Active Hexose Correlated Compound promotes T helper (Th) 17 and 1 cell responses via inducing IL-1β production from monocytes in humans

Abstract

The differentiation of T helper (Th) cells is critically dependent on cytokine milieu. The innate immune monocytes produce IL-1β which can affect the development of Th17 and Th1 cells that predominantly produce IL-17 and IFN-γ, respectively. Oligosaccharides from microorganisms, crops and mushrooms can stimulate innate immune cells. Active Hexose Correlated Compound (AHCC) that contains a large amount of oligosaccharides is a natural extract prepared from the mycelium of the edible Basidiomycete fungus. This compound is reported to modulate immune responses against pathogens although the mechanisms for this effect are largely unknown. Here we show that AHCC could induce high levels of IL-1β production from human monocytes. Furthermore, AHCC-treated monocytes increased the production of IL-17 and IFN-γ from autologous CD4(+) T cells, which was blocked by adding IL-1 receptor antagonist. These finding provide new insight into how food supplements like AHCC could enhance human immunity by modulating monocytes and Th cells.

Fig 1. AHCC induces expression of IL-1β in human monocytes

Monocytes purified from peripheral blood of healthy individuals were incubated for 5 hours with AHCC freeze dried powder (AHCC FD, 500 μg/ml for A and B), α-glucan fraction (500 μg/ml) of AHCC, LPS (100 ng/ml) or vehicle control (control) in the presence or absence of the LPS neutralizer polymyxin B (50 μg/ml). Cells were then fixed, permeabilized and stained with antibodies to IL-1β. Stained cells were analyzed on a flow cytometer. (A) Representative dot plots showing IL-1β expression by monocytes. (B) Polymyxin B (50 μg/ml) inhibited IL-1β expression by monocytes treated with LPS but not AHCC FD (500 μg/ml). Representative data from 6 (A) and 5 (B) independent experiments.

Fig 2. IL-1β and IL-6 are secreted from human monocytes in response to AHCC

Purified human monocytes were treated for 18 hours with AHCC FD (500 μg/ml for A, C; 20 – 500 μg/ml for B, D), α-glucan (500 μg/ml), LPS (100 ng/ml) or vehicle control (control). Cell culture supernatants were collected and analyzed for IL-1β and IL-6 using ELISA. (A, C) Bars and error bars indicate mean (n = 4) and standard error of mean, respectively. *P < 0.05. (B, D) Dose kinetics of AHCC for IL-1β secretion by monocytes. Bars and error bars indicate mean and standard deviation of triplicates, respectively. Representative data from 3 independent experiments.

Fig 3. AHCC-treated monocytes induce high levels of IL-17 and IFN-γ production from CD4⁺ T cells in the presence of anti-CD3 and -CD28 antibodies

Purified human monocytes that had been treated for 5 hours with AHCC FD at high (AHCC FD-H, 500 μg/ml) or low (AHCC FD-L, 100 μg/ml) concentrations, LPS (100 ng/ml) or vehicle control were added to autologous memory CD4⁺ T cells. Cells were then cultured for 7 days in the presence of anti-CD3 and –CD28 antibodies. Culture supernatants were collected and analyzed for IL-17, IFN-γ and IL-13 by multiplex cytokine assay. Bars and error bars indicate mean (n = 8) and standard error of mean, respectively. *P < 0.05.

Fig 4. IL-1 receptor antagonist (IL-1Ra) suppresses IL-17 and IFN-γ production from monocytes stimulated with anti-CD3 and -CD28 antibodies in the presence of AHCC-treated monocytes

Purified human monocytes that had been treated for 5 hours with AHCC FD (500 μg/ml) were added to autologous memory CD4⁺ T cells. Cells were then cultured for 7 days with anti-CD3 and -CD28 antibodies. Recombinant human IL-1Ra (125 ng/ml) was added at days 0 and 2 of cell culture. Culture supernatants were collected and analyzed for IL-17 and IFN-γ by ELISA. Bars and error bars indicate mean (n = 7) and standard error of mean, respectively. *P < 0.05.