Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium

Abstract

We have previously shown that the respiratory syncytial virus [RSV] can productively infect monocyte derived dendritic cells [MoDC] and remain dormant within the same cells for prolonged periods. It is therefore possible that infected dendritic cells act as a reservoir within the airways of individuals between annual epidemics. In the present study we explored the possibility that sub-epithelial DCs can be infected with RSV from differentiated bronchial epithelium and that in turn RSV from DCs can infect the epithelium. A dual co-culture model was established in which a differentiated primary airway epithelium on an Air Liquid Interface (ALI) was cultured on a transwell insert and MoDCs were subsequently added to the basolateral membrane of the insert. Further experiments were undertaken using a triple co-culture model in which in which macrophages were added to the apical surface of the differentiated epithelium. A modified RSV [rr-RSV] expressing a red fluorescent protein marker of replication was used to infect either the MoDCs or the differentiated epithelium and infection of the reciprocal cell type was assessed using confocal microscopy. Our data shows that primary epithelium became infected when rr-RSV infected MoDCs were introduced onto the basal surface of the transwell insert. MoDCs located beneath the epithelium did not become infected with virus from infected epithelial cells in the dual co-culture model. However when macrophages were present on the apical surface of the primary epithelium infection of the basal MoDCs occurred. Our data suggests that RSV infected dendritic cells readily transmit infection to epithelial cells even when they are located beneath the basal layer. However macrophages appear to be necessary for the transmission of infection from epithelial cells to basal dendritic cells.

Figure 1. Schematic representation of dual and triple co-cultures.

pBEC = primary bronchial epithelial cell culture, MoDC = monocyte derived dendritic cells, MDM = monocyte derived macrophages.

Figure 2. Confirmation of differentiation of the ALI cultures.

A. Western blotting of apical cell washes were used to show induction of SPLUNC1/BPIFA1 in primary cells by day 20 compared to day 0 of ALI culture. B. A 20 day ALI culture was stained for MUC5AC as outlined in the methods section. The positive green staining shows the presence of goblet cells within the cell layer after differentiation.

Figure 3. Active RSV infection occurs in both directly and indirectly infected pBECs.

Cell infections and FACS analysis were performed as outlined in the methods section. Direct exposure means pBECs were directly infected with RSV, whereas indirect exposure means that the RSV infected MoDCs were the source of the virus. The image shows a representative example of flow cytometry results for pBECs from a co-culture experiment.

Figure 4. Collated fluorescence data from directly and indirect exposure studies.

Cell infections and FACS analysis were performed as outlined in the methods section. The data represents 6 experiments performed in pBECs with standard error of the mean shown by the error bars.

Figure 5. Active RSV replication is seen in both directly and indirectly infected pBEC cultures.

Cell infections and IF microscopy were performed as outlined in the methods section. The images show active RSV replication (as indicated by red fluorescence) in pBEC images under fluorescence microscopy at the indicated time alongside corresponding phase contrast images of the pBEC cultures.

Figure 6. Active RSV replication is seen in both direct and indirectly infected MoDCs.

Cell infections and FACS analysis were performed as outlined in the methods section. Direct exposure means MoDCs were directly infected with RSV, whereas indirect exposure means that the RSV infected pBECs were the source of the virus. This is shown in an example of flow cytometry results of MoDCs on co-culture from a single experiment.

Figure 7. Collated fluorescence data from direct and indirect exposure studies.

Cell infections and FACS analysis were performed as outlined in the methods section. The data shown represent data from 6 experiments performed in MoDCs with standard error of the mean shown by the error bars.

Figure 8. Active RSV replication is seen in directly infected MoDC cultures by 24 hours.

Cell infections and IF microscopy was performed as outlined in the methods section. The images show active RSV replication (as indicated by red fluorescence), alongside phase contrast images of the cultures.