RSV is the most common cause of viral respiratory infections in infants, which can lead to severe bronchiolitis, hospitalization, and mortality. In murine models, RSV infections leads to induction of TNF-α, IL-6, IL-8, MCP-1, RANTES, and CXCL10 (IP-10) (Graham and others 2000
; Tripp and others 2000
; Jafri and others 2004
; Miller and others 2004
; Estripeaut and others 2008
). In the early steps, the presence of viral infections leads to an innate immune response and there is a rapid (8–24
h) and transient rise in the expression of chemokines and cytokines (Tripp and others 2000
; Estripeaut and others 2008
). Therefore, we hypothesized that PKR, a dsRNA-specific kinase, which is activated early during viral infections could play a role in RSV-induced immunity. To date, there has been no report on the role of PKR in RSV infection, immune responses, and lung injury.
PKR is activated by dsRNA, which is present during the life cycle of most viruses (Jacobs and Langland 1996
). Once activated, PKR phosphorylates eIF-2α, leading to a reduction in translation and thus to an attenuation of viral replication. Several groups using the PKR−/−
mouse have demonstrated that PKR is a key host factor for resistance to viral replication (Balachandran and others 2000
; Stojdl and others 2000
; Guidotti and others 2002
; Carr and others 2006
). Consistent with the role of PKR in viral replication, our data showed that unlike WT mice, PKR−/−
mice (Yang and others 1995
; Baltzis and others 2002
) had a greater load of RSV at 24
h post-infection (). Based on an in vitro
kinetic analysis, we showed that the difference was due to viral replication and not initial infectivity ().
In addition to participating in antiviral responses, PKR also functions as a signaling intermediate in several pathways including MAPK and NF-κB (Kumar and others 1994
; Goh and others 2000b; Zamanian-Daryoush and others 2000
; Zhou and others 2003
). We previously reported that RSV infection of human epithelial cells led to activation of MAPK and subsequently inflammatory cytokine expression (Meusel and Imani 2003
). Also, RSV infection of airway epithelial cells was shown to induce activation of ERK and p38 MAPK (Mastronarde and others 1996
; Pazdrak and others 2002
). In the present study, we report that while activation of the MAPK and NF-κB pathways occurred in WT lung tissue during RSV infection, there was considerably less activation of these factors in the lungs of PKR−/−
mice (). This suggests that in response to RSV, PKR is required for maximal activation of these signaling pathways in vivo.
Several in vitro
studies using the PKR activator, dsRNA, have provided evidence for the involvement of PKR in the development of inflammatory responses. We previously reported that dsRNA activation of PKR was required for induction of inflammatory cytokines in human bronchial epithelial cells (Meusel and others 2002
). Also, treatment of cells with dsRNA led to activation of PKR and increased production of IL-8 and RANTES (Gern and others 2003
). In our experiments, induction of cytokines and chemokines was increased dramatically within the BAL fluid of RSV-infected WT mice as compared to PKR−/−
mice. The induction of cytokines and chemokines was accompanied with a corresponding increase in cellular influx into the lungs ().
Previously, we reported that during intracerebral infection using mouse-adapted polio virus, both PKR−/−
and transgenic mice expressing a trans
-dominant negative mutant form of PKR, displayed a less severe central nervous system inflammation and tissue damage than their WT cohort (Scheuner and others 2003
). Based on these reports and our current data, we believe that PKR is required for both control of RSV replication, and mounting a robust inflammatory immune response.
It is reported that in addition to direct viral pathologies, inflammatory responses can participate in tissue damage during viral infections (Tripp 2004
). Wang et al. reported that the presence of neutrophils enhanced RSV-induced cell damage and detachment from culture dishes, suggesting that neutrophilic infiltrate into the lungs may contribute to the pathogenesis of RSV airway disease (Basler and others 2000
). In this report, we investigated RSV-induced lung injury by comparing the levels of neutrophils, total protein, albumin, and histological score ( and ). Our data showed that the levels of neutrophil influx, total protein, and albumin were greater in BAL fluid collected from RSV-infected WT mice than from PKR−/−
mice. Consistent with these observations, the histological score of WT mice was significantly greater than PKR−/−
mice. These data suggest that PKR is a key factor in RSV-induced lung injury and liquid extravasations into the lungs.
A high stringency comparison of the genes that were induced by RSV in WT and PKR−/−
mice showed that PKR activation was required for induction of 101 genes, 29 of which were associated with early immune responses (See supplementary data at the NCBI Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/projects/geo
). Expectedly several interferon-induced genes were affected by PKR activation (). Also, major histocompatibility complex (MHC) genes such as H2-M2 and H2-Q1 were up-regulated only in WT mice (). This finding is interesting because all of these MHC molecules are considered as the non-classical MHC class-I molecules with low polymorphism. Since members of the non-classical MHC molecules are involved in activation of natural killer (NK) cells (Sambrook and Beck 2007
), it is tempting to speculate that PKR plays a role in viral recognition by these cells. Additional evidence for association of PKR with inflammation and immune responses was revealed in functional analysis of the PKR-regulated genes, which showed that this pathway was associated with inflammatory and immunological disorders ().
In addition to the MHC molecules, several of the antiviral molecules such as PKR, TLR-3, adenosine deaminase acting on RNA (ADAR), and interferon regulatory factor-9 (IRF-9) were up-regulated in WT and not in PKR−/− mice. This further suggests that PKR is critical for antiviral responses during RSV infections.
An interesting observation in our experiments was the induction of p65/p50 heterodimerization and p50/p50 homodimerization of NF-κB complexes (). While activation of the p65/p50 heterodimeric form of NF-κB is required for expression of many genes, the p50 subunit has been shown to be essential for expression of IgE (Delphin and Stavnezer 1995
; Kuchroo and others 1995
). In as much as IgE is a central molecule in allergic responses, these data suggest that PKR may play a role in RSV induction of IgE expression. This is consistent with our previous in vitro
data, which demonstrated that PKR activation regulated IgE class switching in human B lymphocytes (Rager and others 1998
). Previous reports have also shown that IgE levels are higher in RSV-infected mice exposed to ragweed (Leibovitz and others 1988
) and RSV-specific IgE was higher in RSV-infected infants (Welliver and others 1981
; Welliver and Duffy 1993
Collectively, our data establishes a dual role for PKR in RSV infections. First, it is necessary for controlling replication and second, it participates in signal transduction, leading to innate inflammatory responses. These responses, while important for the overall immunity to RSV, likely contribute to lung injury.