<em>In vitro</em> Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Abstract

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

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

“…For neutrophil-AEC coculture experiments, CFBE cells were treated with TNFα for 24 h and exposed to Cif before the addition of neutrophils. For transepithelial neutrophil migration, CFBE cells were seeded on inverted 3-μM pore polycarbonate Transwell filters (51). Calcein AM (Life Technologies)-labeled neutrophils were treated with 15-epi LXA 4 or 14,15-EET in the presence of Cif-WT or Cif-D129S and applied to the basolateral side of CFBE cells.…”

Distinct Cellular Sources of Hepoxilin A3 and Leukotriene B4 Are Used To Coordinate Bacterial-Induced Neutrophil Transepithelial Migration

“…Neutrophils were isolated from whole blood collected from healthy human volunteers (IRB protocol #1999P007782) as previously detailed (33). Briefly, blood was anti-coagulated with acid citrate/dextrose.…”

“…Inverted monolayers were prepared as previously described (33). Briefly, transwell inserts were flipped to an inverted position, collagen coated, then seeded with either mouse lung epithelial cells (MLE12) or human lung epithelial cells (H292) on the underside of the transwell.…”

“…Neutrophils (2×10 5 /96w transwell, 1×10 6 /24w transwell) or whole bone marrow cells (8×10 5 /96w transwell, 2×10 6 /24w transwell) were supplied to the basolateral chamber and incubated for 2h at 37°C. After a 2h migration, the transwells were removed, and the apical well was assayed for neutrophil content by myeloperoxidase assay (33). In cases where different neutrophil or bone marrow sources were used in the same assay, standard curves were used to control myeloperoxidase variation between sources.…”

MicroRNAs Regulate Cytokine Responses in Gingival Epithelial Cells

“…A leukocyte chemotaxis/myeloperoxidase activity assay was carried out as described previously (27), using 24-well Transwell tissue-culture permeable inserts with 3-m pores (Corning, NY, USA). A total of 10 6 induced HL-60 cells in 100 l of Hanks' balanced salt solution (HBSS) buffer (devoid of calcium and magnesium) were added to the upper compartment, and 600 l of supernatant collected from WT and miRNA KD cells stimulated with heat-killed WT P. gingivalis for 6 h was added to the lower compartment.…”