Content uploaded by Mary C. Christman
Author content
All content in this area was uploaded by Mary C. Christman on Dec 26, 2017
Content may be subject to copyright.
This article was downloaded by: [Mary Christman]
On: 14 April 2015, At: 10:07
Publisher: Routledge
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Click for updates
Journal of the American College of Nutrition
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/uacn20
Consuming Lentinula edodes (Shiitake) Mushrooms
Daily Improves Human Immunity: A Randomized
Dietary Intervention in Healthy Young Adults
Xiaoshuang Daia, Joy M. Stanilkaa, Cheryl A. Rowea, Elizabethe A. Estevesb, Carmelo Nieves
Jr.a, Samuel J. Spaisera, Mary C. Christmanc, Bobbi Langkamp-Henkena & Susan S. Percivala
a Department of Food Science and Human Nutrition, University of Florida, Gainesville,
Florida
b Department of Nutrition, Federal University of Jequitinhonha and Mucuri Valleys,
Diamantina, MG, BRAZIL
c MCC Statistical Consulting, Gainesville, Florida
Published online: 11 Apr 2015.
To cite this article: Xiaoshuang Dai, Joy M. Stanilka, Cheryl A. Rowe, Elizabethe A. Esteves, Carmelo Nieves Jr., Samuel
J. Spaiser, Mary C. Christman, Bobbi Langkamp-Henken & Susan S. Percival (2015): Consuming Lentinula edodes (Shiitake)
Mushrooms Daily Improves Human Immunity: A Randomized Dietary Intervention in Healthy Young Adults, Journal of the
American College of Nutrition, DOI: 10.1080/07315724.2014.950391
To link to this article: http://dx.doi.org/10.1080/07315724.2014.950391
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions
Original Research
Consuming Lentinula edodes (Shiitake) Mushrooms
Daily Improves Human Immunity: A Randomized
Dietary Intervention in Healthy Young Adults
Xiaoshuang Dai, Joy M. Stanilka, Cheryl A. Rowe, Elizabethe A. Esteves, Carmelo Nieves, Jr., Samuel J. Spaiser,
Mary C. Christman, Bobbi Langkamp-Henken, and Susan S. Percival
Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida (X.D., J.M.S., C.A.R., C.N., S.J.S.,
B.L.-H., S.S.P.); Department of Nutrition, Federal University of Jequitinhonha and Mucuri Valleys, Diamantina, MG, BRAZIL
(E.A.E.); MCC Statistical Consulting, Gainesville, Florida (M.C.C.)
Key words: mushroom, immunity, gd-T cell, NK-T cell, cytokine
Background: Mushrooms are widely cited for their medicinal qualities, yet very few human intervention
studies have been done using contemporary guidelines.
Objective: The aim of this study was to determine whether consumption of whole, dried Lentinula edodes
(shiitake) mushrooms could improve human immune function. Primary objectives were to ascertain whether L.
edodes consumption would improve gd-T cell proliferation and activation responses, quantify a dose response, and
elicit cytokine secretion patterns. Secondary objectives included determining changes in natural killer T (NK-T) cell
proliferation and activation, secretory immunoglobulin A (sIgA) in saliva, and C-reactive protein (CRP) in serum.
Design: Fifty-two healthy males and females, aged 21–41 years, participated in a 4-week parallel group
study, consuming either 5 or 10 g of mushrooms daily. Each subject had blood drawn before and after 4 weeks
of daily L. edodes consumption. Saliva and serum were also collected. Peripheral blood mononuclear cells were
cultured in autologous serum for 24 hours or 6 days, stained, and examined by flow cytometry.
Results: Eating L. edodes for 4 weeks resulted in increased ex vivo proliferation of gd-T (60% more, p<
0.0001) and NK-T (2-fold more, p<0.0001) cells. Both cell types also demonstrated a greater ability to express
activation receptors, suggesting that consuming mushrooms improved cell effector function. The increase in
sIgA implied improved gut immunity. The reduction in CRP suggested lower inflammation. The pattern of
cytokines secreted before and after mushroom consumption was significantly different; consumption resulted in
increased interleukin (IL)-4, IL-10, tumor necrosis factor (TNF)-a, and IL-1alevels, a decreased macrophage
inflammatory protein-1a/chemokine C-C ligand 3 (MIP-1a/CCL3) level, and no change to IL-6, IL-1b, MIP-1b,
IL-17 and interferon (IFN)-glevels.
Conclusions: Regular L. edodes consumption resulted in improved immunity, as seen by improved cell
proliferation and activation and increased sIgA production. The changes observed in cytokine and serum CRP
levels suggest that these improvements occurred under conditions that were less inflammatory than those that
existed before consumption.
INTRODUCTION
There are thousands of species of mushrooms, although
only about 20 types of mushrooms are cultivated commercially
for culinary purposes. The mushroom Lentinula edodes, com-
monly known as shiitake, is cultivated for both its culinary and
medicinal qualities. In vitro and animal studies have indicated
the potential for immune-modulating activity; however, very
few human studies have been performed.
The research in this lab has focused on the effect of diet on the
function of the gd-T cell. This cell has a T cell receptor (TCR) of
gamma and delta chains that are similar to the alpha and beta
chains of the more common circulating T cells. However, unlike
ab-T cells that recognize protein antigens, gd-T cells recognize
Address correspondence to: Susan S. Percival, Department of Food Science and Human Nutrition, University of Florida, P.O. Box 110370, Gainesville, FL 32611. E-mail:
percival@ufl.edu
This trial was registered at http://www.clinicaltrials.gov as NCT01398176.
Abbreviations: sIgA Dsecretory IgA, CRP DC-reactive protein, PBMC Dperipheral blood mononuclear cells, NK-T Dnatura l killer T cell, MFI Dmean fluores-
cence intensity, MIP-1aDmacrophage inflammatory protein-1a, CCL3 Dchemokine C-C ligand 3.
1
Journal of the American College of Nutrition, Vol. 0, No. 0, 1–10 (2015) ÓAmerican College of Nutrition/
Published by Taylor & Francis Group, LLC
Downloaded by [Mary Christman] at 10:07 14 April 2015
small phospho-antigens that do not require the major histocompat-
ibility complex for processing [1,2]. The majority of gd-T cells are
located in the epithelial lining of the gut, lungs, and genitourinary
tract, where they act as a first line of immunological defense [3,4].
Although they are T cell lymphocytes, they behave more like a
cell of the innate immune system.
The natural killer T (NK-T) cell is also considered an innate
lymphocyte. Unlike the NK cell, the NK-T cells develop in the
thymus and express CD3 on their surface. High NK-T concen-
trations are found in the liver. NK-T cells do not require sensi-
tization to spontaneously lyse tumor cells. Currently, little is
known about the interaction of diet with these cells.
The immunomodulating effect of shiitake mushrooms has
been investigated in many in vitro [5–7] and in vivo models
[8–11]. Agaricus bisporus (white button) mushrooms have also
been shown to influence immunity in mice [12–14].
Reports of immunomodulation by any mushroom in
humans are scarce, and those using L. edodes, even fewer. In a
pilot study, Yamaguchi et al. [15] gave L. edodes mycelia
extract to patients undergoing chemotherapy; an increase in
NK cell activity and a decrease in immunosuppressive acidic
protein levels were observed. One double-blind, crossover, pla-
cebo-controlled intervention study was performed with 42
healthy, elderly individuals. In that study, the soluble b-glucan
extracted from L. edodes (lentinan, b-1,3;1,6-glucan) was con-
sumed for 6 weeks and resulted in an increase in the number of
circulating B cells but no change in immunoglobulins, comple-
ment proteins, or serum levels of cytokines [16].
The aim of this study was to determine whether the con-
sumption of dried L. edodes mushrooms could improve human
immune function, particularly regarding innate lymphocytes.
Primary objectives of the study were to determine whether L.
edodes consumption would improve gd-T cell proliferation
and activation responses, to define any dose response to the
amount of mushrooms consumed, and to obtain data on cyto-
kine networks. Additional objectives included determining
changes to NK-T cell proliferation and activation, secretory
immunoglobulin (sIgA), and serum C-reactive protein (CRP)
levels, and memory (CD45RO
C
) and na€
ıve (CD45RA
C
)gd-T
cell population numbers after mushroom consumption.
SUBJECTS AND METHODS
Subjects
After receiving approval from the University of Florida
Internal Review Board, 66 generally healthy males and
females, 21–41years of age, were recruited and screened, by
phone or in person, to participate in a 4-week, parallel-group
study. To be eligible for enrollment, subjects could not be
vegan or vegetarian and had to agree to have blood drawn and
saliva collected twice; stop consumption of tea, flavonoid-con-
taining supplements, antioxidant supplements, or probiotics in
advance; and, during the study, not consume more than 14
glasses of alcoholic beverages per week or more than 7 serv-
ings of fruits and vegetables per day. In addition, eligible sub-
jects could not be on immunosuppressive drugs or antibiotics,
have hypertension that required medication, chronically use
nonsteroidal anti-inflamatory drugs, or have an ongoing infec-
tion. Women who were pregnant, lactating, or on hormone
therapy did not meet the study inclusion criteria. Subjects were
asked to keep a log of their illnesses during the study. Written
consent was obtained from all enrolled subjects.
Study Design
The study was conducted during the months of September and
October of 2011. Eligible subjects reported to the clinical labora-
tory in the Food Science and Human Nutrition Building on the
University of Florida campus, where consent was obtained and
they were enrolled in the study by trained personnel. Once
enrolled, participants were randomly assigned, using a computer-
ized random number generator, to consume either 5 g (nD26) or
10 g (nD25) of dried L. edodes mushrooms every day for 4
weeks. These dried amounts were equivalent to one (3-oz.) or 2
(6-oz.) servings of fresh mushrooms based on 5 medium mush-
rooms as one serving [17] and a 90%–95% water content.
A 4-week supply of daily packets of dried mushrooms,
either whole or ground, was given to subjects along with
instructions for preparation, which included sufficient rehydra-
tion and cooking. Subjects received half of their mushrooms as
ground and half as whole. The mushrooms were obtained with
the help of a reputable local mushroom grower, who identified
and sourced the dried shiitake mushrooms for the study.
There were 2 blood draws during the study: one before
(baseline) and one after 4 weeks of mushroom consumption.
Blood was drawn from fasting subjects by a trained phleboto-
mist and used for peripheral blood mononuclear cells (PBMC)
isolation and to obtain serum.
In addition, subjects were instructed to complete daily illness
and consumption logs over the course of the study. Illness logs
were used to determine whether the groups differed in the pattern
of illnesses, not as a health outcome. To help ensure compliance
(80% consumption), the study coordinator corresponded weekly
with participants through e-mail. Subjects returned to the clinical
lab at the end of the 4 weeks for the final blood draw, turned in
their illness logs, and completed a questionnaire that asked about
any side effects experienced during the intervention, supplement
use (vitamins and minerals), and self-reported compliance. Over-
all compliance was determined by the quantity of mushrooms
returned, as well as the self-reported data.
PBMC Isolation and Autologous Serum
At each blood draw, 8 mL of blood was collected into a
vacuum tube containing L-heparin; 3 mL was drawn into a
serum activator tube (Vacutainer, BD, Franklin Lakes, NJ) and
processed within 2 hours.
Shiitake Mushrooms Improve Human Immunity
2 VOL. 0, NO. 0
Downloaded by [Mary Christman] at 10:07 14 April 2015
Serum tubes were kept chilled and then centrifuged (2000
£g, 10 minutes, 4C). Serum was separated for use in the
PBMC cultures, and additional aliquots were stored at ¡80C
for CRP analysis.
Whole blood was diluted 1:1 with 0.9% NaCl, layered over
Lympholyte H density gradient separation medium (Cedarlane
Laboratories Ltd., Burlington, NC), and centrifuged (800 £g,20
minutes, 20C). The PBMC band was removed and washed twice
(1250 £g,10minutes,4
C) in RPMI-1640 medium (Mediatech,
Inc., Manassas, VA). The cell pellets were resuspended in clear
RPMI 1640 medium containing 100,000 U/L penicillin, 100 mg/
L streptomycin, 0.25 mg/L fungizone, 50 mg/L gentamicin,
2 mmol/L L-glutamine, and 25 mmol/L HEPES buffer, and cell
counts were acquired using an automated cell counter (Nexcelom
Bioscience LLC, Lawrence, MA).
Cell Culture
PBMC (1 £10
6
/well) were cultured in one well of each of
two 24-well plates, in clear RPMI 1640 medium with 10%
autologous serum and PHA-L (10 mg/mL), interleukin (IL)-2
(1 ng/mL), and IL-15 (1 ng/mL). The plates were incubated
for 24 hours or 6 days at 37C in a humidified 5% CO
2
atmo-
sphere. After 24 hours, the cell culture media was harvested
from the wells of one plate and frozen at ¡80C for cytokine
analysis. Cells were collected and stained for flow cytometry at
24 hours and 6 days.
Flow Cytometry
Before and after mushroom consumption, PBMC were
analyzed by flow cytometry at 3 time points: day 0 (uncul-
tured) and 24 hours and 6 days of culture. Fluorescent anti-
bodies to cell surface markers were obtained from Biolegend
and eBioscience (San Diego, CA). Cells and antibodies were
incubated together at 4C in the dark for 30 minutes and the
cells were washed and fixed with 2% formalydehyde. Cells
were read within 48 hours on an Accuri C6 flow cytometer
(BD Biosciences, San Jose, CA) and data were analyzed
using FlowJo software version 10.0.6 (Tree Star Inc.,
Ashland, OR).
On day 0, freshly isolated PBMC (5 £10
5
) in flow-staining
buffer (phosphate-buffered saline with 2% fetal bovine serum)
were stained with fluorescent antibody panels to determine
baseline population and activation levels for gd-T (CD3-PE,
gdTCR-FITC, CD69-AF647) and NK-T (CD3-PE, CD56-
AF647, CD314-FITC) cells. After 24 hours of culture, PBMC
were stained using the same gd-T and NK-T cell antibody pan-
els to determine the levels of activation for each cell type. After
6 days of culture, PBMC were stained to assess gd-T (CD3-PE,
gdTCR-FITC) and NK-T (CD3-PE, CD56-AF647) cell prolif-
eration and for levels of gd-T na€
ıve and memory cells (gdTCR-
FITC, CD45RA-AF700, CD45RO-PE). Proliferation and
phenotype prevalence results are expressed as a population per-
centage, and activation results are expressed as mean fluores-
cence intensity. The shift in fluorescence intensity of the
population of cells is interpreted as an increase or decrease in
the number of receptors expressed on the cell surface.
Cytokine Determination
The 24-hour cell culture fluids were assayed to determine
the levels of 10 cytokines (IL-1a, IL-1b, IL-4, IL-6, IL-10, IL-
17, interferon [IFN]-g, macrophage inflammatory protein-1a/
chemokine C-C ligand 3 [MIP-1a/CCL3], MIP-1b, and tumor
necrosis factor [TNF]-a), using a Human Cytokine Multiplex
Immunoassay kit, according to manufacturer’s directions
(Millipore Corp., Billerica, MA). The beads were analyzed on
a Luminex 200 instrument (Luminex Corp., Austin, TX) with
xPONENT 3.1 software. Data are expressed in milligrams per
liter.
Saliva Collection and sIgA Determination
At each of the 2 blood draws, subjects were instructed to rinse
their mouth thoroughly with water for 20 seconds. Following the
rinse, subjects waited for 10 minutes and then used a half straw to
transfer their saliva into a 15-mL conical tube during a 2-minute
period. The saliva was kept cold andclarifiedbycentrifugation
(2300 £g, 10 minutes, 4C), and aliquots were stored at ¡80C
until assayed. The amount of sIgA in salivary samples was mea-
sured by ELISA, using a commercially available kit (ALPCO
Immunoassays, Salem, NH) following manufacturer’s instruc-
tions. Determined levels of sIgA are expressed as both micro-
grams per milliliter and micrograms per minute.
Serum CRP
Frozen serum samples were thawed and analyzed for total
CRP using a Human C-Reactive Protein Quantikine ELISA kit
(R&D Systems, Minneapolis, MN), as directed by the manu-
facturer. Total CRP concentrations are expressed in nanograms
per milliliter.
Power Analysis
Based on the proliferative capacity of the gd-T cells in culture
from previous studies [7,9–11], a sample size of 15 individuals
per group would be needed to achieve 80% power at a 5% signifi-
cance level to detect a 15% increase in the percentage of gd-TC,
CD3Ccell population after culturing. To account for dropouts, 25
subjects were recruited for each group.
Statistics
For each subject, the outcomes were measured at baseline
and postintervention. In addition, for those outcomes for which
activation or proliferation was of interest, at each time period
Shiitake Mushrooms Improve Human Immunity
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3
Downloaded by [Mary Christman] at 10:07 14 April 2015
(baseline and postintervention) the initial and 24-hour (6-day)
measurements were also collected. Hence, overall, for some
outcomes we had several repeated observations on each sub-
ject. For those outcomes that had multiple measurements over
time within each time period, the model was a repeated meas-
ures linear model with a non-zero compound symmetry covari-
ance for observations on the same subject and with fixed
effects of treatment, time period, and, where appropriate, cul-
ture time (uncultured and 24 hours or 6 days). To adjust for
variables likely to have an effect on the outcomes but that were
not controlled for during randomization, we included covari-
ates for gender and the continuous variables body mass index
(BMI) and age. The analyses proceeded in a hierarchal manner
starting with the full model with all variables and all interac-
tions of each variable with treatment, time period, and culture
time included. The residuals were checked and, if needed, a
log-transformation of the response was performed and the full
model was rerun on the transformed data.
Cytokines, sIgA, CRP, and memory/na€
ıve outcomes were
analyzed using a general linear mixed model that included
time period (baseline or postintervention) and treatment as
fixed effects and random effect of subject to capture the
repeated measurements on the same subject. Additional covari-
ates included age, gender, and BMI and all 2-way interactions
of these with treatment and time.
For both modeling approaches, nonsignificant terms were
dropped from the model one by one starting with interactions
and those with the largest pvalues. The model was rerun each
time a variable or interaction was removed and checked again
for further removals. The final model included only those cova-
riates (age, BMI, and gender) that were statistically significant,
plus time, culture time, and treatment. Each analysis was done
on the intent to treat population and then repeated on the com-
pliant group. The compliant models never differed from intent
to treat models; therefore, only the intent to treat model is
reported.
Means and SEs were reported for those fixed effects that
were found to be significant and if the outcome required log-
transformation, the means were back-transformed and the SEs
for the back-transformed means were estimated using the Delta
method [18].
RESULTS
Participants
Sixty-six individuals were assessed for eligibility, but 14
were excluded because they either declined participation or
did not meet inclusion/exclusion criteria (Fig. 1). Fifty-two
individuals were randomized to consume either one or 2
servings of mushrooms using a random number generator.
One participant randomized to the 2-serving group was
removed from the study due to participation in a conflicting
study. Two individuals from the one-serving group dropped
out due to a dermatitis associated with undercooked L. edo-
des mushrooms, and one individual was unable to return
for the second blood draw. Three individuals in the 2-serv-
ing group dropped out due to nausea and gastrointestinal
distress, and 3 subjects could not return for the second
blood draw. This resulted in nD23 in the one-serving
group and nD19 in the 2-serving group for analysis. Lack
of compliance was determined for 2 individuals in the one-
serving group and one individual in the 2-serving group.
Intent to treat analysis versus per protocol analysis found
no statistical difference; therefore, analysis on the intent to
treat population has been reported. There was only one
measurement that was statistically different between serv-
ing sizes (IL-4); therefore, the results of the 2 serving sizes
are pooled and the difference between before and after
mushroom consumption was the primary index of statistical
difference. The demographics of the population that were
allocated to intervention are shown in Table 1. There were
no differences seen in the incidence and duration of self-
reported illnesses during the study between individuals con-
suming either one or 2 servings of mushrooms (Table 2).
Cell Proliferation and Activation
Stimulated gd-T cells were able to proliferate to a greater
extent ex vivo, after L. edodes were consumed for 4 weeks
(Table 3). Statistically, the main effect of time (baseline vs 4
weeks) was significantly greater at 4 weeks. Post hoc analysis
showed a significant interaction between time and culture time
(uncultured, 24 hours, 6 days). Before mushroom consump-
tion, stimulated cells proliferated within 24 hours to levels that
were significantly greater than the uncultured cells, but the
increase was not sustained during the 6 days of culture. After
L. edodes were eaten, however, gd-T cells maintained prolifer-
ation levels over the 6-day period.
The expression of CD69 on gd-T cells is an indication of
their potential for activation. Expression of CD69 in uncultured
cells was not statistically different at the 2 time points
(Table 3), whereas stimulation for 24 hours with a mitogen
resulted in an increase both before and after consumption of
L. edodes. However, after 4 weeks of mushroom consumption,
the increase seen was significantly greater than that of baseline
cells.
NK-T cell proliferation was also significantly greater at
4 weeks (Table 3). Post hoc analysis (Time £Culture
time) indicated a significant reduction in the percentage of
this type of cell in the CD3Cpopulation in uncultured
cells. However, following mushroom consumption, a signif-
icant percentage increase was seen over baseline values
after 24 hours and 6 days of culture and stimulation. Sur-
face indicators of NK-T cell activation increased
4 VOL. 0, NO. 0
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
significantly from baseline to 4 weeks. Post hoc analysis of
this data (Time £Culture time) showed significant differ-
ences in the levels of the activation marker. When baseline
levels of marker expression on uncultured cells were com-
pared with uncultured cells at 4 weeks, there was a sub-
stantial reduction in mean fluorescence intensity, whereas
the comparison of cells stimulated and cultured for
24 hours indicated a greater than 10-fold increase in marker
expression between baseline and 4-week values.
Stimulating PBMC with a mitogen in culture for 6 days
after subjects had consumed mushrooms for 4 weeks resulted
in both significantly more na€
ıve gd-T cells and fewer memory
gd-T cells as a percentage of the entire CD3Ccell population
when compared to baseline levels (Table 4).
Fig. 1. Flowchart of subject eligibility, randomization, follow-up, and analysis.
Table 1. Population Demographics of Subjects Allocated to Consume L. edodes Mushrooms
1
One Serving
2
Two Servings
Female (nD16) Male (nD10) Female (nD16) Male (nD9)
Age 22.8 §2.1
3
24.8 §6.4 25.5 §6.3 24.4 §2.9
Weight (kg) 57.3 §8.9 74.0 §6.7 60.1 §6.6 74.7 §7.1
Height (m) 1.6 §0.1 1.7 §0.1 1.6 §0.1 1.8 §0.1
BMI (kg/m
2
) 21.7 §2.3 25.1 §2.2 22.4 §2.2 23.3 §2.4
BMI Dbody mass index.
1
Dried mushrooms were prepared and consumed daily for 4 weeks.
2
Mushroom serving size: One serving is defined as 3 oz. or 85 g fresh weight, equivalent to 5 g dried; 2 servings D10 g dried [17].
3
Mean §SEM. Determined by 2-way analysis of variance.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
Biomarkers of Immunity and Inflammation
The amount of sIgA in the saliva and CRP in the serum are
shown in Table 5. Levels of sIgA were significantly greater in
the saliva after mushrooms were eaten for 4 weeks than at
baseline. The average concentration of serum CRP for all par-
ticipants was within normal range at baseline. After eating
mushrooms for 4 weeks, CRP levels detected in the serum
were reduced significantly by around 30%.
Cytokine Production
Table 6 shows the concentrations of cytokines secreted into
PBMC culture medium during 24 hours of mitogen stimulation.
The quantities of IL-4, IL-10, IL-1a,andTNF-asecreted were
all significantly greater after mushroom consumption, whereas
MIP-1a/CCL3 secretion was significantly lower. The concen-
trations of IL-1b,IL-17,IFN-g, and MIP-1bin 24-hour culture
fluids at 4 weeks were no different from those seen at baseline.
Serving size did not influence cytokine secretion except in the
case of IL-4, where 2 servings of mushrooms were associated
with higher levels than one serving (pD0.045).
Table 2. Self-Reported Illness in Subjects Consuming L.
edodes Mushrooms
1
Group Incidence
2
Duration
3
One serving 18 38
Two servings 18 40
pD0.739
4
pD0.988
zD¡0.333 zD¡0.0146
1
Subjects consumed either one (5 g) or 2 (10 g) servings of dried, prepared
mushrooms daily for 4 weeks.
2
Incidence is the number of times the subject reported at least one symptom asso-
ciated with a cold, the flu, or an allergy.
3
Duration is the total number of days with at least one symptom reported.
4
pdetermined by z-test of proportions; Yates correction applied.
Table 3. Proliferation and Activation of Stimulated
1
Innate Lymphocytes before and after L. edodes Consumption
2
Time £Culture Interactions
Type Time Uncultured 24 Hours 6 Days
gd-T Proliferation % of CD3CBaseline 15.5 §0.6
3
12.4 §0.7
B
19.3 §1.5
A
14.9 §0.8
B
4 weeks 19.2 §0.6 12.7 §0.8
B
22.5 §1.5
A
22.4 §0.8
A
pvalue T: <0.0001 T£C: 0.8150 T£C: 0.1368 T£C: <0.0001
Activation
4
mean
fluorescence intensity
Baseline 4899 §456 755 §61
C
9044 §851
B
4 weeks 7411 §639 998 §85
C
13,824 §1194
A
pvalue T: <0.0001 T£C: 0.1644 T£C: <0.0001
NK-T Proliferation Baseline 10.5 §0.9 12.6 §0.9
C
12.8 §0.9
C
5.9 §0.9
D
% of CD3C4 weeks 14.4 §0.9 5.5 §0.9
D
21.2 §0.9
A
16.6 §0.9
B
pvalue T: <0.0001 T£C: <0.0001 T£C: <0.0001 T£C: <0.0001
Activation
5
Baseline 264 §37 314 §49
C
215 §34
C,D
mean fluorescence intensity 4 weeks 1236 §50 197 §31
D
2275 §183
A
pvalue T: <0.0001 T£C: <0.0001 T£C: <0.0001
NK-T Dnatural killer T cells, T Dtime, T£CDpost hoc interaction.
1
PBMC were cultured in RPMI-1640 with 10% autologous serum, PHA-L (10 mg/mL), IL-2 (1 ng/mL), and IL-15 (1 ng/mL).
2
Subjects consumed either one (5 g) or 2 (10 g) servings of dried, prepared mushrooms daily for 4 weeks. Data from both serving groups were combined for analysis.
3
Mean §SEM. A repeated measures linear model with a non-zero compound symmetry covariance for observations on the same subject and with fixed effects of treat-
ment, time period. Values with different letters are significantly different.
4
Mean fluorescent intensity of CD69 on the gdTCRC, CD3Ccell population.
5
Mean fluorescent intensity of NKG2D (CD314) on the CD56C, CD3Ccell population.
Table 4. Prevalence of Memory and Na€
ıve Phenotypes in Cultured PBMC
1
before and after L. edodes Consumption
2
Phenotype Cell Surface Markers Baseline 4 Weeks pValue
Memory gdTCR-FITC/CD45RO-PE 45.7 §1.5
3
31.0 §1.5 T: <0.0001
Na€
ıve gdTCR-FITC/CD45RA-AF700 52.0 §1.6 67.0 §1.6 T: <0.0001
PBMC Dperipheral blood mononuclear cells, TDtime.
1
PBMC were cultured for 6 d in RPMI-1640 with 10% autologous serum, PHA-L (10 mg/mL), IL-2 (1 ng/mL), and IL-15 (1 ng/mL). Numbe rs for each phenotype are
expressed as a percentage of the gdTCRCcell population.
2
Subjects consumed either one (5 g) or 2 (10 g) servings of dried, prepared mushrooms daily for 4 weeks. Data from both groups were combined for analysis.
3
Mean §SEM.
6 VOL. 0, NO. 0
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
DISCUSSION
The literature suggests that consuming mushrooms will
improve immunity, yet few studies in humans have been done.
This clinical study fed whole mushrooms to humans as a food.
The dried L. edodes mushrooms we used required rehydration
and heat processing before consumption. The 2 individuals in
the one-serving group who dropped out of the study did so
because they acquired shiitake dermatitis, which may have
been due to improper preparation of the dried mushrooms
before they were eaten. The number of subjects that dropped
out of the 2-serving group, 10%, is the normal rate of attrition
for our studies. The fact that it was due to gastrointestinal dis-
tress appears to be related to the consumption of the mush-
rooms; however, symptoms were not severe. In our analyses,
the differences seen between the results for the 2 serving sizes
tended to go in the right direction; that is, if the results for one
serving were lower, then the results for two servings were even
lower. However, only one measure, IL-4, actually achieved sta-
tistical significance. Another limitation of the study was that a
placebo cannot be created when whole food is eaten. With no
placebo, the study lacked a “no treatment” group. Had we
observed a dose effect we would have been able to rule out
changes due to time alone. The self-reported illness data we
collected showed that the 2 groups had indistinguishable illness
patterns; therefore, we feel that illnesses during the study did
not have an effect on our results.
In our study, subjects consumed either 5 or 10 g of mush-
rooms daily for 4 weeks. In order to detect immune changes
after mushroom consumption, we examined the proliferation
and activation of 2 innate lymphocytes, gd-T and NK-T cells.
Before mushrooms were consumed, the proliferation and acti-
vation of these 2 cell types, in the presence of autologous
serum and mitogen stimulation, was minimal. In contrast, after
consumption these lymphocytes responded robustly to the
mitogen stimulation.
The specific mechanism by which mushrooms alter a cell’s
ability to proliferate is not known. We hypothesize that compo-
nents of the mushroom act to prime innate immune cells,
thereby enhancing proliferation and activation responses of
those cells upon stimulation. Priming itself does not evoke a
cellular response but merely prepares the cell, enhancing its
ability to respond to a secondary stimulus. We did find more
na€
ıve gd-T cells in the peripheral blood after mushroom con-
sumption, a cell type that is more likely to be primed than the
memory cell.
Beta-glucans have been linked to immune alterations
[16,19]. Lentinan, the specific b-1,3;1,6-glucan found in L.
edodes mushrooms [19], is known to interact with both toll-
like and dectin receptors [20–22] on macrophages, dendritic
cells, and other immune cells. In the gut, the immune cells that
sample lumen contents interact with the b-glucan and then, as
these cells migrate into lymph and peripheral blood, they com-
municate their information to other immune cells. In addition,
it is likely that b-glucan is fermented by microbes in the large
intestine, and although it is known that compounds derived
from this fermentation are absorbed, the exact mechanism by
which they impact immune cells has not been determined. An
increase in the level of secretory IgA in the saliva is regarded
as evidence of beneficial changes to mucosal immunity [23].
Our findings showed a rise in sIgA levels after consuming L.
edodes mushroom, as has also been reported in humans using
the white button (A. bisporus) mushroom [24].
Table 5. Biomarkers of Immunity Detected in Saliva and
Serum before and after Consumption of L. edodes Mushrooms
1
Baseline 4 Weeks pValue
sIgA
2
(mg/ml) 815.2 §49.3
4
910.7 §81.0 0.049
sIgA (mg/min) 779.1 §58.2 917.9 §94.0 0.045
CRP
3
CRP
3
(mg/L) 1.09 §0.72 0.77 §0.36 0.008
sIgA Dsecretory immunoglobulin A, CRP DC-reactive protein.
1
Subjects consumed either one (5 g) or 2 (10 g) servings of dried, prepared
mushrooms daily for 4 weeks. Data from both groups were combined for
analysis.
2
sIgA in the saliva was detected by ELISA and is expressed as both concentration
(mg/ml) and rate (mg/min).
3
Detection of CRP in the serum was performed using ELISA.
4
Mean §SEM. A general linear mixed model that included time and treatment
as fixed effects.
Table 6. Cytokine Production by Stimulated PBMC
1
before
and after L. edodes Consumption
2
Cytokine Baseline 4 Weeks pValue
IL-4 73.5 §9.63
3
111.3 §11.7 T: <0.0001,
S: 0.045
IL-10 1962.7 §166.6 2618.2 §225.0 T: 0.0001
IL-1a499.1 §40.0 581.7 §45.5 T: 0.0069
TNF-a3029.7 §185.6 3561.3 §243.5 T: 0.0032
MIP1-a26,953.8 §1276.3 8793.2 §176.6 T: <0.0001
IL-1b6103.4 §521.6 6107.2 §704.1 T: NS
IL-6 7943.2 §696.7 6047.2 §411.6 T: NS
IL-17 54.9 §6.4 60.7 §7.6 T: NS
IFN-g2282.1 §214.7 2071.7 §196.8 T: NS
MIP1-b9622.8 §644.1 9645.7 §511.4 T: NS
PBMC Dperipheral blood mononuclear cells, IL Dinterleukin, TNF Dtumor
necrosis factor, MIP Dmacrophage inflammatory protein, IFN Dinterferon,
TDmain effect of time, S Dserving size covariate.
1
PBMC were cultured for 24 hours in RPMI-1640 with 10% autologous serum,
PHA-L (10 mg/mL), IL-2 (1 ng/mL), and IL-15 (1 ng/mL).
2
Subjects consumed either one (5 g) or 2 (10 g) servings of dried, prepared
mushrooms daily for 4 weeks. Data from both groups were combined for
analysis.
3
Mean §SEM. Significance was determined by a general linear mixed model
that included time period and treatment as fixed effects and random effect of sub-
ject to capture the repeated measurements on the same subject. Additional cova-
riates included age, gender, and BMI and all 2-way interactions of these with
treatment and time.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
There are many compounds in L. edodes mushrooms,
besides b-glucan, that have the potential to influence immu-
nity. For example, ergothioneine [25–27] and conjugated lino-
lenic acid [28], as well as vitamins and minerals, are known
bioactive compounds found in most mushrooms. In addition to
priming, L. edodes mushroom consumption appeared to influ-
ence the immune response in such a way as to reduce inflam-
mation. The immune response after priming is more vigorous,
yet the cells produced fewer inflammatory mediators and more
anti-inflammatory mediators. A more vigorous response,
occurring under less detrimental conditions, would seem to
favor the host organism.
CD69, a lectin receptor, is the earliest inducible cell surface
glycoprotein acquired during lymphocyte activation [29].
Knock-out mice lacking CD69 are severely inflamed [30]. Our
results showed that eating mushrooms increased the CD69
expression on the surface of gd-T cells, which would suggest
that there was a lower state of inflammation during the mitogen
stimulation of cell proliferation.
Other evidence for this lower state of inflammation was
seen in the cellular production of cytokines. The cytokines IL-
4 and IL-10 are generally characterized as anti-inflammatory,
because they downregulate the production of inflammatory
cytokines. IL-4 is a regulatory cytokine that is responsible for
the proliferation of B cells and Th2 helper cells. The main
function of IL-10 is to terminate inflammatory responses by
inhibiting the synthesis of IFN-gand TNFa. IL-4 and IL-10
were produced in greater quantity after the subjects consumed
mushrooms. MIP-1a/CCL3 is a chemotactic inflammatory
cytokine produced early in an immune response. It induces the
production of IL-1, IL-6, and TNF-a. Of all of the cytokines
we measured, this was the only one whose production
decreased after mushroom consumption. Less MIP-1a/CCL3
should inhibit the amount of IL-1, IL-6, and TNF-aproduced.
However, we saw TNF-aconcentrations rise, while the cyto-
kines IL-1 and IL-6 remained the same. An elevated TNF-a
concentration has been reported in other in vitro [5–7] and in
vivo [12,13] studies. TNF-ais often classified as an inflamma-
tory cytokine, although one of its functions is to resolve inflam-
mation by causing apoptosis. The fact that TNF-alevels
increased, despite the increase in IL-10 and decrease in MIP-
1a/CCL3, suggests that we still do not know all there is to
know about the regulation of this cytokine.
CRP, the prototypical acute phase protein in humans, par-
ticipates in the nonspecific systemic response and its levels rise
in response to inflammation [31]. The fact that the final con-
centration of CRP was significantly reduced in the subjects
consuming mushrooms, who started with values within the nor-
mal range for that population, again suggests that eating mush-
rooms lowers inflammation.
Changes we saw in the NK-T cells paralleled those of the
gd-T cells, suggesting that other innate lymphocytes may be
primed by dietary compounds. NKG2D (CD314) is a c-type
lectin receptor that is not highly expressed on the surface of
cells under normal resting conditions. At 4 weeks, NKG2D
expression on resting, uncultured cells was significantly
lower than that seen before mushroom consumption. This
lowering of the resting activation state suggests reduced
inflammation.
The expression of NKG2D increases on the cell surface
when cells are activated [32,33]. In this study, mitogen-stimu-
lated NK-T cells expressed greater than 10-fold more NKG2D
on the cell surface after mushroom consumption than cells
stimulated before consumption. An increase in NKG2D
expression may be a part of the priming mechanism, as we
have suggested for CD69 expression on gd-T cells. NKG2D is
part of the machinery used by NK cells to lyse infected or
tumor cells [32]. Increased expression of this molecule on the
cell surface suggests an improvement in the cytolytic ability of
NK-T cells, which may contribute to the resolution of the
immune response by causing apoptosis of cells that are no lon-
ger required when the pathogen threat is eliminated.
In conclusion, we believe that regular L. edodes mushroom
consumption resulted in improved immunity, as seen by increased
cellular proliferation and activation after stimulation and the
higher levels of sIgA produced. We suggest that the mechanism
by which this happens is the priming of innate lymphocytes by L.
edodes, making them able to respond more robustly to a challenge.
In addition, we feel that the expression of activation markers
CD69 and NKG2D on innate T cells, and the pattern of cytokines
secreted by the PBMC, along with the reduction in the level of
CRP, together suggest the presence of an anti-inflammatory envi-
ronment during stimulation by the mitogen. It seems likely that
less inflammation during an immune response may be beneficial
to the host; this results in a less aggressive immune response,
while the ability of the organism to energetically combat a patho-
gen is preserved.
ACKNOWLEDGMENTS
Thank you to the students and staff for their excellent work
in completing this research.
FUNDING
Funding was provided by the U.S. Mushroom Council, Aus-
tralian Mushroom Growers Association, and the University of
Florida Agriculture Experiment Station.
Authors’ Contributions
X.D., J.M.S., B.L.-H., and S.S.P. designed the research. X.D.,
J.M.S., C.A.R., E.A.S., C.N., and S.J.S. conducted the research.
X.D. and J.M.S. analyzed the data, and M.C.C. performed the
8 VOL. 0, NO. 0
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
statistical analysis. X.D., J.M.S., C.A.R., B.L.-H., and S.S.P. wrote
and edited the article. X.D. and J.M.S. contributed equally to the
work in this manuscript. S.S.P. had primary responsibility for final
content. All authors read and approved the final article. None of
the authors had any conflicts of interest.
REFERENCES
1. Kabelitz D, Glatzel A, Wesch D: Antigen recognition by human
gd lymphocytes. Int Arch Allergy Immunol 122:1–7, 2000.
2. Morita CT, Mariuzza RA, Brenner MB: Antigen recognition by
human gamma delta T cells: pattern recognition by the adaptive
immune system. Springer Semin Immunopathol 22:191–217, 2000.
3. Carding SR, Egan PJ: Gammadelta T cells: functional plasticity
and heterogeneity. Nat Rev Immunol 2:336–345, 2002.
4. Ferrarini M, Ferrero E, Dagna L, Poggi A, Zocchi MR: Human
gammadelta T cells: a nonredundant system in the immune-sur-
veillance against cancer. Trends Immunol 23:14–18, 2002.
5. Jin M, Jung HJ, Choi JJ, Jeon H, Oh JH, Kim B, Shin SS, Lee JK,
Yoon K, Kim S: Activation of selective transcription factors and
cytokines by water-soluble extract from Lentinus lepideus. Exp
Biol Med (Maywood) 228:749–758, 2003.
6. Yu LG, Fernig DG, Rhodes JM: Intracellular trafficking and
release of intact edible mushroom lectin from HT29 human colon
cancer cells. Eur J Biochem 267:2122–2126, 2000.
7. Liu M, Li J, Kong F, Lin J, Gao Y: Induction of immunomodulat-
ing cytokines by a new polysaccharide–peptide complex from
culture mycelia of Lentinus edodes. Immunopharmacology
40:187–198, 1998.
8. Chen J, Zhang XD, Jiang Z: The application of fungal beta-glu-
cans for the treatment of colon cancer. Anticancer Agents Med
Chem 13:725–730, 2013.
9. Tanaka K, Ishikawa S, Matsui Y, Tamesada M, Harashima N, Har-
ada M: Oral ingestion of Lentinula edodes mycelia extract inhibits
B16 melanoma growth via mitigation of regulatory T cell–medi-
ated immunosuppression. Cancer Sci 102:516–521, 2011.
10. Chandra L, Alexander H, Traore D, Lucas EA, Clarke SL, Smith
BJ, Lightfoot SA, Kuvibidila S: White button and shiitake mush-
rooms reduce the incidence and severity of collagen-induced arthri-
tis in dilute brown non-agouti mice. J Nutr 141:131–136, 2011.
11. Giese S, Sabell GR, Coussons-Read M: Impact of ingestion of rice
bran and shitake mushroom extract on lymphocyte function and
cytokine production in healthy rats. J Diet 5(Suppl 5):47–61,
2008.
12. Xu Y, Na L, Ren Z, Xu J, Sun C, Smith D, Meydani SN, Wu D:
Effect of dietary supplementation with white button mushrooms
on host resistance to influenza infection and immune function in
mice. Br J Nutr 109:1052–1061, 2013.
13. Yu S, Weaver V, Martin K, Cantorna MT: The effects of whole
mushrooms during inflammation. BMC Immunol 10:12, 2009.
14. Ren Z, Guo Z, Meydani SN, Wu D: White button mushroom
enhances maturation of bone marrow–derived dendritic cells and
their antigen presenting function in mice. J Nutr 138:544–550,
2008.
15. Yamaguchi Y, Miyahara E, Hihara J: Efficacy and safety of orally
administered Lentinula edodes mycelia extract for patients
undergoing cancer chemotherapy: a pilot study. Am J Chin Med
39:451–459, 2011.
16. Gaullier JM, Sleboda J, Ofjord ES, Ulvestad E, Nurminiemi M,
Moe C, Tor A, Gudmundsen O: Supplementation with a soluble
beta-glucan exported from Shiitake medicinal mushroom, Lenti-
nus edodes (Berk.) singer mycelium: a crossover, placebo-
controlled study in healthy elderly. Int J Med Mushrooms 13:
319–326, 2011.
17. FDA.gov: Food Labeling Guide. Accessed at: http://www.fda.gov/
Food/IngredientsPackagingLabeling/LabelingNutrition/default.
htm
18. Casella G, Berger RL: “Statistical Inference.” Pacific Grove, CA:
Duxbury Thomson Learning Inc., 2002.
19. Vetvicka V, Vashishta A, Saraswat-Ohri S, Vetvickova J: Immu-
nological effects of yeast- and mushroom-derived beta-glucans. J
Med Food 11:615–622, 2008.
20. Chen J, Seviour R: Medicinal importance of fungal beta-(1–>3),
(1–>6)-glucans. Mycol Res 111:635–652, 2007.
21. Albeituni SH, Yan J: The effects of beta-glucans on dendritic cells
and implications for cancer therapy. Anticancer Agents Med
Chem 13:689–698, 2013.
22. Ren L, Perera C, Hemar Y: Antitumor activity of mushroom poly-
saccharides: a review. Food Funct 3:1118–1130, 2012.
23. Albers R, Antoine JM, Bourdet-Sicard R, Calder PC, Gleeson M,
Lesourd B, Samartin S, Sanderson IR, Van Loo J, Vas Dias FW,
Watzl B: Markers to measure immunomodulation in human nutri-
tion intervention studies. Br J Nutr 94:452–481, 2005.
24. Jeong SC, Koyyalamudi SR, Pang G: Dietary intake of Agaricus
bisporus white button mushroom accelerates salivary immuno-
globulin A secretion in healthy volunteers. Nutrition 28:527–531,
2012.
25. Weigand-Heller AJ, Kris-Etherton PM, Beelman RB: The bio-
availability of ergothioneine from mushrooms (Agaricus bisporus)
and the acute effects on antioxidant capacity and biomarkers of
inflammation. Prev Med 54(Suppl):S75–S78, 2012.
26. Martin KR: The bioactive agent ergothioneine, a key component
of dietary mushrooms, inhibits monocyte binding to endothelial
cells characteristic of early cardiovascular disease. J Med Food
13:1340–1346, 2010.
27. Ey J, Schomig E, Taubert D: Dietary sources and antioxidant
effects of ergothioneine. J Agric Food Chem 55:6466–7410, 2007.
28. Adams LS, Phung S, Wu X, Ki L, Chen S: White button mush-
room (Agaricus bisporus) exhibits antiproliferative and proapop-
totic properties and inhibits prostate tumor growth in athymic
mice. Nutr Cancer 60:744–756, 2008.
29. Craston R, Koh M, Mc DA, Ray N, Prentice HG, Lowdell MW:
Temporal dynamics of CD69 expression on lymphoid cells. J
Immunol Methods 209:37–45, 1997.
30. Sancho D, Gomez M, Viedma F, Esplugues E, Gordon-Alonso M,
Garcia-Lopez MA, de la Fuente H, Martinez A, Lauzurica P, San-
chez-Madrid F: CD69 downregulates autoimmune reactivity
through active transforming growth factor-beta production in col-
lagen-induced arthritis. J Clin Invest 112:872–882, 2003.
31. Yousuf O, Mohanty BD, Martin SS, Joshi PH, Blaha MJ, Nasir K,
Blumenthal RS, Budoff MJ: High-sensitivity C-reactive protein
and cardiovascular disease: a resolute belief or an elusive link? J
Am Coll Cardiol 62:397–408, 2013.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 9
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
32. Cerwenka A, Lanier LL: NKG2D ligands: unconventional MHC
class I-like molecules exploited by viruses and cancer. Tissue
Antigens 61:335–343, 2003.
33. Rohner A, Langenkamp U, Siegler U, Kalberer CP, Wodnar-
Filipowicz A: Differentiation-promoting drugs up-regulate
NKG2D ligand expression and enhance the susceptibility of acute
myeloid leukemia cells to natural killer cell–mediated lysis. Leuk
Res 31:1393–1402, 2007.
Received May 27, 2014; accepted July 28, 2014.
10 VOL. 0, NO. 0
Shiitake Mushrooms Improve Human Immunity
Downloaded by [Mary Christman] at 10:07 14 April 2015
