Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

CD38 modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells.

Respiratory syncytial virus (RSV) is the most common cause of hospitalization due to bronchiolitis in infants. Although the mechanisms behind this association are not completely elucidated, they appear to involve an excessive immune response causing lung pathology. Understanding the host response to RSV infection may help in the identification of targets for therapeutic intervention. We infected in-vitro human monocyte-derived dendritic cells (DCs) with RSV and analysed various aspects of the cellular response. We found that RSV induces in DCs the expression of CD38, an ectoenzyme that catalyses the synthesis of cyclic ADPR (cADPR). Remarkably, CD38 was under the transcriptional control of RSV-induced type I interferon (IFN). CD38 and a set of IFN-stimulated genes (ISGs) were inhibited by the anti-oxidant N-acetyl cysteine. When CD38-generated cADPR was restrained by 8-Br-cADPR or kuromanin, a flavonoid known to inhibit CD38 enzymatic activity, RSV-induced type I/III IFNs and ISGs were markedly reduced. Taken together, these results suggest a key role of CD38 in the regulation of anti-viral responses. Inhibition of CD38 enzymatic activity may represent an encouraging approach to reduce RSV-induced hyperinflammation and a novel therapeutic option to treat bronchiolitis.

Elevation of Serum Acid Sphingomyelinase Activity in Children with Acute Respiratory Syncytial Virus Bronchiolitis.

Acid sphingomyelinase (ASM) is a lysosomal enzyme that hydrolyzes sphingomyelin into ceramide, a bioactive lipid to regulate cellular physiological functions. Thus, ASM activation has been reported as a key event in pathophysiological reactions including inflammation, cytokine release, oxidative stress, and endothelial damage in human diseases. Since ASM activation is associated with extracellular ASM secretion through unknown mechanisms, it can be detected by recognizing the elevation of secretory ASM (S-ASM) activity. Serum S-ASM activity has been reported to increase in chronic diseases, acute cardiac diseases, and systemic inflammatory diseases. However, the serum S-ASM has not been investigated in common acute illness. This study was designed to evaluate serum S-ASM activity in children with common acute illness. Fifty children with common acute illness and five healthy children were included in this study. The patients were categorized into five groups based on clinical diagnoses: acute respiratory syncytial virus (RSV) bronchiolitis, adenovirus infection, streptococcal infection, asthma, and other infections due to unknown origin. The serum S-ASM activity was significantly elevated at 6.9 ± 1.6 nmol/0.1 mL/6 h in the group of acute RSV bronchiolitis patients compared with healthy children who had a mean level of 1.8 ± 0.8 nmol/0.1 mL/6 h (p < 0.05). In the other illness groups, the serum S-ASM activity was not significantly elevated. The results suggest an association of ASM activation with RSV infection, a cause for common acute illness. This is the first report to describe the elevation of serum S-ASM activity in respiratory tract infection.

Downregulation of matrix metalloproteinase­19 induced by respiratory syncytial viral infection affects the interaction between epithelial cells and fibroblasts.

The present study was designed to examine the expression and function of matrix metalloproteinase­19 (MMP­19), which is downregulated following respiratory syncytial virus (RSV) infection. The diverse expression levels of MMP were examined using a designed cDNA expression array. The expression and secretion of MMP­19 was examined using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis and ELISA, respectively. The proliferation of epithelial cells and lung fibroblasts were examined using flow cytometry. The epithelial­mesenchymal transition (EMT) was also examined by performing western blot and RT­qPCR analyses. The results of the cDNA assay showed that infection with RSV resulted in the abnormal expression of certain metalloproteinases. Among these, the expression of MMP­19 decreased 3 and 7 days following infection. By using flow cytometric, western blot and RT­qPCR analyses, the present study demonstrated that the downregulation of MMP­19 inhibited the proliferation of epithelial cells, promoted the EMT and induced the proliferation of lung fibroblasts. Taken together, the findings of the present study suggested that the downregulation of MMP­19 following RSV infection may be associated with the development of airway hyper­responsiveness.

Increased hydroxymethylglutaryl coenzyme A reductase activity during respiratory syncytial virus infection mediates actin dependent inter-cellular virus transmission.

We have examined the role that hydroxymethylglutaryl coenzyme A reductase (HMGCR) plays during respiratory syncytial virus (RSV) maturation. Imaging analysis indicated that virus-induced changes in F-actin structure correlated with the formation of virus filaments, and that these virus filaments played a direct role in virus cell-to-cell transmission. Treatment with cytochalasin D (CYD) prevented virus filament formation and virus transmission, but this could be reversed by removal of CYD. This observation, together with the presence of F-actin within the virus filaments suggested that newly polymerised F-actin was required for virus transmission. The virus-induced change in F-actin was inhibited by the HMGCR inhibitor lovastatin, and this correlated with the inhibition of both virus filament formation and the incorporation of F-actin in these virus structures. Furthermore, this inhibitory effect on virus filament formation correlated with a significant reduction in RSV transmission. Collectively these data suggested that HMGCR-mediated changes in F-actin structure play an important role in the inter-cellular transmission of mature RSV particles. These data also highlighted the interplay between cellular metabolism and RSV transmission, and demonstrate that this interaction can be targeted using anti-virus strategies.

Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy.

Cyclooxygenases (COXs) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. COX-2 is the inducible isoform that is up-regulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. The roles of cyclooxygenases and their products, PGs, have not been evaluated during respiratory syncytial virus (RSV) infection. In this study we demonstrate that COX-2 is induced by RSV infection of human lung alveolar epithelial cells with the concomitant production of PGs. COX-2 induction was dependent on the dose of virus and the time postinfection. PG production was inhibited preferentially by NS-398, a COX-2-specific inhibitor, and indomethacin, a pan-COX inhibitor, but not by SC-560, a COX-1-specific inhibitor. In vivo, COX-2 mRNA expression and protein production were strongly induced in the lungs and cells derived from bronchioalveolar lavage of cotton rats infected with RSV. The pattern of COX-2 expression in vivo in lungs is cyclical, with a final peak on day 5 that correlates with maximal histopathology. Treatment of cotton rats with indomethacin significantly mitigated lung histopathology produced by RSV. The studies described in this study provide the first evidence that COX-2 is a potential therapeutic target in RSV-induced disease.

IkappaB kinase is a critical regulator of chemokine expression and lung inflammation in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major etiologic agent of severe epidemic lower respiratory tract infections in infancy. Airway mucosal inflammation plays a critical role in the pathogenesis of RSV disease in both natural and experimental infections. RSV is among the most potent biological stimuli that induce the expression of inflammatory genes, including those encoding chemokines, but the mechanism(s) that controls virus-mediated airway inflammation in vivo has not been fully elucidated. Herein we show that the inoculation of BALB/c mice with RSV results in rapid activation of the multisubunit IkappaB kinase (IKK) in lung tissue. IKK transduces upstream activating signals into the rate-limiting phosphorylation (and proteolytic degradation) of IkappaBalpha, the inhibitory subunit that under normal conditions binds to the nuclear factor (NF)-kappaB complex and keeps it in an inactive cytoplasmic form. Mice treated intranasally with interleukin-10 or with a specific cell-permeable peptide that blocks the association of the catalytic subunit IKKbeta with the regulatory protein NEMO showed a striking reduction of lung NF-kappaB DNA binding activity, chemokine gene expression, and airway inflammation in response to RSV infection. These findings suggest that IKKbeta may be a potential target for the treatment of acute or chronic inflammatory diseases of the lung.

Viral induction of inflammatory cytokines in human epithelial cells follows a p38 mitogen-activated protein kinase-dependent but NF-kappa B-independent pathway.

The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-alpha and IL-1beta. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-kappaB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-alpha, but not for IL-1beta. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-alpha and IL-1beta required activation of this signaling pathway. Interestingly, inhibition of NF-kappaB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-kappaB activation may be less important than previously suggested.

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells.

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.

Respiratory syncytial virus infection of human respiratory epithelial cells enhances inducible nitric oxide synthase gene expression.

The induction kinetics of the mRNA of interferon regulatory factor 1 (IRF-1), inducible nitric oxide synthase (iNOS), and proinflammatory cytokines in respiratory syncytial virus (RSV)-infected human type 2 alveolar epithelial cells (A549 cells) were analyzed semiquantitatively by RT-PCR. RSV enhanced IRF-1 and iNOS mRNA expression as early as 4 h after RSV infection and this enhancement lasted several hours. No IFN-gamma gene expression was observed during the whole course of the infection. Expression of IFN-beta, IL-1beta, and TNF-alpha genes was observed slightly at 4 h and became marked 7 h after infection. Addition of neutralizing antibodies to these cytokines to the culture had no effect on the induction of iNOS mRNA. The iNOS transcriptional activity in RSV-infected cells was significantly enhanced by an exogenous cytokine mixture (IL-1beta, TNF-alpha, and IFN-gamma). An apparent nitric oxide (NO) production was identified only when cytokines were added together with RSV infection. A significant increase of iNOS gene expression was observed in nasopharyngeal exudate cells obtained from infants during the acute phase of RSV bronchiolitis. These observations suggest that RSV infection of human respiratory epithelial cells induces the iNOS gene both in vitro and in vivo; this induction may occur rather promptly and involves transcriptional activator IRF-1 induced by the RSV infection itself. The iNOS gene, which is initially induced by RSV infection, may be further enhanced in a paracrine fashion by proinflammatory cytokines released by infection-activated inflammatory cells.