Dual oxic-anoxic co-culture enables direct study of anaerobe–host interactions at the airway epithelial interface

Strict and facultative anaerobic bacteria are widely associated with both acute and chronic airway diseases. However, their potential role(s) in disease pathophysiology remains poorly understood due to inherent limitations of existing laboratory models and conflicting oxygen demands between anaerobes and host cells. To address these limitations, here, we describe a dual oxic-anoxic culture (DOAC) approach that maintains an oxygen-limited microenvironment at the apical epithelial interface while host cells are oxygenated basolaterally. This platform enables epithelial–anaerobe co-culture for ~48 h, and we demonstrate its utility by evaluating reciprocal interactions between the oxygen-sensitive anaerobic bacterium, Fusobacterium nucleatum, and oxygen-demanding airway epithelial cells at the transcriptional level. Using bulk RNAseq, we demonstrate that epithelial colonization results in altered gene expression by F. nucleatum, highlighted by the differential expression of genes associated with virulence, ethanolamine and lysine metabolism, metal uptake, and other transport processes. We also combine DOAC with single-cell RNA sequencing to reveal a cell type-specific transcriptional response of the airway epithelium to F. nucleatum infection, including the increased expression of inflammatory marker genes and cancer-associated pathways. Together, these data illustrate the versatility of DOAC while revealing new insights into anaerobe–host interactions and their mechanistic contributions to airway disease pathophysiology.