Investigating the immuno-modulating properties of Agaricus bisporuson the murine immune system

Mara Grigoraki

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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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Introduction(2)

Kaufmann S. H. E., 2007

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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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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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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 varieties6

Results –Crude extracts

Strongest immunostimulators

#123, #145 and #188

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Results –Heterogeneous extract vs centrifuged

Whole mushroom heterogeneous extracts induce higher TNF-α secretion

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Results –Protein content

A. bisporus’ lectin possibly involved in its immunomodulatory properties

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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 gene10

Results –BMDC’s

Highest interaction : #033 and #188 11

Results –3T3 cell lines

• Something went wrong!

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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

Results –Antibodies

• TLR-2 involved in the recognition of A. bisporus

• Differences not significant

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Results – Inhibiting compounds

• EGTA completely inhibits -> CTL receptors involved

• Mannan has little effect on the recognition of β-glucans

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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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Acknowledgements

• Arjen

• Ruud & Lotte

• Rikus & Debbie

• Lizeth, Savio, Steven & Tom

• Thesis ring members

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Questions?18