Characterisation of lung tissue cell subsets expressing active RSV-RFP
We next characterised the phenotypes of the lung tissue cells containing RSV using the marker RFP, expressed only by actively replicating virus [25] (Figure 5A). The earliest time point at which RSV-RFP could be detected was at 5 dpi, in lung MHC II positive APC populations (Figure 5A); corresponding to the peak viral titre determined by qPCR (see Figure 1C). Although MHC II expression was noticeably high in macrophages in the time-point after RSV infection (see Figure 4B), low levels of RSV-RFP were observed in samples collected across all time points (Figure 5B). RSV-RFP was clearly detected in lung B cells (Figure 5C) and cDCs (Figure 5D) at 5 and 21 dpi of the lung MHC II positive APC populations assessed (Figure 5A).
Lung cDCs had the highest rates of infection, with ~20% of cDCs expressing RSV-RFP at 5 dpi, and ~80% at 21 dpi (Figure 5D). RSV-RFP was not detected in lung pDCs at any time point (data not shown). Further analysis to determine the RSV-RFP positive lung cDC phenotype revealed a cell subset with classical DC markers. These included high CD11c and MHC II, with low F480, as well as high levels of the intra-epithelial cDC marker CD103 (CD103+). These markers were also co-expressed with CD11b at 21 dpi (CD11b+) (Figure 5E). In addition, RSV-RFP was only detected in lung CD103+CD11b+ cDC at 5 and 21 dpi (Figure 5F), with a subset of high RSV-RFP in this population observed at 21 dpi only.
In summary, RSV showed a biphasic replication kinetics in lung tissue, with peak expression at 5 dpi before resolving by 7 dpi and then re-expressing at 21 dpi, with replication primarily restricted to lung CD103+ cDC.