Gabriele Sass

Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice

Concanavalin A (Con A) causes severe TNF-alpha-mediated and IFN-gamma-mediated liver injury in mice. In addition to their other functions, TNF-alpha and IFN-gamma both induce the inducible nitric oxide (NO) synthase (iNOS). Using different models of liver injury, NO was found to either mediate or prevent liver damage. To further elucidate the relevance of NO for liver damage we investigated the role of iNOS-derived NO in the Con A model. We report that iNOS mRNA was induced in livers of Con A-treated mice within 2 hours, with iNOS protein becoming detectable in hepatocytes as well as in Kupffer cells within 4 hours. iNOS-/- mice were protected from liver damage after Con A treatment, as well as in another TNF-alpha-mediated model that is inducible by LPS in D-galactosamine-sensitized (GalN-sensitized) mice. iNOS-deficient mice were not protected after direct administration of recombinant TNF-alpha to GalN-treated mice. Accordingly, pretreatment of wild-type mice with a potent and specific inhibitor of iNOS significantly reduced transaminase release after Con A or GalN/LPS, but not after GalN/TNF-alpha treatment. Furthermore, the amount of plasma TNF-alpha and of intrahepatic TNF-alpha mRNA and protein was significantly reduced in iNOS-/- mice. Our results demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of TNF-alpha production.

The N-Terminus of Nef from HIV-1/SIV Associates with a Protein Complex Containing Lck and a Serine Kinase

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.

TNF-α-Induced Expression of Adhesion Molecules in the Liver Is Under the Control of TNFR1—Relevance for Concanavalin A-Induced Hepatitis

TNF-α has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1−/− and TNFR2−/− mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-γ. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1−/− and TNFR2−/− mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM−/− mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4+ T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.

The heme oxygenase 1 product biliverdin interferes with hepatitis C virus replication by increasing antiviral interferon response.

The anti‐inflammatory and antiapoptotic heme degrading enzyme heme oxygenase‐1 (HO‐1) has been shown recently to interfere with replication of hepatitis C virus (HCV). We investigated the effect of HO‐1 products carbon monoxide (CO), iron and biliverdin on HCV replication using the replicon cell lines Huh‐5‐15 and LucUbiNeo‐ET, stably expressing HCV proteins NS3 through NS5B. Incubation of these cell lines in the presence of the CO donor methylene chloride transiently reduced HCV replication, whereas an increase of iron in cell culture by administration of FeCl3 or iron‐saturated lactoferrin did not interfere with HCV replication. Likewise, depletion of iron by deferoxamine during induction of HO‐1 by cobalt‐protoporphyrin IX did not restore HCV replication. The most prominent effect was observed after incubation of replicon cell lines in the presence of biliverdin. Biliverdin seems to interfere with HCV replication–mediated oxidative stress by inducing expression of antiviral interferons, such as interferon alpha2 and alpha17. Conclusion: The antioxidant biliverdin reduces HCV replication in vitro by triggering the antiviral interferon response and might improve HCV therapy in the future. (HEPATOLOGY 2009.)

Autonomic Renal Denervation Ameliorates Experimental Glomerulonephritis

Increasing evidence indicates that inflammation of visceral organs is significantly affected by the autonomic nervous system. Such neuroimmune interactions have not been studied in the kidney. Here, we show that the rat kidney is innervated by both tyrosine hydroxylase-positive sympathetic efferent nerve fibers and calcitonin gene-related peptide-positive primary afferent nerve fibers, both of which are found in proximity to macrophages and dendritic cells. Complete surgical bilateral renal denervation was performed 2 d before glomerulonephritis was induced by injecting the monoclonal anti-Thy-1.1 antibody OX-7. Denervation significantly reduced albuminuria, mesangiolysis, formation of microaneurysms, deposition of glomerular collagen IV, and expression of TGF-beta compared with sham-operated controls. Accordingly, inflammation, identified by accumulation of interstitial macrophages and renal expression of TNF-alpha, and mesangial cell proliferation were significantly reduced. These findings indicate that autonomic renal denervation ameliorates and, by inference, innervation exacerbates acute inflammation in the kidney; therefore, neurotransmitters or neuropeptides and their receptors might represent novel targets for the treatment of acute glomerulonephritis.

Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice

Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO‐1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO‐1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO‐1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1‐6 and on orthotopic tumor growth in immune‐competent C3H/HeN mice. Our results show that HO‐1 is frequently overexpressed in human HCC. Downmodulation of HO‐1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO‐1 might represent a novel therapeutic approach in HCC therapy.

Cooperative effect of biliverdin and carbon monoxide on survival of mice in immune-mediated liver injury

Induction of the heme‐degrading enzyme heme oxygenase‐1 (HO‐1) has been shown to be beneficial in terms of improvement of liver allograft survival and prevention of CD95‐mediated apoptosis in the liver. In the present study, we investigated the effects of HO‐1, and its products carbon monoxide (CO), biliverdin (BV), and iron/ferritin, in a mouse model of inflammatory liver damage inducible by lipopolysaccharide (LPS) in mice sensitized with the hepatocyte‐specific transcription inhibitor D‐galactosamine (GalN). Our results show that HO‐1 induction by cobalt‐protoporphyrin‐IX (CoPP) reduced cytokine expression, protected mice from liver injury, and prolonged survival. While in contrast to ferritin overexpression, single administration of the CO donor methylene chloride (MC) or of BV also protected mice from liver damage, only coadministration of both HO products prolonged survival and reduced the expression of cytokines, e.g., tumor necrosis factor (TNF) and interferon γ (IFN‐γ). In conclusion, HO‐1–induced prolongation of survival, but not the protection from liver damage, seems to be dependent on down‐regulation of cytokine synthesis. (HEPATOLOGY 2004;40:1128–1135.)

Dissection of the Intracellular Pathways in Hepatocytes Suggests a Role for Jun Kinase and IFN Regulatory Factor-1 in Con A-Induced Liver Failure

Con A administration results in dose-dependent immune-mediated liver injury. Cytokines are important to determine the outcome of liver failure in this model, and especially TNF-α and IFN-γ directly contribute to hepatocyte damage. The intracellular pathways of these two cytokines, which eventually result in tissue destruction, are not well defined. Here we used anti-IFN-γ Abs and adenoviral vectors that express molecules inhibiting distinct TNF-α-dependent pathways in hepatocytes to better understand the relevance of specific intracellular signaling cascades for Con A-induced liver failure. We show that activation of TNF-α- and IFN-γ-dependent intracellular pathways occurs prior to the influx of immune-activated cells into the liver and that anti-TNF-α and anti-IFN-γ neutralizing Abs cannot block infiltration of these cells. Blocking experiments with Abs and adenoviral vectors showed that NF-κB activation and the Fas-associated death domain protein/caspase 8 cascade in hepatocytes during Con A-induced liver failure have no impact on tissue injury. Additionally, STAT1 activation alone after Con A injection in liver cells does not result in liver damage. In contrast, IFN-γ-dependent expression of IFN regulatory factor-1 and TNF-α-dependent activation of c-Jun N-terminal kinase in liver cells correlates with liver cell damage after Con A injection. Therefore, our experiments indicate that IFN regulatory factor-1 and the c-Jun N-terminal kinase pathway are involved in determining hepatocyte damage during Con A-induced liver failure and thus may provide new targets for therapeutic intervention.

Down-regulation of the De-ubiquitinating Enzyme Ubiquitin-specific Protease 2 Contributes to Tumor Necrosis Factor-α-induced Hepatocyte Survival

Tumor necrosis factor-α (TNFα) stimulation of hepatocytes induces either cell survival or apoptosis, which seems to be regulated by the ubiquitin-proteasome system. Here we investigated the role of TNFα-induced down-modulation of the de-ubiquitinating enzyme USP2 for hepatocyte survival. Inhibition of hepatocyte apoptosis by pre-treatment with TNFα (TNFα tolerance) was analyzed in the mouse model of galactosamine/TNFα-induced liver injury and in actinomycin D/TNFα-treated primary mouse hepatocytes. The role of USP2 for TNFα-induced hepatocyte survival was studied using small interference RNA or an expression clone. Injection of mice or preincubation of hepatocytes with TNFα caused a rapid down-regulation of hepatic USP2–41kD, the predominant USP2 isoform in the liver. In vitro an artificial knockdown of USP2 inhibited actinomycin D/TNFα-induced hepatocyte apoptosis, which was associated with elevated levels of the anti-apoptotic protein c-FlipL/S and a concomitant decrease of cellular levels of the ubiquitinligase Itch, a negative regulator of c-Flip. USP2–41kD overexpression abrogated TNFα tolerance in vitro, prevented accumulation of c-FlipL/S and resulted in elevated levels of Itch. Accordingly, c-FlipL/S protein levels were elevated in livers of TNFα-tolerant mice, which correlated to a switch from JNK and ERK to p38 signaling after galactosamine/TNF re-challenge. Our results indicate that TNFα-induced USP2 down-regulation is an effective cytoprotective mechanism in hepatocytes. Hence, USP2 could be a novel pharmacological target, and specific USP2 inhibitors might be potential candidates for the treatment of inflammation-related apoptotic liver damage.

The pan-deacetylase inhibitor panobinostat inhibits growth of hepatocellular carcinoma models by alternative pathways of apoptosis.

Inhibition of deacetylases represents a new treatment option for human cancer diseases. We applied the novel and potent pan-deacetylase inhibitor panobinostat (LBH589) to human hepatocellular carcinoma models and investigated by which pathways tumor cell survival is influenced. HepG2 (p53wt) and Hep3B (p53null) responded to panobinostat treatment with a reduction of cell proliferation and a significant increase in apoptotic cell death at low micromolar concentrations. Apoptosis was neither mediated by the extrinsic nor the intrinsic pathway but quantitative RT-PCR showed an upregulation of CHOP, a marker of the unfolded protein response and endoplasmic reticulum stress with subsequent activation of caspase 12. Dependent on the p53 status, a transcriptional upregulation of p21cip1/waf1, an increased phosphorylation of H2AX, and an activation of the MAPK pathway were observed. In a subcutaneous xenograft model, daily i.p. injections of 10 mg/kg panobinostat lead to a significant growth delay with prolonged overall survival, mediated by reduced tumor cell proliferation, increased apoptosis and reduced angiogenesis in tumor xenografts. Panobinostat increased the acetylation of histones H3 and H4. Panobinostat is a well tolerated new treatment option for HCC that activates alternative pathways of apoptosis, also in p53-deficient tumors.