Trans-epithelial migration is essential for neutrophil activation during RSV infection.

The recruitment of neutrophils to the infected airway occurs early following respiratory syncytial virus (RSV) infection, and high numbers of activated neutrophils in the airway and blood are associated with the development of severe disease. The aim of this study was to investigate whether trans-epithelial migration is sufficient and necessary for neutrophil activation during RSV infection. Here, we used flow cytometry and novel live-cell fluorescent microscopy to track neutrophil movement during trans-epithelial migration and measure the expression of key activation markers in a human model of RSV infection. We found that when migration occurred, neutrophil expression of CD11b, CD62L, CD64, NE, and MPO increased. However, the same increase did not occur on basolateral neutrophils when neutrophils were prevented from migrating, suggesting that activated neutrophils reverse migrate from the airway to the bloodstream side, as has been suggested by clinical observations. We then combined our findings with the temporal and spatial profiling and suggest 3 initial phases of neutrophil recruitment and behavior in the airways during RSV infection; (1) initial chemotaxis; (2) neutrophil activation and reverse migration; and (3) amplified chemotaxis and clustering, all of which occur within 20 min. This work and the novel outputs could be used to develop therapeutics and provide new insight into how neutrophil activation and a dysregulated neutrophil response to RSV mediates disease severity.

ß2-integrin LFA1 mediates airway damage following neutrophil transepithelial migration during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) bronchiolitis is the most common cause of infant hospital admissions, but there is limited understanding of the mechanisms of disease, and no specific antiviral treatment. Using a novel in vitro primary transepithelial neutrophil migration model and innovative imaging methods, we show that RSV infection of nasal airway epithelium increased neutrophil transepithelial migration and adhesion to infected epithelial cells, which is associated with epithelial cell damage and reduced ciliary beat frequency, but also with a reduction in infectious viral load.Following migration, RSV infection results in greater neutrophil activation, degranulation and release of neutrophil elastase into the airway surface media compared to neutrophils that migrated across mock-infected nasal epithelial cells. Blocking of the interaction between the ligand on neutrophils (the ß2-integrin LFA-1) for intracellular adhesion molecule (ICAM)-1 on epithelial cells reduced neutrophil adherence to RSV-infected cells and epithelial cell damage to pre-infection levels, but did not reduce the numbers of neutrophils that migrated or prevent the reduction in infectious viral load.These findings have provided important insights into the contribution of neutrophils to airway damage and viral clearance, which are relevant to the pathophysiology of RSV bronchiolitis. This model is a convenient, quantitative preclinical model that will further elucidate mechanisms that drive disease severity and has utility in antiviral drug discovery.