Aims: The human gastrointestinal tract is relatively anaerobic (physiologic oxygen levels of 4.5%), but despite this the vast majority of cell-culture studies have been performed at atmospheric oxygen levels (21% oxygen). In this study, we aimed to determine whether culturing intestinal epithelial cells at atmospheric oxygen skews responses to pro-inflammatory stimuli compared to culturing at gut physiologic oxygen levels.
Methods: HT-29 human intestinal epithelial cells were incubated in a conventional 21% oxygen incubator, or in a normoxia chamber (Invivo400 Hypoxia Workstation, 4.5% oxygen). Cells were infected with Salmonella typhimurium or purified S. typhimurium flagellin for varying times. The levels of secreted interleukin (IL)-8, a neutrophil-attracting chemokine, in cell culture supernatants were measured using ELISA.
Results: S. typhimurium and its flagellin induced a time – and dose-dependent secretion of IL-8 compared to untreated cells. However, incubator oxygen levels influenced IL-8 secretion, and the levels of S. typhimurium – or flagellin – induced IL-8 were higher at 21% oxygen than at physiologic oxygen levels.
Conclusions: The data indicates that culturing cells at atmospheric rather than physiologic oxygen levels results in elevated IL-8 secretion in response to pro-inflammatory stimuli. This suggests that intestinal epithelial cell responses to other stimuli/environmental stresses may also be overestimated at atmospheric oxygen.
