Respiratory infections are the most common viral illness in human populations. Based on epidemiologic and molecular evidence, there is an association between severe childhood respiratory viral infections and chronic lung immune-mediated diseases such as asthma (3
). Thus far, the underlying molecular mechanisms that are involved in viral induction of chronic respiratory diseases are not completely clear. However, it has been reported that viral load is a critical factor in determining the severity of RSV infections (10
We previously reported that treatment of human respiratory epithelial cells with TGF-β enhanced RSV replication (30
). Recently, a study by Thomas et al. showed that TGF-β also increased rhinovirus replication (47
). A well-characterized function of TGF-β is regulation of cell cycle progression. Activation of the TGF-β1-induced signaling through the Smad 2/3 pathways promotes cell cycle arrest in both the G0
/M phases of the cell cycle, albeit regulation of G0
by TGF-β is much more studied (7
). Our data in Fig. confirm previous findings that TGF-β, depending on the cell type, can affect both G0
Here, we report that the enhancement of RSV replication is associated with cell cycle regulatory properties of TGF-β. Treatment of A549 or PHBE cells with TGF-β inhibited cell proliferation, which was concomitant with inhibition of cell cycle progression and enhancement of RSV replication (Fig. ). The role of cell cycle arrest in RSV replication was further confirmed by using three different pharmacological inhibitors of cell cycle progression, i.e., purvalanol A, olomoucin, or nocodazole, which led to an increase in RSV protein expression and viral titers in both A549 and PHBE cells (Fig. ). A previous report by Kallewaard et al. showed that nocodazole decreased RSV replication in the human epithelial cell line Hep-2 (22
). The reason for the discrepancy between their results and our data is not yet clear, but it could be due to a cell-specific event. It is also possible that the difference may be due to the amounts of nocodazole used in the two studies; we used 100 ng/ml (approximately 0.3 mM), compared to 17 mM used in the studies by Kallewaard et al. (22
Interestingly, infection of epithelial cells with RSV alone resulted in cell cycle arrest (Fig. ). RSV-induced cell cycle arrest was accompanied by a corresponding reduction in Rb phosphorylation, a G0
phase marker, and an increase in His-3 phosphorylation, a G2
-to-M phase transition marker (Fig. ). Previous reports have shown inhibition of cell cycle progression in host cells infected by other RNA viruses. Infection of Vero cells with coronavirus and of fibroblast cells with reovirus resulted in G0
/M cell cycle arrest (4
). Cell cycle arrest at the G0
phase was shown in T lymphocytes after measles virus infection (35
). Our data demonstrated that RSV-infected primary lung epithelial cells underwent G2
/M arrest while the A549 alveolar lung epithelial cell line underwent G0
arrest, which may reflect differences between a lung cancer cell line (A549) and primary cells (PHBE) (Fig. ).
We next address the possible mechanisms of RSV induction of cell cycle arrest. Our hypothesis, which was confirmed by our data, was that RSV infection induced the expression of TGF-β, which may have had an autocrine effect on the cell cycle and subsequently on virus replication. Infection of the epithelial cells with RSV resulted in induction of TGF-β mRNA and protein secretion into the medium (Fig. ). Consistent with the expression data, inhibition of TGF-β using a specific neutralizing antibody or a pharmacological inhibitor of TGF-βR rescued the RSV-induced cell cycle arrest and reduced viral replication (Fig. and ). Since inhibition of TGF-β significantly but incompletely rescued the RSV-induced cell cycle arrest, it is likely that other virus-associated events participate in cell cycle arrest. Nonetheless, it appears that cell cycle arrest in any cycle may be beneficial to RSV replication.
It is not exactly clear how cell cycle arrest enhances virus replication, but a possible mechanism is by the increase in availability of host cellular components for virus replication. Alternatively, it is possible that RSV can take advantage of a cell cycle regulatory molecule that is activated during TGF-β treatment, such as cell division cycle-2 (cdc2), which has been proposed for herpes simplex virus infections (1
). Further experiments to delineate the exact mechanisms of the enhanced RSV replication during cell cycle arrest are under way.
A study by Groskreutz et al. showed that RSV infection reduced the level of p53 tumor suppressor protein. They suggested that RSV infection may be enhanced by the increase in cell survival through lower p53 expression (16
). At this point, the exact correlation between our data and the data published by Groskreutz et al. is not clear. However, since p53 plays a critical role in the cell cycle, it is possible that p53 is involved in our observations. We are currently examining this possibility.
RSV-induced cell cycle arrest benefits viral replication but very likely has adverse effects on lung epithelium. It is plausible that cell cycle arrest of lung cells impedes lung repair during RSV infection, which is known to induce severe bronchiolitis. Bronchiolitis is associated with serious airways injury, including loss of cilia, sloughing of epithelial cells, mucus hypersecretion, and edema. In addition, the interplay between cell cycle arrest and the increase in viral load can lead to a more prolonged and severe infection.
To our knowledge, this is the first report showing that RSV induces expression of TGF-β in human epithelial cells. TGF-β is pluripotent cytokine with profound effects on cell cycle, fibrosis, inhibition of inflammation, and immune regulation. It is established that aberrant expression of TGF-β in lungs, due to genetic polymorphism or environmental insult, leads to fibrosis, airway remodeling, and mucus hypersecretion (2
). These are characteristics that are closely associated with the asthma phenotype. Based on work by others and our data in Fig. , we propose that expression of TGF-β by RSV may contribute to lung remodeling and the asthma phenotype. Currently, we are further delineating the molecular pathways activated during RSV infections that are required for induction of TGF-β.
In addition to the regulation of lung physiology, TGF-β is a pivotal immunoregulatory cytokine. TGF-β can enhance regulatory T-cell development, induce immunoglobulin switching, and act as an anti-inflammatory cytokine (21
). Therefore, it is possible that induction of TGF-β may contribute to the immune deviation that is observed during RSV infections (13
). It is interesting to note that the immunosuppression that is induced during measles virus infections has been attributed to cell cycle arrest in T lymphocytes (31
). Based on our data, it is tempting to speculate that the immunosuppression and T-lymphocyte cell cycle arrest may be mediated through TGF-β expression.
In this study, we have shown for the first time that RSV causes cell cycle arrest of lung epithelial cells in part through the TGF-β autocrine pathway. Furthermore, the cell cycle arrest enhanced viral replication. Our findings provide further mechanistic insight into RSV pathogenesis and RSV-induced pathologies. Further experiments to assess the role of RSV-induced TGF-β in the development of the asthma phenotype in murine experimental models are under way.