Journal of Inflammation Research
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Differential Immune Activating, Anti-Inflammatory,
and Regenerative Properties of the Aqueous, Ethanol,
and Solid Fractions of a Medicinal Mushroom Blend
This article was published in the following Dove Press journal:
Journal of Inflammation Research
Renee Davis 1
Alex Taylor 1
Regan Nally 1
Kathleen F Benson
Paul Stamets 1
Gitte S Jensen 2
2
1
Fungi Perfecti, Olympia, WA 98507,
USA; 2NIS Labs, Klamath Falls, OR
97601, USA
Purpose: To compare three fractions of a medicinal mushroom blend (MMB),
MyCommunity, on immune-activation, inflammation-regulation, and induction of biomarkers
involved in regenerative functions.
Methods: A seventeen-species MMB was sequentially extracted: first, saline solution at
ambient temperature, followed by re-extraction of the solids in ethanol, and finally resuspension of the homogenized ethanol-insoluble solids in cell-culture media. Fractions were tested
on peripheral blood mononuclear cells from three healthy donors. Immunostaining, flowcytometry, and Luminex protein-arrays measured immune-cell activation and cytokine
response. Dose-responses for induction of the CD69 early activation marker and individual
cytokine and growth-factor responses for each donor were evaluated. The CD69 and the
combined cytokine and growth-factor results were subjected to Non-metric Multidimensional
Scaling (NMDS) and multivariate ordination to aid interpretation of the aggregate immune
response and pairwise permutational MANOVA on a distance-matrix to evaluate statistical
differences between treatments on pooled data from all donors.
Results: Differential effects were induced by water-soluble, ethanol-soluble, and insoluble
immunomodulatory compounds of the MMB. The aqueous and ethanol fractions upregulated
expression of CD69 on all tested cell types. Monocyte-activation was correlated with the
ethanol fraction, while NKT and non-NK non-T cell-activation was more closely correlated
with the aqueous fraction. The solid fraction was the most potent inducer of Tumor Necrosis
Factor-α, as well as the anti-viral cytokines interferon-γ, MCP-1 (CCL-2), MIP-1α (CCL-3),
and MIP-1β (CCL-4), and induced G-CSF and b-FGF—growth-factors involved in regenerative functions—and the anti-inflammatory cytokine IL-1ra.
Conclusion: The aqueous, ethanol, and insoluble compounds within MMB induced differential
immune-activating, anti-inflammatory, and regenerative effects. This in vitro data suggests that,
upon consumption, MMB may induce a concerted series of immunomodulatory events based on
the differential solubility and bioavailability of the active constituents. These differential
responses support both immune-activation and resolution of the host defense-induced inflammatory reactions, thus assisting a post-response return to homeostasis.
Keywords: CD69, cytokines, growth factors, anti-viral peptides, medicinal mushrooms,
mycelium, immune support, redundancy analysis, rda, multivariate ordination
Correspondence: Gitte S Jensen
NIS Labs, 1437 Esplanade, Klamath Falls,
OR 97601, USA
Tel +1 541 884-0112
Fax +1403 441-5236
Email gitte@nislabs.com
Introduction
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http://doi.org/10.2147/JIR.S229446
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ORIGINAL RESEARCH
Mushrooms have been embraced for centuries due to their nutritional and medicinal
properties. They have been historically used in the treatment of infectious disease,
gastrointestinal disorders and asthmatic conditions, as well as to support overall
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wellbeing.1 Fungi now occupy their own kingdom, but
they were once considered plants due to their resemblance
and root-like structures. One of many characteristics that
separate fungal from plant organisms is the cell wall
structure. The cell walls of fungi contain chitin,2
a modified form of the polysaccharide cellulose. Chitin is
comprised of β-(1→4)-linked N-acetylglucosamine monomers, whereas cellulose is comprised of β-(1→4)-linked
glucose units. Chitin degrades into a mixture of shorterchained polysaccharides along with monosaccharide
products.3,4 This degradation can occur with a variety of
processing techniques that implement heat and drying.5
Mushroom polysaccharides possess documented immunomodulatory properties, specifically through the activation
of natural killer cells, macrophages, and neutrophils, as well
as induction of innate immune cytokines and interleukins.5
β-glucans are another class of polymers present in the cell
walls of fungi. The generic term β-glucan refers to the
polymeric form of glucose residues connected by
β-(1→3), β-(1→4), and β-(1→6)-linkages. The type of
β-glucans isolated from fungi consist mainly of a linear
backbone of β-(1→3) glucose monomers and side branches
comprised β-(1→3) and β-(1→6)-linked oligosaccharides.6
The most widely studied β-glucans are comprised of
(1→3)-β, and (1,6)-β linkages, which exhibit immunostimulatory and antitumor properties.7,8 These polysaccharides are ligands for the dectin-1 and toll-like receptor 2
(TLR-2) receptor systems expressed on macrophages and
dendritic cells, inducing NK cells, neutrophils, T-cells,
B-cells, as well as TNF-a, IL-4, and IL-6 signaling.9 The
Complement Receptor-3 (CD11b/CD18) in context of
extracellular matrix is also involved in immune responses
to fungal β-glucans.10 Research by Quayle et al demonstrates that other structural components in the fungal cell
wall matrix affect pattern recognition receptor activity.11
The TLR-2 activity of polysaccharide-K (PSK) was
reduced by 81% upon treatment with lipoprotein lipase,
revealing the biological activity of a previously unreported
lipid in the PSK complex. Mushroom β-glucans may contain other functional groups that contribute additional biological effects, particularly regarding antioxidant
activity.12 Mushroom polysaccharide and β-glucan products are common dietary supplements, often available as
whole mushrooms, mycelial powders, dried or liquid
extracts from fruitbodies or from mycelium.
Specialized protein-bound polysaccharide products
have been developed as adjuvant immunotherapies in
oncological clinical settings. Polysaccharide-K (PSK,
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Krestin®, Kureha) and polysaccharide peptide (PSP) are
produced from the cultured mycelium of the turkey tail
mushroom (Trametes versicolor) and are used as antineoplastic agents and immunostimulants in China and
Japan.13–18 Lentinan is a polysaccharide extracted from
the mycelium and fruiting body of the shiitake mushroom
(Lentinula edodes),19 and has demonstrated efficacy as
a biological response modifier and chemotherapy adjuvant
in gastrointestinal and lung cancers.20 Grifolan is another
polysaccharide extracted from maitake (Grifola frondosa)
which enhances production of IL-6, IL-1, and TNF-a.21
Another class of immunologically active compounds
from mushrooms, which are typically much smaller in
size compared to cell-wall polymers, are secondary metabolites. These are compounds that may not be absolutely
required for the growth of the organism but assist in its
survival by offering protection and communication among
other important but not metabolically essential functions.
When consumed, secondary metabolites are associated with
myriad biological effects, including antioxidant, anti-viral,
anti-inflammatory, neuroregenerative, and hepatoprotective
effects. Classes of fungal metabolites with medical significance include sterols, terpenes, and phenols, notable examples of which include ganoderic acid in Ganoderma
lucidum,22 erinacines in Hericium erinaceus,23–25 and betulin in Inonotus obliquus,26 and cordycepin in Cordyceps
militaris.27
A growing body of international scientific and medical
research continues to help define the precise biochemical
pathways leading to improved physiological outcomes.
Cordycepin, 3ʹ-deoxyadenosine, has shown very potent antiinflammatory effects in a spectrum of in vitro and animal
models, specifically via effects on adipose-derived mesenchymal stem cells, where higher doses helped maintain the stemness of the cells and lower doses supported osteogenic
differentiation.28 Isolated compounds from reishi
(Ganoderma lucidum) were tested on the MCF-7 breast cancer
cell line and its non-transformed counterpart MCF10A and
showed selective killing of the transformed cells, both actively
growing, and quiescent slow-cycling cancer stem cells.29
Polysaccharides from reishi30 and maitake31 have also been
shown to promote and enhance the survival/renewal abilities
of primitive hematopoietic stem/progenitor cells. For maitake,
this was specifically linked to β-glucan-mediated increases in
the production of granulocyte-colony stimulating factor.32
Notably, this research demonstrated that this effect supported
stem cell transplantation in a NOD/SCID mouse model. To the
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Davis et al
best of our knowledge, non-β-glucan fractions of maitake
were not previously evaluated in stem cell related studies.
While much prior research on medicinal mushrooms has
focused on the solid, β-glucan-rich fraction, and β-glucanmediated responses are clearly important, focusing on this
compound class in isolation clearly does not reflect the overall
bioactivity of a complex blend when consumed for immune
support. Emerging evidence suggests that a blend of mushrooms may provide additive or synergistic effects on the host
immune response. Preclinical work on a 7-mushroom blend
(Ganoderma resinaceum, Cordyceps sinensis, Trametes versicolor, Hericium erinaceus, Inonotus obliquus, Grifola frondosa, Agaricus brasiliensis f. blazei, Phellinus linteus)
discovered greater NK cell upregulation with a blend over
any one isolated species.33 Findings from another study suggest synergistic radical scavenging activity with a combination
of Boletus edulis and Marasmius oreades mushroom
extracts.34
The purpose of this study was to investigate the differential immunological effects of aqueous, ethanol, and solid
fractions of MMB in vitro, to determine the differences in
biological activity between the soluble and insoluble fractions
of a complex medicinal mushroom blend (MMB), a blend of
17 mushroom species (Table 1) that is used by consumers for
seasonal immune wellness, and by clinicians to enhance
innate immunity. The study of a complex blend rather than
isolated single compounds was of importance. The experimental model included effects on multiple immune cell types
in vitro, because of parallels to events in the gut mucosal
tissue where dendritic cells and macrophages sample antigens
in the gut lumen, and present to lymphocytes in the gutassociated mucosal tissue, leading to local immune cell activation, and cytokine secretion that has systemic effects.
Materials and Methods
Reagents
Phosphate-buffered saline, Roswell Park Memorial Institute
1640 medium, penicillin–streptomycin 100×, interleukin-2
(IL-2), and lipopolysaccharide (LPS) from Salmonella enterica were purchased from Sigma-Aldrich Co. (St Louis, MO,
USA). Lympholyte-Poly was obtained from Thermo-Fisher
Scientific (Waltham, MA, USA). CD69 fluorescein isothiocyanate, CD56 phycoerythrin, CD3 peridinin chlorophyll
protein, and heparin Vacutainer tubes were purchased from
Becton-Dickinson (Franklin Lakes, NJ, USA). Bio-Plex
Pro™ human cytokine arrays were purchased from BioRad Laboratories Inc. (Hercules, CA, USA).
Medicinal Mushroom Blend
The medicinal mushroom blend MyCommunity is sold internationally under the brand Host Defense® MushroomsTM and
was obtained from the manufacturer—Fungi Perfecti, LLC,
Olympia, WA. It is a certified organic freeze-dried blend of
17 medicinal mushroom species (mycelium/fruiting bodies)
for immune system health: Royal sun blazei (Agaricus brasiliensis f. blazei), cordyceps (Cordyceps militaris), enokitake
Table 1 Mushroom Species in the Medicinal Mushroom Blend
Common Name
Botanical Name
Other Names
Material
Primary Medicinal Use(s)
Royal Sun Blazei
Cordyceps
Agaricus blazei
Cordyceps militaris
Himematsutake
M
M
Aox, IM.
IM
M
IM
Tinder conk
Lacriformes
M
M
IM
Aox, IM
Enokitake
Flammulina velutipes
Amadou
Agarikon
Fomes fomentarius
Fomitopsis officinalis
Artist Conk
Ganoderma applanatum s.l.
M
IM, AI
Reishi
Oregon Ganoderma
Ganoderma lucidum s.l. Ling Zhi
Ganoderma oregonense s.l.
M
M
Aox, AI, IM
Aox, AI, IM
Maitake
Grifola frondosa
Hen of the woods
M/FB
IM
Lion’s Mane
Chaga
Hericium erinaceus
Inonotus obliquus
Yamabushitake
M
M
Neurological support
Aox, IM
Shiitake
Lentinula edodes
M
IM
Mesima
Birch Polypore
Phellinus linteus
Piptoporus betulinus
M
M
Aox, IM
Aox, IM
Pearl Oyster
Pleurotus ostreatus
M
IM
Split Gill Polypore
Turkey Tail
Schizophyllum commune
Trametes versicolor
M
M
IM
IM
Abbreviations: M, mycelium; FB, fruiting body; IM, immunomodulator; Aox, antioxidant; AI, anti-inflammatory; SC, stem cell modulator.
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(Flammulina velutipes), amadou (Fomes fomentarius), agarikon (Fomitopsis officinalis), artist conk (Ganoderma applanatum), reishi (Ganoderma lucidum s.l.), Oregon ganoderma
(Ganoderma oregonense s.l.), maitake (Grifola frondosa),
lion’s mane (Hericium erinaceus), chaga (Inonotus obliquus),
shiitake (Lentinula edodes), mesima (Phellinus linteus), birch
polypore (Piptoporus betulinus), pearl oyster (Pleurotus
ostreatus), split gill polypore (Schizophyllum commune),
and turkey tail (Trametes versicolor; Table 1).
Testing for Endotoxins
The MMB powder was tested for endotoxin levels at
Associates of Cape Cod Inc., East Falmouth MA, using
the quantitative kinetic turbidimetric method for the detection of Gram-negative bacterial endotoxin, and reported in
Endotoxin Units (EU).
Testing for Beta-Glucans
The whole MMB powder, as well as the freeze-dried solids
of the aqueous and ethanol MMB fractions, were each tested
for beta-glucan content by the Megazyme® assay at Venture
Laboratories (Lexington, KY). This analysis involves total
glucan hydrolysis by sulfuric acid and alpha-glucan hydrolysis by various enzymes. Total glucan and α-glucan content
are measured spectrophotometrically, and β-glucans are estimated mathematically by the difference of these two values.
All materials submitted for analysis were from the same lot
number of material used in the immunological testing.
Preparation of Mushroom Fractions for
Immune Bioassays
The powder was handled in the following manner, using
pyrogen-free disposables: A 100 mg/mL suspension was
prepared in phosphate-buffered saline (PBS) and allowed
to rehydrate and extract aqueous compounds for 1 hr at
20°C under gentle agitation. The suspension was centrifuged
at 400 g for 10 mins, and the aqueous fraction harvested.
Ethanol (95%) was added to the pellet and vortexed, and
extraction of non-aqueous, ethanol-soluble compounds
allowed for 1 hr at 20°C under gentle agitation. The suspension was centrifuged at 400 g for 10 mins, and the ethanol
fraction harvested. The remaining solid pellet was resuspended in PBS. The aqueous and ethanol fractions were
filtered through a 0.22-micron syringe filter before adding
to cell cultures. The solid fraction was passed through
homogenization spin columns (QIAshredder, Qiagen,
Hercules, CA), but were not filtered through a 0.22-micron
filter. From each fraction, serial dilutions were made in
pyrogen-free physiological saline. See also Figure 1.
Immune Cell Activation
Peripheral venous blood was drawn from three healthy
human donors upon written informed consent, as approved
by the Sky Lakes Medical Center Institutional Review
Board, Federalwide Assurance 2603. The blood was
drawn into heparin vacutainer vials, and the peripheral
blood mononuclear cells (PBMC) isolated using
Lympholyte Poly by centrifugation for 35 mins at
400 g. The PBMC were washed twice in PBS, counted,
and the density adjusted to establish cultures with a cell
density at 106/mL, using Roswell Park Memorial Institute
1640 medium containing penicillin–streptomycin and fetal
bovine serum.
The highly inflammatory lipopolysaccharide (LPS)
from Salmonella enterica was used as a positive control
for immune-cell activation. Serial dilutions of products or
LPS (10 ng/mL) were added to cultures at a volume of
20 μL, and cultures were then incubated at 37°C, 5% CO2
Figure 1 Production of test fractions.
Notes: The medicinal mushroom blend was supplied as a powder, and suspended in PBS at a concentration of 100 g/L. The aqueous extraction was performed over 1 hr at
ambient temperature under constant agitation. The post-aqueous solid fraction was extracted using ethanol under similar conditions. Both the aqueous and post-aqueous
ethanol extracts were filtered through 0.22-micron cellulose acetate filters before adding to bioassays. The remaining post-extraction solids were homogenized but not
filtered prior to testing in bioassays.
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for 24 hrs. In parallel, IL-2 was used as a positive control
for natural killer (NK)-cell activation, at a concentration of
100 IU/mL. Untreated negative control cultures consisted
of PBMC exposed to phosphate-buffered saline in the
absence of test products. All treatments, including each
dose of test product and each positive and negative control, were tested in triplicate. After 24 hrs, cells were
transferred to V-bottom microtiter plates, washed in PBS
containing bovine serum albumin and sodium azide, and
stained for 10 mins with fluorochrome-labeled anti-CD3,
anti-CD56, and anti-CD69 monoclonal antibodies at the
recommended concentration. PBMC were then fixed in
formalin. The fluorescence intensities for CD3, CD56,
and CD69 were measured by flow cytometry, using an
Attune acoustic-focusing flow cytometer (Thermo Fisher
Scientific, Waltham, MA, USA). Data analysis utilized
gating on forward and side scatter to evaluate CD69
expression on lymphocyte versus monocyte/macrophage
subsets. The lymphocyte subpopulation was further analyzed for CD69 expression on CD3-CD56+ NK cells, CD3
+CD56+ NKT cells, CD3+CD56- T cells, and non-NK
non-T lymphocytes.
Production of Cytokines, Chemokines,
and Growth Factors
After 24 hrs of incubation, after transfer to V-bottom
microtiter plates and before cells were stained for flowcytometry analysis, the culture supernatants were harvested from the PBMC cultures described above. Levels
of cytokines, chemokines, and growth factors were quantified using Bio-Plex protein arrays (Bio-Rad Laboratories
Inc., Hercules CA, USA) and utilizing xMAP technology
(Luminex, Austin, TX, USA).
Statistical Analysis
Data organization, exploration, and analysis were conducted
using the statistical computing language R (version 3.5.2)
implemented in the RStudio (version 1.0.143) software
environment.35,36 Most data handling was conducted using
the base package, with additional functions from the dplyr
package (version 0.7.8) used in the reorganization of the raw
data.37 Statistical analysis was implemented using the
R ‘stats’ (version 3.5.2) and “car” (version 3.0–2)
packages,38 as well as the packages “vegan” (version
2.5–4), and “vegan3d” (version 1.1–2) for multivariate ordination plots and Non-metric Multidimensional Scaling
(NMDS).39,40 Multiple comparison test after Kruskal–
Journal of Inflammation Research 2020:13
Davis et al
Wallis used the “pgirmess” package (version 1.6.9), and the
“RVAideMemoire” package (version 0.9–72) was used for
pairwise permutational MANOVA with Bonferroni
correction.41,42
Results
Immune Activation
The aqueous and the ethanol fractions of the MMB blend
were tested for immune cell activation in cultures of peripheral blood mononuclear cells (PBMC) from healthy donors
(Figures 2–4). Whereas the aqueous fraction triggered upregulation of CD69 expression on lymphocytes (Figures 2A
and 4A), the ethanol fraction triggered up-regulation of
CD69 on monocytes (Figures 2B and 4B). Within the lymphocyte subset treated with the aqueous extract of MMB,
only minor increases in CD69 expression were observed on
NK cells (Figures 3A and 4C), NKT (Figures 3B and 4D),
and T cells (Figures 3D and 4F). The most robust increase in
CD69 expression was seen for the CD3- CD56- non-NK
non-T lymphocytes where the increase was statistically significant across all doses tested (Figure 3C) and across all
donors (Figure 4E). This CD3- CD56- population contains
dendritic cells, B lymphocytes, and stem cells, all which may
express CD69.
The CD69 expression across all subpopulations of peripheral blood mononuclear cells was analyzed for the
untreated cultures from all three donors and compared to
CD69 expression in cultures treated with the two highest
doses of MMB (2 mg/mL and 0.4 mg/mL). The analysis
demonstrated the difference in induction of CD69 expression between the aqueous and the post-aqueous ethanolbased extracts (Figure 4). In these univariate box plots,
the implicit statistical assumption is that the response of
each cell type is independent of the response by each other
cell type.
In addition to evaluation of cellular activation and CD69
expression on different cell types by standard univariate
methodology, we also performed non-metric multidimensional scaling (NMDS)—a statistical technique borrowed
from quantitative ecology—and permutational MANOVA.
This is based on the rationale that the PBMC cultures contain
many cell types that interact and affect each other dynamically. These interactions affect the transcriptional landscape
within each culture. This can be analyzed in the same way as
ecologists study changes in flora and fauna abundance across
diverse landscapes. The method was also applied to enable
comparison of responses across the PBMC cultures from all
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Figure 2 Expression of the early activation marker CD69 on lymphocytes and
monocytes.
Notes: *P<0.05; **P<0.01. CD69 expression on lymphocytes (A) and monocytes
(B) in human PBMC cultures treated for 24 hrs with serial dilutions of MMB
aqueous extract in PBS (MMB PBS) and MMB ethanol extract (MMB EtOH).
Results are shown for the 2 g/L dose. Mean fluorescence intensity for CD69
expression is shown. Data presented as mean ± standard from triplicate cultures
and represents data from PBMC cell cultures from one of the three different
healthy human donors.
three donors. In this analogy, each donor is like an ecological
research site, each cytokine, and growth factor is like a plant
or animal type, and each MMB extract treatment is like
a different type of land management practice (e.g. controlled
burn forest vs untended forest vs open range forest). In the
multivariate ordination plots, an assumption of independence
among cell types (in the case of CD69 expression) or
assumed statistical independence among co-induced cytokines is no longer necessary or appropriate. This is because
the contemporaneously collected data is a snapshot of the
aggregate PBMC response and it can be analyzed to account
for covariance of responses among cell types or cytokines.
The differences between the treatment groups is apparent
when the covariance structure of the data set is represented
using NMDS (Figure 5).
In the NMDS ordination plot in Figure 5, each dot represents the “Cartesian coordinate” location of each PBMC
treatment well in a 5-dimensional data space (one dimension
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for each cell type), where the distance between points is
proportional to the dissimilarity between each measurement.
The dissimilarity between measurements was calculated
using Bray-Curtis method to create a statistical distance
matrix.43 This 5-dimensional data space is then projected
into a plot where each axis is an eigenvector of the distance
matrix. An eigenvector is a transect through the data space
that explains the greatest proportion of variance in the data.
Figure 5 is a 3-dimensional projection along the three eigenvectors (NMDS1, NMDS2, and NMDS3) that explain the
greatest proportion of the variances. The overall “goodness
of fit” statistic for this multivariate representation, known as
stress, indicates that the projection in 3-dimensions provides
an excellent representation of the underlying relationships
(stress = 0.043).
Permutational MANOVA and post hoc pairwise comparisons with Bonferroni correction revealed that when considered collectively across cell types, the CD69 expression
induced by MMB PBS was significantly different from the
Untreated PBS (p<0.1), the MMB EtOH was different from
the Untreated EtOH (p<0.05), and the MMB PBS extract
was different from the MMB EtOH extract (p<0.05).
Notably, the proportion of the variance in CD69 expression
levels that can be explained by each treatment type was
distinct for different white blood cell types (Table 2).
The NMDS ordination also confirmed the general
trends identified in the univariate analysis: monocyte activation was strongly associated with the ethanol extract;
non-T non-NK lymphocyte activation strongly correlated
with the aqueous extract; and NKT cell, and T lymphocyte
cell activation having a weaker but detectable activation
by both the aqueous and ethanol extracts.
Immune-Activating Cytokines
Supernatants from the PBMC cultures exposed to various
doses of the MMB fractions for 24 hrs were assayed for
the levels of cytokines, chemokines, and growth factors,
using a magnetic bead-based array and Luminex xMAP
technology. Increases in the levels of immune-activating
cytokines included robust upregulation of specific proinflammatory cytokines, including IL-1β and TNF-α
(Figure 6). The solid fraction of MMB (MMB solids)
showed the most robust immune-activating properties,
but the aqueous extract (MMB PBS), free of insoluble βglucans, also showed induction of immune-activating
cytokines. The post-aqueous ethanol extract (MMB
EtOH) of the solids remaining after aqueous extraction
showed only very minor effects on these cytokines.
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Figure 3 Expression of the early activation marker CD69 on lymphocyte subpopulations.
Notes: *P<0.05; **P<0.01. CD69 expression on NK cells (A), NKT cells (B), non-NK non-T lymphocytes (C), and T cells (D) in human PBMC cultures treated for 24 hrs
with serial dilutions of MMB aqueous extract in PBS (MMB PBS) and MMB ethanol extract (MMB EtOH). Mean fluorescence intensity for CD69 expression is shown. Data
presented as mean ± standard deviation from triplicate cultures and represents one of three separate experiments using PBMC cells from three different healthy human
donors.
Anti-Viral Cytokines
The MMB solid fraction triggered increases in the production of four specific cytokines and chemokines that are
specifically associated with anti-viral activities and cellular
recruitment (Figure 7). Interferon-γ (IFN-γ) was moderately induced by the MMB solid fraction (Figure 7A). The
monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory proteins 1α and 1β were strongly
increased by MMB solid fraction (Figure 7B–D). No
major changes to the biomarkers were apparent in the
univariate analysis of the aqueous or post-aqueous ethanol
extracts.
Anti-Inflammatory Cytokines
The MMB solid fraction was a very strong inducer of the
anti-inflammatory cytokine IL-1ra (Figure 8A), which
functions as an IL-1 receptor antagonist, a strong antiinflammatory protein due to its ability to prevent IL-1 in
engaging in receptor binding and cellular signaling.44 To
a much lesser extent, MMB solids triggered increased
levels of IL-10 (Figure 8B); however, this was not statistically significant in the multivariate data analysis. The
Journal of Inflammation Research 2020:13
aqueous fraction triggered a mild induction of both antiinflammatory cytokines. The post-aqueous ethanol extract
of MMB did not trigger changes to either cytokine.
Cytokines with Effects on Regenerative Functions
The effects of the MMB fractions on three cytokines with
regenerative functions were clearly apparent in the univariate analysis of a representative donor, namely GranulocyteColony stimulating Factor (G-CSF), basic Fibroblast
Growth Factor (bFGF), and Vascular Endothelial Growth
Factor (VEGF) (Figure 9). Both the aqueous and the solid
fractions induced increases in these three cytokines
(Figure 9), with the MMB solid fraction showing the stronger effect.
Multivariate Analysis of Cytokine
Activation
Because the levels of cytokines, chemokines, and growth
factors were all quantified simultaneously using beadbased protein arrays, the meaning of expression levels of
an individual analyte are more appropriately analyzed in
the context of the other contemporaneously induced
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Figure 4 Expression levels of the CD69 early marker across white blood cell types.
Notes: *P<0.05; **P<0.01, ***P<0.001. Mean fluorescence intensity resulting from immunostaining for the CD69 early activation marker for lymphocytes (A), monocytes
(B), NK cells (C), NKT cells (D), non-T non-NK cells (E), and T cells (F). Data were plotted with respect to cell type and treatment group for the two highest
concentrations pooled across all donors. Fluorescence responses that followed a normal distribution (B, D, E) were statistically evaluated with ANOVA followed by a Tukey
Honest Significant Difference post hoc test. Non-normal fluorescence responses that could not be analyzed by ANOVA were evaluated using a Kruskal–Wallis Rank Sum
Test followed by a multiple comparison post hoc test with Bonferroni correction. Statistical significance is indicated as the difference between untreated cells and treatments
with a family-wise error rate indicated by p<0.05 (*); p<0.01 (**); p<0.001 (***).
signaling molecules. Mathematically, this means the variance-covariance structure of the immune analytes must be
taken into account. In the cytokine NMDS, the
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concentrations (pg/mL) of all 27 monitored cytokines
and growth factors from the two highest MMB doses for
all three fractions and including all three donors were
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Davis et al
Figure 5 NMDS ordination plot of CD69 expression by treatment and cell type.
Notes: Ordination plot of NMDS site scores representing Bray-Curtis distance dissimilarities. The three-dimensions of the plot provide an excellent representation of the
data in reduced dimensions (stress < 0.05). The CD69 activation of each cell type is displayed as NMDS species vectors, with the vector direction and length indicating the
strength of the correlation. Ovals represent the standard error of each treatment groups’ data centroid within the multivariate data space.
analyzed simultaneously. The NMDS ordination visualization (Figure 10) and summary statistics (Table 3) illustrate
the strength of the solid fraction in inducing an immune
response. Permutational MANOVA and post hoc pairwise
comparisons with Bonferroni correction revealed that
when considered collectively across cytokines and growth
factor expression, the MMB PBS was different from the
Untreated PBS (p=0.02), and the MMB Solids were different from the Untreated PBS (p=0.02), the MMB PBS
(p=0.02), and MMB EtOH (p=0.03). The net effect of the
MMB EtOH on cytokines and growth factors was not
Table 2 Redundancy Analysis: Summary Statistics
Correlation Between Cell Type and CD69 Induction
Cell Type
r2
p-value
Ordinates
Toward
Monocyte
0.73
<0.001
EtOH
Natural Killer
0.47
<0.001
PBS/Untreated
Natural Killer T
Non-T non-NK Lymphocyte
0.73
0.24
<0.001
<0.001
EtOH
PBS
T Lymphocyte
0.88
<0.001
EtOH
Journal of Inflammation Research 2020:13
for
significantly different from the Untreated PBS, the
Untreated EtOH, or the MMB PBS.
Endotoxin Testing
The level of endotoxin was 3.97 Endotoxin Units (EU)/
mg MMB powder. Using the same assay, purified LPS
results in 10 EU/ng, equivalent to 10,000,000 EU/mg
purified LPS. This 2.5 million-fold difference shows
a much lower content of endotoxin in MMB compared
to LPS and serves as a foundation upon which the
immune activation data can be interpreted, since LPS
was used as a positive control in the immune cell
cultures. In several data sets below (Figures 4C and
8A), the immune-activating properties of MMB fractions exceeded that of a 10 ng/mL dose of LPS, suggesting that endotoxins in MMB would not be a major
contributing factor to the induction of immune cell
activation and cytokine production.
Beta-Glucan Testing
The whole MMB powder was tested for beta-glucan content by the Megazyme® assay and showed 38% (w/w)
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Figure 6 Changes in immune-activating pro-inflammatory cytokine levels in human PBMC cultures.
Notes: Changes in Interleukin-1β (IL-1β), (A) and Tumor Necrosis Factor-α (TNF-α), (B) cytokine levels in human PBMC cultures treated for 24 hrs with serial dilutions of
MMB aqueous extract in PBS (MMB PBS), MMB post-aqueous ethanol extract (MMB EtOH), and the solid fraction (MMB solids). Data are presented as picogram per
milliliter (pg/mL) based on the mean ± standard deviation from triplicate cultures and represents one of three experiments using PBMC from three different healthy human
donors. Cytokine levels for LPS-treated control cultures were as follows: Il-1β: 772±28 pg/mL; TNF-α: 7073±185 pg/mL.
Figure 7 Changes in anti-viral cytokine levels in human PBMC cultures.
Notes: Changes in Interferon-γ (IFN-γ), (A), Monocyte Chemoattractant Protein-1 (MCP-1), (B), Macrophage Inflammatory Protein-α (MIP-1α), (C), and Macrophage
Inflammatory Protein-β (MIP-1β), (D) cytokine levels in human PBMC cultures treated for 24 hrs with serial dilutions of MMB aqueous extract in PBS (MMB PBS), MMB postaqueous ethanol extract (MMB EtOH), and the solid fraction (MMB solids). Data are presented as picogram per milliliter (pg/mL) based on the mean ± standard deviation
from triplicate cultures and represents one of three experiments using PBMC from three different healthy human donors. Cytokine levels for LPS-treated control cultures
were as follows: IFN-γ: 71±2 pg/mL; MCP-1: 2052±35 pg/mL; MIP-1α: 2116±0 pg/mL; MIP-1β: 6497±814 pg/mL.
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Davis et al
Figure 8 Changes in anti-inflammatory cytokines.
Notes: Changes in interleukin-1 receptor antagonist (IL-1ra), (A) and interleukin-10 (IL-10), (B) cytokine levels in human PBMC cultures treated for 24 hrs with serial dilutions of
MMB aqueous extract in PBS (MMB PBS), MMB post-aqueous ethanol extract (MMB EtOH), and the solid fraction (MMB Solids). Data are presented as picogram per milliliter (pg/
mL) based on the mean ± standard deviation from triplicate cultures and represents one of three experiments using PBMC from three different healthy human donors. Please note
the 1000-fold difference in the scales on the Y-axes. Cytokine levels for LPS-treated control cultures were as follows: IL-1ra: 8134±473 pg/mL; IL-10: 301±15 pg/mL.
Figure 9 Changes in cytokines with effects on regenerative functions.
Notes: Changes in Granulocyte-colony stimulating factor (G-CSF), (A), basic Fibroblast Growth Factor (Basic FGF), (B), and Vascular Endothelial Growth Factor (VEGF),
(C) growth factor levels in human PBMC cultures treated for 24 hrs with serial dilutions of MMB aqueous extract in PBS (MMB PBS), MMB post-aqueous ethanol extract
(MMB EtOH), and the solid fraction (MMB Solids). Data are presented as picogram per milliliter (pg/mL) based on the mean ± standard deviation from triplicate cultures and
represents one of three experiments using PBMC from three different healthy human donors. Cytokine levels for LPS-treated control cultures were as follows: G-CSF: 1458
±58 pg/mL; bFGF: 221±8 pg/mL; VEGF: 421±17 pg/mL.
beta-glucan. The water and ethanol fractions used in the
immune assays were measured by the same assay at 2.0%
w/w and 1.1% w/w, respectively. The solid fraction gave
inconclusive results when tested in the Megazyme® assay,
though it assumed that the difference between the whole
MMB powder (38% beta-glucan) and the liquid extract
beta-glucan yields the remaining insoluble beta-glucan in
the solid fraction at approximately 35% w/w.
Discussion
Inflammation is a complex biological response to harmful
stimuli, such as pathogens, as well as trauma or disease
leading to tissue damage. It is a protective response involving immune cells, endothelial cells, and various types of
stem cells, where the various cell types communicate via
secreted biomarkers. The function of inflammation is to 1)
Journal of Inflammation Research 2020:13
increase blood and fluid flow to an area to promote the
circulation of immune and stem cells, 2) eliminate the
initial cause of cell injury, 3) clear out dead cells and
tissues damaged from the original insult and from the
inflammatory process, and 4) initiate tissue repair. Acute
inflammation favors a healing process and return to homeostasis, whereas chronic inflammation does not support
a resolution of the inflammation associated with healing
and regeneration. Efforts to reduce inflammation, increase
stem cell homing, and accelerate repair is thus an active
area of research.45
While the immune-activating, pro-inflammatory
properties of fungal water-insoluble β-glucans are well
documented, the immune-modulating effects of fungal
non-β-glucan-fractions are less recognized. This study
was performed to examine three non-overlapping
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Davis et al
Figure 10 NMDS ordination plot of changes to cytokine and growth factor expression in PBMC cultures.
Notes: Ordination plot of NMDS site scores representing Bray-Curtis distance dissimilarities between each treatment. The three-dimensions of the plot provide an
excellent representation of the data in reduced dimensions (stress < 0.05).
fractions of a medicinal mushroom blend, to compare
the effects of the aqueous fraction to the post-aqueous
ethanol fraction, and to compare both to the solid fraction. The solid fraction contained 38% insoluble betaglucans, while the aqueous and post-aqueous ethanol
fractions contained small amounts of soluble betaglucans accounting for very little content on a mass
basis. The comparison showed that while the aqueous
fraction was able to induce CD69 expression on lymphocytes, the post-aqueous ethanol fraction induced
CD69 on monocytes, thus exhibiting complementary
immune-modulating activities. The solid fraction provided a highly robust effect on cytokine and growth
factor production.
The post-aqueous ethanol fraction showed a more
selective immune regulating activity. It supported
a stronger monocyte activation than the aqueous extract
and comparable induction of IL-6, IL-9, and MIP-1β. This
suggests that hydrophobic ethanol-soluble compounds in
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this fraction have immune activating and anti-viral properties, while also supporting selective aspects of regenerative
functions related to IL-9.
This selective effect of the three MMB fractions is of
interest when discussing the potential differential immune
regulating events after consumption. When a medicinal
mushroom powder is ingested, it is assumed that aqueous
compounds easily dissolve and are absorbed into the gastrointestinal mucosa. Compounds that are not water soluble either remain solids or get broken down by digestive
enzymes and pH changes. The experimental post-aqueous
ethanol fraction used for this project represents a method
to test some compounds that may be released during the
digestive process. The remaining solid fraction represents
material that may get into direct contact with mucosal
immune cells such as dendritic cells, known to extend
trans-mucosal dendrites into the gut lumen, as well as
absorption via transmucosal transport mechanisms to
engage with tissue-residing gut mucosal immune cells.46
Journal of Inflammation Research 2020:13
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Davis et al
Table 3 Multivariate Redundancy Analysis: Summary Statistics by
Cytokine and Growth Factor
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Cytokine/Growth Factor
r2
p-value
Interferon-gamma
IFN-ɣ
0.73
<0.001
Interleukin-1β
IL-1β
0.64
<0.001
Interleukin-5
Interleukin-6
IL-5
IL-6
0.78
0.68
<0.001
<0.001
Interleukin-8
IL-8
0.92
<0.001
Monocyte chemoattractant protein-1
Macrophage inflammatory protein-1α
MCP-1
MIP-1α
0.64
0.95
<0.001
<0.001
Macrophage inflammatory protein-1β
Tumor Necrosis factor- α
MIP-1β
TNF-α
0.85
0.94
<0.001
<0.001
Interleukin-1 receptor antagonist-a
IL-1ra
0.76
0.002
Interleukin-2
Interleukin-4
IL-2
IL-4
0.89
0.82
<0.001
<0.001
Interleukin-9
IL-9
0.69
<0.001
Interleukin-15
Basic Fibroblast Growth Factor
IL-15
bFGF
0.79
0.87
<0.001
<0.001
Granulocyte-Colony Stimulating
G-CSF
0.93
<0.001
Factor
Chemokine (C-C motif) ligand 5
RANTES*
0.33
0.014
VEGF
0.54
<0.001
(CCL-5)
Vascular Endothelial Growth Factor
Notes: *RANTES: “regulated upon activation, normal T cell expressed and
secreted”.
The strong induction of IL-1β, TNF-α, and IFN-γ, in combination with induction of G-CSF, suggests that a cascade of
events is triggered by exposure of cells to MMB fractions,
potentially involving mesenchymal stem cells as a pivotal
regulating cell type. Mesenchymal stem cells can cross the
blood–brain barrier and contribute to repair of brain injuries
such as stroke. Mesenchymal stem cells respond to inflammation in a manner that leads to events to counteract inflammation
and promote homeostasis. When mesenchymal stem cells from
healthy human donors were treated with the inflammatory
cytokines IL-1, TNF-α, and IFN-γ, these cells responded
with a strong increase in G-CSF production.47 This response
was able to reprogram highly inflamed LPS-activated microglial cells to reduce the production of inflammatory mediators.
Further work should include evaluation of temporal events
in cell culture during the cascade when human immune cells
respond to MMB fraction exposure. Future testing should also
include examination of immune activation when cells are
treated with a combination of all three fractions, thereby simulating the exposure of gut mucosal cells to the compounds in
the MMB. Additional work may also include cell cultures of
immune cells isolated from gut mucosal tissue such as lamina
propria and Peyer’s patches, in the presence of bacterial and
viral challenges, as well as characterization of the different
Journal of Inflammation Research 2020:13
mushroom fractions and respective contributions of each of the
17 mushroom species in the MMB. Clinical evaluation of
immune support, for example in acute and chronic viral infections, is also warranted and is currently in the planning stage.
Conclusion
The exposure of human immune cells to fractions of the
medicinal mushroom blend MMB triggered different and distinct immune responses by the hydrophilic, hydrophobic, and
insoluble fractions. The data have documented unique and
potentially synergistic effects, where the aqueous extract activated cells within the non-NK non-T cell fraction, which contains the dendritic cell types involved in immune recognition
and antigen presentation. In contrast, the post-aqueous ethanol
extract contained compounds able to activate monocytes,
T-lymphocytes, and Natural Killer T cells. The solid fraction
was a potent but highly selective inducer of multiple cytokines
with both pro-and anti-inflammatory activity and was also
supportive of induction of growth factors involved in regenerative functions. The results show that the MMB extract
fractions selectively induced immune cell activation and
increased host-defense activity, including anti-viral effects.
These data also provide a mechanistic explanation for the postinflammatory induction of G-CSF and multiple other cytokines
and growth factors involved in tissue repair. Subsequently,
induction of anti-inflammatory cytokines, such as IL-1ra,
help moderate and resolve the immune defense-induced proinflammatory reactions.
Acknowledgments
The study was performed at NIS Labs, an independent
research laboratory focusing on natural products research.
The work was sponsored by Fungi Perfecti, LLC, a grower
and producer of commercially available mushrooms, and
finished consumable products.
Author Contributions
All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be
published, and agree to be accountable for all aspects of
the work.
Disclosure
RD, RN, and AT are employed by the sponsor of the study,
Fungi Perfecti, LLC. PS holds several patents on topics
related to the presented work and is the founder and owner
of Fungi Perfecti, LLC. PS also conducted this research on
a formulation that he has devised which is sold as a
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nutritional supplement. The authors report no other conflicts of interest in this work.
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