Differentiation and immune function of human dendritic cells following infection by respiratory syncytial virus

Abstract

RSV causes annual epidemics of bronchiolitis in winter months resulting in the hospitalization of many infants and the elderly. Dendritic cells (DCs) play a pivotal role in coordinating immune responses to infection and some viruses skew, or subvert, the immune functions of DCs. RSV infection of DCs could alter their function and this could explain why protection after natural RSV infection is incomplete and of short duration. In this study, this interaction between DCs and RSV was investigated using a human primary culture model. DCs were generated from purified healthy adult volunteer peripheral blood monocytes. Effects of RSV upon DC phenotype with RSV primed DCs was measured using flow cytometry. Changes to viability and proliferation of cocultured DCs and T-cells were determined using microscopy with fluorescent dyes (Hoechst 33342 and propidium iodide). DC maturation was not prevented by the RSV challenge. RSV infected a fraction of DCs (10-30%) but the virus replicated slowly in these cells with only small reduction to cell viability. DCs challenged with RSV stimulated T-cell proliferation less well than lipopolysaccharide. This is the first study to demonstrate RSV infection of human monocyte derived DCs and suggests that the virus does not significantly interfere with the function of these cells and potentially may promote cellular rather than humoral responses.

Fig. 1

Flow cytometry data to show the phenotypic differentiation of monocytes (day 0) into immature MoDCs over 7 days of culture. Differentiating cells were stained with FITC conjugated antibodies (□) and matched isotype controls (▪) on day 0, 2, 5 and 7. Results are representative of three independent experiments using different volunteer DCs.

Fig. 2

Maturation of DCs (a) DC maturation by addition of 1 µg/ml LPS. DCs were stained with matched isotype controls and each bar represents the subtracted matched isotype mean fluorescence values for each antibody. The error bars represent the standard error of the mean fluorescence measurements and *significance at P < 0·001 compared to MoDCs cultured with LPS. Results are representative of six independent experiments using different volunteer DCs. (b) DC maturation by addition of 100 µg/ml KLH. DCs were stained with matched isotype controls and each bar represents the subtracted matched isotype mean fluorescence values for each antibody. The error bars represent the standard error of the mean fluorescence measurements and *significance at P < 0·001 compared to MoDCs cultured with KLH. Results are representative of three independent experiments using different volunteer DCs.

Fig. 3

Internalization of 5 × 10⁵ pfu/ml purified RSV by (a) immature DC and (b) mature DC after 30 min of incubation at 37°C (□); DCs incubated with RSV but stained only with the secondary antimouse FITC antibody (minus primary antibody controls)(▪). Results are representative of three independent experiments using different volunteer DCs.

Fig. 4

Flow cytometry data to compare the percentage of DCs positive for intracellular RSV antigens after 48 h and 7 days (a, b) in the absence of RSV and (c, d) in the presence of 1·6 × 10⁵ pfu/ml purified RSV (live RSV); (e, f) 1·6 × 10⁵ pfu/ml UV inactivated purified RSV and (g, h) 1·6 × 10⁵ pfu/ml purified RSV incubated with a humanized anti-F glycoprotein blocking antibody. The plots on the left show DCs after 48 h of incubation compared to the plots on the right that shows DCs after 7 days of incubation. Dead cells and/or cell clusters were excluded from analysis by gating. Results are representative of three independent experiments using different volunteer DCs.

Fig. 5

(a) Allogeneic T-cell proliferation induced by day 7 DCs primed with KLH, LPS and 2·4 × 10⁵ pfu/ml purified RSV. DCs were cocultured with T-cells at a ratio of 1 : 2·5. The error bars represent the standard error between the mean total cell counts derived from counting 3 areas in triplicate wells of a 96 well plate and *significance at P < 0·001 compared to cocultured control MoDCs. (b) Graph to show the percentages of viable, apoptotic and necrotic cells after the T-cell proliferation assay (as shown in (a)) as assessed by staining with Hoechst 33342 and propidium iodide. Results are representative of nine independent experiments.