Respiratory viruses are responsible for most acute respiratory illnesses in humans, including the common cold, bronchiolitis, and pneumonia (Johnston, 2004). These major pathogens include human parainfluenza viruses (PIV), rhinoviruses (RV), respiratory syncytial virus (RSV), and influenza viruses (Monto, 2004). Respiratory infections resulting from PIV, RV, and RSV have also been implicated in the induction and exacerbation of asthma (Folkerts et al., 1998; Stein et al., 1999; Azevedo et al., 2003; Kotaniemi-Syrjänen et al., 2003; Martinez, 2003; Matsuse et al., 2005; Hershenson and Johnston, 2006; Khetsuriani et al., 2007; Kusel et al., 2007). Most children are infected at least once with PIV early in life, but reinfections occur throughout life. Serologic surveys indicate that at least 60% of children have been infected with PIV3 by 2 years of age while approximately 80% have been infected by 4 years of age. PIV are associated with 0–12% of acute lower respiratory tract infections in adults. PIV infections may be associated with not only respiratory infections but also the induction and exacerbation of asthma (virus-induced asthma) (Azevedo et al., 2003; Matsuse et al., 2005). However, many children who wheeze with viral infections during infancy will not go on to develop asthma, suggesting a role of virus- and/or host-specific factors in the pathogenesis of the disease.

Most types of cytokines may be associated with the pathophysiology of virus-induced asthma or excessive virus-induced inflammation (Mallia and Johnston, 2006; Proud and Chow, 2006). In extreme cases, an overproduction of cytokines due to viral infection (e.g., influenza virus infection) may induce a cytokine storm resulting in systemic inflammatory response syndrome (SIRS) (Watanabe et al., 2003). Thus, viral infection-induced overproduction of cytokines in vivo might be closely associated with the pathophysiology of various diseases including asthma (Message and Johnston, 2004; Mallia and Johnston, 2006; Proud and Chow, 2006). However, cytokine profiles of viral infections including PIV infection remain largely unknown.

IκB kinase, p38 MAPK, and Akt are major signaling molecules thought to play a pivotal role in most types of cytokine production in various cells (Hiscott et al., 2001; Adcock et al., 2006). Single strand RNA such as viral RNA binds and activates helicase, which further activates IκB kinase pathways, resulting in the production of certain types of cytokine (Lee and Lau, 2007). In addition, it is well known that phosphorylation of p38 MAPK is associated with the production of cytokines in many types of cells (Suzuki et al., 1999; Wong et al., 2005). Thus, these signaling molecules may be associated with the production of cytokines in virus-infected cells, although the details are not as yet known.

Human lung fibroblasts are reported to be susceptible to various respiratory viruses such as PIV and RV (Bousse et al., 2006; Jang et al., 2009). In addition, lung fibroblasts may be associated with remodeling involved in fibrosis in the small airways (Puxeddu et al., 2006). In asthmatics, viral infections are more likely to result in lower respiratory tract infection (Gern, 2009), and moreover, these infections are not restricted to the respiratory epithelium, but can spread to the structural cells such as smooth muscle cells and fibroblasts (Holgate et al., 2004b). From these background, in the present study we chose human fetal lung fibroblasts to examine the cytokine milieu of the asthmatic airway under chronic viral infection. We hypothesized that PIV infection might induce virus-induced asthma-related cytokine production in fetal lung fibroblasts. To explore this possibility, we profiled cytokines released from PIV-infected fetal lung fibroblasts and examined the role of the signaling pathways (IκB kinase, p38 MAPK, and Akt).

We profiled a number of cytokines from PIV-infected fetal lung fibroblasts. Compared with control (no virus) cells, a significant amount of various cytokines were shown to be released, namely, IL-1β, IL-6, TNF-α, IL-1ra, IFN-γ, IL-2, IL-4, IL-5, IL-10, G-CSF, GM-CSF, IL-8, IP-10, eotaxin, RANTES, PDGF-bb, and VEGF. In addition, we found that most aberrantly released cytokines were associated with phosphorylation of IκB kinase and p38 MAPK. Infection by PIV may induce large-scale production of various cytokines in the airway. Although PIV failed to induce IL-12 and IL-17 production, these results indicate that PIV induces a Th2 cytokine milieu as well as eosinophil and neutrophil recruitment to fibroblasts. As a result, Th2 immunity may be augmented, which is likely to be an important mechanism in the pathophysiology of PIV-induced asthma (virus-induced asthma).

It is suggested that maximal MAP kinase phosphorylation and activity is seen within 60 min after stimulation. In this study, the phosphorylation of IκB kinase and p38 MAP kinase was examined at 24 h after PIV infection. Thus, it is possible that cytokines produced by PIV-infected MRC-5 cells might be responsible for the induction of MAP kinase phosphorylation, since various cytokines produced after PIV infection have been shown to induce IκB kinase and MAP kinase phosphorylation.

We speculate that the effects of PIV on normal fibroblasts can be extrapolated to corresponding cells in asthmatic airways. Previous studies have shown that increased viral replication and impaired IFN response to viral infection occurs in primary epithelial cells and peripheral blood cells from asthmatics compared with normal subjects (Wark et al., 2005; Contoli et al., 2006; Gehlhar et al., 2006). The increase in virus infection-induced inflammation is a probable explanation for these observations. Asthmatics may also be more susceptible to infection (Holgate, 2007). Furthermore, it has also been suggested that infants who are born with poor antiviral responses and/or airway hyper-responsiveness are prone to repetitive severe illnesses (Gehlhar et al., 2006), and these same abnormalities may increase the risk of recurrent wheezing and possibly asthma. Therefore, fibroblasts from those who develop asthma in earlier childhood might possess a defective IFN response to these respiratory viruses. Such partially defective innate immune responses may result in augmented Th2 immunity in asthmatic patients as well as increased and prolonged airway inflammation (Barrett and Austen, 2009).

A direct causative role for these respiratory viruses in the development of asthma has not been proven yet. Lemanske et al. (2005) suggested at least two potential mechanisms for the development of recurrent wheezing in early childhood. First, healthy infants who undergo repetitive severe viral respiratory infections could develop recurrent wheezing because of lung damage and/or airway remodeling. Second, as mentioned above, infants who are born with weak antiviral responses and/or airway hyper-responsiveness are prone to repetitive severe illnesses, which may increase the risk of recurrent wheezing and possibly asthma. A recent study suggests that RSV-induced wheezing is an indicator of genetic predisposition to asthma, but is not associated with allergic sensitization, irrespective of the family history of asthma (Singh et al., 2007). In the present study, PIV-infected human lung fibroblasts produced and released tissue remodeling-related cytokines (PDGF and VEGF) and proinflammatory cytokines (IL-1β, IL-6, and TNF-α) compared with the control. Using animal models, it was also recently suggested that endotracheal inoculation of PIV induces biological and histological changes reminiscent of asthma (Pelaia et al., 2006). In particular, PIV infection elicits an airway influx of inflammatory cells, bronchial hyper-responsiveness, epithelial damage, and bronchiolar fibrosis (Pelaia et al., 2006). These findings suggest that the recurrence of PIV infections in patients with asthma may contribute to prolonged airway inflammation and airway structural changes typical of the disease (Pelaia et al., 2006). Moreover, our results suggest that PIV-induced tissue remodeling-related cytokines and proinflammatory cytokines from human fibroblasts may be associated with respiratory remodeling and prolonged airway inflammation after infection. Human asthma subjects have exhibited significant increases in IFN-γ, in the supernatants of cultured bronchoalveolar cells (Krug et al., 1996). Recently, Magnan et al. (2000) demonstrated increased IFN-γ-producing CD8⁺ T cells in asthmatic airways, and the levels of IFN-γ correlated with asthma severity, bronchial hyper-responsiveness, and blood eosinophilia (Ishihara et al., 1997). IFN-γ has been reported to be associated with severe and chronic asthma. In the present study, PIV-infected human lung fibroblasts produced and released IFN-γ, which might be associated with severity of asthma.

Cytokine production-related signaling pathways related to viral infections have been reported (Santoro et al., 2003; Kohlmeier et al., 2009; Zaheer et al., 2009). For example, Lee et al. suggested that single strand viral RNA of influenza virus binds to the host cellular helicase resulting in activation of signaling pathways including IκB kinase (Lee and Lau, 2007). In addition, viral single strand or double strand RNA/MKK3/6 complex can also activate p38 MAPK (Stephan, 2007). Finally, activation of these pathways may lead to the production and release of cytokines in virus-infected cells as an antiviral response (Stephan, 2007). In the present study, at 24 h after PIV infection, phosphorylation of IκB kinase and p38 MAPK and the release of cytokines were significantly increased in lung fibroblasts. Furthermore, inhibition of phosphorylation of these kinases by specific inhibitors significantly eliminated release of cytokines (Figure 2). At 24 h after infection, PIV genomes (also single strand RNA) may be significantly propagated (De et al., 1990). A possible reason for this is that the increase in phosphorylation of IκB kinase and p38 MAPK after PIV infection might be associated with propagated PIV genomes in the cells, although we did not observe an increase in the PIV genome/helicase or PIV genome MKK3/6 complex.

Fibroblasts have the potential to play a critical role in remodeling of and changes in the airway matrix (Holgate et al., 2004a). In this study, we found that PIV infection-induced significantly higher production of Th1 and Th2 cytokines from fibroblasts compared to control cells. PIV might promote release of not only inflammatory cytokines but also allergy-related cytokines in virus-infected MRC-5 cells. From the present results and previous our results suggest that aberrant release/production of various cytokines (i.e., cytokine storm) due to PIV or RSV infection is also associated with severe respiratory infection such as pneumonia (Ishioka et al., 2010). In addition, we showed that this cytokine production may be closely linked to phosphorylation of signal molecules such as IκB kinase and p38 MAPK. Recurrent RSV infection may damage the lower respiratory airways and induce remodeling. Finally, our findings suggest that PIV infection-induced aberrant phosphorylation of IκB kinase and p38 MAPK in lung fibroblasts might mediate hypersensitive Th1- and Th2-type responses in subjects with asthma, which is likely an important mechanism in virus-induced asthma.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.