Neutrophil: Airway Epithelial Interactions Result in Increased Epithelial Damage and Viral Clearance during RSV Infection

Respiratory syncytial virus (RSV) is a major cause of paediatric respiratory disease. Large numbers of neutrophils are recruited into the airways of children with severe RSV disease. It is not clear whether or how neutrophils enhance recovery from disease or contribute to its pathology. Using an in vitro model of the differentiated airway epithelium, we found that addition of physiological concentrations of neutrophils to RSV infected nasal cultures was associated with greater epithelial damage with lower ciliary activity, cilia loss, less tight junction expression (ZO-1) and more detachment of epithelial cells than seen with RSV infection alone. This was also associated with a decrease in infectious virus and fewer RSV positive cells in cultures after neutrophil exposure compared to pre-exposure. Epithelial damage in response to RSV infection was associated with neutrophil activation (within 1h), and neutrophil degranulation with significantly greater cellular expression of CD11b, MPO and higher neutrophil elastase and myeloperoxidase activity in apical surface medias compared to that from mock-infected AECs. We also recovered more apoptotic neutrophils from RSV infected cultures (>40%), compared to <5% in mock infected cultures after 4h. The results of this study could provide important insights into the role of neutrophils in host response in the airway.


INTRODUCTION 45
Respiratory syncytial virus (RSV) is the major viral cause of pulmonary disease in 46 young infants and the elderly and is responsible for annual epidemics that cause 47 (MPO and matrix metalloproteinase-9 (MMP-9) in the apical surface media. The 120 amount of NE or MPO in apical surface media was measured using commercial activity 121 assay kits (Cayman, USA). MMP-9 release was measured using commercial ELISA kit 122 (Biolegend, USA). All protocols were according to the manufacturers' instructions. AnnexinV apoptosis detection kit (Miltenyl Biotec) was used to carry out this assay. 145 Neutrophils collected from the cell pellet (described above) were resuspended in 50µl 146

CBF and beat pattern 156
Beating cilia were observed via an inverted microscope system (Nikon TiE; Nikon, 157 UK) equipped with an incubation chamber (37°C, 5% CO2) as previously described (3). 158 To determine ciliary beat frequency (CBF), videos were recorded using a ×20 objective 159 using a CMOS digital video camera (Hamamatsu) at a rate of 198 frames per second, 160 and image size of 1024x1024 pixels. CBF (Hz) was calculated using ciliaFA software 161 (19). The number of motile ciliated cells in each sample area was counted (motility 162 index). The dyskinesia index was calculated as the percentage of dyskinetic ciliated 163 cells (those that displayed uncoordinated motile cilia or those that beat with a stiff, 164 flickering or twitching motion) relative to the total number of motile ciliated cells. 165 166

Immunofluorescence microscopy 167
Following fixation, cells were washed three times with PBS, treated with PBS 168 containing 0.1% Triton X-100 for 10 min to permeabilize the cells. Cells were incubated 169 with 5% FCS in PBS for 0.5h at room temperature to block nonspecific interactions, 170 and washed again three times with PBS. All subsequent antibody incubations were 171 carried out in 5% FCS in PBS + 0.1% Triton X-100. Reagents used in this study were 172 rabbit anti-ZO-1 polyclonal antibody (1:200, sc-5562, Santa Cruz) and mouse anti-173 acetylated α-tubulin monoclonal antibody (6-11B-1; 1μg/mL; Sigma). Primary antibody 174 incubations were carried out in a humidified chamber overnight at 4°C, followed by 175 three washes with PBS. Detection of primary antibodies was carried out for 1h using 176 the following reagents: fluorescein FITC (Sigma #F2012) conjugated rabbit anti-mouse 177 (1:64) or AlexaFluor 594-conjugated rabbit anti-donkey antibody (1:250; Invitrogen, 178 Paisley, UK). All secondary antibodies had been tested and found to be negative for 179 cross-reactivity against human epithelial cells. Following three washes in PBS, DNA 180 was stained with Hoechst 33258. After a final wash in distilled water, the insert was cut 181 from the support and mounted under coverslips in 80% (v/v) glycerol, 3% (w/v) n-182 propylgallate (in PBS) mounting medium. Images were captured with confocal laser 183 microscope (Zeiss Observer Z.1) using a 40x water immersion objective. The pinhole 184 was set at 1 airy unit (AU). For Z-stack images the slice thickness was 1μm.  196 197 Neutrophils enhance ciliated epithelial layer disruption and ciliary loss. 198 We found that neutrophils interacted with motile ciliated nAECs almost immediately 199 after introduction and gathered together to in clusters ( Figure 1A). After 1h incubation As RSV has been shown to target ciliated cells for infection, we were especially 213 interested in how ciliary activity is altered during neutrophil interactions with RSV 214 infected epithelial cells. We found that ciliary beat frequency was unaffected by RSV 215 infection or exposure over the entire study period (Table 1). However, RSV infection 216 led to a higher proportion of dyskinetic cilia ( Table 1)

RSV infection, compared to mock-infected cells or following 24h RSV infection with 227
and without neutrophil exposure (Figure 2A/B). However, we found that at 72h post-228 RSV infection, the mean fluorescence intensity for α-tubulin was almost half that of the 229 mock-infected group with neutrophils (P=0.014) ( Figure 2B). This loss α-tubulin 230 staining correlated with a loss in the number of motile cilia, observed by light 231 microscopy (referred to as the mean motility index) at 72h post-RSV infection 232 (57.6±1.89%), which was less compared with the mock-infected controls (70.8±1.97%) 233 (P<0.05). Exposure to neutrophils for 4h lowered the mean motility index of ciliated 234 cells infected with RSV for 72h (50.41±2.77%) compared to pre-neutrophil time point 235 (P<0.05) ( Table 1). As is shown in Figure 2C, the distribution of ciliary beat frequency 236 of the same field of view before and after addition of neutrophils produced a similar  Using flow cytometry ( Figure 4A) we showed that, at 24h post RSV-infection, there 255 was no difference in the percentage apoptotic (PI lo AnnexinV hi ) neutrophils recovered 256 from RSV infected nAECs (11.1±5.1%) compared to 4.8±2.7% in the mock-infected 257 co-cultures ( Figure 4B). However, at 72h post-infection, we detected significantly more 258 apoptotic neutrophils after exposure to RSV infected nAECs with 46.7±11.4% 259 compared to 6.2±0.9% in the mock-infected co-cultures (P<0.0001) ( Figure 4B). 260 Apoptosis appeared to be the dominant form of cell death as we did not detect an increase in dead (PI hi AnnexinV lo ) neutrophils at any time point or test condition. After 262 4h exposure to epithelial cells infected with RSV for 72h we detected 6.43±7.2% dead 263 neutrophils compared to 5.5±7.3% in the mock control (P>0.99) (Figure 4B). We have shown that when RSV infected human primary nasal airway epithelial cells 305 are exposed to neutrophils at physiological concentrations there is increased epithelial 306 layer disruption, ciliary loss and less infectious virus in these cultures. This suggests 307 that neutrophils are helping to eliminate viral infected cells and reduce viral spread. 308 The airway epithelial damage that we observed, consistent with previous studies (20, 309 21), may be a necessary consequence of this anti-viral effect. We found that RSV 310 infection without neutrophils did not reduce CBF, but did increase the number of cilia 311 that presented with an abnormal beat pattern as early as 24h post-infection, which is 312 similar to our previous findings (3, 5). Interestingly, the addition of neutrophils did not 313 further increase ciliary dyskinesia. 314

315
The reduction in number of RSV infected epithelial cells following neutrophil exposure 316 may result from neutrophil degranulation. Neutrophils are known to mediate direct 317 antimicrobial effects and neutralize several influenza A, RSV and vaccinia virus strains 318 through effector mechanisms including degranulation (22)(23)(24). We have shown that 319 neutrophils exposed to RSV infected human primary airway epithelial cells have 320 greater expression of the activation markers CD11B and MPO, and release greater 321 amounts of NE, MPO and MMP9. It is recognized that the inflammatory processes in 322 the airways of infants with RSV bronchiolitis are dominated by an intense neutrophil 323 influx (7, 25) and that neutrophil products such as MPO and NE are released into the 324 airway lumen (26). Indeed, the degree of neutrophilic inflammation correlates with disease severity in patients with RSV-induced bronchiolitis (27). Our study has shown 326 that RSV infected epithelial cells increased NE and MPO and gelatinase (MMP-9) 327 granule populations, at the same time as reducing neutrophil membrane integrity. This 328 appears to be relevant to the pathophysiology of viral respiratory infections (28, 29). 329 Likewise, both NE and MMP-9, which may be necessary for clearance of bacteria, are 330 linked to airway damage and progression of cystic fibrosis (30). Our results are 331 consistent with these clinical findings, suggesting that the cytotoxicity of neutrophil 332 antimicrobial proteases may be important in viral clearance, but may also potentiate 333

RSV-induced lung injury. 334 335
A surprising finding was that at 72h, but not at 24h, post RSV-infection, neutrophil 336 exposure led to an increase in numbers of apoptotic neutrophils. This finding is 337 consistent with a clinical study that found neutrophil apoptosis was accelerated in 338 nasopharyngeal aspirates and peripheral blood of infants with RSV bronchiolitis (31). 339 Neutrophil apoptosis is thought to be associated with loss of degranulation and other 340 pro-inflammatory capacities (32). We found that RSV infected epithelial cells increased 341 neutrophil degranulation in regards to increased neutrophil elastase (NE) and 342 myeloperoxidase (MPO) activity in apical surface media at the same time as we 343 detected the increased neutrophil apoptosis. These data suggest that within this model, 344 there may be at least two subsets of neutrophils that respond differently to RSV 345 infected airway epithelial cells. It is possible that this balance in neutrophil function 346 could be a predictor of disease severity (See Figure 6). 347 348 One limitation to our study was the number of neutrophils used. The exact ratio of 349 interacting neutrophils and airway epithelial cells in the lung of children with RSV 350 bronchiolitis is unknown. We used the equivalent concentration of 5x10 6 /ml neutrophils, 351 which is the upper limit of the number of neutrophils recovered from BAL of infants with 352 RSV bronchiolitis (1.78±3.3x10 6 /ml) that was reported previously by McNamara and 353 colleagues (7). This suggest that our data may indicate airway: immune cell 354 interactions that occur in lungs of infants with large neutrophil infiltrate or severe RSV 355 bronchiolitis. Another limitation of our study was that we exposed epithelial cells to 356 naïve neutrophils directly isolated from peripheral blood. In the lungs, neutrophils are 357 recruited to migrate from the basal sub-epithelial space across the vasculature and 358 epithelium to the airways following RSV infection (33). In this context, the changes that 359 we observed are all the more striking. 360

361
In conclusion, this study has revealed that neutrophils exposed to ciliated epithelial cell 362 cultures infected with RSV have greater degranulation, deploying harmful proteins and 363 proteases to the apical surface media and increasing the capacity for tissue injury. We 364 have shown that neutrophils contribute to RSV-associated ciliary loss combined with 365 epithelial damage, which is likely to result in reduce mucociliary clearance. These 366 effects may contribute to viral clearance and provide important insights into the role of 367 neutrophils in host response in the airway. 368 369 Table I The ciliary beat frequency, dyskinesia index and motility index of healthy nasal respiratory epithelial cells in pseudo-stratified air-liquid interface (ALI) cultures infected with RSV A2 for 24h or 72h and then co-cultured for 1 or 4h with human neutrophils.