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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\u2013anaerobe co-culture for ~48 h, and we demonstrate its utility by evaluating reciprocal interactions between the oxygen-sensitive anaerobic bacterium,\n                      <jats:italic toggle=\"yes\">Fusobacterium nucleatum,<\/jats:italic>\n                      and oxygen-demanding airway epithelial cells at the transcriptional level. Using bulk RNAseq, we demonstrate that epithelial colonization results in altered gene expression by\n                      <jats:italic toggle=\"yes\">F. nucleatum,<\/jats:italic>\n                      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\n                      <jats:italic toggle=\"yes\">F. nucleatum<\/jats:italic>\n                      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\u2013host interactions and their mechanistic contributions to airway disease pathophysiology.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Conflicting oxygen demands between anaerobes and host cells present a significant barrier to\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        modeling of how these cell types interact. To this end, the significance of our dual oxic-anoxic culture (DOAC) approach lies in its ability to maintain anaerobe and epithelial viability during co-culture, paving the way for new insights into the role(s) of anaerobic microbiota in disease. We use DOAC to interrogate reciprocal interactions between the airway epithelium and\n                        <jats:italic toggle=\"yes\">Fusobacterium nucleatum<\/jats:italic>\n                        \u2014an anaerobic commensal with pathogenic potential. Given its link to a range of diseases, from localized infections to various cancers, these data showing how\n                        <jats:italic toggle=\"yes\">F. nucleatum<\/jats:italic>\n                        bacterium re-shapes its metabolism and virulence upon epithelial colonization provide new mechanistic insight into\n                        <jats:italic toggle=\"yes\">F. nucleatum<\/jats:italic>\n                        physiology and how the host responds. We use\n                        <jats:italic toggle=\"yes\">F. nucleatum<\/jats:italic>\n                        as our model, but the DOAC platform motivates additional studies of the gut, lung, and oral cavity, where host\u2013anaerobe interactions and the underlying mechanisms of pathogenesis are poorly understood.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01338-24","type":"journal-article","created":{"date-parts":[[2025,4,9]],"date-time":"2025-04-09T09:00:36Z","timestamp":1744189236000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":7,"title":["Dual oxic-anoxic co-culture enables direct study of anaerobe\u2013host interactions at the airway epithelial interface"],"prefix":"10.1128","volume":"16","author":[{"given":"Patrick J.","family":"Moore","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, University of Minnesota","place":["Minneapolis, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kayla","family":"Hoffman","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Minnesota","place":["Minneapolis, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sara","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, SUNY at Buffalo","place":["Buffalo, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0723-3195","authenticated-orcid":true,"given":"Joshua R.","family":"Fletcher","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, University of Minnesota","place":["Minneapolis, USA"]},{"name":"Department of Population Health and Pathobiology, North Carolina State University","place":["Raleigh, 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