Johannes G. Bode

IFN-α antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3

Eighty percent of patients newly infected with the hepatitis C virus (HCV) develop chronic infection, suggesting that HCV can develop effective strategies to escape the unspecific and specific immune response of the host. Because SOCS molecules have been recognized to be powerful inhibitors of cytokine signaling via the Jak/STAT pathway, virus‐induced expression of these molecules should be an efficient instrument to counteract the cellular response toward interferons (IFNs), an essential part of first line antiviral immune response. This study shows that overexpression of HCV core protein inhibits IFN‐α‐induced tyrosine phosphorylation and activation of STAT1 in hepatic cells. With the use of a STAT1‐YFP fusion protein, further evidence is given that HCV core is capable to inhibit nuclear translocation of STAT1. Inhibition of STATl‐tyrosine phosphorylation was accompanied by the induction of SOCS3‐mRNA expression, suggesting that the HCV core protein impairs IFN‐α‐induced signal transduction via induction of SOCS3 expression. HCV core protein was competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist. These IFN‐antagonistic properties of the HCV core protein may be part of the molecular basis of IFN‐a unresponsiveness in about one‐half of chronically infected HCV‐patients.

Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses

ABSTRACT Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3β and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.

LPS and TNFα induce SOCS3 mRNA and inhibit IL‐6‐induced activation of STAT3 in macrophages

Recent findings indicate that cytokine signaling can be modulated by other mediators of simultaneously activated signal transduction pathways. In this study we show that LPS and TNFα are potent inhibitors of IL‐6‐mediated STAT3 activation in human monocyte derived macrophages, rat liver macrophages and RAW 264.7 mouse macrophages but not in human hepatoma cells (HepG2) or in rat hepatocytes. Accordingly, LPS and TNFα were found to induce the expression of SOCS3 mRNA in each of the investigated type of macrophages but not in HepG2 cells. Using a specific inhibitor, evidence is presented that the p38 MAP kinase might be involved, especially for the inhibitory effect of TNFα.

Hepatic acute phase proteins--regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-κB-dependent signaling.

The function of the liver as an important constituent of the immune system involved in innate as well as adaptive immunity is warranted by different highly specialized cell populations. As the major source of acute phase proteins, including secreted pathogen recognition receptors (PRRs), short pentraxins, components of the complement system or regulators of iron metabolism, hepatocytes are essential constituents of innate immunity and largely contribute to the control of a systemic inflammatory response. The production of acute phase proteins in hepatocytes is controlled by a variety of different cytokines released during the inflammatory process with IL-1- and IL-6-type cytokines as the leading regulators operating both as a cascade and as a network having additive, inhibitory, or synergistic regulatory effects on acute phase protein expression. Hence, IL-1β substantially modifies IL-6-induced acute phase protein production as it almost completely abrogates production of acute phase proteins such as γ-fibrinogen, α(2)-macroglobulin or α(1)-antichymotrypsin, whereas production of for example hepcidin, C-reactive protein and serum amyloid A is strongly up-regulated. This switch-like regulation of IL-6-induced acute phase protein production by IL-1β is due to a complex processing of the intracellular signaling events activated in response to IL-6 and/or IL-1β, with the crosstalk between STAT3- and NF-κB-mediated signal transduction being of particular importance. Recent data suggest that in this context complex formation between STAT3 and the p65 subunit of NF-κB might be of key importance. The present review summarizes the regulation of acute phase protein production focusing on the role of the crosstalk of STAT3- and NF-κB-driven pathways for transcriptional control of acute phase gene expression.

Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNβ gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNβ gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5′ triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-κB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.

The macrophage response towards LPS and its control through the p38MAPK–STAT3 axis

In macrophages detection of gram-negative bacteria particularly involves binding of the outer-wall component lipopolysaccharide (LPS) to its cognate receptor complex, comprising Toll like receptor 4 (TLR4), CD14 and MD2. LPS-induced formation of the LPS receptor complex elicits a signaling network, including intra-cellular signal-transduction directly activated by the TLR4 receptor complex as well as successional induction of indirect autocrine and paracrine signaling events. All these different pathways are integrated into the macrophage response towards an inflammatory stimulus by a highly complex cross-talk of the pathways engaged. This also includes a tight control by several intra- and inter-cellular feedback loops warranting an inflammatory response sufficient to battle invading pathogens and to avoid non-essential tissue damage caused by an overwhelming inflammatory response. Several evidences indicate that the reciprocal cross-talk between the p38(MAPK)-pathway and signal transducer and activator of transcription (STAT)3-mediated signal-transduction forms a critical axis successively activated by LPS. The balanced activation of this axis is essential for both induction and propagation of the inflammatory macrophage response as well as for the control of the resolution phase, which is largely driven by IL-10 and sustained STAT3 activation. In this context regulation of suppressor of cytokine signaling (SOCS)3 expression and the recently described divergent regulatory roles of the two p38(MAPK)-activated protein kinases MK2 and MK3 for the regulation of LPS-induced NF-κB- and IRF3-mediated signal-transduction and gene expression, which includes the regulation of IFNβ, IL-10 and DUSP1, appears to play an important role.

The Mitogen-activated Protein (MAP) Kinase p38 and Its Upstream Activator MAP Kinase Kinase 6 Are Involved in the Activation of Signal Transducer and Activator of Transcription by Hyperosmolarity

Environmental stress (e.g.aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the “stress-responsive” mitogen-activated protein kinases and nuclear factor κB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6.

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.

Cross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1β, TNFα or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

Involvement of CD95 (Apo-1/Fas) ligand expressed by rat Kupffer cells in hepatic immunoregulation

BACKGROUND & AIMS CD95 (Apo-1/Fas) ligand suppresses inflammatory responses in immune-privileged organs. In this study, modulation of the hepatic CD95 receptor/ligand system by interferon gamma and cyclosporin A was investigated. METHODS CD95 receptor and ligand expression were measured at the messenger RNA level by using quantitative reverse-transcription polymerase chain reaction and immunocytochemistry in primary cultures of rat Kupffer cells, hepatocytes, and T lymphocytes. Soluble CD95 in culture supernatants was detected by enzyme-linked immunosorbent assay and apoptosis by the TUNEL method. RESULTS Interferon gamma treatment led to an increase in CD95 ligand messenger RNA levels in Kupffer cells followed by an overexpression of the soluble CD95 receptor. Supernatants derived from 24-hour but not from 48-hour interferon gamma-treated Kupffer cells killed lymphocytes by a CD95-dependent mechanism. Cyclosporin A inhibited CD95 ligand expression in Kupffer cells and lymphocyte killing. In liver parenchymal cells, interferon gamma increased messenger RNA levels of the transmembrane CD95 isoform and sensitivity of these cells toward CD95-mediated apoptosis. CONCLUSIONS The expression pattern of CD95 receptor and ligand in response to interferon gamma points to a coordinated interplay between Kupffer cells, hepatocytes, and T lymphocytes in which Kupffer cells may regulate programmed cell death of T lymphocytes and hepatocytes.