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Influenza and RSV are human viruses responsible for outbreaks in hospitals, long-term care facilities and nursing homes. The present study assessed an air treatment using ozone at two relative humidity conditions (RHs) in order to reduce the infectivity of airborne influenza. Bovine pulmonary surfactant (BPS) and synthetic tracheal mucus (STM) were used as aerosols protectants to better reflect the human aerosol composition. Residual ozone concentration inside the aerosol chamber was also measured. RSV’s sensitivity resulted in testing its resistance to aerosolization and sampling processes instead of ozone exposure. The results showed that without supplement and with STM, a reduction in influenza A infectivity of four orders of magnitude was obtained with an exposure to 1.70 ± 0.19 ppm of ozone at 76% RH for 80 min. Consequently, ozone could be considered as a virucidal disinfectant for airborne influenza A. RSV did not withstand the aerosolization and sampling processes required for the use of the experimental setup. Therefore, ozone exposure could not be performed for this virus. Nonetheless, this study provides great insight for the efficacy of ozone as an air treatment for the control of nosocomial influenza A outbreaks.

Interferon (IFN) &beta, and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFN&beta, is reprogrammed by crosstalk with TNF. IFN&beta, mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFN&beta, and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFN&beta, and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFN&beta, and TNF.

IFNβ typically induces an antiviral and immunoregulatory transcriptional program through the activation of ISGF3 (STAT1, STAT2 and IRF9) transcriptional complexes. The response to IFNβ is context-dependent and is prone to crosstalk with other cytokines, such as TNFα IFNβ and TNFα synergize to drive a specific delayed transcriptional program. Previous observation led to the hypothesis that an alternative STAT1-independent pathway involving STAT2 and IRF9 might be involved in gene induction by the combination of IFNβ and TNFα. Using genome wide transcriptional profiling by RNASeq, we found that the costimulation with IFNβ and TNFα induces a broad antiviral and immunoregulatory transcriptional program independently of STAT1. Additionally, STAT2 and IRF9 are involved in the regulation of only a subset of these STAT1-independent genes. Consistent with the growing literature, STAT2 and IRF9 act in concert to regulate a subgroup of these genes. Unexpectedly, STAT2 and IRF9 were also engaged in specific independent pathways to regulate distinct sets of IFNβ and TNFα-induced genes. Altogether these observations highlight the existence of distinct previously unrecognized non-canonical STAT1-independent, but STAT2 and/or IRF9-dependent pathways in the establishment of a delayed antiviral and immunoregulatory transcriptional program in conditions where elevated levels of both IFNβ and TNFα are present.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveils a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.

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.

The innate immune response is essential to the host defense against viruses, through restriction of virus replication and coordination of the adaptive immune response. Induction of antiviral genes is a tightly regulated process initiated mainly through sensing of invading virus nucleic acids in the cytoplasm by RIG-I like helicases, RIG-I or Mda5, which transmit the signal through a common mitochondria-associated adaptor, MAVS. Although major breakthroughs have recently been made, much remains unknown about the mechanisms that translate virus recognition into antiviral genes expression. Beside the reputed detrimental role, reactive oxygen species (ROS) act as modulators of cellular signaling and gene regulation. NADPH oxidase (NOX) enzymes are a main source of deliberate cellular ROS production. Here, we found that NOX2 and ROS are required for the host cell to trigger an efficient RIG-I-mediated IRF-3 activation and downstream antiviral IFNβ and IFIT1 gene expression. Additionally, we provide evidence that NOX2 is critical for the expression of the central mitochondria-associated adaptor MAVS. Taken together these data reveal a new facet to the regulation of the innate host defense against viruses through the identification of an unrecognized role of NOX2 and ROS., Author Summary The understanding of the mechanisms allowing the host to mount a rapid and efficient innate immune response to RNA viruses has been the subject of intensive research in recent years. Major groundwork allowed the identification of key sensors of virus nucleic acids, including RIG-I and Mda5, which through association with the MAVS adaptor initiate the signaling cascade required for activation of the IRF-3 transcription factor and downstream antiviral genes. Mechanisms of activation and degradation of key signaling molecules allow a tight control of the intensity and duration of the response. Our knowledge of how redox processes regulate signaling cascades is a fast moving field of research. Particularly, the identification of non-phagocytic reactive oxygen species-producing NADPH oxidase (NOX) enzymes revealed new insights into their function in innate immunity. Our endeavor in characterizing the role of NOX in the antiviral response reveals a new facet to the overall picture of antiviral response regulation. Here, we demonstrate that NOX2 is essential for MAVS expression in airway epithelial cells, thereby controlling the capacity of the cell to mount an efficient innate antiviral response following recognition of viruses.

Respiratory syncytial virus (RSV) is the etiological agent of acute respiratory diseases, such as bronchiolitis and pneumonia. The exacerbated production of proinflammatory cytokines and chemokines in the airways in response to RSV is an important pillar in the development of these pathologies. As such, a keen understanding of the mechanisms that modulate the inflammatory response during RSV infection is of pivotal importance to developing effective treatment. The NF-κB transcription factor is a major regulator of proinflammatory cytokine and chemokine genes. However, RSV-mediated activation of NF-κB is far from characterized. We recently demonstrated that aside from the well-characterized IκBα phosphorylation and degradation, the phosphorylation of p65 at Ser536 is an essential event regulating the RSV-mediated NF-κB-dependent promoter transactivation. In the present study, using small interfering RNA and pharmacological inhibitors, we now demonstrate that RSV sensing by the RIG-I cytoplasmic receptor triggers a signaling cascade involving the MAVS and TRAF6 adaptors that ultimately leads to p65ser536 phosphorylation by the IKKβ kinase. In a previous study, we highlighted a critical role of the NOX2-containing NADPH oxidase enzyme as an upstream regulator of both the IκBαSer32 and p65Ser536 in human airway epithelial cells. Here, we demonstrate that inhibition of NOX2 significantly decreases IKKβ activation. Taken together, our data identify a new RIG-I/MAVS/TRAF6/IKKβ/p65Ser536 pathway placed under the control of NOX2, thus characterizing a novel regulatory pathway involved in NF-κB-driven proinflammatory response in the context of RSV infection.

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., Graphical abstract, Highlights • IKKε kinase is amplified in MCF7 and ZR75.1 breast cancer cells. • Brilliant Green, Gentian Violet and Tempol reduce superoxide levels in MCF7 and ZR75.1 cells. • Brilliant Green, Gentian Violet and Tempol inhibit IKKε expression in MCF7 and ZR75.1 cells. • IKKε overexpression in breast cancer cells is dependent on NOX2. • Brilliant Green, Gentian Violet and Tempol reduce MCF7 and ZR75.1 cell viability.

Background IKKε is an oncogenic kinase that was found amplified and overexpressed in a substantial percentage of human breast cancer cell lines and primary tumors using genomic and gene expression analyses. Molecular studies have provided the rational for a key implication of IKKε in breast cancer cells proliferation and invasiveness through the phosphorylation of several substrates. Methods Here, we performed immunohistochemical detection of IKKε expression on tissue microarrays constituted of 154 characterized human breast cancer tumors. We further determined the association with multiple clinicopathological parameters and 5-years overall, disease-free and distant disease free survival. Results We observed expression of IKKε in 60.4% of the breast cancer tumors. IKKε expression status showed no association with a panel of markers used for molecular classification of the tumors, including ER/PR/HER2 status, or with the molecular subtypes. However, IKKε expression was inversely associated with lymph node metastasis status (p = 0.0032). Additionally, we identified a novel association between IKKε and EGFR expression (p = 0.0011). Conclusions The unexpected observation of an inverse association between IKKε and lymph node metastasis advocates for larger scale immunohistochemical profiling of primary breast tumors to clarify the role of IKKε in metastasis. This study suggests that breast cancer tumors expressing EGFR and IKKε may be potential targets for drugs aiming at inhibiting IKKε activity or expression.