Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3

Abstract

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs' effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils' response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs' TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don't upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Keywords: Apoptosis; Eosinophils; Glucocorticoid receptor; Signal transduction.

Figure. 1

Effect of Dexamethasone and IL-5 on eosinophil viability in vitro. Eosinophil apoptosis was assessed by staining with PE-labelled Annexin V and 7 AAD; viable, non-apoptotic cells are represented as Annexin V/7AAD-negative cells. A). Changes in eosinophil viability upon incubation with Dex at a concentration of 0.1 μM. * = p<0.05 vs. control B). Effect of IL-5 at 0.01 ng/ml and 0.1 ng/ml on eosinophil viability upon exposure to Dex at 0.1 μM. IL-5 was added at the same time as Dex, * = p<0.05 vs. cell stimulated with Dex alone, § = p<0.05 vs cells stimulated with IL-5 alone. C). The GR antagonist RU486 (1μM) inhibits the effect of Dex (1 μM), * = p<0.05 vs. control, D) The effect of preincubating eosinophils with Dex or IL-5 on eosinophil viability. Eosinophils were preincubated with Dex for 3 h or 1 h before stimulation with IL-5, or Dex was added at the same time or 1 h after IL-5, followed by viability assessment at 48 h; * = p<0.05 vs. eosinophils stimulated with IL-5 alone. The error bars represent the standard deviation of at least 3 independent experiments.

Figure 2

Effect of the selective GR agonist CpdA on the viability of quiescent and IL-5-stimulated eosinophils. A) Induction of apoptosis in quiescent eosinophils stimulated with CpdA (1 μM) and the antagonistic effect of RU486 (1 μM) for 48 h; * p<0.05 vs. control cells. B). The concentration-dependent effect of CpdA on viability of eosinophils stimulated with IL-5 for 48 h. The error bars represent the standard deviation of at least 3 independent experiments.

Figure 3

Western blot analysis of MKP1, NFIL3 and GR expression. A), Time-course of MKP1 expression shows upregulation by IL-5 and synergistic upregulation by the combination of IL-5 and Dex. There was no detectable expression of MKP1 in CpdA-treated eosinophils. B), Time-course of NFIL3 expression shows upregulation by IL-5 and synergistic upregulation by the combination of IL-5 and Dex. Treatment with CpdA did not produce upregulation of NFIL3 in either quiescent or activated eosinophils. C) Differential effect of Dex and CpdA on phosphorylation of GR on S211. Analysis of the total GR protein level showed a similar content of GR stimulated and control eosinophils within the first 6h of stimulation, and degradation of GR protein at later times. Blots representative of two independent experiments are shown.

Figure 4

SiRNA-mediated inhibition of MKP1 and NFIL3 expression in IL-5-stimulated eosinophils. Eosinophils were transfected with siRNA against MKP1 (A), against NFIL3 (B), control scrambled RNA (A, B) or no RNA (A, B) for 8 h, followed by stimulation with IL-5 (0.1 ng/ml) and Dex (1 μM) for an additional 40 h in the presence of 10% FBS. A), Western blot of MKP1 showed significant inhibition of MKP1 expression in cells transfected with MKP1 siRNA and no effect of MKP1 siRNA on expression of GR or F-actin B), Western blot of NFIL3 showed inhibition of MKP1 expression in cells transfected with NFIL3 siRNA and no downregulation of F-actin or the β chain of IL-5 receptor. Western blots are representative of at least 2 independent experiments.

Figure 5

Effect of inhibition of MKP1 and NFIL3 on eosinophil viability in the presence of IL-5 (0.1 ng/ml) and/or Dex (1 μM) for 40 h. Two controls including eosinophils exposed to transfection reagents and transfected with scrambled RNA were used to exclude a potential effect of RNA on eosinophil viability. Inhibition of MKP1 had no effect on eosinophil viability upon stimulation with IL-5 or Dex, while inhibition of NFIL3 decreased the viability of IL-5-stimulated cells and restored the pro-apoptotic effect of Dex. The error bars represent the standard deviation of 3 experiments from 2 independent donors.

Figure 6

Effect of the Pim1 inhibitor AZD1208 on the expression of NFIL3 (at 24 h) and viability of eosinophils (at 48 h) stimulated with IL-5 and Dex. A) AZD1208 at a concentration specific for inhibition of Pim1 activity (1ng/ml) blocked upregulation of NFIL3 by IL-5 and Dex/IL-5 (24 h) without a significant effect on Pim1 expression. B) Inhibition of Pim1 activity by AZD1208 blocked the anti-apoptotic effect of IL-5 and restored the proapoptotic effect of Dex on activated eosinophils, * p<0.05 vs. control cells. The error bars represent the standard deviation of 4 experiments from 3 independent donors.