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Respiratory syncytial virus is a major cause of respiratory disease. There are conflicting accounts of the response of human epithelial cells to respiratory syncytial virus and a lack of data on its effect on ciliary function. Our aim was to study the early stages of respiratory syncytial virus infection of primary human basal and ciliated cultures. Using high speed videomicroscopy, we found that ciliary beat frequency was unaffected by respiratory syncytial virus infection over 72 h; however, ciliary dyskinesia significantly increased within 24 h of infection (p<0.05). Transmission electron microscopy revealed that ultrastructural abnormalities were confined to ciliated cells, including increased cilia loss and mitochondrial damage. Confocal immunofluorescence microscopy showed that respiratory syncytial virus antigen gradually spread from the cell surface to the ciliary tip of infected cells over 3 days. Interestingly, ciliated cultures secreted fewer viruses than basal (progenitor) cell cultures and produced a chemokine response focused on recruitment of neutrophils.This study highlights differences in infection models and underscores the need to explore further the role of ciliated cells in the establishment of respiratory syncytial virus infection. Introduction Respiratory syncytial virus (RSV) is the major cause of hospitalisation of children with respiratory disease aged <1 year worldwide and it infects almost every child by age 2 years [1]. Recent evidence suggests that the virus initiates infection by targeting human ciliated epithelium lining the nasopharynx [2–4]. We have previously shown that nasal brush biopsies from hospitalised infants with bronchiolitis exhibited significant disruption of the ciliated epithelium during RSV infection, with recovery taking many weeks [5]. More recently ciliated human respiratory epithelium in culture has been used to explore the early effects of RSV infection. These studies have produced conflicting results. While one study ed marked cellular disruption and very rapid ciliostasis after the addition of RSV [6], others no cytopathic effects [2, 7, 8] and no effect on ciliary beat frequency (CBF) [2] over many days, suggesting little effect on the ciliary component of mucociliary clearance. However these studies have lacked evaluation of a crucial aspect of ciliary function, ciliary beat pattern. We have previously shown that in certain phenotypes of primary ciliary dyskinesia, where mucociliary clearance is grossly reduced, all the cilia beat in a dyskinetic fashion, despite CBF being maintained at a normal level [9, 10]. In addition, we have shown that the infection of healthy volunteers with coronavirus caused epithelial damage and gross ciliary dyskinesia, but did not affect CBF [11]. The primary aim of this study was to evaluate the early effect of RSV infection on the beat pattern and frequency of human ciliated epithelial cells in culture, to test the hypothesis that RSV infection would significantly increase ciliary dyskinesia. The second aspect of our study was to investigate the preference of RSV to infect ciliated cells and to compare the effect of RSV in two infection models: human basal cell and ciliated cell cultures. To do this we evaluated the pattern of viral antigen spread in basal and ciliated cell cultures using immunofluorescence microscopy. We also studied the pattern of cytokine and chemokine secretion from the apical surfaces of the basal and ciliated cell cultures. Materials and methods Human nasal epithelial cells were obtained from healthy control subjects (n=9) aged median (range) 21 (19–49) years who had no history of nasal or respiratory disease. None of the subjects was taking medications and none had ed a symptomatic upper respiratory tract infection in the preceding 6 weeks. All individuals gave their consent to be included in the study and all samples were obtained with the individual’s permission and with ethical approval by the University of Leicester Committee for Research Ethics (Leicester, UK). Respiratory epithelial cell culture Human ciliated epithelium was obtained by brushing the inferior nasal turbinate with a 2-mm cytology brush (Keymed, Southend-on-Sea, UK) as previously described [9]. The ciliated epithelial sample was vigorously pipetted into 2 mL 20 mM HEPES-buffered medium 199 (pH 7.4) (Gibco Life Technologies, Paisley, UK), containing penicillin (100 IU•mL−1), streptomycin (100 μg•mL−1) and fungizone (2.5 μg•mL−1) to break-up large cell clumps and kept at 4°C overnight. 1 mL was then placed in a collagen coated well of a 12-well plate (Sigma-Aldrich, Poole, UK) together with 1 mL basal epithelial growth medium (BEGM), containing penicillin (100 IU•mL−1), streptomycin (100 μg•mL−1) and fungizone (2.5 μg•mL−1), at 37°C. The medium bathing the basal cells was replaced every 2–3 days. When the cells were >90% confluent the cells were detached using trypsin/EDTA (Sigma-Aldrich) for 5 min. The cells were then centrifuged (4000×g for 10 min) and the supernatant removed. The pellet was resuspended in BEGM to a concentration of 1×106 cells•mL−1. 400 μL cell suspension was added to each well of a 24-well plate (Sigma-Aldrich) or Lab-TekII Chambered Coverglass slide (for acquiring high-resolution images) and grown until ≥80% confluent. The remaining basal cells were seeded on collagen-coated, semipermeable membrane supports (Transwell-Col; 12 mm in diameter; 0.4-μm pore size; Corning-Costar, Corning) as previously described [12]. At 80% confluence, the apical medium was removed and the cells were maintained at an air–liquid interface (ALI) to allow differentiation of the epithelial subtypes. Well-differentiated cultures were studied ∼4–6 weeks after initiation of an ALI culture, unless otherwise stated. Cells obtained from two individuals produced basal cell and ALI cultures that were used for experimentation. Virus strains and growth conditions Wild-type RSV strain A2 stocks were prepared in monolayers of BSC-1 monkey kidney cells in antibiotic-free Glasgow’s minimum essential medium supplemented with nonessential amino acids and 2% (v/v) fetal calf serum at 5% CO2 and 37°C. Stocks were harvested by disruption with glass beads for 1 min and the supernatant was centrifuged at 1000×g for 5 min to remove cell debris. The filtrate was then purified by centrifugation through a polyethersulfone membrane with a pore size of 1000 kDa molecular weight cut-off (Vivaspin-20; Vivascience, Gloucester, UK), as previously described [13]. The virus fractions were collected and pooled in bronchial epithelial cell basal medium (BEBM; Lonza, Slough, UK), and aliquots containing about 0.5–1×106 plaque-forming units (PFU)•mL−1 were stored at -80°C. Viral infection of primary epithelial cell cultures Frozen aliquots of RSV were thawed immediately prior to use. For basal cell infections, 400 μL viral suspension (multiplicity of infection (MOI) of ∼0.1 PFU per cell) diluted in BEBM was applied for 1 h at 37°C. Control wells received BEBM alone. After this time, the virus inoculum was removed and the cells were incubated in BEGM. The infection was allowed to continue for a further 72 h. At this time, the supernatants were harvested and stored at -70°C for cytokine analyses. For ALI culture infection, the apical surface of the ALI cultures was rinsed with medium (BEBM) and 200 μL viral inoculum (MOI of ∼0.1 PFU per cell) in BEBM was applied to the apical surface for 1 h at 37°C and then removed. Control wells received BEBM alone. All cells were fed basolaterally with fresh ALI medium prior to infection. The infection was allowed to continue for 20–72 h. After this time the apical surface was again rinsed with medium, which was collected and stored at -70°C for cytokine analyses. Cells were fixed overnight with 4% (v/v) paraformaldehyde in PBS for immunostaining. CBF and beat pattern To determine changes in ciliary activity, respiratory cells in culture were placed in an incubation chamber at 37°C and were observed via an inverted microscope system (Nikon TU1000; Nikon, Kingston-upon-Thames, UK) equipped with a motorised stage. Cultures were allowed to equilibrate for 30 min before readings. Prior to infection, the location coordinates of five different ciliated areas from each Transwell insert were saved using NIS Elements AR software (Nikon) to allow repeated observation of the same areas. Beating cilia were recorded using a trouble-shooter digital high-speed video camera (Lake Image Systems, Henrietta, NY, USA) at a rate of 250 frames per second using a 40× objective as previously described [9]. The number of motile ciliated cells in each sample area was counted (motility index) and half were used to determine the average CBF. The CBF of individual ciliated cells was determined by counting the number of frames required for five full sweeps of a clearly observed ciliary tip. This was converted to CBF by a simple calculation (CBF=250/(number frames for five beats)×5) [9]. Ciliary dyskinesia was defined as ciliated cells that displayed uncoordinated motile cilia or those that beat with a stiff, flickering or twitching motion. The dyskinesia index was calculated as the percentage of dyskinetic ciliated cells relative to the total number of motile ciliated cells. The video sequences were analysed by two observers (C. Smith and P. Radhakrishnan); the second observer (P. Radhakrishnan) was blinded to the treatment and time-point recorded. Chemokine and cytokine analysis Chemokines and cytokines were measured using a 96-well multispot assay (Meso Scale Discovery (MSD), Rockville, MD, USA) according to the manufacturer’s instructions. Cytokines were measured using a human T-helper cell (Th1/Th2) type standard 10 spot plate and human chemokines were measured using a high band MS6000 10-spot plate, using SECTOR Imager 6000 (MSD). The lower limit of detection was 1 pg•mL−1. Immunofluorescence microscopy Cells were fixed with 4% (v/v) paraformaldehyde in PBS overnight at 4°C. Following fixation, cells were washed three times with PBS, treated with 3% (w/v) bovine serum albumin (BSA) in PBS for 10 min to block nonspecific interactions, and washed again three times with PBS. All subsequent antibody incubations were carried out in PBS containing 1% (weight/volume) BSA. Reagents used for immunofluorescence in this study were goat anti-RSV polyclonal antibody (ab20745; 40 μg•mL−1; Abcam, Cambridge, UK) and mouse anti-acetylated α-tubulin monoclonal antibody (clone 6-11B-1; 1 μg•mL−1; Sigma). Primary antibody incubations were carried out for 2 h, followed by three washes with PBS. Detection of primary antibodies was carried out for 2 h using the following secondary reagents: fluorescein isothiocyanate-conjugated rabbit anti-goat (1:200; Sigma) or AlexaFluor 594-conjugated rabbit anti-mouse antibody (1:250; Invitrogen, Paisley, UK). All secondary antibodies had been tested and found to be negative for cross-reactivity against human epithelial cells. DNA was stained with Hoechst 33258 (0.2 μg•mL−1). Following three final washes in PBS, the insert was cut from the support and mounted under coverslips in 80% (v/v) glycerol, 3% (w/v) n-propylgallate (in PBS) mounting medium. Low magnification images were acquired using a Nikon TU1000 inverted microscope equipped with NIS Elements software that enabled the acquisition of images of entire wells. High resolution optical sections were obtained using a Leica TCS SP5 confocal microscope equipped with a Leica DMI 6000B inverted microscope (Leica, Milton Keynes, UK) using a 63× oil objective (numerical aperture, 1.4). Images acquired by confocal microscopy were rendered by Imaris Software (Bitplane AG, Zurich, Switzerland) using the blend or maximum intensity projection filters. Transmission electron microscopy In a recent study we used transmission electron microscopy (TEM) to show cilia loss is a feature of patients with severe asthma [14]. To determine whether RSV infection also led to cilia loss, we obtained three additional samples from healthy control subjects (median (range) age 29 (24–58) years) by brushing the bronchus intermedius (by flexible bronchoscopy), and cultured and infected the ciliated cells as before. Following infection, cells were fixed in 4% (v/v) glutaraldehyde and processed as described previously [14]. Statistical analysis Statistical analysis was performed using GraphPad Prism 5 (GraphPad, San Diego, CA, USA). CBF (expressed in hertz) was determined following random selection of cilia. Any difference in the ciliary activity observed for control and RSV was determined using paired t-tests. Nonparametric data were described as median (interquartile range). Within-group comparisons of the magnitude of chemokine/cytokine release were performed using a Wilcoxon signed ranks test. Between groups comparisons were performed using the Mann–Whitney U-test. Results Ciliated nasal epithelial cells showed a higher proportion of dyskinetic cilia following RSV infection (table 1). The median (interquartile range) dyskinesia index was significantly increased (p<0.05) as early as 24 h post-RSV infection (21 (17–25)%) compared with controls (10 (2–13)%). This increased (p<0.05) further to 33 (25–42)% after 72 h of RSV infection compared with controls 13 (11–14)%. Despite changes in ciliary beat pattern, the CBF of ciliated nasal epithelial cells in culture was unaffected by RSV infection over the 72-h study period. The median (interquartile range) CBF of RSV-infected nasal ciliated cells was the same after 72 h (10.8 (10.5–11.7) Hz) as the uninfected controls (10.3 (10.1–12.7) Hz) (p=0.87). The number of ciliated cells with motile cilia, observed by light microscopy, was also unaffected by RSV infection. The median (interquartile range) motility index for ciliated nasal epithelial cells was similar 72 h post-RSV infection (22 (18–29)%) compared with the uninfected controls (20 (18–28)%) (p=0.88). 网站原创范文除特殊说明外一切图文作品权归所有；未经官方授权谢绝任何用途转载或刊发于媒体。如发生侵犯作品权现象，英语论文范文，保留一切法学追诉权。()，英语论文 |
