RESEARCH ARTICLE Plasticity of Airway Epithelial Cell Transcriptome in Response to Flagellin

Airway epithelial cells (AEC) are critical components of the inflammatory and immune re-sponse during exposure to pathogens. AECs in monolayer culture and differentiated epithe-lial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture ver-sus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional re-sponse by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, mono-layer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an un-expectedly muted response to flagellin, both in number of genes expressed and relative en-richment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium