Discussion
This is the first in vivo that studies the evolution of productive RSV infection in monocytes and other immune cells during the course of a primary acute infection. It is also the first to demonstrate persistence of the virus in a subset of pulmonary DCs after the clinical symptoms of infection have resolved. While Infection of the regulatory B cells in vitro has been described previously26 this is the first to demonstrate in vivo productive infection of a small proportion of B-cells in an animal model. These results provide further evidence that RSV has evolved an approach to subverting the host’s immune response which appears to be unique amongst common respiratory viruses. It seems likely that its ability to productively infect monocytes contributes to the characteristic poor long-term immunity generated by its human host and hence poor herd immunity while persistence in pulmonary DCs following the acute infection would provide a niche in which the virus can persist between the characteristic winter epidemics.
This model of primary RSV infection has been used extensively and, reassuringly, the time course of the acute illness and associated neutrophil dominated airways inflammatory response were consistent with that seen in human infants in whom lavage samples from both the upper and lower airways indicate an intense inflammatory response dominated by neutrophils27,28. The neutrophil response peaked at day 5 and had resolved by 21 days post-infection corresponding to the timing of changes in weight, clinical symptom severity scoring and RSV viral load in the lung.
In contrast to the neutrophil dominated inflammatory response observed within the airways flow cytometry analysis showed that antigen presenting cells (APC) were the dominant cells observed in the lung tissue during the acute infection. Again, this is consistent with findings in humans with monocytes being the most common interstitial inflammatory cell found in postmortem specimens from infants dying of RSV29. In contrast to the inflammatory cells recovered from the airways, which peaked at day 5, the lung APC populations increased until day 7 and most remained elevated until 21 dpi. Both cDCs and macrophages numbers remained significantly above baseline at 21 dpi. This is consistent with a previous mouse study that simply assessed lung dendritic cell numbers for 21 days after infection with APC number remaining elevated well beyond the resolution of the acute infection30.
A particular strength of this study is that it was possible to follow RSV replication using the fluorescent labelled RSV as fluorescence is only produced when the virus replicates. The intensity of fluorescence has been shown to directly mirror viral replication in a mouse model of RSV infection31. Unsurprisingly, the data clearly demonstrated RSV replication in lung APC populations though analysis demonstrated that that this was bi-phasic, peaking at 5 and 21 dpi in cDC and B cells. At 21 dpi, when symptoms had completely resolved, the data suggest that RSV replication, and hence persistence, was present in 80% of cDCs. Further subpopulation analysis of cDC showed that CD103 expression was dominant at 5 dpi with co-expression of CD11b observed at 21 dpi. In contrast, pDC numbers did not change significantly and negligible RFP signal (indicating RSV replication) was detected in pDCs. These results are in line with a previous in vitro study which found that pDCs are significantly less susceptible to infection that cDCs33 and a study involving intubated infants in which analysis of BAL samples indicated that cDCs but not pDCs were significantly elevated when compared to samples from controls. The data obtained in this study indicates that RSV preferentially infects and replicates in a subset of inflammatory cDCs and, to lesser extent, B cells, and that RSV replication persisted in these cells well after the clinical symptoms of infection had subsided.
cDCs are known to exist in close proximity to the airway lumen13-15,34 and are likely to be exposed to virus infecting the airways epithelium. Further studies have demonstrated a propensity for CD103 cDCs in carrying and cross-presenting viral antigens, with a likely significant role in induction of antibody memory responses13,35. Given the importance of these cDCs and B cells in certain aspects of immunological memory, productive infection of and persistence within these cells may well prove to be a key factor in the virus’s ability to suppress long-term production of protective antibodies levels which in turn contributes to the annual epidemics of respiratory infection. A number of viruses are known to infect and replicate in DCs, with a suggestion some may have evolved to act like a ‘Trojan horse’ in order to subvert normal immune responses36. Data generated in this and previous studies22 suggests that RSV may be one of those viruses.
The observed replication of RSV in lung cDCs over a prolonged period extending well beyond resolution of clinical symptoms is consistent with a previous in vitro study showing that the virus can continue to replicate within human monocyte derived DCs for many months after the initial infection22. After a period of several months of replication the virus appeared to enter a latent state, which lasted for several months, during which no fluorescence and hence replication was evident before replication re-commence following exposure to nitric oxide22. During the latent period RSV RNA could be identified by PCR even though it was not replicating. In this study replication within a subset of cDCs and some B cells was still ongoing at 21 days post infection. Further in vivo studies with significant longer follow up periods are required to confirm that the virus becomes latent and can be reactivated in an animal model. Previous mouse studies have indicated that this is likely given that in one study RSV RNA could be detected in murine lungs up to 77 days after inoculation in the absence of an evidence of replication37 while in another this period was extended to over 100 days38. Of particular note was the observation that low levels of replicating virus was isolated in a subset of mice who were immunosuppressed with monoclonal antibodies to CD4 and CD8 at 150 days post infection38.
In summary, the mouse model for RSV infection used in this study appeared to be robust in that the most severe clinical symptoms coincided with the peak in RSV viral load and airways neutrophilia as has been observed in human. As has been observed in vitro , the virus infected and replicated in antigen presenting cells and was shown to occur predominantly in CD103+ cDCs and, to a lesser extent, B cells. Importantly, persisting RSV replication was observed in the majority of lung CD103+CD11b+cDCs beyond resolution of the clinical infection and associated acute airways inflammation. Understanding how RSV activity is able to persist in CD103+CD11b+ cDCs without causing an inflammatory response indicative of viral infection, and the role of this subpopulation in antibody production, are likely to assist in novel therapeutic approaches and effective vaccines.