Immunomodulating Activity of Pleurotus eryngii Mushrooms Following Their In Vitro Fermentation by Human Fecal Microbiota

Abstract

Recent studies have revealed the crucial role of several edible mushrooms and fungal compounds, mainly polysaccharides, in human health and disease. The investigation of the immunomodulating effects of mushroom polysaccharides, especially β-glucans, and the link between their anticancer and immunomodulatory properties with their possible prebiotic activity on gut micro-organisms has been the subject of intense research over the last decade. We investigated the immunomodulating effects of Pleurotus eryngii mushrooms, selected due to their high β-glucan content, strong lactogenic effect, and potent geno-protective properties, following in vitro fermentation by fecal inocula from healthy elderly volunteers (>60 years old). The immunomodulating properties of the fermentation supernatants (FSs) were initially investigated in U937-derived human macrophages. Gene expression as well as pro- (TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10, IL-1Rα) were assessed and correlated with the fermentation process. The presence of P. eryngii in the fermentation process led to modifications in immune response, as indicated by the altered gene expression and levels of the cytokines examined, a finding consistent for all volunteers. The FSs immunomodulating effect on the volunteers’ peripheral blood mononuclear cells (PBMCs) was verified through the use of cytometry by time of flight (CyTOF) analysis.

Keywords: PBMCs; cytokines; edible mushrooms; macrophages.

Figure 1

TNF-α gene expression and protein release. Relative expression of TNF-α at 6 h (a) and 24 h (b) of treatment of U937-derived macrophages with FSs, and TNF-α concentration in supernatants of the same cells at 6 h (c) and 24 h (d). Gene expression is exhibited as fold-change of the value in the untreated cells (RPMI) and relative towards GAPDH expression (ΔΔCt) for all samples (a,b). The concentration of the cytokine is expressed in pg/mL of the cell culture supernatants (c,d). Data shown are the means and standard deviation (SD bars) from two technical measurements. TNF-α concentration in RPMI at 6 and 24 h and LPS at 6 h were below detection levels. Asterisks indicate statistical significance as shown by paired-samples t-test, with * p < 0.05. RPMI: baseline, absence of treatment; LPS: treatment with 100 ng/mL lipopolysaccharide, positive control; NC: treatment with FSs from fermentation in the absence of additional carbon source; PE: treatment with FSs from fermentation in the presence of lyophilized mushroom powder of P. eryngii.

Figure 2

IL-10 gene expression and protein release. Relative gene expression of IL-10 at 6 h (a) and 24 h (b) of treatment of U937-derived macrophages with FSs, and IL-10 concentration in supernatants of the same cells at 6 h (c) and 24 h (d). Gene expression is exhibited as fold-change of the value in the untreated cells (RPMI) and relative towards GAPDH expression (ΔΔCt) for all samples (a,b). The concentration of the cytokine is expressed in pg/mL of the cell culture supernatants (c,d). Data shown are the means and standard deviation (SD bars) from two technical measurements. IL-10 concentration in RPMI at 6 h was below detection levels. Asterisks indicate statistical significance as shown by paired-samples t-test, with * p < 0.05. RPMI: baseline, absence of treatment; LPS: treatment with 100 ng/mL lipopolysaccharide, positive control; NC: treatment with FSs from fermentation in the absence of additional carbon source; PE: treatment with FSs from fermentation in the presence of lyophilized mushroom powder of P. eryngii.

Figure 3

IL-1β gene expression and protein release. Relative gene expression of IL-1β at 6 h (a) and 24 h (b) of treatment of U937-derived macrophages with FSs, and IL-1β concentration in supernatants of the same cells at 6 h (c) and 24 h (d). Gene expression is exhibited as fold-change of the value of the value in the untreated cells (RPMI) and relative towards GAPDH expression (ΔΔCt) for all samples (a,b). The concentration of the cytokine is expressed in pg/mL of the cell culture supernatants (c,d). Data shown are the means and standard deviation (SD bars) from two technical measurements. Asterisks indicate statistical significance as shown by paired-samples t-test, with * p < 0.05. RPMI: baseline, absence of treatment; LPS: treatment with 100 ng/mL lipopolysaccharide, positive control; NC: treatment with FSs from fermentation in the absence of additional carbon source; PE: treatment with FSs from fermentation in the presence of lyophilized mushroom powder of P. eryngii.

Figure 4

IL-1Ra gene expression and protein release. Relative gene expression of IL-1Ra at 6 h (a) and 24 h (b) of treatment of U937-derived macrophages with FSs, and IL-1Ra concentration in supernatants of the same cells at 6 h (c) and 24 h (d). Gene expression is exhibited as fold-change of the value in the untreated cells (RPMI) and relative towards GAPDH expression (ΔΔCt) for all samples (a,b). The concentration of the cytokine is expressed in pg/mL of the cell culture supernatants (c,d). Data shown are the means and standard deviation (SD bars) from two technical measurements. Asterisks indicate statistical significance as shown by paired-samples t-test, with * p < 0.05. RPMI: baseline, absence of treatment; LPS: treatment with 100 ng/mL lipopolysaccharide, positive control; NC: treatment with FSs from fermentation in the absence of additional carbon source; PE: treatment with FSs from fermentation in the presence of lyophilized mushroom powder of P. eryngii.

Figure 5

Phenotyping of treated PBMCs with NC/PE fermentation supernatants (FSs) with mass cytometry. (a) viSNE maps presenting the similarities of identified cell types (cell definitions are based on automatic gating performed in Cytobank—each dot represents a single cell) in PBMCs of V4 after different treatments (RPMI, NC, and PE) and (b) viSNE maps presenting the expression of markers CD3, CD4, CD8, CD294, CD28, CD11c, and CD14 on this analysis. (c) Heatmap showing fold changes in abundance of different cell types identified with the Pathsetter analysis in treated PBMCs from all volunteers and (d) boxplots for monocytes and myeloid DCs. RPMI: baseline, absence of treatment; NC: treatment with FSs from fermentation in the absence of additional carbon source; PE: treatment with FSs from fermentation in the presence of lyophilized mushroom powder of P. eryngii. Asterisks indicate statistical significance as shown by Mann–Whitney test, with * p < 0.05.