Physical Interaction of T Cells with Dendritic Cells Is Not Required for the Immunomodulatory Effects of the Edible Mushroom Agaricus subrufescens

Abstract

Mushrooms are well known for their immunomodulating capacities. However, little is known about how mushroom-stimulated dendritic cells (DCs) affect T cells. Therefore, we investigated the effect of mushroom compounds derived from seven edible mushroom species on DCs, their fate in DCs, and the effect of the mushroom-stimulated DCs on T cells. Each mushroom species stimulated DCs in a different manner as was revealed from the DC's cytokine response. Assessing DC maturation revealed that only one mushroom species, Agaricus subrufescens, induced complete DC maturation. The other six mushroom species upregulated MHC-II and CD86 expression, but did not significantly affect the expression of CD40 and CD11c. Nevertheless, mushroom compounds of all investigated mushroom species are endocytosed by DCs. Endocytosis is most likely mediated by C-type lectin receptors (CLRs) because CLR binding is Ca²⁺ dependent, and EGTA reduces TNF-α secretion with more than 90%. Laminarin partly inhibited TNF-α secretion indicating that the CLR dectin-1, among other CLRs, is involved in binding mushroom compounds. Stimulated DCs were shown to stimulate T cells; however, physical contact of DCs and T cells is not required. Because CLRs seem to play a prominent role in DC stimulation, mushrooms may function as a carbohydrate containing adjuvant to be used in conjunction with anti-fungal vaccines.

Keywords: PAMP; PRR; T cell; dendritic cell; immunomodulation; mushroom.

Figure 1

The effect of mushroom compounds of seven mushroom species on cytokine secretion of bone marrow-derived dendritic cells. Both the effects of the complete homogenate (CH) and the alcohol precipitate (AP) were analyzed. Curdlan and zymosan were used as controls. Both the mushroom species and the preparation methods show a differential effect on cytokine secretion by dendritic cells. (A) Tumor necrosis factor α. (B) Interleukin-6. (C) Interleukin-10. (D) Interleukin-12. Asterisk (*) indicates significant difference between CH and AP responses (p < 0.05, n = 4–7). Hashtag (#) indicates significant differences between control and mushroom compounds with regard to IL-10 secretion (p < 0.05, n = 4–7).

Figure 2

Analysis of the maturation state of bone marrow-derived dendritic cells upon stimulation with compounds derived from seven mushroom species. Dendritic cells (DCs) were stimulated with alcohol-precipitated mushroom compounds and curdlan as control. After 24 h, the DCs were labeled with fluorescently labeled antibodies and analyzed with flow cytometry. The obtained mean fluorescence intensity (MFI) is indicated. Only upon stimulation with A. subrufescens, mature DCs are observed. Notably, the upregulation of CD40 and CD86 and the downregulation of CD11c vary. (A) Labeling with anti-CD11c-PE. (B) Labeling with anti-CD40-FITC. (C) Labeling with anti-MHC-II-APC. (D) Labeling with anti-CD86-PE. Asterisk (*) indicates significant difference with the medium control, i.e., unstimulated DCs (p < 0.05, n = 3).

Figure 3

Dendritic cells endocytose mushroom compounds. Alexa Fluor 488-labeled mushroom compounds were added to DCs and analyzed by flow cytometry and confocal microscopy. Fluorescent dextran was used as control. (A) Binding of fluorescently labeled mushroom compounds to DCs as analyzed by flow cytometry. (B) Endocytosis of fluorescent mushroom compounds of six mushroom species analyzed with confocal microscopy. (C) Endocytosis of fluorescent Agaricus subrufescens and Lentinula edodes compounds visualized using confocal microscopy (40× magnification). Plasma membranes and lysosomes were stained as indicated. The merged pictures reveal that labeled compounds are present in the endo-lysosomes of DCs.

Figure 3

Dendritic cells endocytose mushroom compounds. Alexa Fluor 488-labeled mushroom compounds were added to DCs and analyzed by flow cytometry and confocal microscopy. Fluorescent dextran was used as control. (A) Binding of fluorescently labeled mushroom compounds to DCs as analyzed by flow cytometry. (B) Endocytosis of fluorescent mushroom compounds of six mushroom species analyzed with confocal microscopy. (C) Endocytosis of fluorescent Agaricus subrufescens and Lentinula edodes compounds visualized using confocal microscopy (40× magnification). Plasma membranes and lysosomes were stained as indicated. The merged pictures reveal that labeled compounds are present in the endo-lysosomes of DCs.

Figure 4

C-type lectin receptors and notably dectin-1 bind mushroom compounds. Dendritic cells were pre-incubated with either the chelator EGTA or the low molecular weight soluble β-glucan laminarin followed by stimulation with mushroom compounds. The effect of EGTA and laminarin was assessed by measuring the effect on the TNF-α response. EGTA reduced the TNF-α response to 5–10% of the original response and laminarin showed an inhibition to 40–75%. The effect of EGTA indicates that this class of PRRs plays an important role in DC stimulation because C-type lectin receptors (CLRs) are calcium dependent. Laminarin is a dectin-1 antagonist and shows that this CLR is involved in the binding of mushroom compounds.

Figure 5

Mushrooms stimulate T cells through DCs; however, physical contact between these cells is not required. T cells were added to CH stimulated dendritic cells that were physically separated from each other or not. Seven days later the interferon-γ (IFN-γ) and interleukin-17 (IL-17) response was measured. (A) Mushroom induced IL-17 response. (B) Mushroom induced IFN-γ response. (C) Regardless of whether or not T cells were physically separated from DCs (when placed in a ThinCert), the IFN-γ and IL-17 responses do not significantly differ.