title: Permanent culture of macrophages at physiological oxygen attenuates the antioxidant and immunomodulatory properties of dimethyl fumarate.
abstract: We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators.
authors.affiliation: Université Paris-Est, UMR_S955, UPEC, France; INSERM, U955, Equipe 03, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Haas
authors.forename: Sandra
authors.initials: S
authors.lastname: Chrusciel
authors.forename: Sarah
authors.initials: S
authors.lastname: Fayad-Kobeissi
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Dubois-Randé
authors.forename: Francisco
authors.initials: F
authors.lastname: Azuaje
authors.forename: Jorge
authors.initials: J
authors.lastname: Boczkowski
authors.forename: Roberto
authors.initials: R
authors.lastname: Motterlini
authors.forename: Roberta
authors.initials: R
authors.lastname: Foresti
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D000069462:Dimethyl Fumarate; D005650:Fumarates / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000378:metabolism; D007155:Immunologic Factors / Q000494:pharmacology*; D007249:Inflammation / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D008070:Lipopolysaccharides; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D010100:Oxygen / Q000494:pharmacology*; D010101:Oxygen Consumption / Q000187:drug effects / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D000975:Antioxidants; D005650:Fumarates; D007155:Immunologic Factors; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D051547:Heme Oxygenase-1; D000069462:Dimethyl Fumarate; D010100:Oxygen