a-high-resolution-method-to-monitor-phosphorylation-dependent

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon &#946; and antiviral gene expression. ...

Université de Montréal

Innate sensing of viral infection activates a global defense response including type I interferon (IFN) and IFN-stimulated genes (ISGs) expression. We previously reported that HCV NS3/4A protease, an essential protein in viral polyprotein processing, can abrogate antiviral signaling pathways and effectors' response when ectopically expressed in human hepatocytes by cleaving antiviral adaptor CARDIF. However, whether HCV mediates evasion of innate immunity in patients with chronic infection remains unclear.

Targeted impairment of innate antiviral responses in the liver of chronic hepatitis C patients.

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury.

The interferon regulatory factor (IRF)-3 transcription factor plays a central role in the capacity of the host to mount an efficient innate antiviral immune defense, mainly through the regulation of type I Interferon genes. A tight regulation of IRF-3 is crucial for an adapted intensity and duration of the response. Redox-dependent processes are now well known to regulate signaling cascades. Recent reports have revealed that signaling molecules upstream of IRF-3, including the mitochondrial antiviral-signalling protein (MAVS) and the TNF receptor associated factors (TRAFs) adaptors, are sensitive to redox regulation. In the present study, we assessed whether redox regulation of thiol residues contained in IRF-3, which are priviledged redox sensors, play a role in its regulation following Sendai virus infection, using a combination of mutation of Cysteine (Cys) residues into Alanine and thiols alkylation using N-ethyl maleimide. Alkylation of IRF-3 on Cys289 appears to destabilize IRF-3 dimer in vitro. However, a detailed analysis of IRF-3 phosphorylation, dimerization, nuclear accumulation, and induction of target gene promoter in vivo led us to conclude that IRF-3 specific, individual Cys residues redox status does not play an essential role in its activation in vivo.

Individual interferon regulatory factor-3 thiol residues are not critical for its activation following virus infection.

The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB(4) biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG- and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB(4) biosynthesis or by blocking BLT(1). Importantly, neither CB(2) receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ∼100-fold more CB(2) receptor mRNA than purified neutrophils, suggesting that CB(2) receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB(2) expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB(4) promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT(1). Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo.

2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV.

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.

IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of β-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.

Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

Human respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. The innate immune response plays a pivotal role in host defense against RSV, but whether severe outcomes following RSV infection result from excessive or poor innate immune recognition remains unclear. Recent research suggests a situation in which crosstalk between families of pattern recognition receptors (PRRs) occurs in a cell type-dependent manner. The current challenge to empower novel therapeutic approaches and vaccine development is to confirm the role of the individual receptors in RSV pathogenesis in humans.

Pathogen recognition receptor crosstalk in respiratory syncytial virus sensing: a host and cell type perspective.

Cytokine signaling is mediated by the combinatorial usage of seven STAT proteins that form homo- or heterodimers involved in the regulation of specific transcriptional programs. Among STATs, STAT2 is classically known to dimerize with STAT1 and together with IRF9 forms the ISGF3 transcription factor complex that has long been considered a hallmark of activation by type I and type III interferons. However, accumulating evidence reveal distinct facets of STAT2 and IRF9 activity mediated by the segregation in alternative STAT1-independent complexes/pathways that are thought to trigger different transcriptional programs. The goal of this review is to summarize our current knowledge of the stimuli, regulatory mechanisms, and function of these alternative pathways.

STAT2 and IRF9: Beyond ISGF3.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.

Sustained activation of interferon regulatory factor 3 during infection by paramyxoviruses requires MDA5.

Indoleamine 2,3-dioxygenase (IDO) is found in multiple malignancies and exerts immunosuppressive effects that are central in protecting tumors from host T lymphocyte rejection. IDO is an enzyme involved in the catabolism of tryptophan resulting in inhibition of T lymphocyte function. While inhibition of IDO enzymatic activity results in tumor rejection, it is still unknown how we can directly target IDO expression within tumors using drugs. We have chosen to interfere with IDO expression by targeting the key-signaling event signal transducer and activator of transcription 1 (STAT1). We evaluated the efficacy of fludarabine, previously described to inhibit STAT1 phosphorylation. Interestingly, fludarabine was efficient in suppressing protein expression and consequently IDO activity in two different cell lines derived from breast cancer and melanoma when IDO was activated with interferon-gamma (IFN-γ) or supernatants prepared from activated T lymphocytes. However, fludarabine had no inhibitory effect on STAT1 phosphorylation. Other IFN-γ-responsive genes were only marginally inhibited by fludarabine. The level of IDO transcript was unaffected by this inhibitor, suggesting the involvement of post-transcriptional control. Strikingly, we have found that the inhibition of proteasome partially protected IDO from fludarabine-induced degradation, indicating that fludarabine induces IDO degradation through a proteasome-dependent pathway. Currently used in the clinic to treat some malignancies, fludarabine has the potential for use in the treatment of human tumors through induction of IDO degradation and consequently, for the promotion of T cell-mediated anti-tumor response.

Fludarabine downregulates indoleamine 2,3-dioxygenase in tumors via a proteasome-mediated degradation mechanism.

Determining the role of NADPH oxidases in the context of virus infection is an emerging area of research and our knowledge is still sparse. The expression of various isoforms of NOX/DUOX (NADPH oxidase/dual oxidase) in the epithelial cells (ECs) lining the respiratory tract renders them primary sites from which to orchestrate the host defence against respiratory viruses. Accumulating evidence reveals distinct facets of the involvement of NOX/DUOX in host antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity, with individual isoforms mediating co-operative, but surprisingly also opposing, functions. Although in vivo studies in mice are in line with some of these observations, a complete understanding of the specific functions of epithelial NOX/DUOX awaits lung epithelial-specific conditional knockout mice. The goal of the present review is to summarize our current knowledge of the role of individual NOX/DUOX isoforms expressed in the lung epithelium in the context of respiratory virus infections so as to highlight potential opportunities for therapeutic intervention.

Lung epithelial NOX/DUOX and respiratory virus infections.

Oxidative stress is considered a causative factor in carcinogenesis, but also in the development of resistance to current chemotherapies. The appropriate usage of redox-modulating compounds is limited by the lack of knowledge of their impact on specific molecular pathways. Increased levels of the IKKε kinase, as a result of gene amplification or aberrant expression, are observed in a substantial number of breast carcinomas. IKKε not only plays a key role in cell transformation and invasiveness, but also in the development of resistance to tamoxifen. Here, we studied the effect of in vitro treatment with the redox-modulating triphenylmethane dyes, Gentian Violet and Brilliant Green, and nitroxide Tempol on IKKε expression and cell proliferation in the human breast cancer epithelial cell lines exhibiting amplification of IKKε, MCF-7 and ZR75.1. We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide anion levels and inhibit IKKε expression and cell viability. Treatment with Gentian Violet and Brilliant Green was associated with a reduced cyclin D1 expression and activation of caspase 3 and/or 7. Tempol decreased cyclin D1 expression in both cell lines, while activation of caspase 7 was only observed in MCF-7 cells. Silencing of the superoxide-generating NOX2 NADPH oxidase expressed in breast cancer cells resulted in the significant reduction of IKKε expression. Taken together, our results suggest that redox-modulating compounds targeting NOX2 could present a particular therapeutic interest in combination therapy against breast carcinomas exhibiting IKKε amplification.

Nathalie Grandvaux

CRCHUM - Research center,
Centre Hospitalier de l'Université de Montréal,
Department of Biochemistry and Molecular Medicine,
Faculty of Medicine,
CRCHUM - Research center, Centre Hospitalier de l'Université de Montréal

Université de Montréal

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Nathalie Grandvaux

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CRCHUM - Research center,Centre Hospitalier de l'Université de Montréal,Department of Biochemistry and Molecular Medicine,Faculty of Medicine,CRCHUM - Research center, Centre Hospitalier de l'Université de Montréal

Université de Montréal

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

CRCHUM - Research center,
Centre Hospitalier de l'Université de Montréal,
Department of Biochemistry and Molecular Medicine,
Faculty of Medicine,
CRCHUM - Research center, Centre Hospitalier de l'Université de Montréal