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Glucocorticoids Sensitize the Innate Immune System Through Regulation of the NLRP3 Inflammasome

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Glucocorticoids Sensitize the Innate Immune System Through Regulation of the NLRP3 Inflammasome

John M Busillo et al. J Biol Chem.

Abstract

Glucocorticoids have long been recognized as powerful anti-inflammatory compounds that are one of the most widely prescribed classes of drugs in the world. However, their role in the regulation of innate immunity is not well understood. We sought to examine the effects of glucocorticoids on the NOD-like receptors (NLRs), a central component of the inflammasome and innate immunity. Surprisingly, we show that glucocorticoids induce both NLRP3 messenger RNA and protein, which is a critical component of the inflammasome. The glucocorticoid-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of proinflammatory molecules, including mature IL-1β, TNF-α, and IL-6. This effect was specific for glucocorticoids and dependent on the glucocorticoid receptor. These studies demonstrate a novel role for glucocorticoids in sensitizing the initial inflammatory response by the innate immune system.

Figures

FIGURE 1.
FIGURE 1.
Glucocorticoids enhance the expression of NLRP3 in human macrophages. THP-1 (2 × 106) cells were either left untreated (THP) or differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) (THP-MΦ) and treated as described under “Experimental Procedures.” The level of GILZ mRNA (A), NLRP3 mRNA (B), or GR mRNA (C) was normalized to cyclophilin B (PPIB), and data are presented as relative mRNA (mean ± S.E.; n = 4). THP or THP-MΦ were harvested and blotted for total GR or actin (D). Shown are representative blots from four independent experiments. Human monocytes (2 × 106) were differentiated to macrophages and treated as described under “Experimental Procedures.” The level of NLRP3 mRNA (E, left panel) was normalized to cyclophilin B (PPIB), and data are presented as relative mRNA (mean ± S.E.; n = 3). Duplicate samples were processed for Western blot (E, right panel). Shown are representative blots for NLRP3 and actin. Bone marrow monocytes (1.5 × 106) were differentiated to macrophages and treated as described under “Experimental Procedures.” The level of NLRP3 mRNA (F, left panel) was normalized to cyclophilin B (PPIB), and data are presented as relative mRNA (mean ± S.E.; n = 4). Duplicate samples were processed for Western blot (F, right panel). Shown are representative blots for NLRP3 and actin. Veh, vehicle.
FIGURE 2.
FIGURE 2.
Glucocorticoids rapidly and directly increase the levels of NLRP3 mRNA and protein. Following differentiation, THP-MΦ were treated with Dex (100 nm) in the absence of LPS priming (A). Samples were processed for RT-PCR analysis of NLRP3 (mean ± S.E., n = 3) or Western blotting of NLRP3 and GR (right panel). Shown is a representative Western blot from one of three independent experiments. Following differentiation, THP-MΦ were treated with Dex (100 nm) in the presence of 1 μg/ml of LPS (B). Following differentiation, THP-MΦ were treated with Dex (100 nm) or cortisol (Cort; 500 nm) over 24 h (C). Samples were processed for Western blotting of NLRP3. Shown is a representative Western blot from one of three independent experiments. THP-MΦ were incubated with 10 μg/ml cyclohexamide (CHX) for 1 h prior to the addition of Dex (100 nm) for 1 and 6 h. Cellular RNA was isolated and total mRNA for GILZ (D) and NLRP3 (E) was determined as described previously (mean ± S.E., n = 3).
FIGURE 3.
FIGURE 3.
Glucocorticoids specifically regulate NLRP3 but not other components of the inflammasome. Following differentiation, THP-MΦ were left untreated (−) or pretreated with 1 μg/ml of LPS for 1 h prior to the addition of ATP (500 μm), Dex (100 nm), or ATP and Dex. The levels of NLRP3 mRNA (A), IL-1β mRNA (B), and caspase-1 mRNA (C) were determined (mean ± S.E., n = 4). Shown are representative Western blots from four independent experiments for NLRP3, pro-IL-1β (p32), and caspase-1.
FIGURE 4.
FIGURE 4.
Glucocorticoids sensitize the NLRP3 inflammasome to extracellular ATP and enhance inflammasome function. A, THP-MΦ were pretreated for 1 h with 1 μg/ml LPS in medium supplemented with 10% charcoal-stripped FBS. Cells were then stimulated with ATP (500 μm), Dex (100 nm), or ATP and Dex for 6 h. Where indicated, Ru486 was added to the cells 5 min before Dex stimulation. The maturation and release of IL-1β (p17) was determined in cell culture supernatants by Western blot. Shown are representative blots from four independent experiments. B, upper panel: THP-MΦ were treated with Dex (100 nm), cortisol (Cort; 500 nm), aldosterone (Aldo; 1 μm), estradiol (Est, 10 nm), testosterone (Test, 10 nm), or ethanol (Veh) for 6 h in the presence of ATP (500 μm), and maturation and release of IL-1β was determined. Shown are representative blots from three independent experiments. Lower panel: LPS-primed THP-MΦ were left untreated (−) or treated with 0, 1, 10, 33.3, and 100 nm Dex in the presence of ATP (500 μm) for 6 h, and the maturation and release of IL-1β (p17) was determined. Shown are representative blots from three independent experiments. C, upper panel: LPS-primed THP-MΦ were pretreated with Veh (H2O) or Dex (100 nm) for 0, 3, 4, 5, and 5.5 h prior to the addition of ATP (500 μm). IL-1β release was determined as described above. Shown are representative blots from three independent experiments. Lower panel: LPS-primed THP-MΦ were concomitantly treated with Veh (H2O) or Dex and either 100 or 50 μm ATP for 6 h. Shown are representative blots from four independent experiments. D, 96 h following electroporation of either non-targeting control (NTC) or NLRP3-specific siRNA, LPS-primed THP-MΦ were left untreated (−) or treated with Veh (H2O) or Dex (100 nm) in the presence of ATP (500 μm) for 6 h. Shown are representative blots from four independent experiments. E, LPS-primed THP-MΦ were pretreated for 30 min with either Veh (dimethyl sulfoxide) or YVAD (10 μm) prior to the addition of ATP, Dex, or ATP+Dex. Shown are representative blots from three independent experiments.
FIGURE 5.
FIGURE 5.
Glucocorticoids enhance the release of the proinflammatory cytokines, TNF-α, and IL-6 from THP-MΦ. A, THP-MΦ were left untreated (−) or primed with LPS for 1 h prior to stimulation with ATP, Dex, or ATP+Dex for 6 h. Cell culture supernatants were harvested, and cytokine levels were determined by flow cytometry using a cytometric bead array as described under “Experimental Procedures.” Shown are representative flow cytometric plots from five individual experiments. B, cytokine levels were quantitated as described under “Experimental Procedures.” Data are expressed as mean ± S.E. (n = 5).

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