In vitro and in vivo studies of the immunomodulatory effect of Echinacea purpurea on dendritic cells

Abstract

Background: Extracts of Echinacea have been used traditionally for the treatment of diverse types of infections and wounds. They have become very familiar immunostimulant herbal medicine. However, the specific immunomodulatory effect of Echinacea remains to be elucidated.

Aim: In our study, the effect of Echinacea purpurea extract on the generation of immature DCs from monocytes was described, as well as its effect on DC differentiation. In addition, an in vivo experiment was conducted to investigate whether treatment of mice with extracts derived from E. purpurea has immunomodulatory effect on murine splenic DCs.

Methods: Immature DCs were generated by incubating peripheral blood monocytes with cytokine cocktail (GM-CSF + IL-4) and matured by tumor necrosis factor-α (TNF-α). The cells were randomized to 5 groups to investigate E. purpurea effect in different stages. Phenotypic analysis of cell marker CD83-expressed on DCs was performed by flow cytometry. Mice were randomly divided into 3 groups; control, E. purpurea treated and E. purpurea-TNF-α treated group. The murine splenic DCs were isolated and phenotyped for CD83 and CD11c by flow cytometry.

Results: Treatment of monocytes with E. purpurea prior to addition of the maturation factor TNF-α resulted in a significant decrease in the yield of DC expressing CD83. On the other hand, immature DCs generated in the culture in the presence of GM-CSF and IL-4, when treated simultaneously with E. purpurea and TNF-α, exhibited an insignificant change in the yield of CD83-expressing DCs compared with untreated control. The in vivo experiments showed that splenic DCs obtained from mice treated with E. purpurea with or without TNF-α did not exhibit significant changes in CD83 or CD11c compared with those obtained from control mice.

Conclusion: Our findings suggest that the immunomodulatory mechanisms of E. purpurea impact generation fate of DCs rather than differentiation stages. The results obtained in the in vivo study utilizing murine splenic DCs supported those observed in vitro.

Keywords: CD11c; CD83; Dendritic cells; Echinacea purpurea; Murine splenic cells.

Figure 1

Morphological changes during generation and differentiation of control untreated DCs (40×). (A): Day 0: adherent monocytes. (B): Day 7: immature DCs. (C): Day 9: mature DCs.

Figure 2

Echinacea purpurea effect on DCs morphology on day 9 (100×). (A): DCs nucleated with branched projections (control without treatment). (B): DCs in E. purpurea treated culture, there were no observable changes in all E. purpurea treated groups compared with control.

Figure 3

Effect of E. purpurea on CD83 expression in various cell cultures. (A): Flow cytometry showing the expression of CD83. Monocyte-derived DCs were generated in presence or absence of E. purpurea. (Group A) control group; (Group B) E. purpurea (500 μg/ml) on day 7; (Group C) TNF-α (10 ng/ml) on day 7 + E. purpurea (500 μg/ml) on day 8; (Group D) E. purpurea (500 μg/ml) on day 7 + TNF-α (10 ng/ml) on day 8; (Group E) E. purpurea (500 μg/ml) on day 0 + TNF-α (10 ng/ml) on day 7. (B): Percentage of DCs expressing CD83 in different groups. Expression of CD83 cell marker is measured by BD FACSCalibur®. Each value is the mean of 3 experiments ± SE. ^*P < 0.05.

Figure 4

Flow cytometry of cellular markers CD83 and CD11c of murine splenic DCs obtained from all animal groups. Mice of each group (n = 10) were treated with E. purpurea with or without TNF-α, then splenocytes were analyzed by flow cytometry. T cells and B cells (CD83⁻) were gated out. Major CD11c⁻CD83⁺ and CD11c⁺CD83⁺ populations are circled. Total CD11c⁻CD83⁺ cells are gated by R1, whereas CD11c⁺CD83⁺ population is gated by R2. SSC-Height is referred to side scattered pulse height. FSC-Height is referred to forward scattered light height.

Figure 5

Effect of E. purpurea on the percentage of CD11c⁻CD83⁺ and CD11c⁺CD83⁺ murine splenic DCs. E. purpurea was administered orally, whereas TNF-α was given i.p. n = 10 for each group. Expression of CD11c and CD83 cell marker was measured by BD FACSCalibur®. Each value is the mean ± SE.