Transmigration and phagocytosis of macrophages in an airway infection model using four-dimensional techniques

During infection, recruited phagocytes transmigrate across the epithelium to remove the pathogens deposited on the airway surface. However, it is difficult to directly observe cellular behaviors (e.g., transmigration) in single-cell layer cultures or in live animals. Combining a three-dimensional (3D) cell coculture model mimicking airway infection with time-lapse confocal imaging as a four-dimensional technique allowed us to image the behaviors of macrophages in 3D over time. The airway infection model was moved to a glass-bottomed dish for live-cell imaging by confocal laser scanning microscopy. Using time-lapse confocal imaging, we recorded macrophages transmigrating across the polyethylene terephthalate (PET) membrane of the inserts through the 5-μm pores in the PET membrane. Macrophages on the apical side of the insert exhibited essentially three types of movements, one of which was transmigrating across the epithelial cell monolayer and arriving at the surface of monolayer. We found that adding Staphylococcus aureus to the model increased the transmigration index but not the transmigration time of the macrophages. Only in the presence of S. aureus were the macrophages able to transmigrate across the epithelial cell monolayer. Apical-to-basal transmigration of macrophages was visualized dynamically. We also imaged the macrophages phagocytizing S. aureus deposited on the surface of the monolayer in the airway infection model. This work provides a useful tool to study the cellular behaviors of immune cells spatially and temporally during infection.