Figure 1.
RSV infection of both multi-ciliated and airway basal cells.
Apical exposure of HBEC to RSV-A2-GFP led to the infection of multi-ciliated cells. GFP+ (infected) cells were observed throughout the epithelium. These were identified as an acetylated α-tubulin+, ciliated cell (#, orange) by confocal imaging (A–B). Under these conditions, the sub-apical p63+ basal cells (red) did not become infected by RSV (B). Removal of the surface of the epithelium immediately before exposure to RSV resulted in numerous p63+GFP+ (*) cells 16 h later (C–D), consistent with infection of the airway basal cell. When well-differentiated cells were subjected to a mechanical scratch injury (E) followed by RSV infection multiple infected cells were clearly visible within the wound (F). A majority of the infected cells were determined as basal cells (p63+) by confocal microscopy (G–I). Presented are representative images from three independent experiments in two different donors. White bar indicates 1,000 µm in A, C, 50 um in B, D, 500 µm in E–F and 20 µm in G–I.
Figure 2.
RSV infection of basal cells influences differentiation.
The effects of RSV-A2-GFP infection of basal cells (100 pfu/insert, 3 h after seeding) were investigated over the duration of epithelial growth and differentiation. Infection of the epithelium was monitored by live cell imaging of GFP+ objects every 2–3 days (A; dotted line) whilst viral release was determined by plaque assay at day 3, 6, 13 and 21 (A; filled bars). Mean data ± SEM from 3 independent studies are shown. Representative planar and transverse views by confocal microscopy showing RSV-A2-GFP infected cells at day 6 (B), infected non-ciliated cells at day 13 (C) and infected ciliated cells at day 20 (D). GFP was used to visualize RSV-infection (green), acetylated α-tubulin stain for cilia (orange) and p63 for basal cells (red). Scale bars indicate 50 µm.
Figure 3.
Epithelial composition following RSV infection.
The effects of RSV-A2-GFP infection of basal cells on epithelial differentiation were assessed by quantitative immunofluorescence. Representative, images of HBEC cultures (21 days after seeding/infection) that were either uninfected (A) or infected with 1, 10, 100, or 1,000 pfu of RSV-A2-GFP (B–E, respectively). MUC5AC+ goblet cells and acetylated a-tubulin+ ciliated cells are pseudo-colored green and orange respectively. Staining for each of the cell types was quantified by image analysis (F). An alternative RSV strain, RSV-A2-MOT0972, was also assessed for effects on epithelial differentiation by quantitative immunofluoresence (G) as described above. For each study, mean data ± SEM from 6–9 inserts over three independent experiments are shown. Statistical significance was determined with a one-way ANOVA with post-hoc Dunnetts test compared to untreated control cells. * indicates p<0.05, **p<0.01 and ***p<0.001.
Figure 4.
RSV infection induces an IFN-β and Type III interferon response in basal cells.
The induction of an innate host response following RSV-A2-GFP infection of basal cells (100 pfu/insert, 3 h after seeding) was investigated over the duration of epithelial growth and differentiation. The expression of immune modulators was assessed at days 3, 7 and 14 after infection. qRT-PCR data are expressed relative to the time-matched, uninfected control (A). At day 3, 7, 14 and 21 media was collected and the levels of secreted IL-28A and IL-28B/IL-29 were determined by a sandwich ELISA (B). Quantitative immunofluorescence was used to assess the effects of IL-28A (30 ng/mL), IL-28B (30 ng/mL), IL-29 (30 ng/mL) or IFN-β (2.5 ng/mL) on epithelial differentiation (C). Mean data ± SEM from 9 inserts over 3 independent experiments are shown, except for panel B, in which 14 inserts over 5 independent experiments were analyzed. Statistical significance was determined with a one way ANOVA statistical test with post-hoc Dunnetts test compared to untreated control cells. *p<0.05, **p<0.01, and ***p<0.001.
Figure 5.
Neutralization of the IFN response during RSV-A2-GFP infection.
The effects of neutralization of IL-28A, IL-28B/IL-29 and IFN-β following RSV infection were assessed. GFP+ surface area was used to assess the degree of RSV infection (A) and quantitative immunofluorescence was used to determine the balance of acetylated α-tubulin+ and MUC5AC+ cells. Mean data ± SEM are shown from 10–14 inserts over 4–6 independent experiments, respectively. Statistical significance was determined with a one way ANOVA statistical test with post-hoc Dunnetts test compared to untreated control cells, ***p<0.001.
Figure 6.
Proposed model for the impact of RSV infection of the airway basal cell.
The multi-potent airway basal cell plays a central role in epithelial repair following injury, resulting in an epithelium composed of both ciliated and goblet cells (A). In patients with an impaired epithelial barrier, resulting from either the natural history of an RSV infection (epithelial sloughing) or because of a pre-existing respiratory disease, the airway basal cell can now become exposed to inhaled RSV particles (B). Infection of the basal cells by RSV results in the release of IL-28A, IL-28B, IL-29 and IFN-β that influences basal cell differentiation towards a hypersecretory phenotype i.e. gain of goblet cells and loss of ciliated cells that has the potential to contribute towards a disease exacerbation.