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The Journal of Nutrition
Nutritional Immunology
Dietary Supplementation with White Button
Mushroom Enhances Natural Killer Cell Activity
in C57BL/6 Mice
1,2
Dayong Wu,
3
* Munkyong Pae,
3
Zhihong Ren,
3,5
Zhuyan Guo,
3
Donald Smith,
4
and Simin Nikbin Meydani
3
3
Nutritional Immunology Laboratory,
4
Comparative Biology Unit, Jean Mayer USDA Human Nutrition Research Center on Aging at
Tufts University, Boston, MA 02111; and
5
State Key Laboratory for Infectious Disease Prevention and Control, National Institute
of Communicable Disease Control and Prevention, China CDC, 102206 Beijing, China
Abstract
Mushrooms are reported to possess antitumor, antiviral, and antibacterial properties. These effects of mushrooms are
suggested to be due to their ability to modulate immune cell functions. However, a majority of these studies evaluated the
effect of administering extracts of exotic mushrooms through parental routes, whereas little is known about the immu-
nological effect of a dietary intake of white button mushrooms, which represent 90% of mushrooms consumed in the U.S. In
this study, we fed C57BL/6 mice a diet containing 0, 2, or 10% (wt/wt) white button mushroom powder for 10 wk and
examined indices of innate and cell-mediated immunity. Mushroom supplementation enhanced natural killer (NK) cell activity,
and IFNgand tumor necrosis factor-a(TNFa) production, but only tended to increase IL-2 (P¼0.09) and did not affect IL-10
production by splenocytes. There were significant correlations between NK activity and production of IFNg(r¼0.615, P,
0.001) and TNFa(r¼0.423, P¼0.032) in splenocytes. Mushroom supplementation did not affect macrophage production of
IL-6, TNFa, prostaglandin E
2
, nitric oxide, and H
2
O
2
, nor did it alter the percentage of total T cells, helper T cells (CD4
1
),
cytotoxic or suppressive T cells (CD8
1
), regulatory T cells (CD4
1
/CD25
1
), total B cells, macrophages, and NK cells in spleens.
These results suggest that increased intake of white button mushrooms may promote innate immunity against tumors and
viruses through the enhancement of a key component, NK activity. This effect might be mediated through increased IFNg
and TNFaproduction. J. Nutr. 137: 1472–1477, 2007.
Introduction
Immune function, particularly innate and T cell–mediated im-
mune response, is critical to preventing and controlling micro-
bial infection (1–4) and neoplasia (5,6). There are, however,
limited strategies available to efficiently modulate the immune
response. Nutritional interventions that involve optimizing the
intake of essential nutrients and utilizing promising functional
foods have become an increasingly favored approach to the mod-
ulation of immune cell function. Mushrooms have long been
suggested to possess immunomodulatory properties (7–11).
The substances present in mushrooms with immunomodula-
tory and antitumor activity are mainly polysaccharides (in
particular b-D-glucans), polysaccharopeptides, and polysac-
charide proteins (10,12–14). Mushrooms also contain a spec-
trum of nutrients at varied levels, such as B vitamins, vitamin D,
and minerals (potassium, copper, magnesium, selenium, and
zinc). In recent years, the consumption of mushrooms, either as
whole mushrooms or extracted supplements, has increased. Al-
though historically, mushroom consumption has been associated
with the maintenance of health and warding off disease, sci-
entific data supporting their health benefits are limited. Epide-
miological data regarding the health benefits of mushroom
consumption is essentially nonexistent.
Studies relating the mushroom’s medicinal use started ;4
decades ago, and some clinical and experimental studies thus far
have suggested that mushrooms and their polysaccharide com-
ponents inhibit tumor growth. This effect of mushrooms may be
due to their ability to modulate immune system function rather
than having a direct effect on cancer cells (8,9,11). In the major-
ity of early studies, purified mushroom polysaccharides were
administered through a parental route or added to cell cultures.
More recent studies, however, reported that oral supplementa-
tion with a variety of different mushroom species are effective in
modulating certain immune functions (7,9,15,16).
Mushroom products have been evaluated for their pharma-
ceutical potential, and studies thus far have predominantly
focused on tumor-bearing animals or cancer patients. Whether
1
Supported by a grant from the Mushroom Council and the USDA, Agriculture
Research Service under contract 58-1950-9-001. Any opinions, findings, conclu-
sions, or recommendations expressed in this article are those of the authors and
do not necessarily reflect the view of the USDA.
2
Author disclosures: D. Wu, M. Pae, Z. Ren, Z. Guo, D. Smith, and S. N.
Meydani, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: dayong.wu@tufts.
edu.
1472 0022-3166/07 $8.00 ª2007 American Society for Nutrition.
Manuscript received 8 February 2007. Initial review completed 5 March 2007. Revision accepted 3 April 2007.
at TUFTS UNIVERSITY on February 27, 2013jn.nutrition.orgDownloaded from
an increase in the dietary consumption of mushrooms or mush-
room supplements can also have an impact on healthy animals
or subjects has not been established. This is despite the fact that
mushrooms and their subcomponents are widely consumed by
the general population as part of a normal diet. A majority of
studies on mushrooms have been conducted outside of the U.S
and almost all have used exotic mushrooms. In contrast, few
studies have evaluated the potential health benefits of white
button mushrooms (Agaricus bisporus), a strain of mushroom
that constitutes 90% of mushrooms consumed in the U.S.
Furthermore, little is known about their potential immunolog-
ical effects. Because the mushrooms of different strains have a
varied composition of nutrients and other functional ingredi-
ents, the results obtained from studies of these strains might
not be applicable to white button mushrooms. Our objective
in this study, therefore, was to determine the effect of white
button mushrooms on innate and T cell–mediated immune
functions.
Materials and Methods
Diets. Fresh white button mushrooms were provided by Franklin Farms
through the Mushroom Council. Mushroom stems were first cut off at
level of fruit body (crown) and each mushroom was cut into quarters.
The cut pieces of mushroom were freeze-dried for 5 d and then ground to
powder in a grinder (Norton). Dry matter was 7.5% of fresh weight.
Mushroom powder was added at 2 or 10% (wt:wt) to AIN-93M diet
(17), purchased from Research Diets, and thoroughly mixed. A control
mix was added to the control diet at 10% (wt:wt) and to 2% mushroom
diet at 8% (wt:wt) to equalize the levels of total energy and macronu-
trients in the experimental diets. The control mix was calculated to best
match the mushroom powder in energy, total carbohydrates, dietary
fiber, and protein and was prepared by mixing 25% casein, 37.5% corn
starch, and 37.5% cellulose. We chose the 2 and 10% levels of sup-
plementation to represent the high intake of fresh mushrooms achievable
through diet (2%) and supplementation (10%).
Animals. Male C57BL/6JNIA mice (4 mo of age) were purchased from
the National Institute on Aging colonies at Harlan Sprague Dawley.
After a 2-wk acclimation period, all mice were weight-matched and
placed into 1 of 3 experimental groups. Mice were housed individually
and maintained in an environmentally controlled atmosphere (temper-
ature 23C, relative humidity 45%) with a 12:12-h light:dark cycle.
Mice were given free access to water and group pair fed their respective
diet. To do this, mice were initially given a weighed portion of food daily.
If any mouse did not eat the entire portion of food, the weighed portion
for all mice was decreased to the amount that the mouse ate the previous
day. If all the mice consumed all of the diet, then the quantity of diet
given was increased until an individual mouse did not consume all the
food. By group feeding, we decreased the variability among the mice
both within each diet group and among the diet groups. All mice were
observed daily for clinical signs of disease, and body weight was
recorded at wk 0 (start), 1, 2, 4, 6, 8, and 10 (end). At the end of study,
mice were killed by CO
2
asphyxiation and exanguination. All conditions
and handling of the animals were approved by the Animal Care and Use
Committee of the Jean Mayer USDA Human Nutrition Research Center
on Aging at Tufts University and conducted according to the NIH
Guidelines for the Care and Use of Laboratory Animals.
Splenocyte isolation. Spleens were aseptically removed and placed
in sterile RPMI 1640 (Biowhittaker) medium-supplemented with 25
mmol/L HEPES (Invitrogen Gibco), 2 mmol/L glutamine (Gibco), 100
kU/L penicillin and 100 mg/L streptomycin (Gibco). Medium, supple-
mented as described, was referred to as complete RPMI. Single cell sus-
pensions were prepared as previously described (18). All the experiments
were conducted under a condition of 37C, atmosphere of 5% CO
2
, and
a 95% humidity, unless indicated otherwise.
Cellular composition of splenocytes. The percentages of major com-
ponent cell types in spleen were determined using fluorescent-activated
cell sorting (FACS)
6
analysis. Splenocytes (1 310
6
cells/sample) were
stained with following anti-mouse antibodies: FITC-conjugated anti-
CD3 (T cells), PE-conjugated anti-CD19 (B cells), FITC-conjugated anti-
CD4 [T helper (Th) cells], PE-conjugated anti-CD8 (T suppressor or
cytotoxic cells), APC-conjugated anti-CD25 (IL-2 receptor achain),
FITC-conjugated anti-natural killer-1.1 (NK-1.1 cells), and APC-conju-
gated anti-mouse F4/80 [macrophages (Mf)]. F4/80 antibody was from
Caltag and all the other antibodies were from BD PharMingen. Stained
cells were analyzed on a FACSCalibur (BD Biosciences) and the results
were analyzed using the Summit software,version 4.0 (DakoCytomation).
Natural killer cell activity assay. NK cell activity was assessed using
radioisotope
51
Cr release assay. YAC-1 cells, a murine lymphoma cell
line purchased from ATCC, were incubated with
51
Cr (Perkin Elmer Life
and Analytical Sciences) (25 mCi/1 310
6
cells) for 90 min at 37C.
Labeled YAC-1 cells were added to appropriately diluted splenocytes in a
round-bottomed 96-well plate (Nunc) for final effector:target cell ratios
of 10:1, 25:1, 50:1, and 100:1. The cocultured cells were incubated
in triplicate for 4 h at 37C. Spontaneous release was measured as the
amount of
51
Cr released by target cells alone, and maximum release was
measured by the amount of
51
Cr released after the addition of 5% Triton
X solution. Supernatant was collected after brief centrifugation (250 3g
for 1 min) and counted in a Cobra II gamma counter (Packard In-
struments) for radioactivity as counts per min (cpm). NK cell activity
was expressed as a specific lysis percentage: (sample cpm – spontaneous
cpm)/(maximum cpm – spontaneous cpm) 3100.
Mitogenic response of splenocytes. Splenocytes (1 310
5
cells/well),
in 96-well, flat-bottom plates (Becton Dickinson Labware), were
cultured in the presence or absence of the T cell mitogen concanavalin
A (Con A; Sigma) at 0.5, 1.5, or 3 mg/L, phytohemagglutinin P (PHA;
Difco Laboratories) at 2, 5, or 20 mg/L, or LPS (Sigma) at 0.1, 1, or 10
mg/L for 72 h. Cultures were pulsed with 0.5 mCi [
3
H]-thymidine (Perkin
Elmer) during the final 4 h of incubation. The cells were harvested onto
glass fiber filter mats (Wallac) by a Tomtec harvester (Wallac) and cell
proliferation was quantified as the amount of [
3
H]-thymidine incorpo-
rated into DNA as determined by liquid-scintillation counting in a 1205
Betaplate counter (Wallac). The counter had an efficiency of .50% for
3
H. Results are expressed as Bq.
Cytokine and prostaglandin E
2
production by splenocytes.
Splenocytes (4 310
6
cells/well) in 24-well culture plates (Becton
Dickinson Labware) were cultured in the presence of Con A (1.5 mg/L)
or LPS (1 mg/L) for 24 h for inflammatory IL-6, tumor necrosis factor-a
(TNFa), IL-12p70, and prostaglandin E
2
(PGE
2
) production or in the
presence of Con A (1.5 mg/L) or PHA (5 mg/L) for 48 h for IFNg, IL-2,
and IL-10 production. Cell-free supernatants were collected at the end of
incubation and stored at 270C for later analysis. All of the cytokines
were measured using ELISA. The reagents for IL-6, TNFa, IFNg, IL-2,
IL-10, and IL-12p70 assays were from BD PharMingen. PGE
2
was mea-
sured using radioimmunoassay as previously described (19).
Cytokine, PGE
2
, NO, and H
2
O
2
production by Mf.Peritoneal
exudate cells were obtained by peritoneal lavage and enriched for
Mfusing the method of Kumagai et al. (20). Peritoneal Mfprepared in
this manner were at least 90% pure, as assessed by the expression of
Mac-1 or F4/80 cell surface antibody.
Mf(;5310
5
cells/well) were incubated in 24-well culture plates
(Becton Dickinson Labware) in the presence of LPS (1 mg/L) for 24 h.
Cell-free supernatants were analyzed for cytokine, PGE
2
, and NO pro-
duction. Cells were lysed in 1 mol/L NaOH for total cell protein analysis
using the bicinchoninic acid protein assay kit (Pierce). IL-6, TNFa, IL-12,
and PGE
2
were analyzed as described above and NO was measured using
6
Abbreviations used: Con A, concanavalin A; cpm, counts per minute; FACS,
fluorescent-activated cell sorting; Mf, macrophages; NK, natural killer; PGE
2
,
prostaglandin E
2
; PHA, phytohemagglutinin; Th, helper T cells; TNFa, tumor
necrosis factor-a.
Mushroom and immunity 1473
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Griess assay as previously described (21). All the concentrations were
normalized with total cell protein.
For measurement of H
2
O
2
production, Mf(;5310
5
cells/well)
were incubated in 96-well culture plates (Becton Dickinson Labware) in
the presence of phorbol 12-myristate 13-acetate (Sigma) at 10 mg/L or
zymosan (Sigma) at 1 g/L for 1 h. Supernatants were collected and
analyzed for H
2
O
2
production using Amplex Red hydrogen peroxide/
peroxidase assay kit (Molecular Probes). Total cell protein was measured
as described above and used to normalize H
2
O
2
concentrations.
Statistical analysis. All results were expressed as means 6SEM.
Statistical analysis was conducted using Systat 10 statistical software.
Significant differences were determined using ANOVA for overall effect
of mushroom consumption and was followed by Fisher’s least signifi-
cance difference (LSD) post-hoc test for individual comparisons. Pearson
correlation was used to determine associations between NK activity and
production of IFNgor TNFa. Significance was set at P,0.05.
Results
General condition of mice. Mice in all diet groups remained
healthy throughout the experiment. Daily food intakes of group
pair-fed mice varied from 3 to 3.6 g/d. Body weight did not differ
among the diet groups at the start of the study and increased
from 1.6 to 1.7 g in the 3 dietary groups after the 10-wk feeding
period with no difference in body weight gain among the diet
groups (data not shown).
Subpopulations of splenocytes. The percentage of total B
cells (CD191), total T cells (CD31), Th cells (CD41), suppres-
sive/cytotoxic T cells (CD81), regulatory T cells (CD41/
CD251), NK cells (NK 1.11), and Mf(F4/801)didnotdiffer
among the 3 diet groups (data not shown).
NK cell activity. For all the effector:target cell ratios tested, NK
cell activity was significantly enhanced by mushroom supple-
mentation in a dose-dependent manner (Fig. 1). Similar results
(data not shown) were obtained when NK activity was expressed
as killing activity per given number of NK cells and calculated
based on the total splenocytes used in this assay and the
percentage of NK cells in the splenocytes obtained from the
aforementioned FACS assay.
Th 1 and Th 2 cytokine production. Con-A stimulated se-
cretion of Th 1 cytokine IFNgwas significantly greater in the
group fed the 10% mushroom diet (25.9 64.2 kU/L) than in the
control group (16.5 61.9 kU/L) and was intermediate in the 2%
mushroom diet group (22.0 62.9 kU/L). TNFaproduction was
significantly higher in the group fed the 10% mushroom diet
(60 618 ng/L) than in those fed the control diet (30 65 ng/L).
The 2% mushroom-fed mice tended to have higher (P¼0.09)
IL-2 production compared with the control mice (data not
shown). IL-10 (Th 2 cytokine), IL-12p70, IL-6, and PGE
2
(lipid
inflammatory mediator and T cell suppressor) production did
not differ among diet groups (data not shown).
Correlations between NK activity and IFNgand TNFa
production. Con A- (Fig. 2A) and PHA-stimulated (Fig. 2B)
IFNgproduction and NK activity in splenocytes were signifi-
cantly and positively correlated. In addition, NK activity and
TNFalevels were weakly but significantly correlated (Fig. 2C).
The results in Figure 2 were obtained at a 100:1 effector:target
cell ratio; results were similar at all the other effector:target cell
ratios tested (data not shown).
Mfsecretion of inflammatory mediators and H
2
O
2
.Mush-
room supplementation did not affect peritoneal Mfproduction
of IL-6, TNFa, PGE
2
, NO, and H
2
O
2
(data not shown), sug-
gesting that macrophage-derived products do not contribute to
the mushroom-induced enhancement of NK activity.
T and B cell proliferation. Mushroom supplementation did
not affect the proliferative response of splenocytes upon stim-
ulation with T cell mitogen Con A or PHA, or B cell mitogen
LPS (Fig. 3).
Discussion
This study demonstrates that dietary supplementation with
white button mushrooms enhances NK cell activity and IFNg
and TNFaproduction. The results suggest that the consumption
of white button mushrooms, a strain constituting 90% of
mushrooms consumed in the U.S., can modulate the body’s
innate immunity. To our knowledge, for the first time, these
results demonstrate that dietary supplementation with white
button mushrooms significantly enhances the ability of spleen
NK cells to lyse the target tumor cells in a dose-dependent
manner. This increased NK capacity mainly represents a more
vigorous cytolytic activity of NK cells toward target cells and is
not due to an increase in the number of total NK cells because
the percentage of NK cells in the spleen was not altered by
mushroom supplementation. Other strains of edible mushrooms
have also been shown to increase NK activity; however, these
studies were mostly conducted in tumor-bearing or virus-
infected animals (15,22–24).
NK cells are a group of specialized lymphocytes characterized
by their ability to spontaneously kill pathogen-infected cells and
tumor cells and are thus a key component of the innate immune
system (25). This function of NK cells is mediated and regulated
by the immunoregulatory cytokines produced by NK cells
themselves as well as other cells such as T cells and Mf. IFNgis
the most important cytokine in mediating NK activity. IFNgis
thought to play an essential role in NK cell-mediated suppres-
sion of viral and bacterial infection (26–28) and the inhibition of
tumor initiation and metastasis (26,29). To a lesser degree,
TNFamay also contribute to the antiviral and antitumor effects
of NK cells (26,28). In this study, the enhancement of NK
FIGURE 1 Effect of mushroom intake on NK activity of splenocytes
from mice. Isolated splenocytes were incubated with
51
Cr-labeled
YAC-1 cells (NK target cells) at the ratios as indicated. NK cell activity
was assessed using
51
Cr release assay and expressed as specific lysis
(%): (sample cpm – spontaneous cpm)/(maximum cpm – spontaneous
cpm) 3100. Values are mean 6SEM, n¼14/group. Means at a ratio
without a common letter differ, P,0.05.
1474 Wu et al.
at TUFTS UNIVERSITY on February 27, 2013jn.nutrition.orgDownloaded from
activity by mushroom supplementation was accompanied by an
increase in splenocyte production of IFNg. Furthermore, there
were significant positive correlations between NK activity and
IFNgproduction. We also found a higher TNFaproduction, but
only in mice fed 10% mushroom and when compared with
controls. Accordingly, a weak but significant correlation was
found between NK activity and TNFaproduction. These results
are consistent with a previous study in which i.p. injection of
D-Fraction extracted from maitake mushroom increased IFNg
and TNFaproduction by whole spleen cells in carcinoma-
bearing C3H/HeN mice (24). Because IFNgand TNFaare
produced by different cell types found in spleen (T cells, NK
cells, and Mf), further studies using intracellular cytokine
staining and flow cytometry are needed to determine the cellular
origin of the mushroom-induced increase in these cytokines. Our
results using peritoneal macrophages, however, indicate that the
effect of mushroom is not mediated through Mf.
Helper T cells are designated as Th 1 and Th 2 subpopula-
tions based on the patterns of their cytokine secretion. Th 1 cells,
characterized by IL-2, IFNg, and TNFaproduction, are mainly
involved in cell-mediated immunity to intracellular infections.
Th 2 cells, characterized by IL-4, IL-5, and IL-10 production, are
mainly involved in humoral immunity against extracellular
infections. Mushroom polysaccharides have been shown to
induce polarization of Th response but with a varied manner.
Although some of these mushroom-derived compounds induce
Th 1 response, others favor Th 2 response. It has been suggested
that the effect of mushrooms on Th cell polarization is de-
pendent upon the type of the mushroom-derived polysaccha-
rides and animal strain used (7). In this study, mushroom
supplementation increased IFNgand TNFaproduction and
tended to increase in IL-2 production, but did not affect IL-10
production. These results indicate that white button mushrooms
may induce a shift toward Th 1 response. This is further sup-
ported by our in vitro experiment in which extract of white button
mushroom promoted maturation and the antigen-presenting
function of bone marrow-derived dendritic cells (Z. Ren, Z.
Guo, S. N. Meydani, and D. Wu, unpublished results), which
have been shown to induce Th 1 response (30,31). Whereas
enhanced Th 1 response is beneficial for antimicrobial and anti-
tumor defense, it may be undesirable in certain autoimmune and
inflammatory disorders where overactive Th 1 response plays a
role. Thus, further studies using appropriate animal models are
needed to determine the clinical significance of the observed
immunomodulatory effects of white button mushrooms.
The polysaccharides, particularly b-1,3-glucans, are believed
to be the active components in mushrooms and are therefore
most frequently used in the studies to determine the effect of
mushrooms. It is still not well understood how polysaccharides
affect systemic immune function after oral ingestion given the
fact that they are dietary fibers. However, several studies have,
indeed, observed a tumor-inhibitory effect after oral adminis-
tration of certain glucans (16,24,32–34). We previously reported
that feeding mice a diet containing extract of the mycelia of
Coriolus versicolor increased delayed-type hypersensitivity skin
response, an in vivo measure of cell-mediated immunity, in old
mice (18). Together with the findings of our dietary supplemen-
tation study, it is reasonable to suggest that, in contrast to
common thought, immunomodulatory effects of mushrooms
may not entirely depend on the distribution of these polysac-
charides into the target tissues. Alternatively, because the cells
(dendritic cells, Mf, lymphocytes) in gut-associated lymphoid
tissues have direct access to gut content and continuously
recirculate in the blood and lymphatic systems, they can be
activated by mushroom polysaccharides in the gut and move to
blood and other systemic immune organs. It is also possible that
when whole mushroom extracts are used, some nonpolysac-
charide molecules present in mushrooms, such as lipids,
FIGURE 2 Correlations between mouse splenocyte NK activity and
IFNgor TNFaproduction after mushroom supplementation. Spleno-
cytes from the mice (n¼14/group) were stimulated with Con A (A, C)
or PHA (B).
Mushroom and immunity 1475
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vitamins, flavonoids, minerals, and trace elements, with the
immunomodulating property might contribute to the observed
effects. However, the 2 and 10% mushroom powder added to
the diets in this study do not substantially increase the level of
nutrients that are known to have immunostimulatory effects
above those present in the basal AIN-93M diet. For example,
based on the food intake of mice and nutrient composition of
raw white button mushrooms (35), we calculated that the 2 and
10% mushroom powder in diets provided only 2.4 and 12% of
more zinc and 7 and 35% more selenium than those found in the
basal diet, respectively. Therefore, it seems unlikely that the
nonpolysaccharide nutrients present in mushrooms play a major
role in inducing the enhancement of NK activity following
mushroom consumption.
In conclusion, the results of this study demonstrate that die-
tary supplementation with white button mushrooms enhances
NK cell activity. This effect of mushrooms may be mediated
through increased production of IFNgand TNFa. These results
suggest that consumption of white button mushrooms may in-
crease innate immunity to tumors and viral infections. Future
studies are needed to determine the clinical significance of these
findings, particularly in those with impaired immune functions,
such as elderly, and in those with cancer.
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LPS, at the concentrations as indicated for 72 h.
Values are means 6SEM, n¼14/group.
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