Effect of prostaglandin I2 analogs on cytokine expression in human myeloid dendritic cells via epigenetic regulation

Abstract

Prostaglandin I(2) (PGI(2)) analog is regarded as a potential candidate for treating asthma. Human myeloid dendritic cells (mDCs) play a critical role in the pathogenesis of asthma. However, the effects of PGI(2) analog on human mDCs are unknown. In the present study, circulating mDCs were isolated from six healthy subjects. The effects of PGI(2) analogs iloprost and treprostinil on cytokine production, maturation and T-cell stimulatory function of human mDCs were investigated. Tumor necrosis factor (TNF)-α and interleukin (IL)-10 were measured by enzyme-linked immunosorbent assay. The expression of costimulatory molecules was investigated by flow cytometry. T-cell stimulatory function was investigated by measuring interferon (IFN)-γ, IL-13 and IL-10 production by T cells cocultured with iloprost-treated mDCs. Intracellular signaling was investigated by Western blot and chromatin immunoprecipitation. We found that iloprost and treprostinil induced IL-10, but suppressed TNF-α production in polyinosinic-polycytidylic acid (poly I:C)-stimulated mDCs. This effect was reversed by the I-prostanoid (IP), E-prostanoid (EP) receptor antagonists or intracellular free calcium (Ca(2+)) chelator. Forskolin, an adenyl cyclase activator, conferred a similar effect. Iloprost and treprostinil increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels, and iloprost also increased intracellular Ca(2+). Iloprost suppressed poly I:C-induced mitogen-activated protein kinase (MAPK) phospho-p38 and phospho-activating transcription factor (ATF)2 expression. Iloprost downregulated poly I:C-induced histone H3K4 trimethylation in the TNFA gene promoter region via suppressing translocation of histone 3 lysine 4 (H3K4)-specific methyltransferases MLL (mixed lineage leukemia) and WDR5 (WD repeat domain 5). Iloprost-treated mDCs inhibited IL-13, IFN-γ and IL-10 production by T cells. In conclusion, PGI(2) analogs enhance IL-10 and suppress TNF-α expression through the IP/EP2/EP4 receptors-cAMP and EP1 receptor-Ca(2+) pathway. Iloprost suppressed TNF-α expression via the MAPK-p38-ATF2 pathway and epigenetic regulation by downregulation of histone H3K4 trimethylation.

Figure 1

Iloprost and treprostinil enhanced IL-10– and poly I:C–induced IL-10 expression and suppressed poly I:C–induced TNF-α expression in human mDCs. Isolated mDCs were pretreated with iloprost or treprostinil for 24 or 48 h, or for 2 h, and then stimulated with poly I:C for 6, 24 or 48 h. Supernatants were collected for cytokine measurement. Iloprost alone (10⁻⁸ to 10⁻⁷ mol/L) (A) or in the presence of poly I:C (10⁻⁹ to 10⁻⁷ mol/L) (B) enhanced IL-10 expression in human mDCs. Treprostinil alone (10⁻⁹ to 10⁻⁷ mol/L) (C) also enhanced IL-10 expression. Iloprost (10⁻⁸ to 10⁻⁷ mol/L) (D) and treprostinil at higher concentration (10⁻⁶ mol/L) (E) suppressed poly I:C–induced TNF-α expression. Results presented are the mean ± SD of six independent experiments using mDCs from six subjects. *P < 0.05 compared with vehicle-treated cells (A, C) or poly I:C–treated cells (B, D, E). (A, B) □, 6 h; , 24 h; ■, 48 h.

Figure 2

Iloprost enhanced IL-10 expression and suppressed poly I:C–induced TNF-α expression in mDCs via the IP and EP receptors in human mDCs. Isolated human mDCs were pretreated with IP receptor antagonist CAY 10449 or EP receptor antagonists 1 h before treatment of iloprost. (A) CAY 10449 reversed iloprost-enhanced IL-10 expression. (B) The EP1 receptor antagonist SC19220 (10⁻⁵ mol/L), but not EP2 or EP4 receptor antagonists, also reversed iloprost-enhanced IL-10 expression. CAY 10449 (C) and EP1, EP2 and EP4 receptor antagonists (D) all significantly reversed iloprost-suppressed poly I:C–induced TNF-α expression. Results presented are the mean ± SD of six independent experiments using mDCs from six subjects. *P < 0.05 compared with iloprost-treated cells (A, B) or iloprost plus poly I:C–treated cells (C, D).

Figure 3

Treprostinil enhanced IL-10 expression via the EP4 receptor and suppressed poly I:C–induced TNF-α expression via the IP receptor in human mDCs. Isolated human mDCs were pretreated with IP receptor antagonist CAY 10449 or EP receptor antagonists 1 h before treatment of treprostinil. (A) CAY 10449 was unable to reverse treprostinil-enhanced IL-10 expression. (B) EP4 receptor antagonist GW627368X (10⁻⁵ mol/L), but not EP1 or EP2 receptor antagonists, reversed treprostinil-enhanced IL-10 expression. (C) The IP receptor antagonist CAY 10449, but not the EP1, EP2 or EP4 receptor antagonist, reversed treprostinil-suppressed poly I:C–induced TNF-α expression in mDCs. Result represented the mean ± SD of six independent experiments using the mDCs of six subjects. *P < 0.05 compared with treprostinil-treated cells (B) or treprostinil plus poly I:C–treated cells (C).

Figure 4

The modulatory effects of PGI₂ analogs on IL-10 and TNF-α expression is indepen dent of PPARs in human mDCs. To investigate the involvement of PPARs in the modulatory effect of PGI₂ analogs on IL-10 and TNF-α expression in human mDCs, the cells were pre-treated with PPAR-α antagonist (GW 6741) and PPAR-γ antagonists (GW9662) 1 h before the treatment of iloprost or treprostinil. Neither PPAR-α antagonist nor PPAR-γ antagonist changed the enhancing effect of iloprost (A) or treprostinil (B) on IL-10 expression, and the suppressive effect of iloprost (C) or treprostinil (D) on poly I:C–induced TNF-α expression. Results represented are the mean ± SD of three independent experiments using mDCs from three subjects.

Figure 5

PGI₂ analogs modulate IL-10 and TNF-α expression via the IP-EP2/EP4-cAMP pathway, and iloprost also modulated IL-10 and TNF-α expression via the EP1-Ca²⁺ pathway in human mDCs. Isolated human mDCs were treated with iloprost or treprostinil for 24 h. Iloprost (10⁻⁸ to 10⁻⁷ mol/L) (A) and treprostinil (10⁻⁷ mol/L) (B) increased intracellular cAMP levels in human mDCs. Forskolin, an adenyl cyclase activator, enhanced IL-10 expression (C) and suppressed poly I:C–induced TNF-α expression (D). To verify the involvement of EP1-Ca²⁺ pathway in the modulatory effect of iloprost, cells were treated with iloprost (10⁻⁷ mol/L) for 2 h with or without pretreatment of EP1 receptor antagonist SC 19220 (10⁻⁵ mol/L) and were detached and labeled with Fluo-3-AM. The change of intracellular Ca²⁺ was analyzed using flow cytometry. One experiment representative of three is shown as a histogram plot graph (E). (F) Iloprost increased cytosol Ca²⁺ level by the measurement of the mean fluorescence intensity (MFI), and the EP1 receptor antagonist SC 19220 partly reversed the effect. BAPTA-AM, an intracellular free Ca²⁺ chelator, partly reversed the modulatory effects of iloprost on IL-10 (G) and poly I:C–induced TNF-α expression (H). Results represent the mean ± SD of three independent experiments using mDCs from three subjects for the cAMP assay, the flow cytometry analysis for intracellular Ca²⁺ and the BAPTA-AM experiment, and of six independent experiments using mDCs from six subjects for the forskolin experiment. *P < 0.05 compared with vehicle-treated cells (A, B, C, F, G) or poly I:C–treated cells (D, H). ^#P < 0.05 compared with iloprost-treated cells.

Figure 6

Iloprost suppressed poly I:C–induced TNF-α expression in human mDCs via the MAPK-p38-ATF2 pathway. Isolated human mDCs were pretreated with MAPK-p38 inhibitor (SB203580), MAPK-JNK inhibitor (SP600125) or MAPK-ERK inhibitor (PD98059) 1 h before poly I:C stimulation. Supernatants were collected 24 h after poly I:C stimulation for TNF-α measurement. For Western blotting analysis, mDCs were pretreated with or without iloprost (10⁻⁷ mol/L) for 2 h and then stimulated with poly I:C for 1 h, and cell lysates were collected. (A) All MAPK inhibitors suppressed poly I:C–induced TNF-α expression. Iloprost suppressed poly I:C–induced phospho-p38 (B) and phospho-ATF2 (C) expression. Result of TNF-α expression represented the mean ± SD of six independent experiments using mDCs from six subjects. For Western blotting analyses, one experiment representative of three is shown. *P < 0.05 compared with poly I:C–treated cells.

Figure 7

Iloprost suppressed poly I:C–induced TNF-α expression in mDCs via histone H3K4 trimethylation in the TNFA gene promoter region. Isolated mDCs were pretreated with iloprost for 2 h and then stimulated with poly I:C for 1 h and were used for ChIP assay using anti-H3K4 antibodies. The cells were also used for cytosolic and nuclear protein analysis by Western blotting using anti-MLL and anti-WDR5 antibodies. The nuclear and cytosolic fractionation technique was verified using anti–α-tubulin and anti–histone H3 antibodies. (A) Iloprost downregulated poly I:C–induced H3K4 trimethylation in the TNFA gene promoter region. (B) Iloprost suppressed poly I:C–induced translocation of cytosolic H3K4-specific methyltransferases MLL and WDR5 proteins into nucleus. The nuclear fraction is lack of α-tubulin, and the cytosolic fraction is lack of H3. To investigate whether MAPK-p38 signaling is responsible for the translocation of MLL and WDR5 from cytoplasm to nucleus, mDCs were pretreated with MAPK-p38 inhibitor SB203580 for 2 h and then stimulated with poly I:C for 1 h. (C) SB203580 suppressed poly I:C–induced translocation of MLL and WDR5 from cytoplasm to nucleus. (D) Densitometry analysis of the Western blot data shown in (B). Results represented are the mean ± SD of three independent experiments using mDCs from three subjects for the ChIP assay and densitometry analysis. For Western blotting analyses, one experiment representative of three is shown. *P ≤ 0.05 compared with poly I:C–treated cells (A and D). OD, optical density. A: □, Control; ■, poly I:C; , iloprost + poly I:C.

Figure 8

Iloprost-treated mDCs suppressed IL-13, IFN-γ and IL-10 production by CD4⁺ T cells. Autologous CD4⁺ T cells were isolated and then cocultured with vehicle-treated or iloprost-treated mDCs (10⁵ mDCs/10⁶ T cells) for 5 d in the presence of anti-CD3 and anti-CD28 antibodies with or without the addition of IL-10 neutralizing antibody. Supernatants were collected for IL-13, IFN-γ and IL-10 measurement. Iloprost-treated mDCs significantly suppressed IL-13 (A), IFN-γ (B) and IL-10 production (C) by CD4⁺ T cells. (D) The addition of IL-10 neutralizing antibody did not change the effect of iloprost-treated mDCs on IL-13 and IFN-γ production by T cells. Results represent the mean ± SD of six independent experiments using mDCs from six subjects in the experiments without the addition of IL-10 neutralizing antibody and of three independent experiments using mDCs from three subjects in the experiments with the addition of IL-10 neutralizing antibody. *P ≤ 0.05 compared with the group of vesicle-treated mDC/T cell coculture. ^§P ≤ 0.05 compared with the group of iloprost-treated mDC/T-cell coculture without the addition of IL-10 neutralizing antibody. (D) □, Control; ■, iloprost (10⁻⁷ mol/L); , iloprost (10⁻⁷ mol/L) + anti–IL-10.