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.