Abstract
Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial 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 versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response 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, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment 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.
Highlights
The paradigm of the lung epithelium as a physical barrier to injurious substances and infectious agents has evolved with the recognition that airway epithelial cells (AECs) are important modulators of the host’s acute inflammatory and immune response to pathogens [1]
air liquid interface (ALI)-cultured human AEC and large airway AECs freshly obtained from human subjects may share similar transcriptional profiles [9,10], we recently reported significant differences in microRNA expression between primary cells and ALI cultures [11]
Variability in AEC transcriptome is strongly influenced by culturing method We applied correspondence analysis to segregate the AEC samples based on culture system, gene expression interrogation method, and exposure
Summary
The paradigm of the lung epithelium as a physical barrier to injurious substances and infectious agents has evolved with the recognition that airway epithelial cells (AECs) are important modulators of the host’s acute inflammatory and immune response to pathogens [1]. Evidence for the pleotropic role of human epithelial cells in lung defense, immune and inflammatory processes and responses to lung injury has been based, in large part, from in vitro studies, including cell lines and primary lung epithelial cell cultures in both submerged. Lung Epithelial Cell Response to Flagellin monolayer and air liquid interface (ALI) systems [4]. Murine models, especially those involving genetically modified mice, have supported biological inferences derived from studies of AECs in culture [5,6,7,8]. Previous reports using microarray analysis indicated that AEC gene expression profiles representing numerous biological processes undergo extensive changes during differentiation in ALI culture [12,13]
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