Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases. Video LinkThe video component of this article can be found at

A model of neutrophil migration across epithelia is desirable to interrogate the underlying mechanisms of neutrophilic breach of mucosal barriers. A co-culture system consisting of a polarized mucosal epithelium and human neutrophils can provide a versatile model of trans-epithelial migration in vitro, but observations are typically limited to quantification of migrated neutrophils by myeloperoxidase correlation, a destructive assay that precludes direct longitudinal study. Our laboratory has recently developed a new isotropic 1-μm resolution optical imaging technique termed micro-optical coherence tomography (μOCT) that enables 4D (x,y,z,t) visualization of neutrophils in the co-culture environment. By applying μOCT to the trans-epithelial migration model, we can robustly monitor the spatial distribution as well as the quantity of neutrophils chemotactically crossing the epithelial boundary over time. Here, we demonstrate the imaging and quantitative migration results of our system as applied to neutrophils migrating across intestinal epithelia in response to a chemoattractant. We also demonstrate that perturbation of a key molecular event known to be critical for effective neutrophil trans-epithelial migration (CD18 engagement) substantially impacts this process both qualitatively and quantitatively.

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