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Clinical Trial
. 2014 May;124(5):2219-33.
doi: 10.1172/JCI72948. Epub 2014 Apr 8.

RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

Rachael M Liesman, Ursula J Buchholz, Cindy L Luongo, Lijuan Yang, Alan D Proia, John P DeVincenzo, Peter L Collins, Raymond J Pickles
Clinical Trial

RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

Rachael M Liesman et al. J Clin Invest. 2014 May.

Abstract

Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.

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Figures

Figure 1
Figure 1. RSV-GFP infection, replication, and clearance in HAE cultures.
(A) Representative en face fluorescent images of GFP-positive cells in HAE infected with RSV expressing GFP at days 1, 3, 5, and 7 pi. Scale bar: 200 μm. (B) The number of RSV-infected cells in HAE over time, quantified as the percentage of the epithelium surface area positive for GFP fluorescence. (C) Virus titers present in the airway surface fluid over time. Data (mean ± SEM) are representative of independent experiments utilizing at least 4 cultures per donor obtained from 3 different donors.
Figure 2
Figure 2. Morphologic and structural changes in RSV-infected ciliated columnar cells.
(A) Representative histologic sections of HAE inoculated with RSV expressing GFP and counterstained with H&E or immunoprobed for GFP (green) and β-tubulin IV (red) at days 1, 3, and 5 pi. Yellow outlines at top left depict the different ciliated cell morphologies seen in RSV-infected HAE, where noninfected ciliated cells are columnar and RSV-infected ciliated cells exhibit rounded morphology. Arrowheads indicate robust apical terminal web in noninfected ciliated cells (top left), which thins in RSV-infected ciliated cells with rounded morphology (top right). Images are representative of independent experiments with 4 different donor cultures. Scale bar: 10 μm. (B) Representative TEM of cross-sections of the apical surfaces of noninfected (left) and RSV-GFP–infected (center, right) HAE. Arrowhead indicates particulates showing virus-like morphology. Asterisk indicates disorganized basal bodies. Scale bars: 1 μm.
Figure 3
Figure 3. Ciliated cell shedding and loss of cilia activity during RSV-GFP infection of HAE.
(A) Representative images of histological cross-sections of noninfected HAE (top) or HAE infected with RSV expressing GFP (bottom) fixed at day 5 pi using a fixation protocol to preserve the content and depth of airway surface secretions. Scale bar: 20 μm. (B) Concentration of dsDNA as an index for numbers of shed cells present in apical washes harvested every 24 hours from noninfected (white circles) or RSV-infected (black squares) HAE. Data (mean ± SEM) were derived from 4 cultures per donor for 4 different donors. (C) Fluorescent bead velocity as an index of mucus transport rates on the apical surfaces of noninfected (white circles) or RSV-infected (black squares) HAE at indicated times pi. Data (mean ± SEM) represent 3–6 cultures per donor for 3 different donors. (D) Surface area of active cilia beat on noninfected (white circles) or RSV-infected (black squares) HAE. Data (mean ± SEM) represent 4 cultures. Data in B–D show significant differences at day 3 pi (P < 0.05) and at all time points thereafter (P < 0.001), as determined by unpaired t test.
Figure 4
Figure 4. RSV-induced ciliated cell rounding is unique to RSV infection and is due to expression of the RSV NS2 protein.
(A) Representative images of histologic cross-sections of fixed and paraffin-embedded HAE infected with recombinant RSV-GFP, low-passage clinical isolate RSV Memphis 37, or recombinant PIV3-GFP. Infected cells were detected at day 3 pi using an anti-RSV antibody or anti-PIV3 antibody, as appropriate. Scale bar: 10 μm. (B) Representative images of histologic cross-sections of HAE infected with RSV, or gene deletion mutants RSVΔNS1 and RSVΔNS2. All viruses express GFP. (C and D) Height of infected ciliated cells (mean ± SD) was determined using confocal microscopy of fixed but unprocessed HAE infected with RSV, PIV3, HMPV, PIV5, or SeV or the RSV gene deletion mutants RSVΔNS1, RSVΔNS2, and RSVΔNS1/2. All viruses expressed GFP. At least 100 infected cells in cultures obtained from 3 different donors were measured. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s post-hoc test.
Figure 5
Figure 5. Expression of RSV NS2 in ciliated cells using PIV3 results in infected cell rounding.
(A) Growth kinetics of recombinant PIV3 (white circles) and PIV3 expressing either RSV NS1 (PIV3-NS1, black squares), NS2 (PIV3-NS2, white triangles) or both NS1 and NS2 (PIV3-NS1/2, black triangles) in HAE. All viruses expressed GFP. Virus titers in apical washes were assessed at 24-hour intervals. Data (mean ± SEM) represent 4 cultures per donor with cultures from 3 different donors. (B) Representative images of histologic cross-sections of HAE infected with PIV3, PIV3-NS1, PIV3-NS2, or PIV3-NS1/2. Infected cells were detected with an anti-PIV3 antibody. Scale bar: 10 μm. (C) Height of infected cells (mean ± SD) was determined as described in Figure 4. *P < 0.05, **P < 0.01, 1-way ANOVA with Tukey’s post-hoc test.
Figure 6
Figure 6. Expression of RSV NS2 by PIV3 in HAE cultures mimics RSV-induced cytopathology at the apical surface of ciliated cells.
(A) Representative images of histologic sections of noninfected HAE, or HAE infected by RSV, PIV3, PIV3-NS1, PIV3-NS2, or PIV3-NS1/2 fixed at day 2 pi showing similar apical membrane disruption in ciliated cells infected by RSV, PIV3-NS2, and PIV3-NS1/2. Richardson’s counterstain was used. Scale bar: 10 μm. (B) Surface area of active cilia beat on noninfected HAE (black circles, dotted line) or HAE infected with PIV3 (white circles), PIV3-NS1 (black squares), PIV3-NS2 (white triangles), or PIV3-NS1/2 (black triangles). Data (mean ± SEM) represent quadruplicate cultures. All viruses expressed GFP.
Figure 7
Figure 7. PIV3-NS2 causes ciliated cell rounding in the hamster nasal respiratory epithelium in vivo.
(A) The nasal epithelium of Golden Syrian hamsters was inoculated with 106 PFU of PIV3 (black circles), PIV3-NS1 (white squares), or PIV3-NS2 (black triangles), and virus titers in the nasal turbinates were determined at day 3 pi. Data (mean ± SEM) represent 3 independent experiments with a total of 9 to 12 animals. (B) Representative images of histologic cross-sections of hamster nasal respiratory epithelium infected with PIV3 (top), PIV3-NS1 (center), or PIV3-NS2 (bottom). Virus antigen was detected at day 3 pi with an anti-PIV3 antibody. Scale bar: 10 μm. (C) Height of infected ciliated cells (mean ± SD) was determined by measuring height of virus antigen–positive cells from fixed tissue sections, with at least 200 cells analyzed across 3 individual animals. All viruses expressed GFP. ***P < 0.001, 1-way ANOVA with Tukey’s post-hoc test.
Figure 8
Figure 8. Distal airway obstruction and accelerated clearance of virus infection in hamsters infected by PIV3-NS2.
(A and B) Representative images of histologic cross sections of hamster large airways (A) and small airways (B) 3 days and 5 days after infection with PIV3 or PIV3-NS2. Note the rapid loss of PIV3-NS2–infected cells from airways at 5 days pi, a time when PIV3 infection remained robust. Scale bar: 100 μm. (C) Morphometric quantitative analysis of distal airway cell accumulation in hamsters infected with PIV3 or PIV3-NS2 by measuring the percentage of cross-sectional airway lumen surface area occupied by virus antigen–positive cells at day 3 pi. Histologic, antigen-stained whole lung sections from 6 animals were measured. Each column represents an individual animal, and each symbol represents occlusion of an individual airway. Statistical analysis of the mean occlusion values of the two groups was used to determine significance. P < 0.01, Mann-Whitney U test. (D) Whole lung virus loads in hamsters infected with PIV3 (black circles) or PIV3-NS2 (white squares) measured over time, demonstrating a more rapid clearance of PIV3 infection when the virus expressed RSV NS2. Data (mean ± SEM) represent 3 independent experiments with a total of 4 to 9 animals per time point. All viruses expressed GFP. ***P < 0.0001, unpaired t test.
Figure 9
Figure 9. Shed epithelial cells infected by PIV3-NS2 into hamster lower conducting airways resembles shed epithelial cells in RSV-infected human infants.
(A) Representative images from histologic cross sections of hamster distal airways infected with PIV3 and PIV3-NS2. Sections were stained with H&E, or with antibody against PIV3 to identify PIV3-infected cells, or with an antibody against cytokeratin 18 (CK18) to identify epithelial cells at day 3 pi. Scale bar: 50 μm. (B) Sample images of histologic cross sections of human lower airways obtained postmortem from a patient naturally infected by RSV. Sections were stained with H&E, or with an RSV-specific antibody to detect infected cells, or with a CK8/18 antibody to detect epithelial cells. Scale bar: 50 μm. The smaller-diameter distal airways of both hamster and human demonstrate virus antigen–positive cells that have shed and accumulated in the lumen, often sufficient to have caused airway occlusion.
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