Peroxisome proliferator-activated receptor-γ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages

Abstract

Objective: 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyzes the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome proliferator- activated receptor-γ (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism, and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPARγ therein.

Methods and results: 11β-HSD1 gene expression is higher in proinflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages, whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages but not in resting macrophages or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPARγ induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes.

Conclusion: Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity.

Figure 1. 11β-HSD1 gene expression is higher in M2 macrophages

Primary human macrophages were cultured for 7 days in the absence (RM) or in the presence of IL-4 (15 ng/ml) (M2). Pro-inflammatory M1 macrophages were obtained by activating RM macrophages with LPS (100ng/ml) during 4h. 11β-HSD1 (A), MR (B) and AMAC1 (C) mRNA levels measured by Q-PCR and normalized to those of cyclophilin. Results are expressed as the mean value ± SD of triplicate determinations, representative of three independent experiments. Statistically significant differences are indicated (**p<0.01,***p<0.001).

Figure 2. 11β-HSD1 enzyme activity is highest in M2 macrophages leading to pronounced anti-inflammatory effects of cortisone

RM (A), M1 (B) and M2 macrophages (C) were incubated with radio-labeled cortisone (E) for the indicated time periods. Production of cortisol (F) was then measured. 11β-HSD1 reductase activity was determined as the percentage conversion of cortisone to cortisol. Results are representative of two independent experiments. Statistically significant differences are indicated (*p<0.05;**p<0.01,***p<0.001). RM and M2 macrophages were treated with cortisone or dexamethasone (Dexa, 1μM) for 2h before stimulation with LPS (100ng/ml) for 4h. mRNA levels of IL-6 (D), TNFα (E) and IL-1β (F) were measured by Q-PCR. Results are representative of three independent experiments. Statistically significant differences between control and treated cells are indicated (*p<0.05,**p<0.01).

Figure 3. Cortisone more pronouncedly induces the expression of GR target genes in M2 macrophages

RM and M2 macrophages were incubated with cortisone (1 μM) for different time points. GILZ (A), ANGPTL4 (B), and PDK4 (C) mRNA levels were measured by Q-PCR. Statistically significant differences between control and treated cells are indicated (*p<0.05;**p<0.01;***p<0.001).

Figure 4. 11β-HSD1 expression is induced by PPARγ in RM, M1 and M2 macrophages

(A) RM, M2 and M1 macrophages were treated in the absence or in the presence of GW1929 (600nM); 11β-HSD1 mRNA level was measured by Q-PCR. Results are representative of three independent experiments. Statistically significant differences between control and treated cells (*p<0.05;**p<0.01;***p<0.001) and basal RM or M1 and M2 macrophages are indicated (^§p<0.05;^§§p<0.01). (B) Intracellular 11β-HSD1 and β-actin protein expression analyzed by western blot and immunoreactive band intensity was quantified. Results are representative of two independent experiments. (C) RM were treated with vehicle or the PPARγ antagonist GW9662 (1μM) in the absence or presence of GW1929 (600nM) for 24h. (D) RM were transfected with scrambled or human PPARγ siRNA and subsequently treated with GW1929 (600nM) or DMSO during 24h. (E) RM were infected with Ad-GFP or Ad-PPARγ and subsequently stimulated for 24h with or without GW1929 (600nM). 11β-HSD1 mRNA levels were measured by Q-PCR and normalized to those of cyclophilin. Results are expressed as the mean value ± SD of triplicate determinations, representative of three independent experiments. Statistically significant differences are indicated (*p<0.05;**p<0.01;***p<0.001).

Figure 5. PPAR binds more avidly to the 11β-HSD1 PPRE in M2 macrophages

(A) EMSA was performed using the end-labeled DR1-consensus-PPRE or 11β-HSD1-PPRE oligonucleotide in the presence of unprogrammed reticulocyte lysate or in vitro translated hPPARγ and hRXRα. Supershift assays were performed using an anti-PPARγ antibody. (B) RM were transfected with the indicated reporter constructs (DR1-11β-HSD1 PPRE)₆ or (DR1-consensus PPRE)₆ (C), in the presence of pSG5-empty or pSG5-PPARγ vector, treated or not with GW1929 (600nM) and luciferase activity was measured. Statistically significant differences are indicated (*p<0.05;**p<0.01;***p<0.001). (D) EMSA was performed using in vitro produced RXR and PPARγ or nuclear extracts from RM, M1 and M2 in the absence or in the presence of exogenous hRXRα and supershift assays performed using an anti-PPARγ antibody. (E) ChIP assays were performed and quantified using chromatin from RM and M2 macrophages, immunoprecipitated with rabbit IgG or PPARγ-specific antibodies and then subjected to PCR using primer pairs covering either the 11β-HSD1 gene promoter or the β-actin gene. (F) H3K9ac ChIP-seq data for RM and M2 macrophages. The Y-axis shows the number of mapped tags sequenced on ChIP DNA from both RM and M2 macrophages. Promoters P1 and P2 as well as the identified PPRE are indicated. ChIP experiments were performed on H3K9-immunoprecipitated chromatin from two independent donors using primers covering the two identified 11β-HSD1 PPRE sites on the P1 and P2 promoters. Relative fold enrichments relative to a negative control region (set at 1) are shown. (G) RM and M2 macrophages were transfected with the (DR1-11β-HSD1 PPRE)₆ construct, treated or not with GW1929 (600nM). Statistically significant differences are indicated (*p<0.05;**p<0.01).

Figure 6. PPARγ activation increases 11β-HSD1 activity in M2 macrophages leading to the induction of GR-target genes by cortisone

RM (A), M1 (B) and M2 (C) macrophages were treated for 24h in the absence or in the presence of GW1929 (600nM) and subsequently incubated with radio-labelled cortisone (E) for the indicated time periods. Production of cortisol (F) was then measured. 11β-HSD1 reductase activity was determined as the percentage conversion of cortisone to cortisol. Results are representative of two independent experiments. Statistically significant differences between control and treated cells are indicated (*p<0.05). RM and M2 macrophages were activated or not with GW1929 (600nM) for 24h and subsequently treated for another 24h with cortisone (1μM). PDK4 (D), GILZ (E) and ANGPTL4 (F) mRNA levels were measured by Q-PCR. Statistically significant differences between control and treated cells are indicated (*p<0.05;*p<0.01;***p<0.001).