N. Beraza

Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function

Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined. Using a mouse model of polymicrobial sepsis, we show that abrogation of APP production by hepatocyte-specific gp130 deletion, the signaling receptor shared by IL-6 family cytokines, strongly increased mortality despite normal bacterial clearance. Hepatic gp130 signaling through STAT3 was required to control systemic inflammation. Notably, hepatic gp130–STAT3 activation was also essential for mobilization and tissue accumulation of myeloid-derived suppressor cells (MDSCs), a cell population mainly known for antiinflammatory properties in cancer. MDSCs were critical to regulate innate inflammation, and their adoptive transfer efficiently protected gp130-deficient mice from sepsis-associated mortality. The hepatic APPs serum amyloid A and Cxcl1/KC cooperatively promoted MDSC mobilization, accumulation, and survival, and reversed dysregulated inflammation and restored survival of gp130-deficient mice. Thus, gp130-dependent communication between the liver and MDSCs through APPs controls inflammatory responses during infection.

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

BACKGROUND & AIMSnDisruption 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.nnnMETHODSnWe 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.nnnRESULTSnHepatocyte-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.nnnCONCLUSIONSnInhibition 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.

Pharmacological IKK2 inhibition blocks liver steatosis and initiation of non-alcoholic steatohepatitis

Background: Non-alcoholic-steatohepatitis (NASH) leading to fibrosis, end-stage cirrhosis and hepatocellular carcinoma is an increasing health problem in the Western world. Thus, the need for new therapeutic approaches is increasing. IKK2 plays a key role in the development of NASH by mediating inflammation and insulin resistance. Aim: Here the beneficial effects of a pharmacological IKK2 inhibitor (AS602868) on initial stages of NASH progression were tested. Methods: Mice were fed with a high sucrose diet (HSD) and daily-administered AS602868 and vehicle. The impact of AS602868 on NASH progression was studied using biochemical, histological and molecular markers. Results: AS602868 treatment prevented HSD-induced weight gain and visceral fat accumulation. In adipose tissue, AS602868-treated mice exhibited a lower degree of infiltrated macrophages along with reduced proinflammatory cytokine production. Further analysis demonstrated that AS602868 treatment efficiently inhibited nuclear factor (NF)-κB activation in liver non-parenchymal cells and as a consequence attenuated the inflammatory response in the liver. Accordingly, in HSD/AS602868 mice, liver and adipose tissue adiponectin levels remained at levels comparable with those of control chow-fed mice, while they were decreased in HSD/vehicle animals. Additionally, AS602868 improved lipid β-oxidation mediated by peroxisome proliferator-activated receptor (PPAR) α and PPARγ. Systemic pharmacological IKK2 inhibition by AS602868 treatment efficiently prevented liver steatosis and inflammation, and improved antioxidant response. All this contributed to attenuation of NASH progression as evidenced by lower hepatocyte apoptosis and early stages of liver fibrosis. Conclusion: The data demonstrate that AS602868-mediated IKK2 inhibition represents a new therapeutic approach to prevent dietary-induced NASH progression.

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.

Hepatocyte‐specific inhibitor‐of‐kappaB‐kinase deletion triggers the innate immune response and promotes earlier cell proliferation during liver regeneration

Nuclear factor κB (NF‐κB) is one of the main transcription factors involved in liver regeneration after partial hepatectomy (PH). It is activated upon IκB phosphorylation by the IκB kinase (IKK) complex comprising inhibitor of kappaB kinase 1 (IKK1), inhibitor of kappaB kinase 2 (IKK2), and nuclear factor‐B essential modifier (NEMO). We studied the impact of hepatocyte‐specific IKK2 deletion during liver regeneration. A 70% PH was performed on IKK2f/f (wild‐type) and IKK2ΔLPCmice (hepatocyte‐specific IKK2 knockout mice). PH in IKK2ΔLPC compared with IKK2f/f mice resulted in weaker and delayed NF‐κB activation in hepatocytes, while nonparenchymal liver cells showed earlier NF‐κB activation and higher tumor necrosis factor expression. Additionally, these animals showed increased and earlier serum amyloid A and chemotactic cytokine L‐1 levels followed by enhanced polymorphonuclear cell recruitment to the liver. These results correlated with earlier Jun kinase activity, c‐myc expression, and matrix metalloproteinase‐9 activity, suggesting earlier priming in IKK2ΔLPC mice after PH. These data preceded a more rapid cell cycle progression and earlier hepatocyte proliferation as evidenced through cyclin and 5‐bromo‐2‐deoxyuridine analysis. Interestingly, despite faster G1/S progression, IKK2ΔLPC mice exhibited an enduring mitosis phase, because mitotic bodies were still observed at later stages after PH. Conclusion: We demonstrate that PH in IKK2ΔLPC mice triggers a more rapid and pronounced inflammatory response in nonparenchymal liver cells, which triggers earlier hepatocyte proliferation. (HEPATOLOGY 2008.)

Lack of gp130 expression results in more bacterial infection and higher mortality during chronic cholestasis in mice.

Chronic cholestasis is associated with increased bacterial infections and sepsis resulting in higher mortality in humans. In the current study, we investigated the relevance of gp130‐dependent pathways after bile duct ligation (BDL). BDL was performed in conditional gp130 knockout (loxP/Cre system) mice and respective controls. Liver injury, regulation of the acute phase response, and the impact on survival and bacterial infections were determined. Acute BDL resulted in increased IL‐6 levels, Stat3 activation, and an increase in acute‐phase proteins (serum‐amyloid‐A [SAA]), which was blocked in gp130‐deleted animals. In addition, the antimicrobial gene hepcidin was regulated in a gp130‐dependent manner after BDL. During chronic cholestasis Stat3 activation was dramatically reduced, while high SAA levels were maintained via gp130‐dependent signaling. Inhibition of gp130‐dependent pathways resulted in higher mortality and liver damage, which was associated with higher infiltration of immune‐activated cells and increased germ number in the liver. In conclusion, during acute and chronic cholestasis, the gp130 system is essential for controlling the acute‐phase response. Lack of gp130 expression results in pronounced bacterial growth in bile and liver after BDL, which is associated with higher mortality. Activation of gp130‐dependent pathways after BDL is essential and appears to be a therapeutic target during cholestasis. (HEPATOLOGY 2005;42:1082–1090.)

Nor-ursodeoxycholic acid reverses hepatocyte-specific nemo-dependent steatohepatitis

Background Hepatocyte-specific NEMO/NF-κB deleted mice (NEMOΔhepa) develop spontaneous non-alcoholic steatohepatitis (NASH). Free fatty acids and bile acids promote DR5 expression. TRAIL/NK cell-mediated activation of TRAIL-R2/DR5 plays an important role during acute injury in NEMOΔhepa mice. Aim To inhibit the progression of NASH in the absence of hepatocyte-NEMO/NF-kB signaling. Methods NEMOf/f and NEMOΔhepa mice were fed with a low-fat diet, and with two anticholestatic diets; UDCA and NorUDCA. The impact of these treatments on the progression of NASH was evaluated. Results We show that high expression of DR5 in livers from NEMOΔhepa mice is accompanied by an abundant presence of bile acids (BAs), misregulation of BA transporters and significant alteration of lipid metabolism-related genes. Additionally, mice lacking NEMO in hepatocytes spontaneously showed ductular response at young age. Unexpectedly, feeding of NEMOΔhepa mice with low-fat diet failed to improve chronic liver injury. Conversely, anti-cholestatic treatment with nor-ursodeoxycholic acid (NorUDCA), but not with ursodeoxycholic acid (UDCA), led to a significant attenuation of liver damage in NEMOΔhepa mice. The strong therapeutic effect of NorUDCA relied on a significant downregulation of LXR-dependent lipogenesis and the normalisation of BA metabolism through mechanisms involving cross-talk between Cyp7a1 and SHP. This was associated with the significant improvement of liver histology, NEMOΔhepa/NorUDCA-treated mice showed lower apoptosis and reduced CyclinD1 expression, indicating attenuation of the compensatory proliferative response to hepatocellular damage. Finally, fibrosis and ductular reaction markers were significantly reduced in NorUDCA-treated NEMOΔhepa mice. Conclusions Overall, our work demonstrates the contribution of bile acids metabolism to the progression of NASH in the absence of hepatocyte-NF-kB through mechanisms involving DR5-apoptosis, inflammation and fibrosis. Our work suggests a potential therapeutic effect of NorUDCA in attenuating the progression of NASH.

NF-κB Essential Modifier Is Required for Hepatocyte Proliferation and the Oval Cell Reaction After Partial Hepatectomy in Mice

BACKGROUND & AIMSnThe transcription factor nuclear factor κB (NF-κB) is activated by the IκB kinase complex. The regulatory subunit of this complex, NF-κB essential modifier (NEMO or IKBKG), is a tumor suppressor. Hepatocyte-specific deletion of NEMO induces chronic liver inflammation that leads to apoptosis, oxidative stress, development of nonalcoholic steatohepatitis, and hepatocarcinogenesis.nnnMETHODSnWe performed partial hepatectomies in mice with hepatocyte-specific disruption of NEMO (Nemo(Δhepa)). Some mice were fed a diet that contained the antioxidant butylated hydroxyanisole (BHA), and others were given daily intraperitoneal injections of the oxidant phenetyl isothiocyanate (PEITC).nnnRESULTSnNemo(Δhepa) mice had impaired liver regeneration after partial hepatectomy and 50% mortality, indicating that NEMO is required for the regenerative response. Liver cells of the mice had a strong oxidative stress response; these cells down-regulated the NF-κB-dependent antioxidant response and reduced levels of proteins that repair DNA double-strand breaks. However, the impairments to hepatocyte proliferation were compensated by a response of oval cells in Nemo(Δhepa) mice. Oval cells expressed low levels of albumin and thereby expressed normal levels of NEMO. Repopulation of the liver with oval cells that expressed NEMO reversed liver damage in Nemo(Δhepa) mice. Interestingly, these mice still developed hepatocellular carcinomas 6 months after partial hepatectomy, whereas Nemo(Δhepa) mice fed the BHA diet were protected from carcinogenesis.nnnCONCLUSIONSnIn livers of mice, expression of NEMO and activation of NF-κB are required for hepatocyte proliferation and liver regeneration. These mechanisms require control of oxidative stress and DNA integrity.

Restoration of p53 function: A new therapeutic strategy to induce tumor regression?

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

The gut‐brain‐liver axis: A new option to treat obesity and diabetes?

Energy and glucose homeostasis are regulated by food intake and liver glucose production, respectively. The upper intestine has a critical role in nutrient digestion and absorption. However, studies indicate that upper intestinal lipids inhibit food intake as well in rodents and humans by the activation of an intestine–brain axis. In parallel, a brain–liver axis has recently been proposed to detect blood lipids to inhibit glucose production in rodents. Thus, we tested the hypothesis that upper intestinal lipids activate an intestine–brain–liver neural axis to regulate glucose homeostasis. Here we demonstrate that direct administration of lipids into the upper intestine increased upper intestinal long-chain fatty acylcoenzyme A (LCFA-CoA) levels and suppressed glucose production. Co-infusion of the acyl-CoA synthase inhibitor triacsin C or the anaesthetic tetracaine with duodenal lipids abolished the inhibition of glucose production, indicating that upper intestinal LCFA-CoAs regulate glucose production in the preabsorptive state. Subdiaphragmatic vagotomy or gut vagal deafferentation interrupts the neural connection between the gut and the brain, and blocks the ability of upper intestinal lipids to inhibit glucose production. Direct administration of the N-methyl-D-aspartate ion channel blocker MK-801 into the fourth ventricle or the nucleus of the solitary tract where gut sensory fibres terminate abolished the upper-intestinal-lipid-induced inhibition of glucose production. Finally, hepatic vagotomy negated the inhibitory effects of upper intestinal lipids on glucose production. These findings indicate that upper intestinal lipids activate an intestine– brain–liver neural axis to inhibit glucose production, and thereby reveal a previously unappreciated pathway that regulates glucose