Effect of Thymoquinone on Mouse Dendritic Cells

Abstract

Thymoquinone, a component of Nigella sativa is known to confer protection against tumour growth due to stimulation of tumour cell apoptosis. Moreover, thymoquinone has remarkable anti-inflammatory potency. Surprisingly, despite its powerful influence on inflammation and its immunomodulatory effects, little is known about its effect on dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC maturation and cytokine release is triggered by bacterial components such as lipopolysaccharides (LPS). The present study explored whether thymoquinone modifies LPS-induced DC maturation, survival and cytokine release. To this end, mouse bone marrow derived DCs were treated with LPS and different concentrations of thymoquinone and the surface expression of CD11c, CD86, MHCII, CD54 and CD40 was determined by FACS analysis, the formation of the interleukins 10 (IL-10) and 12 (IL-12p70) as well as TNF-α by ELISA, caspase activation by FITC-labelled antibodies (FACS), cell membrane scrambling by annexin V binding (FACS) and Akt and ERK1/2 phosphorylation by Western blotting. LPS increased the percentage of CD11c⁺CD86⁺, CD11c⁺MHCII⁺, CD11c⁺CD40⁺ and CD11c⁺CD54⁺ cells and stimulated the release of IL-10, IL-12p70 and TNF-α. These effects were blunted by thymoquinone in a concentration dependent manner (1-20 µM). Moreover, LPS decreased and thymoquinone increased caspase 3 and caspase 8 activation and annexin V binding. Moreover, LPS-induced phosphorylation of prosurvival kinases Akt and ERK1/2 was abrogated by thymoquinone. In conclusion, thymoquinone compromises the maturation, cytokine release and survival of DCs.

Effect of Bacterial Lipopolysaccharide on Na⁺/H⁺ Exchanger Activity in Dendritic Cells

“…Apoptotic cell membrane scrambling was evidenced from annexin V binding to phosphatidylserine at the cell surface [33]. To this end, the percentage of phosphatidylserinetranslocating cells was evaluated by staining with FITC-conjugated Annexin V. In brief 4 x 10 5 cells were harvested and washed twice with Annexin washing buffer (AWB, 10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl 2 ).…”

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

“…Cultures were supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF; 35 ng/ml, Preprotech, Hamburg, Germany) and fed with fresh medium containing GM-CSF on days 3 and 6. On day 7, Ͼ85% of the cells expressed CD11c, which is a marker for mouse DCs (85)(86)(87). Experiments were performed on days 7-8 of DC culture.…”

Effect of Thymoquinone on Cytosolic pH and Na⁺/H⁺ Exchanger Activity in Mouse Dendritic Cells

“…Sham experiments were conducted by adding plain DMSO to the respective solutions at a final concentration of 0.1 %. Thymoquinone was used at a final concentration of 10 µM, which has previously been shown to be an effective concentration [23].…”

“…The antinflammatory effect of thymoquinone is at least partially due its influence on maturation, cytokine formation and survival of dendritic cells (DCs) [23], antigen-presenting cells involved in the initiation of both innate and adaptive immunity and thus critically important for the regulation of the immune response [24][25][26]. Stimulation of DCs by bacterial components such as lipopolysaccharides (LPS) triggers DCs maturation [27][28][29][30][31] and the release of several cytokines such as IL-10, IL-12 and TNF-α [32][33][34].…”