Respiratory syncytial virus binds and undergoes transcription in neutrophils from the blood and airways of infants with severe bronchiolitis

Abstract

Background: Neutrophils are the predominant cell in the lung inflammatory infiltrate of infants with respiratory syncytial virus (RSV) bronchiolitis. Although it has previously been shown that neutrophils from both blood and bronchoalveolar lavage (BAL) are activated, little is understood about their role in response to RSV infection. This study investigated whether RSV proteins and mRNA are present in neutrophils from blood and BAL of infected infants.

Methods: We obtained blood and BAL samples from 20 infants with severe RSV bronchiolitis and 8 healthy control infants. Neutrophil RSV F, G, and N proteins, RSV N genomic RNA, and messenger RNA (mRNA) were quantified.

Results: RSV proteins were found in BAL and blood neutrophils in infants with RSV disease but not in neutrophils from healthy infants. BAL and blood neutrophils from infants with RSV disease, but not those from healthy infants, expressed RSV N genomic RNA, indicating uptake of whole virus; 17 of 20 BAL and 8 of 9 blood neutrophils from patients expressed RSV N mRNA.

Conclusions: This work shows, for the first time, the presence of RSV proteins and mRNA transcripts within BAL and blood neutrophils from infants with severe RSV bronchiolitis.

Figure 1.

Respiratory syncytial virus (RSV) F, G, and N protein expression by bronchoalveolar lavage (BAL) and blood neutrophils from patient and control samples. A, Flow cytometry analysis of total (black line) and cell surface expression only (gray line) of RSV protein binding to BAL or blood neutrophils from infants with and without RSV infection. Representative histograms are shown after gating for viable cells by ethidium monoazide bromide staining, CD16 (a neutrophil marker), and respective anti-RSV monoclonals or isotype controls (shown shaded in gray). Numbers shown represent the percentage of positive cells above isotype control values following permeabilization. B, Summary data for percentage of positive cells for each patient (n = 20) or controls (n = 8). Each point represents an individual value, while the bar represents the sample mean. C, Summary data for mean fluorescence intensity of each sample analyzed by FACS. Each point represents an individual sample value. The bar represents the median value. B and C, the open dot highlights the corresponding data for the RSV-positive infant BAL neutrophils shown in Figure 3.

Figure 2.

Correlation between expression of different RSV F, G, and N protein levels in BAL and blood neutrophils. A, Correlation in percentage of cells positive between F (y-axis) and G (x-axis) (BAL r ² = .899, P < .001; blood r ²= .96, P < .001); F (y-axis) and N (x-axis) (BAL r ² = .835, P < .001; blood r ² = .982, P < .001) and G (y-axis) and N (x-axis) (BAL r ² = .911, P < .001; blood r ² = .957, P < .001). Each point represents an individual sample value. B, Correlation in mean fluorescence intensity (MFI) between F (y-axis) and G (x-axis) (BAL r ² =0.86, P < .001; blood r ² =.899, P < .001); F (y-axis) and N (x-axis) (BAL r ² = .86, P < .001; blood r ² = .86, P < .001) and G (y-axis) and N (x-axis) (BAL r ² = .83, P < .001; blood r ² = .911, P < .001). Each point represents an individual sample.

Figure 3.

Detection of RSV F, G, and N proteins within the cytoplasm of neutrophils by immunofluorescence. A representative BAL sample (shown in Figures 1B and 1C by an open dot with percentage of positive cells F, G, and N [87, 80, 86, respectively] and MFI for F, G, and N [50, 43, 50, respectively]) was stained with isotype control (IgG1 and IgG2a) or RSV-specific F, G, and N monoclonal antibodies. Positive staining (PE) is shown in red while nuclei that were counterstained by DAPI are shown in blue.