Rhinovirus (RV) is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations, and primarily infects bronchial epithelial cells. Immune responses from BECs to RV infection are critical in limiting viral replication, and remain unclear in COPD. The objective of this study is to investigate innate immune responses to RV infection in COPD primary BECs (pBECs) in comparison to healthy controls.
Primary bronchial epithelial cells (pBECs) from subjects with COPD and healthy controls were infected with RV-1B. Cells and cell supernatant were collected and analysed using gene expression microarray, qPCR, ELISA, flow cytometry and titration assay for viral replication.
COPD pBECs responded to RV-1B infection with an increased expression of antiviral and pro-inflammatory genes compared to healthy pBECs, including cytokines, chemokines, RNA helicases, and interferons (IFNs). Similar levels of viral replication were observed in both disease groups; however COPD pBECs were highly susceptible to apoptosis. COPD pBECs differed at baseline in the expression of 9 genes, including calgranulins S100A8/A9, and 22 genes after RV-1B infection including the signalling proteins pellino-1 and interleukin-1 receptor associated kinase 2. In COPD, IFN-β/λ1 pre-treatment did not change MDA-5/RIG-I and IFN-β expression, but resulted in higher levels IFN-λ1, CXCL-10 and CCL-5. This led to reduced viral replication, but did not increase pro-inflammatory cytokines.
COPD pBECs elicit an exaggerated pro-inflammatory and antiviral response to RV-1B infection, without changing viral replication. IFN pre-treatment reduced viral replication. This study identified novel genes and pathways involved in potentiating the inflammatory response to RV in COPD.
COPD; Immune response; Viral infection; Gene expression
IL-5 is a pleiotropic cytokine that promotes eosinophil differentiation and survival. While naïve bronchial epithelial cells (BEC) produce low levels of IL-5, the role of BEC-derived IL-5 in allergic airway inflammation is unknown. We now show that BEC, isolated from mice with OVA-induced allergic airway disease (AAD), produced elevated levels of IL-5 mRNA and protein as compared to BEC from naïve mice. To determine the contribution of BEC-derived IL-5 to effector responses in the airways, IL-5 deficient bone marrow chimeric mice were generated in which IL-5 expression was restricted to stromal (e.g. BEC) or hematopoietic cells. When subjected to AAD, IL-5 produced by BECs contributed to mucous metaplasia, airway eosinophilia, and OVA-specific IgA levels. Thus, IL-5 production by BEC can impact the microenvironment of the lung, modifying pathologic and protective immune responses in the airways.
IL-5; asthma; mucous metaplasia; airway eosinophilia; IgA
Respiratory syncytial virus (RSV)-induced bronchiolitis in infants, although inflammatory in nature, is not responsive to glucocorticoids. We have recently shown that RSV-infected lung epithelial cells have impaired glucocorticoid receptor (GR)-mediated transactivation. In this study, we show that the N-terminal region of GR is required for RSV repression of GR transactivation and that RSV infection of lung epithelial cells reduces ligand-dependent GR phosphorylation at serine 211 and serine 226. However, we also show that these changes in GR phosphorylation do not account for the RSV repression of GR transactivation suggesting other regions of the GR N-terminus must also be involved.
•GR N-terminus is essential for RSV suppression of GR-induced transactivation.•RSV infection inhibits dexamethasone-induced GR phosphorylation.•Protein phosphatase inhibitor reverses RSV suppression of GR phosphorylation.•Protein phosphatase inhibition does not reverse RSV effects on GR function.•Mutation of GR phosphorylation sites does not reverse RSV effects on GR function.
Respiratory syncytial virus; Glucocorticoid receptor; Transactivation; Phosphorylation; Protein phosphatase
Innate antiviral responses in bronchial epithelial cells (BECs) provide the first line of defense against respiratory viral infection and the effectiveness of this response is critically dependent on the type I interferons (IFNs). However the importance of the antiviral responses in BECs during influenza infection is not well understood. We profiled the innate immune response to infection with H3N2 and H5N1 virus using Calu-3 cells and primary BECs to model proximal airway cells. The susceptibility of BECs to influenza infection was not solely dependent on the sialic acid-bearing glycoprotein, and antiviral responses that occurred after viral endocytosis was more important in limiting viral replication. The early antiviral response and apoptosis correlated with the ability to limit viral replication. Both viruses reduced RIG-I associated antiviral responses and subsequent induction of IFN-β. However it was found that there was constitutive release of IFN-β by BECs and this was critical in inducing late antiviral signaling via type I IFN receptors, and was crucial in limiting viral infection. This study characterizes anti-influenza virus responses in airway epithelial cells and shows that constitutive IFN-β release plays a more important role in initiating protective late IFN-stimulated responses during human influenza infection in bronchial epithelial cells.
Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor–dependent inflammatory responses, largely through P2X7. Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X7 to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X7 messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X7 was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2′-3′-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro–IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X7 in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing.
P2X7; IL-1β; human rhinovirus; respiratory epithelium; danger signaling
Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells.
Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication.
RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity.
Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.
It is unknown whether nutritional deficiencies affect the morphology and function of structural cells, such as epithelial cells, and modify the susceptibility to viral infections. We developed an in vitro system of differentiated human bronchial epithelial cells (BEC) grown either under selenium adequate (Se+) or selenium deficient (Se-) conditions, to determine whether selenium deficiency impairs host defense responses at the level of the epithelium. Se- BECs had normal SOD activity, but decreased activity of the selenium-dependent enzyme GPX1. Interestingly, catalase activity was also decreased in Se- BECs. Both Se- and Se+ BECs differentiated into a mucociliary epithelium; however, Se- BEC demonstrated increased mucus production and increased Muc5AC mRNA levels. This effect was also seen in Se+ BEC treated with 3-aminotriazole, and inhibitor of catalase activity, suggesting an association between catalase activity and mucus production. Both Se- and Se+ were infected with influenza A/Bangkok/1/79 and examined 24 hours post-infection. Influenza-induced IL-6 production was greater while influenza-induced IP-10 production was lower in Se- BECs. In addition, influenza-induced apoptosis was greater in Se- BEC as compared to the Se+ BECs. These data demonstrate that selenium deficiency has a significant impact on the morphology and influenza-induced host defense responses in human airway epithelial cells.
Influenza; selenium; bronchial epithelial cells; in vitro
Maternal smoking during pregnancy increases the risk of respiratory disease in offspring, but surprisingly little is known about the underlying mechanisms. Nicotinic acetylcholine receptors (nAChRs) expressed in bronchial epithelial cells (BECs) mediate the effects of nicotine on lung development and function. Recently, BECs were also shown to express a GABAergic paracrine loop that was implicated in mucus overproduction in asthma. We therefore investigated the interactions between cholinergic and GABAergic signaling in rhesus macaque BECs, and found that nicotine upregulated GABA signaling in BECs through the sequential activation of BEC nAChR and GABA receptors. The incubation of primary cultures of rhesus BECs increased concentrations of GAD, GABAA receptors, and mucin mRNA. The nicotine-induced increase in glutamatic acid decarboxylase (GAD) and GABAA receptor mRNA resulted in increased GABA-induced currents and increased expression of mucin. The ability of nicotine to increase mucin expression was blocked by nicotinic and GABAA antagonists. These results implicate GABA signaling as a middleman in nicotine's effects on mucus overproduction. Similar effects of nicotine on GABA signaling and the expression of mucin were seen in vivo after chronic exposure of rhesus monkeys to nicotine. These data provide a new mechanism linking smoking with the increased mucin seen in asthma and chronic obstructive pulmonary disorder, and suggest a new paradigm of communication between non-neuronal transmitter systems in BECs. The existence of neural-like transmitter interactions in BECs suggests that some drugs active in the central nervous system may possess previously unexpected utility in respiratory diseases.
smoking; nicotine; GABA; nicotinic receptor; mucin
Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.
Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.
Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.
Respiratory syncytial virus is a leading cause of lower respiratory tract illness among infants, the elderly and immunocompromised individuals. Currently, there is no effective vaccine or disease modifying treatment available and novel interventions are urgently required. Cathelicidins are cationic host defence peptides expressed in the inflamed lung, with key roles in innate host defence against infection. We demonstrate that the human cathelicidin LL-37 has effective antiviral activity against RSV in vitro, retained by a truncated central peptide fragment. LL-37 prevented virus-induced cell death in epithelial cultures, significantly inhibited the production of new infectious particles and diminished the spread of infection, with antiviral effects directed both against the viral particles and the epithelial cells. LL-37 may represent an important targetable component of innate host defence against RSV infection. Prophylactic modulation of LL-37 expression and/or use of synthetic analogues post-infection may represent future novel strategies against RSV infection.
Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-α/β) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-α/β receptor.
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. Premature infants, immunocompromised individuals and the elderly exhibit the highest risk for the development of severe RSV-induced disease. Murine studies demonstrate that CD8 T cells mediate RSV clearance from the lungs. Murine studies also indicate that the host immune response contributes to RSV-induced morbidity as T-cell depletion prevents the development of disease despite sustained viral replication. Dendritic cells (DCs) play a central role in the induction of the RSV-specific adaptive immune response. Following RSV infection, lung-resident DCs acquire viral antigens, migrate to the lung-draining lymph nodes and initiate the T-cell response. This article focuses on data generated from both in vitro DC infection studies and RSV mouse models that together have advanced our understanding of how RSV infection modulates DC function and the subsequent impact on the adaptive immune response.
chemokine; cytokine; dendritic cell; respiratory syncytial virus; T cell; vaccine
Respiratory syncytial virus (RSV) is an important cause of acute respiratory disease in infants, immunocompromised subjects and the elderly. However, it is unclear why most primary RSV infections are associated with relatively mild symptoms, whereas some result in severe lower respiratory tract infections and bronchiolitis. Since RSV hospitalization has been associated with respiratory bacterial co-infections, we have tested if bacterial Toll-like receptor (TLR) agonists influence RSV-A2-GFP infection in human primary cells or cell lines. The synthetic bacterial lipopeptide Pam3-Cys-Ser-Lys4 (Pam3CSK4), the prototype ligand for the heterodimeric TLR1/TLR2 complex, enhanced RSV infection in primary epithelial, myeloid and lymphoid cells. Surprisingly, enhancement was optimal when lipopeptides and virus were added simultaneously, whereas addition of Pam3CSK4 immediately after infection had no effect. We have identified two structurally related lipopeptides without TLR-signaling capacity that also modulate RSV infection, whereas Pam3CSK4-reminiscent TLR1/2 agonists did not, and conclude that modulation of infection is independent of TLR activation. A similar TLR-independent enhancement of infection could also be demonstrated for wild-type RSV strains, and for HIV-1, measles virus and human metapneumovirus. We show that the effect of Pam3CSK4 is primarily mediated by enhanced binding of RSV to its target cells. The N-palmitoylated cysteine and the cationic lysines were identified as pivotal for enhanced virus binding. Surprisingly, we observed inhibition of RSV infection in immortalized epithelial cell lines, which was shown to be related to interactions between Pam3CSK4 and negatively charged glycosaminoglycans on these cells, which are known targets for binding of laboratory-adapted but not wild-type RSV. These data suggest a potential role for bacterial lipopeptides in enhanced binding of RSV and other viruses to their target cells, thus affecting viral entry or spread independent of TLR signaling. Moreover, our results also suggest a potential application for these synthetic lipopeptides as adjuvants for live-attenuated viral vaccines.
Respiratory syncytial virus (RSV) infections are an important cause of hospitalization of infants during the winter season. However, RSV is often not the only detectable pathogen, but co-infections with respiratory bacteria are common. It has been hypothesized that this results from epithelial damage caused by the virus, facilitating colonization by pathogenic bacteria such as Streptococcus pneumoniae. However, an inverse order of events is not impossible: bacterial infections may activate respiratory epithelial cells through TLR signaling, resulting in increased susceptibility to virus infections. We tested this hypothesis by screening bacterial TLR agonists for their capacity to modulate RSV infection in different cell types, and identified the lipopeptide and prototype TLR1/2 agonist Pam3CSK4 as an enhancer of RSV infections. However, to our surprise this proved independent of TLR activation, but was mediated by enhancement of binding between virus and target cell. Two structurally related lipopeptides unable to stimulate TLR responses were identified that enhanced infections with RSV, but also with other enveloped viruses including HIV-1, human metapneumovirus, and measles virus. We speculate that bacterial infections may influence the pathogenesis of virus infections by facilitating binding to target cells.
Respiratory syncytial virus (RSV) is responsible for significant morbidity and mortality, particularly in infants younger than 18 months and in the elderly. To date, there are few effective treatment options available to prevent or treat RSV infections. Attractive therapeutic strategies include targeting host epithelial adhesion molecules required for RSV infection, enhancing localized cell-mediated immunity, interfering with RSV viral gene expression and developing a multigene DNA vaccine. The most recent data supporting the advantages and limitations of each of these approaches are discussed in detail. Several promising strategies offer hope for safe and effective prophylaxis and treatment of RSV infection.
respiratory syncytial virus infection; allergic disease; chitosan; nanoparticles
Respiratory viruses, including respiratory syncytial virus (RSV), can cause asthma exacerbations and bronchiolitis. Both conditions are associated with enhanced cognate immune responses and inflammation and reduced immune regulation. Lung epithelial cells (LECs) can contribute to antiviral and allergic immune responses while gut epithelial cells can inhibit effector T cell responses. A study was performed to determine whether healthy LECs regulate antigen-specific T cell responses and if this regulation is lost during RSV infection.
LA4 cells, a murine LEC line, infected with RSV or primary murine LECs were co-cultured with ovalbumin-specific T cell receptor transgenic CD4+ T cells from DO11.10 mice and ovalbumin-pulsed bone marrow-derived dendritic cells (DC) to assess T cell proliferation by flow cytometry and cytokine production.
The presence of LECs abrogated DC-induced T cell proliferation and significantly reduced T cell cytokine release. These effects of LECs were predominantly contact-dependent, primarily affected T cells directly and were partly mediated by transforming growth factor β. Soluble factors and DC-mediated effects also contributed to T cell inhibition. RSV infection of LECs reduced their inhibitory capacity in an infection dose-dependent manner. This was independent of proinflammatory cytokines released by infected LECs, but in part due to Toll-like receptor activation and to infection-induced cell death.
Healthy LECs are potent inhibitors of T cell activation, but this regulatory function is lost after RSV infection. These findings suggest a central role for LECs in maintaining the tolerogenic environment of healthy lungs. Loss of this regulatory capacity after viral infection may allow development of excessive cognate immune responses and pulmonary inflammation.
Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality in infants and the elderly and is a continuing challenge for vaccine development. A murine T helper cell (Th) type 2 response associates with enhanced lung pathology, which has been observed in past infant trials using formalin-inactivated RSV vaccine. In this study, we have engineered an optimized plasmid DNA vector expressing the RSV fusion (F) protein (DNA-F). DNA-F was as effective as live RSV in mice at inducing neutralizing antibody and cytotoxic T lymphocyte responses, protection against infection, and high mRNA expression of lung interferon γ after viral challenge. Furthermore, a DNA-F boost could switch a preestablished anti-RSV Th2 response towards a Th1 response. Critical elements for the optimization of the plasmid constructs included expression of a secretory form of the F protein and the presence of the rabbit β-globin intron II sequence upstream of the F-encoding sequence. In addition, anti-F systemic immune response profile could be modulated by the route of DNA-F delivery: intramuscular immunization resulted in balanced responses, whereas intradermal immunization resulted in a Th2 type of response. Thus, DNA-F immunization may provide a novel and promising RSV vaccination strategy.
respiratory syncytial virus; DNA vaccine; vector design; F protein; immune modulation
Respiratory syncytial virus (RSV) infection is the leading viral cause of severe lower respiratory tract illness in young infants. Clinical studies have documented that certain polymorphisms in the gene encoding the regulatory cytokine IL-10 are associated with the development of severe bronchiolitis in RSV infected infants. Here, we examined the role of IL-10 in a murine model of primary RSV infection and found that high levels of IL-10 are produced in the respiratory tract by anti-viral effector T cells at the onset of the adaptive immune response. We demonstrated that the function of the effector T cell -derived IL-10 in vivo is to limit the excess pulmonary inflammation and thereby to maintain critical lung function. We further identify a novel mechanism by which effector T cell-derived IL-10 controls excess inflammation by feedback inhibition through engagement of the IL-10 receptor on the antiviral effector T cells. Our findings suggest a potentially critical role of effector T cell-derived IL-10 in controlling disease severity in clinical RSV infection.
IL-10 is a major anti-inflammatory protein that plays an essential role in regulating the balance between pathogen clearance by the immune response and immune mediated injury resulting from the immune response to pathogen infection. In this report, we demonstrate that anti-viral effector T cells, a critical cell type responsible for respiratory syncytial virus clearance, are able to produce a large quantity of IL-10. The function of IL-10 is to control the immune response in order to avoid the development of excessive pulmonary inflammation associated with the clearance of infectious virus. We further identified a likely mechanism that T cell-derived IL-10 operates to control inflammation and describe a novel potential target of IL-10 action in the RSV infected lungs. Our data thus may lay the ground for the future studies exploring the application of IL-10 in therapeutic approaches to modulate pulmonary inflammation and injury in young infants suffering severe respiratory syncytial virus induced diseases.
Singh D, McCann KL, Imani F. MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption. Am J Physiol Lung Cell Mol Physiol 293: L436–L445, 2007. First published June 8, 2007; doi:10.1152/ajplung.00097.2007.—Respiratory syncytial virus (RSV) is the major cause of bronchiolitis in infants, and a common feature of RSV infections is increased lung permeability. The accumulation of fluid in the infected lungs is caused by changes in the endothelial and epithelial membrane integrity. However, the exact mechanisms of viral-induced fluid extravasation remain unclear. Here, we report that infection of human epithelial cells with RSV results in significant epithelial membrane barrier disruption as assessed by a decrease in transepithelial electrical resistance (TEpR). This decrease in TEpR, which indicates changes in paracellular permeability, was mediated by marked cellular cytoskeletal rearrangement. Importantly, the decrease in TEpR was attenuated by using p38 MAPK inhibitors (SB-203580) but was partially affected by JNK inhibitor SP-600125. Interestingly, treatment of A549 cells with MEK1/2 inhibitor (U-0126) led to a decrease in TEpR in the absence of RSV infection. The changes in TEpR were concomitant with an increase in heat shock protein 27 (Hsp27) phosphorylation and with actin microfilament rearrangement. Thus our data suggest that p38 MAPK and Hsp27 are required for RSV induction of human epithelial membrane permeability.
lung permeability; mitogen-activated protein kinase
Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants worldwide. Severe RSV infections in infants cause bronchiolitis, wheeze, and/or cough and significantly increase the risk of developing asthma. RSV pathogenesis is thought to be due to a Th2-type immune response initiated in response to RSV infection specifically in the infant. Using a neonatal mouse system as an appropriate model for human infants, we sought to determine if local inhibition of IL-4Rα expression during primary RSV infection in the neonate would prevent Th2-skewed responses to secondary RSV infection and improve long-term pulmonary function. To reduce IL-4Rα expression, antisense oligonucleotides (ASO) specific for IL-4Rα were administered intranasally to neonatal mice at the time of primary infection. Mice were initially infected with RSV at one week of age and reinfected at six weeks of age. Administration of IL-4Rα ASO during primary RSV infection in neonatal mice abolished the pulmonary dysfunction normally observed following reinfection in the adult. This ablation of pulmonary dysfunction correlated with a persistent rebalancing of the Th cell compartment with decreased Th2 responses (i.e. reduced goblet cell hyperplasia and Th2 cells and cytokine secretion) and increased Th1 responses (i.e. elevated Th1 cell numbers and type I antibodies and cytokines). Our data support our hypothesis that a reduction in the Th2 immune response during primary infection in neonates prevents Th2-mediated pulmonary pathology initially and upon reinfection; and further suggest that vaccine strategies incorporating IL-4Rα ASO may be of significant benefit to infants.
Asthma inception is associated with respiratory viral infection, especially infection with respiratory syncytial virus (RSV) and/or human rhinovirus (HRV), in the vast majority of cases. However, the reason why RSV and HRV induce the majority of bronchiolitis cases during early childhood and why only a small percentage of children with RSV- and HRV-induced bronchiolitis later develop asthma remains unclear. A genetic association study has revealed the important interaction between viral illness and genetic variants in patients with asthma. Severe RSV- and HRV-induced bronchiolitis may be associated with a deficiency in the innate immune response to RSV and HRV. RSV and HRV infections in infants with deficient innate immune response and the dysfunction of regulatory T cells are considered to be a risk factor for the development of asthma. Sensitization to aeroallergens, beginning in the first year of life, consistently predisposes children to HRV-induced wheezing illnesses, but the converse is not true. Some evidence of virus specificity exists, in that allergic sensitization specifically increased the risk of wheezing in individuals infected with HRV, but not RSV. Administration of Palivizumab, a humanized monoclonal antibody that targets the A antigenic site of the Fusion-protein of RSV, decreases the risk of hospitalization in high-risk infants and the risk of recurrent of wheezing. However, palivizumab did not have any effect on subsequent recurrent wheezing in children with a family history of atopy. These findings suggest that infection with RSV and infection with HRV might predispose individuals to recurrent wheezing through an atopy-independent and an atopy-dependent mechanism, respectively. Respiratory virus-induced wheezing illnesses may encompass multiple sub-phenotypes that relate to asthma in different ways.
respiratory syncytial virus; human rhinovirus; virus-induced asthma; cellular immunity; humoral immunity
Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract illness in infants, the effect of RSV on human airway smooth muscle (HASM) has not been studied. We hypothesized that RSV has direct effects on cAMP formation and β2-adrenergic receptor (ADRB2) density and that ADRB2 haplotype influences this response. A recombinant green-fluorescent protein (rg) expressing RSV was used to determine whether RSV could infect cultured HASM. Influence of RSV infection on β2-adrenergic responsiveness was determined by measuring differences in isoproterenol (ISO)-induced cyclic AMP (cAMP) formation, ADRB2 density, and Gi expression in HASM cells challenged with RSV, with ultraviolet-inactivated RSV, and with mock infection. The rgRSV efficiently infected cultured HASM cells. ISO-induced cAMP formation was significantly reduced in cells infected with RSV, compared with mock-infected and ultraviolet-inactivated RSV, in a time- and concentration-dependent manner. Forskolin-induced cAMP formation and Gi expression were not altered in cells infected with RSV, suggesting that the influence of RSV on β2-adrenergic relaxation was upstream of cAMP formation. ADRB2 density was reduced in cells infected with RSV, compared with mock infection, and the Arg16Gln27 ADRB2 haplotype was associated with decreased ISO-induced cAMP formation (P < 0.05) and with decreased ADRB2 density at baseline (P < 0.05). The implications of these results are that limitations of β2-agonists in the treatment of any airway obstruction associated with RSV infection may be related to direct effects of RSV on HASM, and ADRB2 genotype may predict β2-adrenergic responses.
β2-adrenergic receptor; haplotype; human airway smooth muscle; isoproterenol; respiratory syncytial virus
Respiratory syncytial virus (RSV) is a major cause of morbidity in infants, young children, and the elderly worldwide. Currently, there is no effective vaccine, and antiviral drugs to control infection are limited. RNA interference is a powerful tool amenable to development of antiviral drugs. Using small interfering RNA (siRNA) targeting the RSV P gene (siRNA-P), RSV replication can be silenced both in vitro and in a BALB/c model of RSV infection. In this study, we examine the effect of siRNA prophylaxis on the primary and memory immune response to RSV infection in mice. We show that mice prophylactically treated with siRNA-P to decrease but not eliminate RSV replication exhibit reduced pulmonary inflammation and lung pathogenesis and produce a robust anti-RSV memory response when subsequently challenged with RSV. The pulmonary T-cell memory response was characterized by high numbers of CD44hi CD62Llo CD4+ and CD8+ T cells, M2 peptide tetramer+ CD8+ T cells expressing gamma interferon, and an RSV-specific antibody response. The results support the hypothesis that siRNAs can be developed as effective antiviral drugs that can be used to reduce the viral load and parameters of pathogenesis without limiting the induction of the memory immune response.
Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and viral pneumonia in young children and a serious health risk in immunocompromised individuals and the elderly. Immunity to RSV is not completely understood. In this work, we established a method for monitoring RSV infection by real-time PCR and applied this method for analysis of RSV replication in vivo in the cotton rat model in naïve animals and in animals rendered immune to RSV by prior RSV infection. We found that even though no virus could be isolated from the lungs of RSV-challenged immune animals, RSV infection in fact took place and an accumulation of viral RNA transcripts was observed. This type of replication, therefore, can be termed “abortive,” as RSV is capable of entering the cells in the lungs of immune animals, yet the production of progeny viruses is impaired. Similar patterns of RSV gene expression gradient were observed between naïve and reinfected animals, indicating that the skewing of mRNA gradient of viral gene expression, a mechanism documented during latent infection by other viruses, is not likely to be responsible for abortive replication of RSV during reinfection. We found that passive administration of antibodies to RSV prevents productive infection normally accompanied by viral release in the lung, but it does not prevent abortive replication of the virus. To the best of our knowledge, this is the first evidence of abortive replication of RSV in vivo.