Cystathionine γ-lyase deficiency enhances airway reactivity and viral-induced disease in mice exposed to side-stream tobacco smoke.

BACKGROUND: Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV comorbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract. METHODS: We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H2S-generating enzyme in the lung to determine the contribution of H2S to airway disease in response to side-stream tobacco smoke (TS), and to TS/RSV co-exposure. RESULTS: Following a 2-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared with wild-type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited a more severe clinical disease, airway obstruction and AHR, enhanced viral replication, and lung inflammation, compared with TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage fluid and increased levels of inflammatory cytokines and chemokines. CONCLUSION: This study demonstrates the critical contribution of the H2S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

Hydrogen Sulfide: A Novel Player in Airway Development, Pathophysiology of Respiratory Diseases, and Antiviral Defenses.

Hydrogen sulfide (H2S) is a biologically relevant signaling molecule in mammals. Along with the volatile substances nitric oxide (NO) and carbon monoxide (CO), H2S is defined as a gasotransmitter. It plays a physiological role in a variety of functions, including synaptic transmission, vascular tone, angiogenesis, inflammation, and cellular signaling. The generation of H2S is catalyzed by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). The expression of CBS and CSE is tissue specific, with CBS being expressed predominantly in the brain, and CSE in peripheral tissues, including lungs. CSE expression and activity are developmentally regulated, and recent studies suggest that CSE plays an important role in lung alveolarization during fetal development. In the respiratory tract, endogenous H2S has been shown to participate in the regulation of important functions such as airway tone, pulmonary circulation, cell proliferation or apoptosis, fibrosis, oxidative stress, and inflammation. In the past few years, changes in the generation of H2S have been linked to the pathogenesis of a variety of acute and chronic inflammatory lung diseases, including asthma and chronic obstructive pulmonary disease. Recently, our laboratory made the critical discovery that cellular H2S exerts broad-spectrum antiviral activity both in vitro and in vivo, in addition to independent antiinflammatory activity. These findings have important implications for the development of novel therapeutic strategies for viral respiratory infections, as well as other inflammatory lung diseases, especially in light of recent significant efforts to generate controlled-release H2S donors for clinical therapeutic applications.

Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infant and elderly populations worldwide. Currently, there is no efficacious vaccine or therapy available for RSV infection. The molecular mechanisms underlying RSV-induced acute airway disease and associated long-term consequences remain largely unknown; however, experimental evidence suggests that the lung inflammatory response plays a fundamental role in the outcome of RSV infection. High-mobility group box 1 (HMGB1) is a nuclear protein that triggers inflammation when released from activated immune or necrotic cells and drives the pathogenesis of various infectious agents. Although HMGB1 has been implicated in many inflammatory diseases, its role in RSV-induced airway inflammation has not been investigated. This study investigates the molecular mechanism of action of extracellularly released HMGB1 in airway epithelial cells (A549 and small airway epithelial cells) to establish its role in RSV infection. Immunofluorescence microscopy and Western blotting results showed that RSV infection of human airway epithelial cells induced a significant release of HMGB1 as a result of translocation of HMGB1 from the cell nuclei to the cytoplasm and subsequent release into the extracellular space. Treating RSV-infected A549 cells with antioxidants significantly inhibited RSV-induced HMGB1 extracellular release. Studies using recombinant HMGB1 triggered immune responses by activating primary human monocytes. Finally, HMGB1 released by airway epithelial cells due to RSV infection appears to function as a paracrine factor priming epithelial cells and monocytes to inflammatory stimuli in the airways. IMPORTANCE: RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection.

Hydrogen sulfide (H2S) is an endogenous gaseous transmitter whose role in the pathophysiology of several lung diseases has been increasingly appreciated. Our recent studies in vitro have shown, we believe for the first time, that H2S has an important antiviral and antiinflammatory activity in respiratory syncytial virus (RSV) infection, the leading cause of bronchiolitis and viral pneumonia in children. Our objective was to evaluate the therapeutic potential of GYY4137, a novel slow-releasing H2S donor, for the prevention and treatment of RSV-induced lung disease, as well as to investigate the role of endogenous H2S in a mouse model of RSV infection. Ten- to 12-week-old BALB/c mice treated with GYY4137, or C57BL/6J mice genetically deficient in the cystathionine γ-lyase enzyme, the major H2S-generating enzyme in the lung, were infected with RSV and assessed for viral replication, clinical disease, airway hyperresponsiveness, and inflammatory responses. Our results show that intranasal delivery of GYY4137 to RSV-infected mice significantly reduced viral replication and markedly improved clinical disease parameters and pulmonary dysfunction compared with the results in vehicle-treated control mice. The protective effect of the H2S donor was associated with a significant reduction of viral-induced proinflammatory mediators and lung cellular infiltrates. Furthermore, cystathionine γ-lyase-deficient mice showed significantly enhanced RSV-induced lung disease and viral replication compared with wild-type animals. Overall, our results indicate that H2S exerts a novel antiviral and antiinflammatory activity in the context of RSV infection and represent a potential novel pharmacological approach for ameliorating virus-induced lung disease.

Role of hydrogen sulfide in paramyxovirus infections.

UNLABELLED: Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. IMPORTANCE: RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

Oxidative stress in Nipah virus-infected human small airway epithelial cells.

Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.

Mitochondrial antiviral-signalling protein plays an essential role in host immunity against human metapneumovirus.

Human metapneumovirus (hMPV) is a common cause of respiratory tract infection in the paediatrics population. Recently, we and others have shown that retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) are essential for hMPV-induced cellular antiviral signalling. However, the contribution of those receptors to host immunity against pulmonary hMPV infection is largely unexplored. In this study, mice deficient in mitochondrial antiviral-signalling protein (MAVS), an adaptor of RLRs, were used to investigate the role(s) of these receptors in pulmonary immune responses to hMPV infection. MAVS deletion significantly impaired the induction of antiviral and pro-inflammatory cytokines and the recruitment of immune cells to the bronchoalveolar lavage fluid by hMPV. Compared with WT mice, mice lacking MAVS demonstrated decreased abilities to activate pulmonary dendritic cells (DCs) and abnormal primary T-cell responses to hMPV infection. In addition, mice deficient in MAVS had a higher peak of viral load at day 5 post-infection (p.i.) than WT mice, but were able to clear hMPV by day 7 p.i. similarly to WT mice. Taken together, our data indicate a role of MAVS-mediated pathways in the pulmonary immune responses to hMPV infection and the early control of hMPV replication.

Alveolar macrophages contribute to the pathogenesis of human metapneumovirus infection while protecting against respiratory syncytial virus infection.

Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of upper and lower respiratory tract infections in young children and among elderly and immunocompromised patients. The pathogenesis of hMPV-induced lung disease is poorly understood. The lung macrophage population consists of alveolar macrophages (AMs) residing at the luminal surface of alveoli and interstitial macrophages present within the parenchymal lung interstitium. The involvement of AMs in innate immune responses to virus infections remains elusive. In this study, BALB/c mice depleted of AMs by intranasal instillation of dichloromethylene bisphosphonate (L-CL2MBP) liposomes were examined for disease, lung inflammation, and viral replication after infection with hMPV or RSV. hMPV-infected mice lacking AMs exhibited improved disease in terms of body weight loss, lung inflammation, airway obstruction, and hyperresponsiveness compared with AM-competent mice. AM depletion was associated with significantly reduced hMPV titers in the lungs, suggesting that hMPV required AMs for early entry and replication in the lung. In contrast, AM depletion in the context of RSV infection was characterized by an increase in viral replication, worsened disease, and inflammation, with increased airway neutrophils and inflammatory dendritic cells. Overall, lack of AMs resulted in a broad-spectrum disruption in type I IFN and certain inflammatory cytokine production, including TNF and IL-6, while causing a virus-specific alteration in the profile of several immunomodulatory cytokines, chemokines, and growth factors. Our study demonstrates that AMs have distinct roles in the context of human infections caused by members of the Paramyxoviridae family.

CDK9-dependent transcriptional elongation in the innate interferon-stimulated gene response to respiratory syncytial virus infection in airway epithelial cells.

Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3(-/-) MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser(2) carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-ß) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.

Human metapneumovirus glycoprotein G inhibits TLR4-dependent signaling in monocyte-derived dendritic cells.

Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells (DCs) are potent APCs and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived DCs (moDCs), as downregulation of its expression by small interfering RNA significantly blocked hMPV-induced chemokine and type I IFN expression. Similar results were found in bone marrow-derived DCs from TLR4-deficient mice. moDCs infected with a virus lacking G protein expression produced higher levels of cytokines and chemokines compared with cells infected with wild-type virus, suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as infection of moDCs with recombinant hMPV lacking G protein inhibited LPS-induced production of cytokine and chemokines significantly less than did wild-type virus, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.

Micro-CT Features of Lung Consolidation, Collagen Deposition and Inflammation in Experimental RSV Infection Are Aggravated in the Absence of Nrf2.

Severe respiratory syncytial virus (RSV) infections in early life have been linked to the development of chronic airway disease. RSV triggers the production of reactive oxygen species (ROS), which contributes to inflammation and enhanced clinical disease. NF-E2-related factor 2 (Nrf2) is an important redox-responsive protein that helps to protect cells and whole organisms from oxidative stress and injury. The role of Nrf2 in the context of viral-mediated chronic lung injury is not known. Herein, we show that RSV experimental infection of adult Nrf2-deficient BALB/c mice (Nrf2-/-; Nrf2 KO) is characterized by enhanced disease, increased inflammatory cell recruitment to the bronchoalveolar compartment and a more robust upregulation of innate and inflammatory genes and proteins, compared to wild-type Nrf2+/+ competent mice (WT). These events that occur at very early time points lead to increased peak RSV replication in Nrf2 KO compared to WT mice (day 5). To evaluate longitudinal changes in the lung architecture, mice were scanned weekly via high-resolution micro-computed tomography (micro-CT) imaging up to 28 days after initial viral inoculation. Based on micro-CT qualitative 2D imaging and quantitative reconstructed histogram-based analysis of lung volume and density, we found that RSV-infected Nrf2 KO mice developed significantly greater and prolonged fibrosis compared to WT mice. The results of this study underscore the critical role of Nrf2-mediated protection from oxidative injury, not only in the acute pathogenesis of RSV infection but also in the long-term consequences of chronic airway injury.

Hypoxia-inducible-factors differentially contribute to clinical disease and the control of viral replication during RSV infection.

Hypoxia-inducible-factors (HIF) are transcription factors that regulate cellular adaptation to hypoxic conditions, enabling cells to survive in low-oxygen environments. Viruses have evolved to stabilize this pathway to promote successful viral infection, therefore modulation of HIFs could represent a novel antiviral strategy. In previous in vitro studies, we found that respiratory syncytial virus (RSV), a leading cause of respiratory illness, stabilizes HIFs under normoxic conditions, with inhibition of HIF-1α resulting in reduced viral replication. Despite several HIF modulating compounds being tested/approved for use in other non-infectious models, little is known about their efficacy against respiratory viruses using relevant animal models. This study aimed to characterize the disease modulating properties and antiviral potential of anti-HIF-1α (PX478) and anti-HIF-2α (PT2385) in RSV-infected BALB/c mice. We found that inhibition of HIF-1α worsen clinical disease parameters, while simultaneously improving airway function. Additionally, anti-HIF-1α results in significantly reduced viral titer at early and peak time points of RSV replication, followed by a loss in viral clearance when given every day, but not every-other-day. In contrast, inhibition of HIF-2α was associated with improved clinical parameters, with no changes in airway function, and amelioration of interstitial pneumonia. Furthermore, anti-HIF-2α reduced early and peak lung viral replication, with no impairment of viral clearance. Analysis of lung cells found significant modification in the T cell compartment that correlated with changes in lung pathology and viral titers in response to each HIF inhibitor administration. These data underscore the complex role of HIFs in RSV infection and highlight the need for careful therapeutic consideration.

The impact of RSV/SARS-CoV-2 co-infection on clinical disease and viral replication: insights from a BALB/c mouse model.

RSV and SARS-CoV-2 are prone to co-infection with other respiratory viruses. In this study, we use RSV/SARS-CoV-2 co-infection to evaluate changes to clinical disease and viral replication in vivo. To consider the severity of RSV infection, effect of sequential infection, and the impact of infection timing, mice were co-infected with varying doses and timing. Compared with a single infection of RSV or SARS-CoV-2, the co-infection of RSV/SARS-CoV-2 and the primary infection of RSV followed by SARS-CoV-2 results in protection from SARS-CoV-2-induced clinical disease and reduces SARS-CoV-2 replication. Co-infection also augmented RSV replication at early timepoints with only the low dose. Additionally, the sequential infection of RSV followed by SARS-CoV-2 led to improved RSV clearance regardless of viral load. However, SARS-CoV-2 infection followed by RSV results in enhanced SARS-CoV-2-induced disease while protecting from RSV-induced disease. SARS-CoV-2/RSV sequential infection also reduced RSV replication in the lung tissue, regardless of viral load. Collectively, these data suggest that RSV and SARS-CoV-2 co-infection may afford protection from or enhancement of disease based on variation in infection timing, viral infection order, and/or viral dose. In the pediatric population, understanding these infection dynamics will be critical to treat patients and mitigate disease outcomes.

SARS-CoV-2 Inhibits NRF2-Mediated Antioxidant Responses in Airway Epithelial Cells and in the Lung of a Murine Model of Infection.

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

Antioxidant mimetics modulate oxidative stress and cellular signaling in airway epithelial cells infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is one of the most common causes of bronchiolitis and pneumonia among infants and young children worldwide. In previous investigations, we have shown that RSV infection induces rapid generation of reactive oxygen species (ROS), which modulate viral-induced cellular signaling, and downregulation of antioxidant enzyme (AOE) expression, resulting in oxidative stress in vitro and in vivo, which plays a pathogenetic role in RSV-induced lung disease. In this study, we determined whether pharmacological intervention with synthetic catalytic scavengers could reduce RSV-induced proinflammatory gene expression and oxidative cell damage in an in vitro model of infection. Treatment of airway epithelial cells (AECs) with the salen-manganese complexes EUK-8 or EUK-189, which possess superoxide dismutase, catalase, and glutathione peroxidase activity, strongly reduced RSV-induced ROS formation by increasing cellular AOE enzymatic activity and levels of the lipid peroxidation products F(2)-8-isoprostane and malondialdehyde, which are markers of oxidative stress. Treatment of AECs with AOE mimetics also significantly inhibited RSV-induced cytokine and chemokine secretion and activation of the transcription factors nuclear factor-κB and interferon regulatory factor-3, which orchestrate proinflammatory gene expression. Both EUKs were able to reduce viral replication, when used at high doses. These results suggest that increasing antioxidant cellular capacities can significantly impact RSV-associated oxidative cell damage and cellular signaling and could represent a novel therapeutic approach in modulating virus-induced lung disease.

Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis.

RATIONALE: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known. OBJECTIVES: To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection. METHODS: Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays. MEASUREMENTS AND MAIN RESULTS: RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice. CONCLUSIONS: RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.

Respiratory syncytial virus infection changes the piwi-interacting RNA content of airway epithelial cells.

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (sncRNAs) of about 26-32 nucleotides in length and represent the largest class of sncRNA molecules expressed in animal cells. piRNAs have been shown to play a crucial role to safeguard the genome, maintaining genome complexity and integrity, as they suppress the insertional mutations caused by transposable elements. However, there is growing evidence for the role of piRNAs in controlling gene expression in somatic cells as well. Little is known about changes in piRNA expression and possible function occurring in response to viral infections. In this study, we investigated the piRNA expression profile, using a human piRNA microarray, in human small airway epithelial (SAE) cells infected with respiratory syncytial virus (RSV), a leading cause of acute respiratory tract infections in children. We found a time-dependent increase in piRNAs differentially expressed in RSV-infected SAE cells. We validated the top piRNAs upregulated and downregulated at 24 h post-infection by RT-qPCR and identified potential targets. We then used Gene Ontology (GO) tool to predict the biological processes of the predicted targets of the most represented piRNAs in infected cells over the time course of RSV infection. We found that the most significant groups of targets of regulated piRNAs are related to cytoskeletal or Golgi organization and nucleic acid/nucleotide binding at 15 and 24 h p.i. To identify common patterns of time-dependent responses to infection, we clustered the significantly regulated expression profiles. Each of the clusters of temporal profiles have a distinct set of potential targets of the piRNAs in the cluster Understanding changes in piRNA expression in RSV-infected airway epithelial cells will increase our knowledge of the piRNA role in viral infection and might identify novel therapeutic targets for viral lung-mediated diseases.

Role of human metapneumovirus glycoprotein G in modulation of immune responses.

Human metapneumovirus (hMPV) is an important pathogen responsible for acute respiratory tract infections in children, the elderly, and immunocompromised patients, with no effective treatment or vaccine currently available. Knowledge of virus- and host-specific mechanisms contributing to the pathogenesis of hMPV infection is still limited. Studies have shown that hMPV surface glycoprotein G is an important virulence factor, by inhibiting innate immune signaling in airway epithelial cells and immune cells. In this study, we investigated the role of G protein in modulating innate and adaptive immune responses in mice infected with a recombinant virus with deletion of G protein (rhMPV-ΔG). Results show that rhMPV-ΔG was strongly attenuated, as it did not induce significant clinical disease, airway obstruction and airway hyperresponsiveness (AHR), compared to infection with a control strain (rhMPV-WT). By analysis of cells in bronchoalveolar fluid and lung tissue, as well as cytokine production, we found that G protein mediates aspects of both innate and adaptive immune responses, including neutrophils, dendritic cells, natural killer cells and B cells. Lung T cells recruited in response to rhMPV-ΔG had a significantly higher activated phenotype compared to those present after rhMPV-WT infection. Despite highly attenuation characterized by low levels of replication in the lung, rhMPV-ΔG was able to induce neutralizing antibodies and to protect mice from a secondary hMPV challenge. However, challenged mice that had received rhMPV-ΔG as primary infection showed some signs of lung disease at the earliest time points, which were less evident in mice that had received the rhMPV-WT strain as primary infection. These results demonstrate some of the mechanisms by which G protein could contribute to airway disease and modulate immune response to hMPV infection.

Antiviral activity of extracellular vesicles derived from respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children and elderly. No vaccine or effective treatment is currently available for RSV. Extracellular vesicles (EVs) are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins (i.e. cargo). Viral infections can induce profound changes in EV cargo, and the cargo can modulate cellular responses of recipient cells. We have recently shown that EVs isolated from RSV-infected cells were able to activate innate immune response by inducing cytokine and chemokine release from human monocytes and airway epithelial cells, however, we did not investigate the potential antiviral contribution of EVs to a subsequent infection. The objective of this study was to assess the presence of innate immune mediators, including type I and III interferons (IFNs) in EVs released from airway epithelial cells infected with RSV, and their potential role in modulating viral replication in recipient cells. EV-derived from cells infected with RSV were associated with significant amounts of cytokine and chemokines, as well as IFN-ß and -λ, compared to EVs isolated from mock-infected cells. Cells treated with RSV-EVs showed significantly lower levels of viral replication compared to untreated or mock-EV-treated RSV infected cells. Cellular pretreatment with Cerdulatinib, an IFN receptor signaling inhibitor, inhibited the antiviral activity of RSV-EVs in recipient airway epithelial cells. Furthermore, treatment of A549 cells with RSV-EVs induced the expression of IFN-dependent antiviral genes, supporting the idea that RSV-EVs exerts their antiviral activity through an interferon-dependent mechanism. Finally, we determined the concentrations of soluble and EV-associated IFN-ß and IFN-λ in five nasopharyngeal secretions (NPS) of children with viral infections. There were significant levels of IFN-λ in NPS and NPS-derived EVs, while IFN-ß was not detected in either of the two types of samples. EVs released from RSV-infected cells could represent a potential therapeutic approach for modulating RSV replication in the airways.

Hydrogen Sulfide Donor GYY4137 Rescues NRF2 Activation in Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) can cause severe respiratory illness in infants, immunocompromised, and older adults. Despite its burden, no vaccine or specific treatment is available. RSV infection is associated with increased reactive oxygen species (ROS) production, degradation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), and decreased antioxidant enzymes (AOEs), leading to oxidative damage and lung injury. Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays a physiological role in numerous cellular processes and a protective role in multiple pathological conditions, displaying vasoactive, cytoprotective, anti-inflammatory, and antioxidant activities. H2S can promote NRF2 activation through the sulfhydration of Kelch-like ECH-associated protein 1, the cytoplasmic repressor of NRF2. Here we investigated whether increasing cellular H2S levels could rescue NRF2 and NRF2-dependent gene expression in RSV-infected primary airway epithelial cells. We found that treatment with the H2S donor GYY4137 significantly increased NRF2 levels and AOEs gene expression by decreasing KEAP1 levels, and by modulating pathways involved in RSV-induced NRF2 degradation, such as NRF2 ubiquitination, and promyelocytic leukemia (PML) protein levels. These results suggest that the administration of exogenous H2S can positively impact the altered redox balance associated with RSV infection, which represents an important determinant of RSV-induced lung disease.