Host-pathogen-immune interactions in an air-liquid interface airway model

Background Air–liquid interface (ALI) cell culture systems have improved the study of host-pathogen interactions in respiratory infections. However, most ALI models lack immune components, limiting their ability to capture epithelial–immune crosstalk. To address this, we developed a dual-cell ALI model incorporating human peripheral blood monocyte-derived macrophages beneath differentiated airway epithelial cells. Methodology Macrophages were seeded on the basolateral side of transwell inserts using fibronectin coating. Model characterization included transepithelial electrical resistance (TEER) to assess epithelial barrier integrity, IL-8 secretion as a marker of epithelial inflammatory signaling, and confocal microscopy to evaluate cellular architecture before and after infection. Mono- and dual-cell cultures were infected with the laboratory strain Pseudomonas aeruginosa PAO1. Results Macrophages adhered stably to the basolateral surface without compromising epithelial barrier integrity. Following infection, IL-8 secretion was elevated in epithelial monocultures compared to dual-cell cultures, suggesting early immune modulation in the presence of macrophages. While overall bacterial burden was comparable, confocal imaging revealed clustered bacterial growth in monocultures and a more dispersed spatial distribution in dual-cell cultures. Conclusions This dual-cell ALI model enables investigation of early epithelial–immune interactions, inflammatory modulation, and bacterial colonization dynamics during airway infection. The system provides a versatile and human-relevant platform for studying respiratory host–pathogen interactions.