, and cell death mechanisms were studied in primary isolated hepatocytes. RESULTS Overexpression of activated caspase 3, CASP8 and RIPK3 was characteristic of liver explants from patients with primary biliary cholangitis. Twenty-eight days after BDL, Casp8Δhepamice showed decreased necrotic foci, serum aminotransferase levels and apoptosis along with diminished compensatory proliferation and ductular reaction. These results correlated with a decreased inflammatory profile and ameliorated liver fibrogenesis. A similar phenotype was observed in Ripk3-/- mice. IDN-7314 treatment decreased CASP8 levels but failed to prevent BDL-induced cholestasis, independently of CASP8 in hepatocytes. CONCLUSION These findings show that intervention against CASP8 in liver parenchymal cells - specifically in cholangiocytes - might be a beneficial option for treating obstructive cholestasis, while broad pan-caspase inhibition might trigger undesirable side effects. LAY SUMMARY Loss of caspase 8 - a protein involved in programmed cell death - in liver parenchymal cells protects against experimental cholestasis. Therefore, specific pharmacological intervention against caspase 8 might be a valid alternative for the treatment of obstructive cholestasis in the clinic, whereas broad pan-caspase inhibiting drugs might trigger undesirable side effects.

From NAFLD to HCC: Is IL-17 the crucial link?

Nonalcoholic fatty liver disease (NAFLD) has an increasing prevalence worldwide and is relevant for liver-related as well as overall morbidity and mortality. Patients with active inflammatory NAFLD, termed nonalcoholic steatohepatitis (NASH), are at particular risk for developing hepatocellular carcinoma (HCC), even in noncirrhotic livers. Innovative basic science studies have therefore been designed to elucidate the molecular and cellular mechanisms linking metabolic liver disease and inflammation to HCC development. One of the most prominent findings is a strong activation of the innate immune system, especially macrophages, in NASH that perpetuates hepatic inflammation, links hepatic to systemic (e.g., adipose tissue) inflammation, and provides a protumorigenic microenvironment characterized by excess angiogenesis and T-cell suppression. More recently, the role of the adaptive immune cells in cancer surveillance and metabolic liver disease has been increasingly recognized. It has become evident that adaptive immunity provides an important component of tumor surveillance protecting against HCC. Novel experimental HCC models in mice either combine a specific diet (like high-fat diet [HFD] or methionine choline deficient [MCD] diet) with a genotoxic agent like diethylnitrosamine or use a long-term choline-deficient high-fat diet (CD-HFD), thereby recapitulating the NASH-HCC transition in vivo. Dysregulation of lipid metabolism in NAFLD was recently found to cause oxidative damage to tumorsuppressive CD4T cells in mouse models, thereby promoting a selective loss of intrahepatic antitumoral CD4T lymphocytes. Furthermore, CD8T cells and natural killer (NK) T (NKT) cells, alongside tumor necrosis factor/interleukin (IL)-6 signaling cascades, drive NASH and HCC. A recent publication in Cancer Cell highlights now a role of a specific subset of CD4T cells, the IL-17expressing T-helper (Th) 17 cells, in metabolic inflammation, insulin resistance, and HCC development. Gomes et al. show that excessive nutrients induce expression of unconventional prefoldin RPB5 interactor (URI) in the liver of dietary steatohepatitis models in mice (Fig. 1). URI has been previously described by the same group to promote HCC development through inhibition of nicotinamide adenine dinucleotide synthesis and triggering DNA damage. In the current study, the same group investigated the impact of URI on HCC in the presence of NAFLD. Mice expressing the human URI in hepatocytes (referred to as “mutants”) spontaneously developed steatohepatitis on chow diet, CD-HFD, or MCD diet. This was accompanied by an increase in Kupffer cell (KC) infiltration in these mutant mice. Mutants also showed a decrease in total body fat with an increase in neutrophil infiltration in white adipose tissue (WAT) and impaired glucose tolerance. DNA damage was increased in mutants, and IL-17 blood levels were elevated. Accordingly, Th17 cells were up-regulated in blood and liver. Mice that were hemizygous for hepatic URI showed a partial protection from steatohepatitis and no difference in total T-cell count, but significantly reduced Th17 cells. Next, the group inhibited Th17 differentiation with digoxin by blocking the transcriptional activity of retinoic acid receptor-related orphan receptor gamma t (RORct), a regulator for Th17 differentiation. The treatment reduced Th17 cells as well as IL-17 and white blood cell counts in mutant mice. Digoxin also rescued insulin sensitivity, lipid dysregulation, and tumor development. Conversely, administration of recombinant IL17 (rIL-17) into wild-type (WT) mice led to hepatic DNA damage, hepatic steatosis, and increased circulating Th17 cells. In WAT, rIL-17 increased neutrophil infiltration as well as signs of WAT insulin resistance. Blocking IL-17 with a neutralizing antibody had opposite effects. Genetic ablation of the IL-17 receptor subunit, IL-17RA, in myeloid cells improved steatohepatitis, insulin sensitivity, and reduced the number of infiltrating T cells in liver in WT and mutant mice, suggesting that myeloid cells were the prime responders to IL-17 (Fig. 1). Finally, the investigators correlated their findings using patient material. URI expression was associated with hepatic steatosis and high IL-17 levels in patients with NASH. Thus, this article provides experimental evidence that nutrient surplus triggers hepatic expression of oncogenic URI, driving genotoxic stress and IL-17

135 Jnk1 IN HEPATIC STELLATE CELLS MODULATES LIVER FIBROGENESIS IN VIVO AND IN VITRO

Chronic liver damage may eventually progress to end-stage liver cirrhosis and hepatocellular carcinoma. Cytoglobin (Cygb) is a 21 kDa globin expressed in hepatic stellate cells and functions as a hypoxia sensor and a gas carrier. It serves as local peroxidase by degrading H2O2. However, its pathophysiological role in vivo remains undetermined. Here, we report the promotion of liver cancer development in Cygb-deficient (KO) mice administrated with either diethylnitrosamine (DEN) or choline-deficient amino acid-defined (CDAA) diet that induces hepatosteatosis. Methods: Cygb KO mice and corresponding wild-type (WT) mice at 8 week-old were treated with either DEN, CDAA, or CSAA diet for 8–32 weeks. Macroscopic and microscopic observations were performed. Gene expressions and intracellular signaling pathways were analyzed. Oxidative stress was determined by the formation of 8-OHdG, DHE, and nitrotyrosine. Results: Model 1; 25 or 0.05 ppm DEN treatment for 25 or 36 weeks induced liver tumor formation in 100% or 44%, respectively, in Cygb KO mice compared to 44% or 0%, respectively, in WT mice. Background liver developed fibrosis together with the augmented expression of mRNA of TGF-beta 3, collagen 1a1, and TIMP-1. Inflammatory gene expressions and augmented oxidative stress formation were evident in KO mice. Model 2; as early as 8 weeks of CDAA treatment, Cygb KO mice exhibited dominant steatohepatitis, which resulted in advanced fibrosis at 16 week-point, compared with WT as assessed by pathological NASH scores, collagen deposition, alpha-smooth muscle actin expression, and hepatic hydroxyproline content. Surprisingly, after 32 weeks under CDAA administration, 100% of both male and female Cygb KO mice developed liver cancers, compared to 0% in corresponding WT mice. Analyses at 32 week-point showed histologically severe inflammatory reactions concomitant with increased mRNA expression of Tnfa, Tgfb1, Il-1b and Il-6 in Cygb KO mice compared with WT mice. Cygb KO mice showed increased hepatocyte proliferation (Ki67 staining) and expression of AFP. Oxidative stress and antioxidant defense PCR array identified altered expression of 31 genes involved in the metabolism of reactive oxygen species in Cygb KO mice. Conclusion: Deficiency of Cygb promotes liver cancer development through activating inflammatory reaction and oxidative stress pathway.