Viral infections trigger nearly 80% of asthma exacerbations, and RV accounts for the majority of virus-induced exacerbations (1
). RV is also an important trigger of COPD exacerbations (3
). In the respiratory tract, RV replicates mainly in the ciliary epithelial cells of the nasal mucosa and, to a lesser extent, oral cavity and throat (34
). In the common cold, typical symptoms such as coryza and cough climax on Day 2 or 3 and usually resolve by Day 5, although occasionally they may persist for longer. However, little is known about infection of the lower respiratory tract with RV. Until recently, rhinoviruses had not been reliably cultured from lower airway secretions (35
). RV RNA has been detected by PCR in lower airway cells from volunteers experimentally infected with RV16 (5
), and rhinovirus capsid protein has been found in airway epithelial cells, albeit sporadically (6
). Together these findings suggest that rhinoviruses can grow in the lower airways, although the extent of RV replication in these locations is unknown.
Because of species-specific variations in the ICAM-1 D1 extracellular immunoglobulin domain, mouse models of major group RV have not been implemented. However, the LDL-R family of proteins is highly conserved between human and mice, providing a possible means of infection for minor group viruses. In the present article, we show evidence that RV1B infects mouse airway epithelial cells in vivo
. After intranasal inoculation with RV1B, we isolated positive-strand and negative (replicative)-strand viral RNAs from the lungs of C57BL/6 mice up to 7 and 4 days after exposure, respectively. We also detected RV1B protein in airway epithelial cells 1 day after inoculation. Although specific to the airway epithelium, RV1B protein expression was patchy and limited to the larger airways, similar to patients experimentally infected with RV16 (6
). We also showed that lung homogenate from RV1B-exposed mice infects HeLa cells. RV1B exposure induced airway inflammation, as demonstrated by lung histology, increased BAL neutrophils and lymphocytes, and increased MPO activity. RV1B also induced the production of KC, MIP-2, RANTES, MIP-1α, and JE. The observed neutrophilic inflammation is similar to that found in human subjects after experimental RV16 infection (13
). Neutrophil number, IL-8, and epithelial-derived neutrophil activating peptide-78 are also increased in the sputum and airways of patients with exacerbations of asthma (17
) and COPD (19
). Airway inflammation was accompanied by a functional state of hyperresponsiveness that persisted 4 days after viral exposure. RV1B exposure induced a robust interferon response, further evidence of viral replication (36
). Finally, inoculation with UV-irradiated virus had significantly reduced effects on airway inflammation, cytokine expression, and methacholine responsiveness compared with intact virus. Together, these data suggest that mouse lower airways may be infected with RV1B.
On the other hand, several observations speak against infection. First, although the extent and time course of viral replication in the human lung is unknown, the steep reduction in viral RNA we observed is inconsistent with the time course of viral replication in the upper respiratory tract of humans (37
). Second, positive staining of the airway epithelium with RV1B antiserum does not prove replicative infection; the use of an antibody targeting nonstructural viral proteins would better address this issue. Third, because UV irradiation may partially inhibit picornavirus attachment (38
), the effects of viral irradiation on RV-induced responses may overestimate the role of viral replication, and may instead represent an inhibition of RV binding to the airway epithelium.
Moreover, it should be noted that exposure to UV-irradiated virus, but not sham HeLa cell lysate, caused modest airway neutrophilic inflammation and short-lived airway cholinergic responsiveness. In addition, UV-irradiated RV induced the production of a mouse IL-8 homolog, MIP-2, providing a possible mechanism for the observed neutrophilic inflammation. Finally, UV-irradiated virus was sufficient to induce airway epithelial cell Akt phosphorylation (see below
). These data suggest that early events before viral replication, for example, viral attachment and internalization, may be sufficient for a subset of RV-induced epithelial cell responses. Because, as noted above, UV irradiation may partially inhibit picornavirus attachment (38
), and therefore UV inactivation, experiments may underestimate the sufficiency of virus attachment and internalization for cellular responses. Numerous studies have demonstrated the sufficiency of UV-irradiated virus to induce IL-8 expression in cultured airway epithelial cells (24
). Bafilomycin, an inhibitor of vacuolar proton ATPases, which promotes the low endosomal pH needed for viral uncoating, decreases RV14-induced ICAM-1 but not IL-8 expression in human tracheal epithelial cells (40
). If indeed events before viral replication such as attachment and internalization were sufficient for a subset of RV-induced epithelial cell responses, this could explain how RV enhances lower airway inflammation in the absence of abundant viral replication (35
RV attachment and endocytosis promote phosphorylation of the p85 regulatory subunit of PI 3-kinase, as well as activation of PI 3-kinase and Akt phosphorylation, in cultured human bronchial epithelial cells (24
). In the present study, we found that RV1B colocalizes with phosphorylated Akt in the airway epithelium of exposed mice, and increases the phosphorylation of Akt in whole lung extracts. Pretreatment of RV1B-infected mice with the PI 3-kinase chemical inhibitor LY294002 decreased BAL neutrophils and lung KC, MIP-2, MIP-1α, and IFN-γ production. Administration of UV-irradiated replication-deficient virus also increased Akt phosphorylation, but induced only modest neutrophilia and lung KC expression. Taken together, these data suggest that activation of PI 3-kinase/Akt signaling is required but not sufficient for maximal RV-induced airway inflammation. We have previously shown in cultured human bronchial epithelial cells that PI 3-kinase activity is required for viral internalization (24
), and therefore this is likely the mechanism by which LY294002 blocks RV1B-induced airway responses. PI 3-kinase may therefore represent a therapeutic target for RV-induced exacerbations of chronic airway disease.
As expected, RV39, a major subgroup RV, failed to induce airway inflammation in C57BL/6 mice, likely due to species-specific variations in ICAM-1. We therefore could not compare the signaling events and inflammatory events initiated by major group RV with those found in the present study after minor group (RV1B) exposure. However, infection with RV1B and RV39 induced similar levels of Akt phosphorylation and IL-8 expression in cultured 16HBE14o−
human bronchial epithelial cells, and inhibition of PI 3-kinase blocked RV1B-induced IL-8 expression (data not shown), just as it blocks RV1B-induced KC expression in mouse cells. Although the downstream signaling events after ligation of LDL-R have not been extensively studied, LDL-R family members bind ligands and internalize them for lysosomal degradation by clathrin-mediated endocytosis, similar to ICAM-1 (43
). Ligation of LDL-R by apoprotein E and LDL induces Akt activation (44
). Lipoprotein particles induce NADPH oxidase-mediated production of superoxide in endothelial cells via an LDL-R family receptor (45
), as has been reported after RV16 infection of A549 cells (46
). Finally, one study has shown that RV1B and RV16, a major group serotype, induce nearly identical patterns of gene expression in primary cultured airway epithelial cells (47
). Thus, there are ample data suggesting that major and minor subgroup RVs elicit similar airway epithelial cell signaling pathways and inflammatory responses.
We conclude that RV1B exposure induces airway inflammation and hyperresponsiveness in C57BL/6 mice. Although our results are suggestive, we cannot conclusively state that the observed airway changes are due to replicative infection, rather than simply binding and endocytosis of the virus. Indeed, nonreplicative UV-irradiated virus was sufficient to induce a subset of RV-induced responses. Nevertheless, our model, especially when combined with established animal models of asthma, COPD, and cystic fibrosis, could provide important insight into the pathogenesis of RV-induced exacerbations of chronic airway disease. Finally, on the basis of our observation that activation of PI 3-kinase/Akt signaling is required for maximal RV-induced neutrophilic airway inflammation, future studies examining the role of PI 3-kinase inhibitors in the treatment of RV-induced airway disease may be warranted.