Erwin Daniel Brenndörfer

Regulation of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA Stability by TNF-α Involves Activation of the MKK6/p38MAPK/MK2 Cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-α. In contrast to IL-6, TNF-α increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38MAPK-cascade is required for TNF-α-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38MAPK cascade, basal SOCS3-expression is strongly reduced and TNF-α-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38MAPK-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3′ untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-α. In summary, these data indicate that TNF-α regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38MAPK cascade and that the activation of MK2, a downstream target of p38MAPK, is important for the regulation of SOCS3 expression.

The hepatitis C virus and immune evasion: non-structural 3/4A transgenic mice are resistant to lethal tumour necrosis factor α mediated liver disease

Background: The hepatitis C virus (HCV) establishes chronic infection by incompletely understood mechanisms. The non-structural (NS) 3/4A protease/helicase has been proposed as a key complex in modulating the infected hepatocyte, although nothing is known about the effects this complex exerts in vivo. Aim: To generate mice with stable and transient hepatocyte expression of the HCV NS3/4A proteins to study its effects in vivo. Methods: NS3/4A expression was determined by western blot and immunohistochemistry. Two independent pathologists determined the liver histology. Hepatic immunity was characterised by quantifying intrahepatic immune cell subsets. Liver damage was induced using carbon tetrachloride (CCl4), lipopolysaccaride (LPS), tumour necrosis factor α (TNFα), and anti-Fas antibody. Results: Expression of NS3/4A was restricted to the cytoplasm of hepatocytes, and did not cause liver cancer or any spontaneous liver pathology. However, the presence of NS3/4A modulated the intrahepatic immunity, as follows: first, the CD4+ T cell and type I/II dendritic cell subsets were reduced in transgenic livers; second, NS3/4A protected hepatocytes from liver damage mediated in vivo by CCl4, LPS, TNFα, but not FAS; and third, both stable and transiently NS3/4A transgenic mice were resistant to lethal doses of liver targeted TNFα, and the resistance could be reverted by treatment with a p38 mitogen activated protein kinase inhibitor (MAPK). Conclusions: Hepatic expression of NS3/4A does not induce spontaneous liver disease. NS3/4A does, however, alter the intrahepatic immune cell subsets and protects hepatocytes against TNFα induced liver damage in vivo. The TNFα resistance can be reverted by treatment with a p38 MAPK inhibitor. This represents a new immune evasion strategy conferred by NS3/4A.

Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor–induced signal transduction by cleavage of the T‐cell protein tyrosine phosphatase

The hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability, viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication, on the one hand, and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. The study describes a novel mechanism by which HCV influences the activation of the epithelial growth factor receptor/Akt pathway through a nonstructural (NS)3/4A‐dependent down‐regulation of the ubiquitously expressed tyrosine phosphatase T cell protein tyrosine phosphatase (TC‐PTP). NS3/4A is demonstrated to cleave TC‐PTP protease‐dependently in vitro at two cleavage sites. The in vivo relevance of this finding is supported by the fact that down‐regulation of TC‐PTP protein expression could also be demonstrated in HCV‐infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. Conclusion: This down‐regulation of TC‐PTP results in an enhancement of epithelial growth factor (EGF)‐induced signal transduction and increases basal activity of Akt, which is demonstrated to be essential for the maintenance of sufficient viral replication. Hence, therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects, further diminishing viral replication. (HEPATOLOGY 2009;49:1810–1820.)

Hepatitis C virus (HCV) employs multiple strategies to subvert the host innate antiviral response.

Abstract Hepatitis C virus (HCV) is a serious global health problem which accounts for approximately 40% of chronic liver diseases worldwide. HCV frequently establishes a persistent infection, although it is recognized and targeted by innate immunity as well as cellular and humoral immune mechanisms. This suggests that HCV has developed powerful strategies to escape elimination by innate and adaptive immunity. HCV-induced liver injury is thought to be mainly immune-mediated rather than due to direct cytopathic effects of the virus. Hence, therapeutic strategies should target those mechanisms favoring viral persistence since unspecific enhancement of host antiviral immunity may theoretically also promote liver injury. The present review summarizes our current understanding of how the hepatitis C virus interferes with the innate antiviral host-response to establish persistent infection.

Anti–tumor necrosis factor α treatment promotes apoptosis and prevents liver regeneration in a transgenic mouse model of chronic hepatitis C

Tumor necrosis factor α (TNFα) has been implicated in a variety of inflammatory diseases, and anti‐TNFα has been shown to improve therapy when added to standard of care in chronic hepatitis C virus (HCV) infection. In addition, patients with chronic HCV have increased serum levels of TNFα and the macrophage‐attracting chemokine (C‐C motif) ligand 2 (CCL2). A mouse model of chronic HCV with hepatic nonstructural (NS) 3/4A protein expression mimics the human infection through a reduced response to double‐stranded RNA and cleavage of the T cell protein tyrosine phosphatase. The mice also display a resistance to TNFα in vivo. We therefore analyzed the relationship between NS3/4A and TNFα. Wild‐type and NS3/4A‐transgenic (Tg) mice were treated with TNFα/D‐galactosamine (D‐galN), acting through the TNF receptor 1 on hepatocytes and macrophages, or lipopolysaccharide (LPS)/D‐galN, acting through Toll‐like receptor 4 on sinusoidal endothelial cells, macrophages, and dendritic cells. Mice were analyzed for hepatic signaling, liver damage, TNFα, and CCL2. Similar to HCV‐infected humans, NS3/4A‐Tg mice displayed elevated basal levels of TNFα and CCL2. Treatment of NS3/4A‐Tg mice with TNFα/D‐galN or LPS/D‐galN led to increased hepatic nuclear factor kappa B (NFκB) activation, increased TNFα and CCL2 levels, decreased apoptosis, and increased hepatocyte regeneration. Importantly, blocking NFκB activation (bortezomib) or administering anti‐TNFα (infliximab) 4 hours after LPS/D‐galN injection reversed the resistance of NS3/4A‐Tg mice to TNFα‐induced liver injury. Conclusion: Resistance to TNFα seen in NS3/4A‐Tg mice is explained by a hepatoprotective effect of NFκB and TNFα. Hence, anti‐TNFα agents block these effects and are antiviral by promoting hepatocyte apoptosis and preventing hepatocyte regeneration. (HEPATOLOGY 2010;.)

Interplay between host cell and hepatitis C virus in regulating viral replication.

Abstract Viral life cycle as that of the hepatitis C virus (HCV) completely relies on host cell infrastructure, presupposing that the virus has evolved mechanisms to utilize and control all cellular molecules or pathways required for viral life cycle. Hence, HCV must have acquired the ability to gain access to key pathways controlling processes, such as cell growth, apoptosis and protein synthesis, which are all considered to also be crucial for liver regeneration. This occurs in a balanced way permitting persistent replication of viral genomes and production of infectious particles without endangering host cell viability and survival. In particular during the last decade, accumulating evidence indicates that HCV utilizes signaling pathways of the host with major impact on cellular growth, viability, cell cycle or cellular metabolism, such as epidermal growth factor-receptor mediated signals, the PI3K/Akt cascade or the family of Src kinases. Furthermore, HCV specifically interacts with parts of the cellular machinery involved in protein translation, processing, maturation and transport, such as components of the translation complex, the heat shock protein family, the immunophilins or the vesicle-associated membrane protein-associated proteins A and B. The present review focuses on the interplay between viral proteins and these factors of the host cell enabling the virus to utilize host cell infrastructure.

Heterologous T Cells Can Help Restore Function in Dysfunctional Hepatitis C Virus Nonstructural 3/4A-Specific T Cells during Therapeutic Vaccination

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting “healthy” heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2–restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.

TCR-Redirected Human T Cells Inhibit Hepatitis C Virus Replication: Hepatotoxic Potential Is Linked to Antigen Specificity and Functional Avidity

Virus-specific CTL with high levels of functional avidity have been associated with viral clearance in hepatitis C virus (HCV) infection and with enhanced protective immunity. In chronic HCV infection, lack of antiviral CTL is frequently observed. In this study, we aim to investigate novel HCV TCRs that differ in Ag specificity. This involved isolating new HCV-specific murine TCRs that recognize a conserved HLA-A2–restricted CTL epitope within the nonstructural protein (NS) 5A viral protein and comparing them with TCRs recognizing another conserved CTL target in the NS3 viral protein. This was done by expressing the TCRs in human T cells and analyzing the function of the resulting TCR-transduced T cells. Our result indicates that these TCRs are efficiently assembled in transduced human T cells. They recognize peptide-loaded targets and demonstrate polyfunctional features such as IL-2, IFN-γ, and TNF-α secretion. However, in contrast to NS3-specific TCRs, the NS5A TCR-transduced T cells consist of a smaller proportion of polyfunctional T cells and require more peptide ligands to trigger the effector functions, including degranulation. Despite the differences, NS5A TCRs show effective inhibition of HCV replication in human hepatoma cells with persistent HCV RNA replication. Moreover, cellular injury demonstrated by aspartate aminotransferase release and cell death is less significant in the hepatoma cells following coincubation with NS5A TCR-transduced T cells, which is a property consistent with noncytotoxic antiviral CTLs. Our results suggest that HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

Hepatitis C virus non-structural 3/4A protein interferes with intrahepatic interferon-γ production

Background The non-structural (NS) 3/4A protease/helicase of the hepatitis C virus is known to modulate signalling pathways in the infected hepatocyte by cleaving CARD adaptor inducing IFNβ (Cardif), T-cell protein tyrosine phosphatase (TC-PTP) and TIR domain-containing adaptor inducing IFNβ (TRIF), but the effects of NS3/4A in vivo still remain unclear. Aim To investigate the influence of NS3/4A on intracellular and intercellular signalling in vivo by analysing the intrahepatic inflammatory response of naïve, lipopolysaccharide (LPS)/d-galactosamine (d-galN) or tumour necrosis factor-α (TNFα)/d-galN-treated NS3/4A-transgenic (Tg) mice. Methods The intrahepatic immunity of naïve and LPS/d-galN- or TNFα/d-galN-treated NS3/4A-Tg mice was determined using western blot, ELISA, real-time PCR, flow cytometry and survival monitoring. The injection of cytokines or antibodies against signalling components was performed to analyse the relevance of the respective pathways for the investigated issues. A Tg mouse lineage expressing an inactivated NS3/4A protease (NS3/4AIle1073Ala-Tgs) was generated to examine if protective effects were NS3/4A protease dependent. Results The activation of hepatic signal transducer and activator of transcription 1 and 2 was impaired in NS3/4A-Tg mice after treatment with LPS/d-galN or TNFα/d-galN. This was paralleled by a reduction in hepatic interferon-γ (IFNγ). Reconstitution of IFNγ reverted the resistance to LPS/TNFα in NS3/4A-Tg mice. Subsequently, blocking IFNγ in vivo rendered wild-type mice resistant against treatment with LPS/TNFα. A new Tg mouse expressing an inactivated NS3/4A protease had the same phenotype as wild-type mice with respect to hepatic IFNγ levels and sensitivity to LPS/d-galN. Finally, the chemokine profile was altered in the NS3/4A-Tg mice towards an anti-inflammatory state, which helps to explain the altered immune cell subsets and reduction in hepatic IFNγ production. Conclusions Our data demonstrate that the NS3/4A protease reduces the intrahepatic production of IFNγ and alters TNFα-mediated effects, thereby impairing the hepatic inflammatory response. This may contribute to viral persistence.

Cleavage of the T Cell Protein Tyrosine Phosphatase by the Hepatitis C Virus Nonstructural 3/4A Protease Induces a Th1 to Th2 Shift Reversible by Ribavirin Therapy

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus–mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α–induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ+ T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.