In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Abstract

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Fig. 1.

Infectivity of RSV A2 and BT2a in WD-PBECs. WD-PBEC cultures (n = 5 donors) were infected with RSV A2 or BT2a (MOI ∼4). (A) Virus growth kinetics determined by titrating RSV in apical washes at 24-h intervals following infection. Data are presented as mean + SEM log₁₀ TCID₅₀/mL. (B) At 144 hpi, these cultures were fixed with 4% paraformaldehyde (PFA) (vol/vol) and stained with Alexa Fluor 488-conjugated anti-RSV F protein mAb (green); nuclei were counterstained with DAPI (blue). Representative en face micrographs of RSV A2- or BT2a-infected cultures are presented. (Magnification: 10×.) (Scale bars: 200 μm.)

Fig. 2.

RSV infection does not cause gross CPE but is restricted to apical ciliated and occasional nonciliated cells. WD-PBECs were infected as indicated in Fig. 1. (A) At 144 hpi, RSV- and mock-infected cultures were observed by phase-contrast microscopy for evidence of CPE. The white-cloud appearance is caused by mucus secretion. (Magnification: 10×.) (B) Confocal orthogonal sections of WD-PBECs, fixed at 144 hpi and stained for RSV F protein (green); nuclei were counterstained with DAPI (blue). (Magnification: 40×.) (C) En face and orthogonal (Insets) confocal images of RSV-infected WD-PBECs at 144 hpi, fixed and stained for RSV F protein (green) and ZO-1 (red). White arrowheads show tight junctions. (Magnification: 63×.) At 144 hpi, WD-PBECs were fixed, permeabilized, and stained for (D) RSV F protein (green) and β-tubulin (red) or (E) RSV F (green) and Muc5Ac (red). (Magnification: 63×.). Lower panels show orthogonal sections. Images are representative of five different donors.

Fig. 3.

RSV causes syncytia formation, apical cell sloughing, and apoptosis. WD-PBEC cultures were infected as indicated in Fig. 1. (A) At 144 hpi, cultures were fixed and stained with DAPI and anti-RSV F mAb to visualize nuclei (blue) and RSV-infected cells (green), respectively. Confocal data from RSV A2-infected (A) and BT2a-infected (B) cultures revealed syncytia formation after RSV infection (white arrows). Two different planes (main panel and Inset) are presented for BT2a infection. (Magnification: 63×.) (C) Large syncytium following BT2a infection. The orthogonal section (Lower) shows the fusion (white arrow) between two adjacent syncytia. (Magnification: 63×.) Images are representative of five different donors. (D) Cytospins of apical washes were performed 72 hpi, and slides were stained for DAPI to visualize apical cell sloughing. RSV infection induced considerable sloughing of cells compared with noninfected control slides, with BT2a > A2 >> uninfected cultures. (Magnification: 63×.) (E) Quantification of nuclei following cytospins of apical washes of RSV A2-, BT2a-, and mock-infected cultures (n = 3 donors). Values are means ± SEM. Areas under the curve were calculated for each donor and compared. **P < 0.01. (F) Cytospins were performed at 72 hpi and stained for evidence of apoptosis using the TUNEL assay. BT2a-infected > A2-infected >> uninfected cultures in terms of apoptosis induction. (Magnification: 63×.)

Fig. 4.

RSV causes the destruction of ciliated cells. WD-PBEC cultures were infected as indicated in Fig. 1. (A) Cultures were trypsinized 144 hpi, and cytospins were performed. Slides were stained for β-tubulin to visualize ciliated cells left in the culture after infection. (B) Percentages of ciliated cells remaining in WD-PBEC cultures following RSV or mock infection (n = 3 donors) were determined by counting β-tubulin–positive cells among a total of 1,500 cells. Values are means + SEM. **P < 0.01. (C) Cytospins were performed 144 hpi with apical washes and stained for β-tubulin to visualize detached ciliated cells (arrowheads).

Fig. 5.

RSV induces enhanced mucus secretion and goblet cell hyperplasia/metaplasia in WD-PBECs. WD-PBEC cultures were infected as indicated in Fig. 1. (A) Cytospins of apical washes 144 hpi. Slides were stained for MUC5AC to visualize mucus secretion following A2, BT2a, and mock infection. BT2a-infected > A2-infected >> uninfected cultures in terms of mucus secretion. (Magnification: 10×.) (B) Cultures were fixed with 4% PFA (vol/vol) and stained for MUC5AC (red) and RSV F protein (green) 144 hpi. Higher numbers of goblet cells were evident in BT2a- than in A2-infected cultures, and both infected cultures had demonstrably higher goblet cell content than uninfected cultures. (Magnification: 60×.) (C) Quantification of goblet cell hyperplasia for each condition. Values are means + SEM and represent the area covered by goblet cells in five different fields of each condition from three donors. Each condition was normalized to the mock-infected value.

Fig. 6.

Basolateral chemokine secretion induced following RSV infection. WD-PBECs were infected as indicated in Fig. 1. Chemokine secretions in the basolateral medium of RSV- and mock-infected cultures harvested at 24, 96, 120, and 144 hpi were measured (n = 5 donors). Because the medium was replaced every day, the data correspond to chemokine secretions within the preceding 24 h. Values are means + SEM. *P < 0.05, **P < 0.01, ***P < 0.001.