An in vitro transepithelial migration assay to evaluate the role of neutrophils in Respiratory Syncytial Virus (RSV) induced epithelial damage

Large numbers of neutrophils migrate into the lungs of children with severe Respiratory Syncytial Virus (RSV) disease. It is unclear how these cells contribute to viral clearance and recovery from infection or whether they contribute to disease pathology. We have developed a novel in vitro model to study neutrophil migration through airway epithelial cells (AECs), the main cellular target of RSV infection. Our model reproduces a physiologically relevant cell polarity and directionality of neutrophil migration. Using this model, we found that RSV infected AECs induced rapid neutrophil transepithelial migration. We also detected increased AEC damage associated with RSV infection, with a further increase in epithelial cells shedding from the Transwell membrane following neutrophil migration. This was not observed in the mock infected controls. Neutrophils that migrated through the RSV infected AECs showed increased cell surface expression of CD11B and MPO compared to neutrophils that had not migrated. In conclusion, our in vitro co-culture assay can be used to identify critical mechanisms that mediate epithelial cell damage and promote inflammation in children with severe RSV disease.