TNF-α and Macrophages Are Critical for Respiratory Syncytial Virus-Induced Exacerbations in a Mouse Model of Allergic Airways Disease.

Viral respiratory infections trigger severe exacerbations of asthma, worsen disease symptoms, and impair lung function. To investigate the mechanisms underlying viral exacerbation, we established a mouse model of respiratory syncytial virus (RSV)-induced exacerbation after allergen sensitization and challenge. RSV infection of OVA-sensitized/challenged BALB/c mice resulted in significantly increased airway hyperresponsiveness (AHR) and macrophage and neutrophil lung infiltration. Exacerbation was accompanied by increased levels of inflammatory cytokines (including TNF-α, MCP-1, and keratinocyte-derived protein chemokine [KC]) compared with uninfected OVA-treated mice or OVA-treated mice exposed to UV-inactivated RSV. Dexamethasone treatment completely inhibited all features of allergic disease, including AHR and eosinophil infiltration, in uninfected OVA-sensitized/challenged mice. Conversely, dexamethasone treatment following RSV-induced exacerbation only partially suppressed AHR and failed to dampen macrophage and neutrophil infiltration or inflammatory cytokine production (TNF-α, MCP-1, and KC). This mimics clinical observations in patients with exacerbations, which is associated with increased neutrophils and often poorly responds to corticosteroid therapy. Interestingly, we also observed increased TNF-α levels in sputum samples from patients with neutrophilic asthma. Although RSV-induced exacerbation was resistant to steroid treatment, inhibition of TNF-α and MCP-1 function or depletion of macrophages suppressed features of disease, including AHR and macrophage and neutrophil infiltration. Our findings highlight critical roles for macrophages and inflammatory cytokines (including TNF-α and MCP-1) in viral-induced exacerbation of asthma and suggest examination of these pathways as novel therapeutic approaches for disease management.

Lipopolysaccharide induces IL-6 production in respiratory syncytial virus-infected airway epithelial cells through the toll-like receptor 4 signaling pathway.

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis in young children. Microbial agents such as endotoxin and RSV are implicated in airway inflammation during the development of reactive airway disease (RAD) later in childhood. Toll-like receptors (TLRs) are involved in an inflammation cascade through pathogen-associated molecular pattern recognition including lipopolysaccharide (LPS) and viral components. In this study, we investigated the expression of TLRs and cytokine-chemokine production profiles of RSV-infected epithelial cells. In live-RSV infected human tracheal epithelial cell line (9HTEo), TLRs 1-10 mRNA levels were up-regulated in a time-dependent manner compared with ultraviolet (UV)-inactivated RSV. RSV was shown to alter TLR4 membrane and cytosolic location in epithelial cells. Stimulating RSV-infected epithelial cells with TLR4 agonist LPS increased synthesis of IL-6, IL-8, and reduced regulated on activation, normal T cell expressed and secreted (RANTES) production. TLR4 neutralizing antibody HTA125 and TLR4-targeting RNA interference experiments revealed that TLR4 signaling pathway played a predominant role in mediating LPS-induced-IL-6 production of RSV infected epithelial cells. Altogether, our studies indicated that TLR4 play a critical role in leading LPS mediated-IL-6 response in RSV infected-epithelial cells and might be an important factor influencing the cytokine-chemokine profile of epithelial cells interacting with virus and endotoxin, which is correlated with phenotypes of RSV diseases.

Interleukin-8 mRNA synthesis and protein secretion are continuously up-regulated by respiratory syncytial virus persistently infected cells.

The aim of this study was to investigate whether respiratory syncytial virus persistence regulates interleukin 8 (IL-8) mRNA synthesis and protein secretion in a human lung epithelial cell line (A549). Therefore, we established RSV persistence in these cells (A549per) and determined the levels of interleukin-8 mRNA by RT-PCR and of protein through ELISA. Interleukin-8 mRNA synthesis and protein secretion were continuously up-regulated in A549per cells during passages and in A549 cells that had been incubated with supernatants (cA549per) obtained from A549per passages. These results suggested that the enhancement of interleukin-8 was stimulated either by the presence of the RSV genome in the cell or by soluble mediator(s) induced by RSV, which, in turn, increased interleukin-8 mRNA synthesis and protein secretion. Soluble RSV F and G proteins were identified as mediators. Moreover, interleukin-8 enhancement was observed after 1-min incubation with the soluble mediators, thus suggesting that interleukin-8 up-regulation was triggered by receptor-ligand interaction.

Replication-dependent potent IFN-alpha induction in human plasmacytoid dendritic cells by a single-stranded RNA virus.

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-alpha induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-alpha induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-alpha induction in PDC. HRSV-induced IFN-alpha production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-alpha by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-alpha induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.

Unilateral nasal infection of cotton rats with respiratory syncytial virus allows assessment of local and systemic immunity.

An in vivo model for the study of local and systemic effectors of immunity to respiratory syncytial virus (RSV) is described. Cotton rats (Sigmodon fulviventer) inoculated in one nostril with a small volume (2 microliters) of virus suspension contracted a unilateral nasal infection which did not extend to the contralateral nasal turbinates, nor to the lungs. Immunity to subsequent RSV challenge could be induced by small priming doses ( < 10 p.f.u. per animal), but was dependent upon viral replication, as virus inactivated by UV light was not immunogenic. Immunity occurred in the absence of detectable neutralizing serum antibody. The onset of resistance to viral challenge occurred simultaneously in ipsilateral nasal, contralateral nasal and pulmonary tissues. However, low levels of transient viral replication occurred in contralateral nasal turbinates and in lungs following virus challenge, thus indicating that local components of immunity acting at the ipsilateral site of infection were more effective than systemic components acting at the other sites. Further evidence is provided to suggest that three types of immunological effectors - local, persistent, systemic and transient systemic - participate in the immune response to RSV infection.