Julia Freimuth

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-specific NEMO deletion promotes NK/NKT cell- and TRAIL-dependent liver damage.

Nuclear factor κB (NF-κB) is one of the main transcription factors involved in regulating apoptosis, inflammation, chronic liver disease, and cancer progression. The IKK complex mediates NF-κB activation and deletion of its regulatory subunit NEMO in hepatocytes (NEMOΔhepa) triggers chronic inflammation and spontaneous hepatocellular carcinoma development. We show that NEMOΔhepa mice were resistant to Fas-mediated apoptosis but hypersensitive to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) as the result of a strong up-regulation of its receptor DR5 on hepatocytes. Additionally, natural killer (NK) cells, the main source of TRAIL, were activated in NEMOΔhepa livers. Interestingly, depletion of the NK1.1+ cells promoted a significant reduction of liver inflammation and an improvement of liver histology in NEMOΔhepa mice. Furthermore, hepatocyte-specific NEMO deletion strongly sensitized the liver to concanavalin A (ConA)–mediated injury. The critical role of the NK cell/TRAIL axis in NEMOΔhepa livers during ConA hepatitis was further confirmed by selective NK cell depletion and adoptive transfer of TRAIL-deficient−/− mononuclear cells. Our results uncover an essential mechanism of NEMO-mediated protection of the liver by preventing NK cell tissue damage via TRAIL/DR5 signaling. As this mechanism is important in human liver diseases, NEMOΔhepa mice are an interesting tool to give insight into liver pathophysiology and to develop future therapeutic strategies.

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature NF-?B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Loss of caspase‐8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

109 DEPLETION OF CASPASE-8 IN MICE MODULATES TNF-INDUCED COMPLEX FORMATION AND CELL CYCLE SIGNALING IN HEPATOCYTES FOLLOWING PARTIAL HEPATECTOMY

an inflammatory process, initiated by the production of TNF alpha and interleukin-6 (IL-6). Since cannabinoid receptors 2 (CB2) play a major role in the regulation of inflammation, we investigated herein whether they regulate liver regeneration. Liver regeneration was studied in a model of acute hepatitis induced by a single injection of carbon tetrachloride (CCl4) or following two-third hepatectomy. Experiments were performed in CB2 knockout (CB2−/−) and wild type (WT) mice, or in WT mice pretreated with the CB2 agonist JWH-133. Hepatic CB2 receptor expression was markedly induced following partial hepatectomy and CCl4-induced liver injury. Liver injury was increased in CB2−/− mice compared to WT mice following acute CCl4 administration, as assessed by serum ALT and TUNEL labelling. In addition, PCNA expression was significantly delayed in CB2−/− animals as compared to WT mice (72 vs 48 hrs). Conversely, treatment of WT mice with a single dose of CB2 specific agonist, JWH-133, reduced liver injury and accelerated liver regeneration in response to CCl4 exposure. Similar delay in PCNA expression was observed in CB2−/− animals submitted to partial hepatectomy. Impaired regeneration in CB2−/− mice was associated with a reduction in the expression of IL-6; consistent with these data, IL-6 administration to CB2−/− mice partially restored PCNA expression. Finally, MMP-2 activity was enhanced in CB2−/− mice injected with CCl4, in keeping with the reported effects of MMP-2 in liver injury. These data demonstrate that the CB2 receptors reduce liver injury and promote liver regeneration following acute insult, by a mechanism involving IL-6 production and inhibition of MMP-2 activity. These results suggest that CB2 agonists elicit useful hepatoprotective properties, in addition to their antifibrogenic effects.