Spatial and temporal analysis of neutrophil trans-epithelial migration in response to RSV infection

In the airways, recruitment and activation of neutrophils occurs early following respiratory virus (RSV) infection and is associated with the development of severe disease. We investigated whether activated neutrophils selectively migrate across virus infected airway epithelial cells, or whether trans-epithelial migration is sufficient and necessary for neutrophil activation. We profiled the movement and adherence of fluorescently labelled human neutrophils during migration across primary human airway epithelial cells (AECs) infected with RSV in vitro. In RSV infected AECs neutrophil adherence, with clustering occurs after 15-18 minutes. Using flow cytometry, we found that, when migration occurred, expression of CD11b, CD62L, CD64, NE and MPO were increased in all compartments of our system and RSV infection further increased CD11b and NE expression. We found evidence suggesting that migrated neutrophils can migrate in reverse to the basolateral membrane. Our study provides novel insights into how airway activated neutrophils mediate systemic disease in respiratory virus infection. Graphical Abstract LegendAir-liquid interface (ALI) models for studying neutrophil trans-epithelial migration in response to RSV infection. Left panel shows primary airway epithelial cells cultured at ALI on membrane inserts with 3m pore size that permits neutrophils to migrate through. 1) unmigrated neutrophils expressing baseline levels of CD11b, 2) neutrophils migrate across infected AECs and some remain adherent to the infected AECs, 3) neutrophils are shown to increase expression of CD11b and other activation associated markers, 4) some activated neutrophils undergo reverse migration as 5) neutrophils with the increased expression of CD11b are detected on the basolateral side of the insert. Right panel shows primary airway epithelial cells cultured at ALI on membrane inserts with 0.4m pore size that prevents neutrophils migration. Here, after 1h incubation with RSV infected AECs, neutrophils on the basolateral side 1) and 2) were shown to express the same level of CD11b markers, indicating that neutrophil migration is key process for increasing expression of these activation markers. Drawing created using BioRender.com. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY