This study investigated the immune-modulating properties of Agaricus bisporus (common button mushroom) on the murine immune system. The aim was to study how A. bisporus modulates immune responses and acts as an adjuvant in cases of allergy. Crude extracts from five A. bisporus varieties were tested for their ability to stimulate cytokine production from bone marrow dendritic cells. The varieties #123, #145 and #188 induced the highest immune responses. Protein content analysis suggested that A. bisporus lectin may be involved in its immunomodulatory effects. Recognition studies found that C-type lectin receptors and TLR2 play a role in the immune cells' recognition of A. bispor

1. Investigating the immuno-modulating
properties of Agaricus bisporus on the
murine immune system
Mara Grigoraki
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2. Introduction
• Rise in atopic disorders
• “Hygiene hypothesis” : decrease in infectious diseases in the
industrialized countries
• Allergies : abnormal immune reactions to allergens -> atopy
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3. Introduction(2)
Kaufmann S. H. E., 2007
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4. Introduction(3)
• Mushrooms: medicinal and nutritional properties
• Immunomodulation via β-glucan binding to PRRs
• Dectin-1, TLR-2 and CR3 and possibly other PRRs
Lee D. H. & Kim H. W., 2014
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5. Aim
Agaricus bisporus as an adjuvant modulates the
immune responses
• Study immune cells with key role in the adjuvant function of A.
bisporus in case of allergy
• Determine the receptors involved in the recognition of A. bisporus
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6. Approach – Cytokine secretion
• Re-stimulate frozen mouse spleen cells with OVA and aCD3 and
measure IFN-γ, IL-17A and IL-10 secretion
a. Few viable cells
b. No positive control
c. Two orders of magnitude difference with
previous results
• Stimulate BMDC’s with crude extracts from five A. bisporus varieties
and measure TNF-α, IL-6 and IL-10
• Determine protein content of the five A. bisporus varieties
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7. Results – Crude extracts
Strongest immunostimulators
#123, #145 and #188
80
60
40
20
0
Concentration(ng/ml)
60
40
20
0
Concentration(ng/ml)
6
4
2
0
Concentration(ng/ml)
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8. Results – Heterogeneous extract vs
centrifuged
Whole mushroom heterogeneous extracts induce higher TNF-α
secretion
25
20
15
10
5
0
Concentration(ng/ml)
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9. Results – Protein content
A. bisporus’ lectin possibly involved in its immunomodulatory
properties
3
2
1
0
ProteinConcentration(mg/ml)
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10. Approach – Dectin-1 recognition
• Label A. bisporus whole crude extracts
• Stimulate BMDC’s and NIH 3T3 parental and dectin-1 cells
• Measure fluorescence
3T3 parental: embryonic fibroblast
3T3 dectin-1: transformed parental expressing dectin-1 gene
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11. Results – BMDC’s
Highest interaction : #033 and #188 11

12. Results – 3T3 cell lines
• Something went wrong!
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13. Approach – PRR recognition
• Neutralization Assays:
a. Antibodies binding TLR-2, dectin-1 and CR3
b. Inhibiting compounds : EGTA, Laminarin, Mannan
• Measure TNF-α
EGTA : calcium specific chelator
Laminarin : soluble β-glucan
Mannan : polysaccharide consisting of mannose units 13

14. Results – Antibodies
• TLR-2 involved in the recognition of A. bisporus
• Differences not significant
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15. Results – Inhibiting compounds
• EGTA completely inhibits -> CTL receptors involved
• Mannan has little effect on the recognition of β-glucans
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16. Conclusions & Recommendations
• Strains #123, #145 and #188 induce higher immune responses
• Lectin is a potential immunomodulator
• CTL and TLR2 receptors play a role in the recognition of A. bisporus
• Repeat neutralization assays with β-glucan extracts
• Block dectin-1 on 3T3 with antibodies and stimulate with β-glucan
extracts
• Conjugate lectin to laminarin and target dectin-1 on DC’s and 3T3
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17. Acknowledgements
• Arjen
• Ruud & Lotte
• Rikus & Debbie
• Lizeth, Savio, Steven & Tom
• Thesis ring members
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18. Questions?
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