A Proinflammatory Effect of the β-Glucan from Pleurotus cornucopiae Mushroom on Macrophage Action

Abstract

PCPS from P. citrinopileatus mushroom extract is a β-1,6-glucan possessing a proinflammatory effect on innate immune cells. The PCPS stimulated THP-1 macrophages to secrete significant levels of TNF. Moreover, the mRNA expressions of TNF and IL-1β were significantly enhanced by PCPS treatment. However, the PCPS did not induce to express both IL-12 and IL-10 mRNA in the macrophages. Next, the P. cornucopiae extract (containing mainly PCPS) treatment against mice showed significant increases in TNF and IL-1β mRNA expressions in the peritoneal macrophages of them. In this study, the expression levels of IFNγ mRNA in the spleen were almost the same between the extract- (PCPS-) treated group and control group. However, the expression of IL-4 mRNA showed a lower level in the extract-treated group than that in the control. Our results suggested that the PCPS could induce proinflammatory action in the immune response. In addition, the proinflammatory effect of the PCPS on THP-1 was enhanced by 5'-GMP-Na, while it was reduced by vitamin D₂. These two compounds are majorly contained in the P. citrinopileatus mushroom. Therefore, these results suggested that the P. citrinopileatus mushroom might contain other immune regulative compounds, such as vitamin D₂, as well as PCPS.

Figure 1

P. citrinopileatus polysaccharide PCPS induces secretion of proinflammatory cytokine, TNF, in THP-1 macrophages. Human monocyte cell line THP-1 was differentiated into macrophages by PMA (160 nM) for 48 hrs., and then they were stimulated with the different concentrations of the 450 kD polysaccharide (PCPS) purified from a hot water extract of P. citrinopileatus fruiting bodies, or LPS (10 ng/ml, as a positive control). After 24 hours, the secretion of TNF in the cell supernatant was determined by ELISA. The data shown are the average of 3 independent experiments. The depicted error bars represent the SEM of these 3 experiments. For statistical analysis, a one-way ANOVA followed by the Dunnett's t test was used; ^∗p < 0.05 and ^∗∗∗p < 0.001 when compared to the RPMI medium control.

Figure 2

The P. citrinopileatus polysaccharide PCPS induces some proinflammatory cytokine expressions in THP-1. Human monocyte cell line THP-1 was differentiated into macrophages by PMA (160 nM) for 48 hrs., and then they were stimulated with the different concentrations of the 450 kD polysaccharide (PCPS) purified from a hot water extract of P. citrinopileatus fruiting bodies, or LPS (10 ng/ml, as a positive control). After 2 hours, the mRNA expression levels of TNF (a), IL-1β (b), IL-12 (c), and IL-10 (d) in the THP-1 were determined by using quantitative real-time PCR. Expression was determined relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and then each expression level was compared to the level of the RPMI medium control. The experiments shown are an average of 3 experiments. The depicted error bars represent the SEM of these 3 experiments. For statistical analysis, a one-way ANOVA followed by the Dunnett's t test was used; ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 when compared to the RPMI medium control.

Figure 3

Effects of the P. citrinopileatus extract on body weights and relative spleen weights. The depicted error bars represent the SEM (n = 5/group). After acclimation for 7 days, the animals were randomly divided into 2 groups (n = 5 for each group): normal control (C), P. citrinopileatus extract group (PCPS). The experimental extracts dissolved in distilled water were orally administered every day for two weeks. Normal control mice were administered an equal volume of distilled water. Body weights were monitored every two days during the period. Spleen weights were calculated as a proportion of body weight (g/100 g bw) at day 14.

Figure 4

Effects of the P. citrinopileatus extract on cytokine expression in the mice peritoneal cells. After acclimation for 7 days, the animals were randomly divided into 2 groups (n = 5 for each group): normal control (C), P. citrinopileatus extract group (PCPS). The experimental extracts dissolved in distilled water were orally administered every day for two weeks. Normal control mice were administered an equal volume of distilled water. Peritoneal cells in the treated mice were collected after thioglycollate injection. After 2 hours incubation, the mRNA expression levels of TNF (a), IL-1β (b), IL-12 (c), and IL-10 (d) in the adherent cells from the peritoneal cells were determined by using quantitative real-time PCR. Expression was determined relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then each expression level was compared to the level of the normal control group. The depicted error bars represent the SEM, and Student's t test was used for the statistical analysis; ^∗∗∗p < 0.001 when compared to the normal control.

Figure 5

Influences of the P. citrinopileatus extract on cytokine expression in the mice spleen cells. After acclimation for 7 days, the animals were randomly divided into 2 groups (n = 5 for each group): normal control (C), P. citrinopileatus extract group (PCPS). The experimental extracts dissolved in distilled water were orally administered every day for two weeks. Normal control mice were administered an equal volume of distilled water. The mRNA expression levels of IFNγ (Figure 5(a)) and IL-4 (Figure 5(b)) in the mice spleen cells were determined by using quantitative real-time PCR. Expression was determined relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and then each expression level was compared to the level of the normal control group. The depicted error bars represent the SEM, and Student's t test was used for the statistical analysis; ^∗p < 0.05 when compared to the normal control.

Figure 6

Combination effect of guanyl acid/vitamin D₂ with PCPS on proinflammatory cytokines secretion from the stimulated macrophages THP-1. Human monocyte cell line THP-1 was differentiated into macrophages by PMA (160 nM) for 48 hrs., and then they were stimulated with 1 μg/ml of the 450 kD polysaccharide (PCPS) purified from a hot water extract of P. citrinopileatus fruiting bodies plus the different concentrations of 5′-GMP-Na (Figure 6(a)) or ergocalciferol as vitamin D₂ (Figure 6(b)). After 24 hours, the secretion of TNF in the cell supernatant was determined by ELISA. The data shown are the average of 3 independent experiments. The depicted error bars represent the SEM of these 3 experiments. For statistical analysis, a one-way ANOVA followed by the Dunnett's t test was used; ^∗∗∗p < 0.001 when compared to the RPMI medium control.