Immuno-modulatory activity of Ganoderma lucidum-derived polysacharide on human monocytoid dendritic cells pulsed with Der p 1 allergen

Abstract

Background: Ganoderma lucidum-derived polysaccharide (PS-G) can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs), suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs) from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10) were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile.

Results: PS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1) cell cytokine (INF-γ) production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone.

Conclusion: The presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.

Figure 1

PS-G increased co-stimulatory molecule expression on MD-DCs. Immature MD-DCs were pulsed either with Der p 1 (1 μg/ml) or PS-G (10 or 25 μg/ml), or both for 24 h and analyzed by flow cytometry for CD80, CD86, CD83, and HLA-DR expression. DCs stimulated with LPS (100 ng/ml) were positive maturation controls. Values shown are the mean fluorescence intensity (MFI) indexes. Dotted line, isotype control; solid line, specific mAbs. One representative of three independent experiments is shown.

Figure 2

The effect of PS-G on the phagocytic capacity of MD-DCs. Immature MD-DCs (1 × 10⁶ cells/1 ml per well) were incubated for 24 h either with Der p 1 (1 μg/ml) or PS-G (10 or 25 μg/ml) or both. DCs stimulated with LPS (100 ng/mL) were positive maturation controls. Cells were then incubated with FITC-dextran for 1 h at 4°C (dotted lines) or 37°C (solid lines). Values shown in the flow cytometry profiles are the MFI indexes. One representative of three independent experiments is shown.

Figure 3

PS-G increased IL-12 p40 production in Der p 1-pulsed MD-DCs from five healthy children (A) and six children with allergic asthma (B). MD-DCs were incubated for 24 h with Der p 1, PS-G, or both. The supernatants were collected for cytokines. Data are represented as the mean ± SE for three determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 4

PS-G increased IL-12 p70 production in Der p 1-pulsed MD-DCs from five healthy children (A) and six children with allergic asthma (B). MD-DCs were incubated for 24 h with Der p 1, PS-G, or both. The supernatants were collected for cytokines. Data are represented as the mean ± SE for three determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 5

PS-G increased IL-6 production in Der p 1-pulsed MD-DCs from five healthy children (A) and six children with allergic asthma (B). MD-DCs were incubated for 24 h with Der p 1, PS-G, or both. The supernatants were collected for cytokines. Data are represented as the mean ± SE for three determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 6

PS-G increased IL-23 production in Der p 1-pulsed MD-DCs from five healthy children (A) and six children with allergic asthma (B). MD-DCs were incubated for 24 h with Der p 1, PS-G, or both. The supernatants were collected for cytokines. Data are represented as the mean ± SE for three determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 7

PS-G increased IL-10 production in Der p 1-pulsed MD-DCs from five healthy children (A) and six children with allergic asthma (B). MD-DCs were incubated for 24 h with Der p 1, PS-G, or both. The supernatants were collected for cytokines. Data are represented as the mean ± SE for three determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 8

PS-G affected T cell cytokine response. MD-DCs from five children with allergic asthma were pulsed with Der p, PS-G, or both for 24 h and cultured with autogenic naïve T cell. Supernatants were analyzed for IFN-g (A) and IL5 (B), which were produced by activated T cells after 2 days of culture. Data are represented as means ± SEM of triplicates and representative of three independent experiments. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)

Figure 9

PS-G affected T cell proliferation response. MD-DCs from five children with allergic asthma were pulsed with Der p, PS-G, or both for 24 h and cultured with autogenic naïve T cell. Autologous T-cell proliferation was measured after 3 days of co-culture with MD-DC. The stimulation index (SI) was calculated as a mean counts per minute (cpm) of stimulated wells divided by the mean cpm of control wells. Values are presented as the mean stimulation index for triplicate wells. Data are represented as the mean ± SE of five determinations. Statistical analysis focused on DCs with or without PS-G in the presence of Der p 1. *p < 0.05. (Cont = control)