Martina Buck

Amplification of liver injury is mediated by macrophages but the signaling by which the macrophage inflammasome enhances liver injury is not completely understood. The CCAAT/Enhancer Binding Protein-β (C/EBPβ) is a critical signaling molecule for macrophages because expression of a dominant inhibitor of C/EBPβ DNA-binding sites or a targeted deletion of C/EBPβ results in impaired macrophage differentiation. We reported that expression of the phosphorylation-mutant C/EBPβ-Glu217, which mimics phosphorylated C/EBPβ-Thr217, was sufficient to confer macrophage survival to Anthrax lethal toxin. Here, using primary hepatocytes, primary liver macrophages, dominant positive and negative transgenic mice of the C/EBPβ-Thr217 phosphoacceptor, macrophage ablation and an inhibitory peptide of C/EBPβ-Thr217 phosphorylation, we determined that this phosphorylation is essential for the activation of the inflammasome in liver macrophages and for the hepatocyte apoptosis induced by hepatotoxins that results in liver injury. Similar findings were observed in the livers of patients with acute injury induced by Toxic Oil Syndrome.

Although C/EBPβ^ko mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-β on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice. Mice expressing the non-phosphorylatable C/EBPβ-Ala217 transgene had a marked reduction in lung injury on day-13 after Bleomycin exposure, compared to C/EBPβ^wt mice, judging by the decrease of CD68⁺ activated monocytes/macrophages, bone marrow-derived CD45⁺ cells and lung cytokines as well as by the normal surfactant protein-C expression by lung pneumocytes. On day-21 after Bleomycin treatment, C/EBPβ^wt mice but not mice expressing the dominant negative C/EBPβ-Ala217 transgene developed severe lung fibrosis as determined by quantitative collagen assays. All mice were of identical genetic background and back-crossed to the parental wild-type inbreed FVB mice for at least ten generations. Treatment of C/EBPβ^wt mice with a cell permeant, C/EBPβ peptide that inhibits phosphorylation of C/EBPβ on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPβ on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis. These data suggest that the RSK-C/EBPβ phosphorylation pathway may contribute to the development of lung injury and fibrosis.

Muscle wasting is a critical feature of patients afflicted by acquired immune deficiency syndrome (AIDS), cancer, or chronic inflammatory diseases. In a mouse model of muscle wasting, TNF-α induces oxidative stress and nitric oxide synthase-2 (NOS2) and decreases myogenin, Jun-D, and creatinine kinase muscle isoform (CKM) expression. Here, we studied 12 patients with muscle wasting due to cancer (N = 10) or AIDS (N = 2) and 4 control subjects. We show that in skeletal muscle of cachectic patients there is 1) increased expression and activity of the TNF-α signaling, including TNF-α mRNA, activation of TNFR1, and TNF-α-associated to TNFR1; 2) increased oxidative stress, as determined by the presence of malondialdehyde-lysine adducts; 3) increased NOS2 mRNA and protein; 4) decreased expression of Jun-D, myogenin, myosin, and CKM mRNA and protein; 5) impaired CKM-E box binding activities, associated with decreased Jun-D/myogenin activities; and 6) oxidative modification and ubiquitination of Jun-D. These studies show that these molecular pathways are modulated in association with muscle wasting in patients with cancer or AIDS, and whether or not they cause muscle wasting remains to be determined.

Goals To describe the mechanisms of severe hepatocellular injury with apoptosis in 2 patients receiving hepatitis C virus (HCV)-796. Background HCV-796 is a hepatitis C polymerase inhibitor approved by the US Food and Drug Administration for a phase 2 study of the treatment of hepatitis C in combination with PEG-Interferon and ribavirin. Results The injury occurred after more than 12 weeks of treatment, with a >20-fold increase in serum alanine aminotransferase and aspartate aminotransferase, and a marked increase in total (and direct) bilirubin in the absence of cholestasis. There was no evidence of autoimmune or viral hepatitis. Involvement of the mitochondrial apoptotic pathway was demonstrated by (1) release of cytochrome C into the cytosol; (2) association of cytochrome C with apoptotic protease activating factor-1 in the cytosol; (3) activation of initiator caspase 9; (4) activation of effector caspase 3; (5) increased serum caspase-3 cleaved cytokeratin-18 peptide; (6) nuclear fragmentation; (7) mitochondrial structural abnormalities; (8) expression of light chain 3 B, an indicator of autophagy; (9) probable autophagy of mitochondria by autophagosomes; and (10) probable phagocytosis of apoptotic hepatocytes by activated macrophages. Immunoglobulin G immune complexes were identified in the hepatocytes and localized to the endoplasmic reticulum and Golgi of these patients after the drug-induced liver disease, reflecting a primary or secondary target. Hepatitis C treatment was discontinued at weeks 15 and 19 in patients 1 and 2, respectively. After more than 6 months off the medication, both patients normalized the serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin with undetectable HCV RNA. Conclusions HCV-796 may cause severe hepatocellular injury and apoptosis, with a marked immune reaction in susceptible patients.

Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes. This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.

Activation of hepatic stellate cells (HSC) is responsible for the development of liver fibrosis in chronic liver diseases of all causes, and remarkably, HSC clearance by apoptosis may allow recovery from liver injury and reversal of liver fibrosis. Because in preclinical studies it has been shown that activation of ribosomal S-6 kinase (RSK) and phosphorylation of the CCAAT/enhancer-binding protein (C/EBP) beta in activated HSC is critical for the progression of liver fibrosis, RSK has been considered as a therapeutic target for the liver fibrosis. Unexpectedly, preclinical studies documented a strong anti inflammatory effect of RSK inhibitors, decreasing liver injury induced by hepatotoxins. Therefore, RSK inhibitors reduce liver fibrosis directly by inducing apoptosis of activated HSC, and indirectly by preventing liver injury and inflammation. The activation of RSK and C/EBP beta phosphorylation also occurs in human liver fibrosis. Thus, appropriate RSK inhibitors may be beneficial in the prevention and treatment of liver injury and liver fibrosis. These issues will be discussed in this review. (C) 2008 Prous Science, S.A.U. or its licensors. All rights reserved.

In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis. Chronic treatment with the hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ^+/+ mice with CCl₄-induced severe liver fibrosis, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis. These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.

Bacillus anthracis lethal toxin (LT) impairs innate and adaptive immunity. Anthrax lethal factor stimulates cleavage of MAPK kinases, which prevents the activation of antiapoptotic MAPK targets. However, these MAPK targets have not been yet identified. Here, we found that LT induces macrophage apoptosis by enhancing caspase 8 activation and by preventing the activation of ribosomal S6 kinase-2 (RSK), a MAPK target, and the phosphorylation of CCAAT/enhancer binding protein-β (C/EBPβ) on T²¹⁷, a RSK target. Expression of the dominant positive, phosphorylation mimic C/EBPβ-E²¹⁷ rescued macrophages from LT-induced apoptosis by blocking the activation of procaspase 8. LT inhibited macrophage phagocytosis and oxidative burst and induced apoptosis in normal mice but not in C/EBPβ-E²¹⁷ transgenic mice. These findings suggest that C/EBPβ may play a critical role in anthrax pathogenesis, at least in macrophages.