Respiratory syncytial virus infection disrupts monolayer integrity and function in cystic fibrosis airway cells

Abstract

Background: Respiratory Syncytial Virus (RSV) infection is a common contributor to pulmonary symptoms in children with cystic fibrosis (CF). Here we examined RSV infection in immortalized bronchial epithelial cells (CFBE41o-) expressing wild-type (wt) or F508del cystic fibrosis transmembrane conductance regulator (CFTR), for monolayer integrity and RSV replication.

Methods: CFBE41o- monolayers expressing wt or F508del CFTR were grown on permeable supports and inoculated with RSV A2 strain. Control experiments utilized UV-inactivated RSV and heat-killed RSV. Monolayer resistance and RSV production was monitored for up to six days post-infection.

Results: Within 24 h, a progressive decrease in monolayer resistance was observed in RSV infected F508del CFBE41o- cells, while the monolayer integrity of RSV infected wt CFTR CFBE41o- cells remained stable. RSV replication was necessary to disrupt F508del CFBE41o- monolayers as UV-irradiated and heat killed RSV had no effect on monolayer integrity, with an earlier and much more pronounced peak in RSV titer noted in F508del relative to wt CFTR-expressing cells. RSV infection of wt CFBE41o- monolayers also resulted in blunting of CFTR response.

Conclusions: These findings identify an enhanced sensitivity of CFBE41o- cells expressing F508del CFTR to RSV infection, replication and monolayer disruption independent of the cellular immune response, and provide a novel mechanism by which cystic fibrosis airway epithelia are susceptible to RSV-dependent injury.

Figure 1

Resistance measurements across wt and F508del CFTR expressing CFBE41o- monolayers post RSV inoculation. Transepithelial resistance was measured daily (up to 6 days) in CFBE41o- cells transduced to overexpressed wild type (wt) CFTR or F508del CFTR. The white circles represent uninfected CFBE41o- cells expressing F508del CFTR while the black circles represent uninfected CFBE41o- cells expressing wt CFTR. The triangles represent CFBE41o- cells expressing wt CFTR (black) and F508del CFTR (white) infected with RSV A2 at a MOI of 0.1. Each time point represents the mean data obtained from 12 separate wells.

Figure 2

Resistance measurement across F508del CFTR expressing CFBE41o- cells post inoculation with live and killed RSV. Transepithelial measurements were obtained in uninfected CFBE41o- cells expressing F508del CFTR (white circles), as well as F508del CFTR cells infected with UV-irradiated RSV (grey circles), heat-killed RSV (black circles) and live RSV (MOI = 0.01 white triangles, MOI = 0.1 grey triangles and MOI = 1 black triangles). Monolayer resistance was measured daily from time of infection through Day 6 post-RSV.

Figure 3

CFTR current measurements in control uninfected and RSV infected wt CFBE41o- cells. To measure CFTR dependent short circuit (I_SC), CFBE41o- cells were cultured on 6 mm diameter permeable inserts and mounted into modified Ussing chambers. Cell monolayers were stimulated with adenosine (10 μM) and genistein (50 μM) to activate CFTR ion transport and blocked with CFTR_INH172, (10 μM, mucosal) to confirm the CFTR dependence of I_SC changes.

Figure 4

RSV titer in media of F508del and wt CFBE41o- cells with time. CFBE41o- cells transduced to overexpressed F508del (black square) or wt CFTR (black diamond) were inoculated with RSV (MOI = 0.1) at time point 0. Media from both cell lines were collected from the apical compartment at Days 2, 4 and 6 post RSV infection, and used to determine changes in viral load over time.