Cilostazol inhibits plasmacytoid dendritic cell activation and antigen presentation

Abstract

Background: Cilostazol, an anti-platelet drug for treating coronary heart disease, has been reported to modulate immune cell functions. Plasmacytoid dendritic cells (pDCs) have been found to participate in the progression of atherosclerosis mainly through interferon α (IFN-α) production. Whether cilostazol influences pDCs activation is still not clear. In this study, we aimed to investigate the effects of cilostazol on cell activation and antigen presentation of pDCs in vitro in this study.

Methods: Peripheral blood mononuclear cells isolated by Ficoll centrifugation and pDCs sorted by flow cytometry were used in this study. After pretreated with cilostazol for 2 h, cells were stimulated with CpG-A, R848 or virus for 6 h or 20 h, or stimulated with CpG-B for 48 h and then co-cultured with naïve T cell for five days. Cytokines in supernatant and intracellular cytokines were analyzed by ELISA or flow cytometry respectively.

Results: Our data indicated that cilostazol could inhibit IFN-α and tumor necrosis factor α (TNF-α) production from pDCs in a dose-dependent manner. In addition, the ability of priming naïve T cells of pDCs was also impaired by cilostazol. The inhibitory effect was not due to cell killing since the viability of pDCs did not change upon cilostazol treatment.

Conclusion: Cilostazol inhibits pDCs cell activation and antigen presentation in vitro, which may explain how cilostazol protects against atherosclerosis.

Keywords: Antigen presentation; Cilostazol; Interferon α; Plasmacytoid dendritic cell; Tumor necrosis factor α.

Figure 1.. Cilostazol inhibits CpG-A-induced IFN-α and TNF-α production from PBMCs.

(A): The reduction of IFN-α in supernatant. 2×10⁵ PBMCs in each well of 96-well plates were pretreated with cilostazol at indicated doses for 2 h before stimulated by CpG-A for 20 h. IFN-α level in supernatant was measured by ELISA. (B): The gating strategy of flow cytometry. (C): The inhibition of cilostazol on intracellular IFN-α and TNF-α production. 2×10⁵ PBMCs in each well of 96-well plates were pretreated with 40 µmol/L cilostazol for 2 h before stimulated by CpG-A for 6 h and BFA was added for the last 3 h. The amount of intracellular IFN-α and TNF-α was tested by flow cytometry. Data were representative of four independent experiments. (D): Statistical data of Figure 1C. In particular, paired two-tail t-test was used to analyze the differences between each donor in different groups. *P < 0.05, **P < 0.01, vs. control group. CD: cluster of differentiation; FSC: forward scatter; HLA-DR: human leucocyte antigen-D region; IFN-α: interferon-α; Lin: linear amplification; PBMCs: peripheral blood mononuclear cells; SSC: side scatter; TNF-α: tumor necrosis factor-α.

Figure 2.. R848-induced IFN-α and TNF-α production are also decreased by cilostazol in a dose-dependent manner.

PBMCs were pretreated with cilostazol at indicated concentrations for 2 h before stimulated with 2 µg/mL R848 for 6 h and BFA was added for the last 3 h. Intracellular IFN-α and TNF-α was examined by flow cytometry. Data were representative of two independent experiments. CD: cluster of differentiation; IFN-α: interferon-α; PBMCs: peripheral blood mononuclear cells; TNF-α: tumor necrosis factor-α.

Figure 3.. Cilostazol inhibits CpG-A and virus-induced cytokine production.

(A): The gating strategy and purity of pDCs. (B): Cilostazol inhibited CpG-A-induced IFN-α production from pDCs. Purified pDCs were pretreated with cilostazol at indicated concentrations for 2 h before stimulated by CpG-A for 20 h. The supernatant was used to measure IFN-α by ELISA. (C): Cilostazol inhibited CpG-A-induced TNF-α production from pDCs. Samples were randomly chosen from three independent experiments in Figure 3B, and the levels of TNF-α were determined by FlowCytomix™. (D): Cilostazol directly reduced HSV-induced IFN-α production from pDCs. pDCs were treated with cilostazol and HSV (5 MOI) as in Figure 3B, and the levels of IFN-α were tested by ELISA. One representative of two independent experiments is shown. *P < 0.05, **P < 0.01, vs. control group. CD: cluster of differentiation; FSC: forward scatter; HLA-DR: human leucocyte antigen-D region; HSV: herpes simplex virus; IFN-α: interferon-α; Lin: linear amplification; pDCs: plasmacytoid dendritic cells; SSC: side scatter; TNF-α: tumor necrosis factor-α.

Figure 4.. The viability of pDC does not change upon cilostazol treatment.

3×10⁴ pDCs were pretreated with or without cilostazol (40 µmol/L) for 2 h, before stimulated by 2 µmol/L CpG-A for 8 h. Cell viability was determined by Guava Viacount (A) and Trypan Blue staining (B). Data were representative of two independent experiments. Guava: guava easyCyte; pDC: plasmacytoid dendritic cell.

Figure 5.. Cilostazol inhibits pDCs to prime naïve T cells.

PDCs were pretreated with cilostazol (40 µmol/L) or DMSO for 2h before stimulated with CpG-B for 48 h, then co-cultured with fresh naïve T cells for 5 d, and pulsed with 0.5 µCi of [³H]-TdR (2 Ci/mmol) for 18 h. Cells were harvested onto glass fiber filters and counted for radioactivity. The result was representative of two independent experiments. CPM: counts per minute; pDCs: plasmacytoid dendritic cells; [³H]TdR: ³H –thymidine.