Investigating the function of neutrophils in RSV Bronchiolitis using a novel in vitro translational model of transepithelial immune cell migration

Background: Respiratory Syncytial Virus (RSV) infects almost all children before the age of 2. In most children, RSV infection causes a mild to moderate illness, but a small percentage of children develop severe RSV bronchiolitis and require hospitalisation. A significant proportion of these acutely unwell children were previously otherwise healthy. Clinical studies of infants with severe bronchiolitis have shown a massive infiltrate of neutrophils to the airway, comprising over 80% of all cells recovered. In this thesis I explore further whether the effect of neutrophil infiltration is necessary for viral clearance or detrimental to clinical disease, by exploring the interaction between neutrophils and RSV infected airway epithelial cells (AECs). Aims: The aims of this thesis were to (i) describe neutrophil movement across RSV infected AECs, (ii) measure the effect of interaction with RSV infected AECs on neutrophil apoptosis, (iii) activation, then (iv) compare these effects between adult and cord blood neutrophils. Methods: To examine the complex relationship between the RSV infected airway and neutrophil function, this study optimises a novel in-vitro transepithelial migration model of RSV infected AECs, comprising human neutrophils, ciliated epithelial cells cultured at air-liquid interface and RSV. This has allowed real-time video microscopy of neutrophil transepithelial migration, examination of dynamic neutrophil activation and assessment of neutrophil viability and apoptosis during infection. Results and Conclusion: Three populations of neutrophils interacting with AECs were described, those basolateral to AECs, those apical and those adhered to AECs. Imaging neutrophil migration revealed adhered neutrophils appear to kill virally infected AECs during migration. Neutrophil apoptosis and activation were defined by their interaction with AECs and evidence presented in this thesis suggest physiological differences between adult and cord blood neutrophil response to RSV infected AECs may predispose to a more severe disease picture in neonates