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.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…3) of our physioxic condition indicates that hypoxia was not induced, but the possible existence of an O 2 gradient within our cultures should be considered in future work. Haas et al [76] reported that the long-term culture of macrophages under 5% O 2 attenuated the immunomodulatory response to dimethyl fumarate and cells cultured under air exhibited a "pro-oxidant" phenotype (i.e. lower GSH:GSSG ratio), which is in agreement with our present observations.…”

Inflammasome Priming in Sterile Inflammatory Disease

“…Dimethyl fumarate has been suggested to exert its beneficial effects largely by decreasing neuroinflammation [28] . It has also been shown to decrease ROS production in a mouse macrophage cell line [29] , most likely as a consequence of its upregulation of antioxidants such as heme oxygenase-1, superoxide dismutase-2, and nuclear factor erythroid 2-related factor 2 (Nrf2), as well as decreasing IL-1 (Il1b) mRNA expression, as shown in primary rat microglial cells [28] .…”

Regulator Versus Effector Paradigm: Interleukin-10 as Indicator of the Switching Response

“…These conditions include physical and psychological stressful events like exercise, exposure to extreme thermal conditions, different kinds of pain, depression, sleep deprivation, hypoxia, etc. [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. In general, an increased IL-10 level during these events is associated with better outcome and adaptation.…”