Medicinal Mushroom Ganoderma lucidum (Leyss: Fr) Karst. Triggers Immunomodulatory Effects and Reduces Nitric Oxide Synthesis in Mice

Abstract

This study investigated the effect of Ganoderma lucidum supplementation on lymphocytes and peritoneal macrophages from mice. Our results show that G. lucidum in vivo was able to increase interferon-γ (IFN-γ) concentration but reduced CD3⁺ and CD8⁺ spleen lymphocytes. Ex vivo, IFN-γ; and interleukin-10 levels were increased and the tumor necrosis factor-α (TNF-α) level was reduced by peritoneal macrophages from mice fed with G. lucidum. In the absence of stimuli nitric oxide production was reduced in mice fed with G. lucidum, and with lipopolysaccharide stimulation nitric oxide production was increased but was lower than control values (P < .05). G. lucidum was grown by solid-state culture in wheat grain, and a chow containing 10% G. lucidum mycelium was formulated (G10). Swiss male mice were divided into two groups, termed G10 and control groups according to the diet, and were fed for 3 months. Peritoneal macrophages were obtained and investigated with regard to phagocytosis, lysosomal volume, hydrogen peroxide, superoxide anion, and cytokines ex vivo. In the plasma we investigated concentrations of cytokines, and in the spleen we determined subsets of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ lymphocytes.

Introduction

M any reports about the pharmacological effect of extracts and metabolites from medicinal mushrooms have attracted considerable global interest, particularly from the pharmaceutical industry. As a consequence, some mushrooms, such as Ganoderma lucidum (“Ling Zhi” or “Reishi), Agaricus brasiliensis (Sun mushroom), and Lentinus edodes (Shiitake), among many others, have been commercialized mainly as dried fruiting bodies, capsules, or aqueous extract or as dietary supplements for the prevention and treatment of a wide variety of diseases.¹

Therapeutic effects of mushrooms have been associated with their actions on immune cells such as lymphocytes and macrophages as well as in the modulation of production of interleukins (ILs), interferon (IFN), and tumor necrosis factor (TNF) and also upon other biological mediators.^1,2 Mushroom metabolites are able to drive the adaptive immune system activating B cells (CD19⁺) or T cells, including T-helper (Th) cells (CD4⁺ Th) and cytotoxic T cells (CD8⁺ Tc). Some mushroom polysaccharides have been shown to induce the differentiation of CD4⁺ T cells into Th1 or Th2 cells and also regulate cell-mediated or humoral immunity. In fact, such broad immunomodulatory effects exerted by mushroom metabolites justify their use when an increase of cell-mediated immunity is desirable and also in situations when its activity must be down-regulated, e.g., autoimmune disease and tissue transplantation.³

G. lucidum contains several active compounds such as polysaccharides, triterpenes, lectins, proteins, amino acids, nucleotides, steroids, lactones, fatty acids, enzymes, and other components that can carry out a large range of effects in the organism with distinct pharmacological properties.⁴ For example, polysaccharides mainly as β-d-glucans-(1→3) branching at the C-6 position have an antitumoral effect by stimulating the host defense mechanism. Furthermore, ganoderic acids composed of highly oxygenated triterpenes are able to act directly on neoplasic cells.⁵ Many studies have reported that G. lucidum metabolites can modulate the immune responses and also have antioxidant properties.^6
–8 Macrophages and other cells from the immune system produce mediators involved in the inflammation response such as cytokines, nitric oxide (NO), and reactive oxygen species, including superoxide anion (O₂ ^•−) and hydrogen peroxide (H₂O₂). Because an increase in the oxidative burst can induce severe tissue damage, foods that present antioxidant properties have been extensively screened by nutraceutical companies. In this context the use of mushrooms with immunomodulatory and antioxidant properties is of great interest as an adjuvant in cancer management regimens, to reduce anticancer therapy side effects, and to enhance immune defense.^9,10

Based on its antioxidant and immunomodulatory effects, here we investigated whether a regular chow supplemented with G. lucidum mycelium, produced by solid-state cultivation in wheat grain, given to mice would modify the immune response by peritoneal macrophages and spleen lymphocytes. To do so we measured populations of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ spleen cells and concentrations of IL-12p70, monocyte chemoattractant protein (MCP)-1, TNF-α, IL-10, IL-6, and IFN-γ in the plasma (in vivo) and in the supernatant of cultivated peritoneal macrophages (ex vivo). In addition, peritoneal macrophages were evaluated with regard to superoxide anion, H₂O₂, and NO production, phagocytosis, and lysosomal volume.

Materials and Methods

Mushroom culture

The microorganism used was G. lucidum strain CG 144, from Fuzhou, China, provided by Embrapa (The Brazilian Agency for Farming and Cattle Raising Research Center, Brasilia, Federal District, Brazil). The strain was kept by subcultivation in potato dextrose agar, once every 2 months, at 30°C for 10 days and subsequently stored at 4°C.

Wheat grains (Triticum aestivum L.) were washed and wetted for a 12-hour period in clean water. Trays of polyethylene (28 × 18 × 3 cm) containing 300 g of wet wheat grains were autoclaved at 120°C and 1 atmosphere pressure for 45 minutes. G. lucidum was cultivated in Erlenmeyer flasks (1 L) containing 500 mL of the following medium: glucose, 35 g/L; peptone, 5 g/L; yeast extract, 2.5 g/L; KH₂PO₄, 0.883 g/L; and MgSO₄ · 7H₂O, 0.5 g/L (pH 5.5).¹¹ The flasks were incubated with agitation (120 rpm) at 30°C for 10 days. Each flask produced about 3.5 g (dry weight) of the mycelium, which was separated by centrifugation (1,000 g, 15 minutes), suspended in 200 mL of sterile deionized water, and used as inoculum. This inoculum was sown in the proportion of 40 mL per 100 g of the wheat grains. The trays were further incubated at 30°C in 90% relative humidity in air for 20–30 days. The wheat grains completely covered by mycelium of G. lucidum were removed from trays and dried at 50°C with circulating air and then ground up. The meal obtained is referred as “fermented wheat flour.”

G. lucidum mycelium content

Analysis of fermented wheat flour was performed according to the AOAC's official methodology.¹² Mycelium content of the fermented wheat flour mass was determined indirectly through the amount of ergosterol by using high-performance liquid chromatography. Phytochemical analysis was done as described by Wagner et al.¹³ and Matos.¹⁴

Diet preparation

A regular chow containing protein (22%), ether extract (4%), mineral material (10%), fiber (8%), calcium (1.4%), phosphorus (0.8%), and vitamins (Nuvital CR1™, Curitiba, PR, Brazil) was supplemented with 10% fermented wheat flour by G. lucidum, subsequently referred as G10 chow.

Study design

All procedures involving animals were approved by the Federal University of Paraná Committee for Animal Welfare. Fourteen male Swiss mice (Mus musculus) healthy, 35 days old (±3 days) weighing 18.85 g (±1.25 g) were divided into two groups (seven per group). One group was fed with regular chow and subsequently referred as the control group, and the other was fed with G10 chow and subsequently referred to as the G10 group. The mice were kept in the animal house at a temperature of 24 ± 2°C and relative humidity of 55 ± 10% with a 12-hour/12-hour light/dark cycle for 12 weeks and fed with the respective diets and water ad libitum. Body weight was measured weekly during the whole feeding period. At the end of the 12^th week, with the animal under ether anesthesia, blood samples were collected from the mice by cardiac puncture, and the plasma concentrations of IL-12p70, MCP-1, TNF-α, IL-10, IL-6, and IFN-γ were measured by flow cytometry (FACSCalibur™) by using BD Biosciences Pharmigen reagents (Becton Dickinson, Franklin Lakes, NJ, USA). Then the mice were killed through ether inhalation, and peritoneal macrophages were obtained by intraperitoneal lavage with 5 mL of sterile phosphate-buffered saline (PBS) (pH 7.4). The spleen was removed in order to measure the population of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ cells by flow cytometry (FACSCalibur and CELL Quest software [BD Biosciences]). In brief, the spleen was placed in 60- × 15-mm Petri dishes containing 5 mL of PBS, and by using the plunger of a 10-mL syringe the spleen was disintegrated to obtain a homogeneous cell suspension. The residual cell clumps and debris were removed by filtration and centrifugation (200 g, 8 minutes). The pellet was resuspended in 20 mL of PBS. Cell counting was performed in a Neubauer chamber by optical microscopy, using a trypan blue solution (1%). Immunofluorescence staining was carried out as described by Coligan et al.¹⁵ In order to identify lymphocyte subsets, the monoclonal antibodies anti-mouse CD3⁺ (17A2) conjugated with fluorescein isothiocyanate and anti-mouse CD4⁺ (H129.19), CD8⁺ (53-6.7), and CD19⁺ (1D3) conjugated with R-phycoerythrin (Becton Dickinson) were used.

Ex vivo experiment

Peritoneal macrophages were obtained by centrifugation (200 g for 8 minutes at 4°C), washed, and counted using a trypan blue solution (1%). Peritoneal cells were characterized by flow cytometry, and about 50% were macrophages. Then the peritoneal macrophages were resuspended (1 × 10⁶cells/mL) in PBS (pH 7.4) or for culture in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal calf serum containing 10 U/mL streptomycin and 20 U/mL penicillin.

Cytokines

Aliquots (500 μL) of peritoneal macrophages suspension in RPMI-1640 medium were added to wells of a 24-well tissue culture plate and incubated for 2 hours at 37°C. Then the wells were washed twice with PBS to remove the nonadherent cells. Then, RPMI-1640 medium (2 mL) was added to the wells, and the plate was incubated for 24 hours. In the supernatant of the culture medium the IL-12p70, MCP-1, TNF-α IL-10, IL-6, and IFN-γ concentrations were measured by flow cytometry (FACScalibur) by using BD Biosciences Pharmigen reagents. The results were expressed as pg/mL.

NO

NO production was measured as nitrite (NO₂ ^-) by the Griess reaction. Aliquots of peritoneal macrophages (100 μL) were added into wells of a 96-well flat-bottomed tissue culture plates, incubated for 2 hours at 37°C, and then washed twice with PBS to remove the nonadherent cells. The remaining cells (2 × 10⁵ in a final volume of 200 μg) were incubated for 24 hours in the absence or presence of lipopolysaccharide (LPS) (final concentration, 10 μg/mL). Equal volumes of cell culture supernatant and Griess reagent were incubated for 10 minutes at room temperature, and the absorbance was measured at 550 nm (Benchmark microplate reader, Bio-Rad, Hercules, CA, USA). Nitrite concentration was determined from a standard curve generated using NaNO₂ and expressed as μmol/L.¹⁶

To determine the O₂ ^•- and H₂O₂ production, phagocytosis, and lysosomal volume, aliquots (100 μL) of peritoneal macrophages were added to the wells of 96-well flat-bottomed tissue culture plates and left for 1 hour. The plates were washed with PBS to remove the nonadherent cells. Then 100 μL of PBS was added into the wells (cell density, 1 × 10⁶ cells/mL).

O₂ ^•−

O₂ ^•− production was estimated by the nitro blue tetrazolium (NBT) (Sigma) reduction assay.¹⁷ Peritoneal macrophages (100 μL) were incubated at 37°C in the absence and presence of 10 μL of phorbol myristyl acetate (final concentration, 4 μM) and 0.2% NBT. After 1 hour the mixture was centrifuged (453 g for 5 minutes), the supernatant was discarded, and the peritoneal macrophages were fixed by adding 100 μL of methanol (50%) for 10 minutes. The plate was centrifuged again, the supernatant was discarded, and the plate was dried. Then 120 μL of KOH (2 M) and 140 μL of dimethyl sulfoxide were added to the wells. After 30 minutes the reduction of NBT resulted in the formation of blue formazan, which was detected spectrophotometrically (550 nm). The results were expressed as absorbance (per 10⁶ cells/mL).

H₂O₂

H₂O₂ production was measured as described by Pick and Mizel.¹⁸ This assay is based on the horseradish peroxidase-dependent conversion of phenol red into a colored compound by H₂O₂. Peritoneal macrophages (100 μL) were incubated in the presence of glucose (5 mM), phenol red solution (0.56 mM), and horseradish peroxidase (8.5 U/mL) in the dark for 1 hour at 20°C. H₂O₂ production was detected spectrophotometrically (620 nm). The results were determined from a standard curve and expressed as μmol (per 10⁶ cells/mL).

Phagocytosis

Phagocytosis was determined by adding 10 μL of neutral red-stained zymosan (1 × 10⁸ particles/mL) to each well containing 100 μL of peritoneal macrophages (1 × 10⁶ cells/mL). After incubation (37°C for 30 minutes) the peritoneal macrophages were fixed with Baker's formol-calcium (4% formaldehyde, 2% sodium chloride, and 1% calcium acetate) for 30 minutes. The cells were then washed twice by centrifugation (453 g for 5 minutes). The neutral red stain was solubilized by adding 100 μL of acidified alcohol (10% acetic acid and 40% ethanol in distilled water) to each well. After 30 minutes, the absorbance at 550 nm was measured, and the phagocytosis was calculated from a standard curve constructed from known amounts of stained zymosan. The results were expressed as absorbance (per 10⁵ cells).¹⁹

Lysosomal volume

The uptake of the cationic dye neutral red, which concentrates in cell lysosomes, was used to assess the volume of the peritoneal macrophages lysosomal system. Twenty microliters of 2% neutral red in PBS was added to 100 μL of peritoneal macrophages per microplate well and incubated for 30 minutes. The cells were then washed twice with PBS by centrifugation (453 g for 5 minutes). Neutral red was solubilized by a 30-minute incubation adding 0.1 mL of 10% acetic acid plus 40% ethanol solution. The absorbance was read at 550 nm, and lysosomal volume was expressed as absorbance (per 10⁶ cells/mL).²⁰

Statistical analysis

The data are presented as mean ± SEM values. Statistical analysis was performed by a two-tailed unpaired Student's t test (GraphPad [San Diego, CA, USA] Prism version 5 software). The value of P < .05 was taken to indicate statistical significance.

Results

G. lucidum mycelium composition

The mycelial growth in the fermented wheat flour was estimated according to the ergosterol content and was about 0.628 mg/g with the following composition: 73.3%, 15.0%, 1.7%, 8.0%, and 2.0% by gram weight of carbohydrates, proteins, lipids, humidity, and ash, respectively. Phytochemical screening revealed the presence of cardiac glycosides and saponins. On the other hand, flavonoids, anthraquinones, alkaloids, and tannins were not detected.

Effect on populations of spleen lymphocytes

Populations of spleen lymphocytes from mice fed the fermented wheat flour diet (G10 group) were significantly reduced. The CD3⁺ population was 12.83% (P < .05), and that of CD8⁺ was 14.30% (P < .05) compared to the control group. There was a slight reduction in the population of CD4⁺ spleen cells, but this was not significant (P > .05 versus control). In the G10 group (Table 1), in spite of an increase in the CD4⁺:CD8⁺ ratio this was not different from the control group (P > .05).

Table 1.

Populations of Spleen Lymphocytes from Mice Fed Regular Chow (Control Group) or G. lucidum-Supplemented Chow (G10 Group)

Spleen lymphocytes (%)	Control group	G10 group
CD3⁺	46.91 ± 1.66	40.89 ± 1.30^a
CD4⁺	32.24 ± 0.80	30.57 ± 1.2
CD8⁺	17.69 ± 0.63	15.16 ± 0.15^a
CD4⁺:CD8⁺	1.83 ± 0.05	2.02 ± 0.09
CD19⁺	34.93 ± 1.15	34.97 ± 1.20

The control group received diet without G. lucidum mycelium, whereas the G10 group received a diet supplemented with 10% G. lucidum mycelium. After 12 weeks subsets of populations of spleen lymphocytes were measured by flow cytometry. Data are mean ± SEM values of seven mice per treatment group.

P < .05 compared to control.

Plasma concentrations of cytokines

In the plasma from mice fed fermented wheat flour supplemented with G. lucidum mycelium (G10 group) there was a significant increase in the IFN-γ concentration of 15.36% (P < .05 vs. control), but the increase in the IL-12p70 concentration was not different from the control group (P > .05). In the G10 group the IL-10 plasma concentration was lower than the control group, but again it was not significantly different (P > .05). TNF-α MCP-1, and IL-6 plasma concentrations (Table 2) were similar to control values (P > .05).

Table 2.

Concentrations of Plasma Cytokines from Mice Fed Regular Chow (Control Group) or G. lucidum-Supplemented Chow (G10 Group)

Cytokine (pg/mL)	Control group	G10 group
IFN-γ	7.00 ± 0.35	8.07 ± 0.24^a
TNF-α	25.40 ± 0.48	26.95 ± 0.96
MCP-1	54.63 ± 2.87	52.08 ± 2.22
IL-10	35.33 ± 3.34	29.38 ± 0.32
IL-12p70	35.88 ± 1.37	42.53 ± 3.39
IL-6	10.00 ± 0.41	10.15 ± 0.44

The control group received diet without G. lucidum mycelium, whereas the G10 group received a diet supplemented with 10% G. lucidum mycelium. After 12 weeks concentrations of plasma cytokines were measured by flow cytometry. Data are mean ± SEM values of seven mice per treatment group.

P < .05 compared to control.

Concentrations of cytokines ex vivo

Peritoneal macrophages were cultivated in culture medium, and concentrations of cytokines were determined in the supernatant. Peritoneal macrophages obtained from the G10 group presented an increase of 14.10% in IFN-γ concentration (P < .05) and 41.89% in IL-10 concentration (P < .0001) compared to the control group. The TNF-α concentration was 17.44% lower compared to the control group (P < .05). MCP-1, IL-12p70, and IL-6 concentrations (Table 3) were not different from control values (P > .05).

Table 3.

Production of Cytokines by Peritoneal Macrophages from Mice Fed Regular Chow (Control Group) or G. lucidum-Supplemented Chow (G10 Group)

Cytokine (pg/mL)	Control group	G10 group
IFN-γ	9.75 ± 0.40	11.13 ± 0.36^a
TNF-α	354.30 ± 19.40	292.50 ± 13.45^a
MCP-1	214.8 ± 50.21	214.20 ± 42.46
IL-10	57.20 ± 1.11	81.17 ± 3.50^b
IL-12p70	34.98 ± 1.13	32.55 ± 1.20
IL-6	5,062.00 ± 114.4	4,680.00 ± 162.2

The control group received diet without G. lucidum mycelium, whereas the G10 group received a diet supplemented with 10% G. lucidum mycelium. After 12 weeks concentrations of cytokines in supernatant of peritoneal macrophages were assayed by flow cytometry. Data are mean ± SEM values of seven mice per treatment group.

P < .05, ^b P < .0001 compared to control.

NO production, under nonstimulated condition (Fig. 1), was lower by peritoneal macrophages obtained from mice fed fermented wheat flour supplemented with G. lucidum (G10 group) compared to the control group (P < .001). The stimulation with LPS induced an increase in NO production (P < .05) in both groups, but the increase was lower in the G10 group (P < .001).

FIG. 1.

NO production by peritoneal macrophages from mice fed regular chow (C) or G. lucidum-supplemented chow (G10). Peritoneal macrophages were cultured for 24 hours in the absence (open columns) or presence (solid columns) of 10 μg/mL bacterial LPS. Data are mean ± SEM values of seven mice per treatment group. ^a P < .05 compared to corresponding group without LPS; ^b P < .001 compared to C group without LPS; ^c P < .001 compared to C group with LPS stimulation.

O₂ ^•− and H₂O₂ production by peritoneal macrophages obtained from the G10 and control groups were the same (P > .05), as well as the phagocytosis and lysosomal volume (P > .05) (Table 4).

Table 4.

Phagocytosis, Lysosomal Volume, and H₂O₂ and O₂ ^•− Production by Peritoneal Macrophages from Mice Fed Regular Chow (Control Group) or G. lucidum-Supplemented Chow (G10 Group)

Parameter	Control group	G10 group
Phagocytosis of zymosan × 10⁻⁷ (absorbance per 10⁵ cells)	0.6184 ± 0.1048	0.6192 ± 0.0423
Lysosomal volume by neutral red uptake (absorbance by 10⁶ cells/mL)	0.0612 ± 0.018	0.079 ± 0.0046
H₂O₂ (μmol per 10⁶ cells/mL)	0.0738 ± 0.0165	0.0728 ± 0.0141
O₂ ^•− (absorbance per 10⁶ cells/mL)	0.1236 ± 0.0393	0.1100 ± 0.0051

Data are mean ± SEM values of seven mice per treatment group (P > .05).

Discussion

G. lucidum has been shown to have immunomodulatory, antitumor, antilipidemic, antiglycemic, and antioxidative actions.²¹ Considering that G. lucidum cultivation in nature is a long-term process that can take up to 6 months to form the fruiting body, the mycelial cultivation by submersible liquid fermentation or by solid-state fermentation allows us to obtain standardized nutraceutical substances in a higher yield.²² Most of the studies about this mushroom have been focused on hot water-soluble fractions or ethanol-soluble fractions and also on its biological effects in vitro. Here we extend these data by testing the effect of the whole G. lucidum mycelium in vivo and ex vivo.

Cytokines play a key role in the development, differentiation, and regulation of immune cells. Dysregulation in the production of cytokines or in their activity is thought to have a central role in the development of autoimmune and neoplastic diseases. Except for IFN-γ concentration, all the other cytokines (Table 2) quantified in the plasma have similar concentrations (P > .05 vs. control). There was a significant increase in the IFN-γ concentration in both supernatant of peritoneal macrophages (ex vivo) and plasma (in vivo) from the G10 group. These results corroborate previous studies reporting similar effects of polysaccharides from G. lucidum in cultured cells.^6,23 The meaning of these findings can be interpreted as a pro-inflammatory response by IFN-γ, which will facilitate the antitumor activity of G. lucidum extracts through activation of macrophages and stimulation of T lymphocytes as has been suggested previously.¹

The ex vivo approach was quite important because it allowed us to investigate the G. lucidum activity on peritoneal macrophages regardless of the other immune cells. IL-10 is an anti-inflammatory cytokine that attenuates the cell-mediated immunity response and triggers allergic and anti-helminthic response.²⁴ Down-regulation of IL-10 production can result in severe inflammation and perhaps autoimmune disease.²⁵ In our experiment a significant increase in IL-10 concentration was detected in the supernatant culture of peritoneal macrophages obtained from mice fed fermented wheat flour supplemented with G. lucidum (Table 3), but it was slightly reduced in the plasma of the control group (Table 2). The difference between the results ex vivo and in vivo perhaps might be explained by other chemical mediators secreted by other immune cells in vivo, which would reduce the stimulatory effect of G. lucidum metabolites on IL-10 production in plasma.

Another immunosuppressive effect found was a significant reduction in the TNF-α concentration in the supernatant of peritoneal macrophages in culture obtained from mice fed fermented wheat flour (the G10 group). Previous studies have reported the anti-ulcerogenic effect of G. lucidum polysaccharides in indomethacin-induced lesions in rats by suppression of TNF-α gene expression.²⁶ Wasting of skeletal muscle (cachexia) is a common feature of different illnesses such as cancer, sepsis, chronic heart failure, rheumatoid arthritis, and other inflammatory and chronic diseases. Cytokines from the immune system play an important role in the development of cachexia because elevation of circulating pro-inflammatory cytokines, especially TNF-α (cachectin), is a common feature of such conditions.^27,28 In addition, some studies have suggested that IFN-γ can interfere with the signal transduction machinery inducing down-regulation of TNF-α expression, which has being proposed as the target for a new strategy against cachexia.²⁹ Our results show that G. lucidum induced an increase in IFN-γ production and a reduction in TNF-α production by peritoneal macrophages. We suggest that IFN-γ overproduction triggered by G. lucidum down-regulated TNF-α expression by peritoneal macrophages.

The downstream immune response is chosen depending on which subtype of CD4⁺ Th cells is activated. For Th1 type, IL-12 is necessary,³⁰ whereas for Th2 type, IL-4 and IL-10 are the cytokines critical for differentiation.³¹ Mice fed fermented wheat flour supplemented with G. lucidum (the G10 group) showed a slight increase in the IL-12p70 plasma concentration compared to the control group, but it was not enough to promote the Th1 response. Conversely, there was a decrease in the populations of CD3⁺ and CD8⁺ spleen cells from mice of the G10 group (Table 1). These results corroborate the findings reported by other authors showing the immunosuppressive effect of “Ling-Zhi 8,” a protein extracted from G. lucidum mycelium, in transplanted mice.³² It is well recognized that Th1 cells promote cell-mediated immunity and host defense against intracellular organisms, but they also contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, Crohn's disease, and multiple sclerosis. In turn, Th2 cells attenuate cell-mediated immunity but can promote allergic responses.²⁴ Our results show that G. lucidum down-regulates the Th1 response but keeps the Th2 response unaltered in comparison to the control group. This feature greatly promotes the immunotherapeutic potential of G. lucidum as an adjuvant in the treatment of autoimmune diseases. In a recent previous study we showed that the consumption of fermented wheat flour rich in mycelium of G. lucidum induced an immunomodulatory effect characterized by T-cell dominance in tumor-bearing mice, increasing the resistance against sarcoma 180.³³ It is possible that G. lucidum metabolites can promote the selective induction of distinct populations of T cells exhibiting a wide range of therapeutic effects.

Among the different substances released by macrophages and other immune system cells, H₂O₂, TNF-α and NO play a special role not only because of their tumoricidal effect but also because they are able to induce tissue damage. In regular situations, NO plays a protective role in vascular compartment via its antioxidant and vasorelaxant effects. However, at high levels, NO is cytotoxic because of its ability to induce oxidative stress through the formation of peroxynitrite with O₂ ^•−.³⁴ The NO synthesis in macrophages is regulated by inducible NO synthase, which is mainly expressed under stimulus of the pro-inflammatory cytokines, especially TNF-α and bacterial LPS.³⁵ Here we found a significant reduction in NO production under basal and stimulated condition by peritoneal macrophages from mice fed G. lucidum-supplemented fermented wheat flour diet (Fig. 1). Our results corroborate the findings from other authors that reported the down-regulated effect of G. lucidum components diminishing the LPS-induced NO release.^7,8

The oxidative stress caused by reactive oxygen species production is recognized as one of the most important mechanisms underlying cardiovascular diseases.³⁶ Previous reports have described the free radical scavenging activity of G. lucidum extracts both in vitro ³⁷ and in vivo. ¹⁰ Here we also investigated the possibility of the consumption of fermented wheat flour by G. lucidum leading to an oxidative stress that could alter the reactive oxygen species release in healthy organisms and in the absence of stimulation. What we found was no change in the H₂O₂ and O₂ ^•− production by peritoneal macrophages from the G10 group compared to the control group (Table 4). Also, another important innate immune response such as phagocytosis and lysosomal volume were not different between the groups (P > .05).

Our phytochemical screening showed the presence of cardiac glycosides and saponins in the G. lucidum-fermented wheat. The class of steroid-like compounds designated cardiac glycosides includes drugs such as digoxin, digitoxin, and ouabaian, whose efficacy in the treatment of cardiac failure as anti-arrhythmic agents is well known. However, recent findings have revealed that these compounds can block a complex of cell signal transduction mechanisms dependent on TNF-α resulting in anti-inflammatory and anticancer effects.^38,39 The presence of cardiac glycosides in G. lucidum-fermented wheat flour suggests that they may be among the agents responsible for some of the immunomodulatory activity found in this work. However, this hypothesis must be tested.

Conclusions

Here we show that feeding of fermented wheat flour supplemented with G. lucidum for 3 months caused both immunostimulatory and immunosuppressive effects in peritoneal macrophages and spleen lymphocytes from mice. The immunostimulatory effect was characterized by an increase in the IFN-γ concentration both in vivo and ex vivo. The G. lucidum immunomodulatory effect on peritoneal macrophages (ex vivo) was evidenced through an increase in IL-10 release and a decrease in TNF-α production. Cell immunity was suppressed by a selective decrease in the population of CD3⁺ and CD8⁺ T spleen cells. Also, G. lucidum caused a decrease in NO production by peritoneal macrophages, showing its anti-inflammatory properties. These results suggest that G. lucidum metabolites can act not only to enhance a specific immune response against tumor cells or pathogenic microorganisms, but also to mitigate the adverse effects of the immune system such as immune-induced wasting, autoimmune diseases, and the inflammatory process.

Footnotes

Acknowledgments

The authors thank the National Research Council (CNPq), Brazil for financial support, the Paraná Tecnologia for technical support, and Dr^a. Arailde Fontes Urben (The Brazilian Agency for Farming and Cattle Raising Research Center, Embrapa, Brazil) who kindly provided the G. lucidum strain.

Author Disclosure Statement

No competing financial interests exist. We certify that we have no commercial associates (consultancies, stock ownership, equity interest, patent-licensing arrangements) that might pose a conflict of interest in connection with the submitted article and that we accept full responsibility for the conduct of the trial, had full access to all the data, and controlled the decision to publish.

References

Lull

, Wichers

, Savelkoul

HFJ

. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediators Inflamm, 2005; 2005,2:63–80.

Zaidman

, Yassin

, Mahajna

, Wasser

. Medicinal mushroom modulators of molecular targets as cancer therapeutics. Appl Microbiol Biotechnol, 2005; 67:453–468.

Borchers

, Keen

, Gershwin

. Mushrooms, tumors, and immunity: an update. Exp Biol Med, 2004; 229:393–406.

Jong

, Birmingham

. Medicinal benefits of the mushroom Ganoderma. Adv Appl Microbiol, 1992; 37:101–134.

Wasser

, Weis

. Therapeutic effects of substances occurring in higher basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol, 1999; 19:65–96.

Wang

, Hsu

, Tzeng

, Lee

, Shiao

, Ho

. The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int J Cancer, 1997; 70:699–705.

Song

, Kim

, Sa

, Jin

, Lim

, Park

. Anti-angiogenic and inhibitory activity on inducible nitric oxide production of the mushroom Ganoderma lucidum. J Ethnopharmacol, 2004; 90:17–20.

Woo

CWH

, Man

RYK

, Siow

, Choy

, Wan

EWY

, Lau

, Karmin

. Ganoderma lucidum inhibits inducible nitric oxide synthase expression in macrophages. Mol Cell Biochem, 2005; 275:165–171.

Kidd

. The use of mushroom glucans and proteoglycans in cancer treatment. Altern Med Rev, 2000; 5:4–27.

10.

Sheena

, Ajith

, Janardhanan

. Prevention of nephrotoxicity induced by the anticancer drug cisplatin, using Ganoderma lucidum, a medical mushroom in South India. Curr Sci, 2003; 85:478–482.

11.

Tang

, Zhong

. Fed-batch fermentation of Ganoderma lucidum for hyperproduction of polysaccharide and ganodermic acid. Enzyme Microb Tech, 2002; 31:20–28.

12.

Official Methods of Analysis of AOAC International 17 ^th. Association of Official Analytical Chemists (AOAC): Gaithersburg, MD, 2000.

13.

Wagner

, Bladt

, Zgainski

. Plant Drug Analysis. Springer Verlag: Berlin, 1984.

14.

Matos

FJA

. Introdução à Fitoquímica Experimental. Universidade Federal do Ceará: Fortaleza, Brazil, 1988.

15.

Coligan

, Kruisbeck

, Margulies

, Shevach

, Strober

. Current Protocols in Immunology. John Wiley & Sons: New York, 1992.

16.

Stuehr

, Marletta

. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci USA, 1985; 82:7738–7742.

17.

Madhavi

, Das

. Effects of n-3 and n-6 fatty acids on survival of vineristine sensitive and resistant human cervical carcinoma cells, in vitro. Cancer Lett, 1994; 84:31–41.

18.

Pick

, Mizel

. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. J Immunol, 1981; 46:211–226.

19.

Bonatto

JRS

, Folador

, Aikawa

, Yamazaki

, Pizatto

, Oliveira

HHP

, Vecchi

, Curi

, Calder

, Fernandes

. Lifelong exposure to dietary fish oil alters macrophage responses in Walker 256 tumor-bearing rats. Cell Immunol, 2004; 231:56–62.

20.

Pizato

, Bonatto

, Piconcelli

, de Souza

, Sassaki

, Naviwaiko

, Nunes

, Curi

, Calder

, Fernandes

. Fish oil alters T-lymphocyte proliferation and macrophage responses in Walker 256 tumor-bearing rats. Nutrition, 2006; 22:425–432.

21.

Cheung

, Ng

, Hood

. Identification and quantification of base and nucleoside markers in extracts of Ganoderma lucidum, Ganoderma japonicum and Ganoderma capsules by micellar electrokinetic chromatography. J Chromatography A, 2001; 911:119–126.

22.

Soccol

, Vandenberghe

LPS

. Overview of applied solid-state fermentation in Brazil. Biochem Eng J, 2003; 13:205–218.

23.

Kim

, Eo

, Oh

, Lee

, Han

. Antiherpectic activities of acid protein bound polysaccharide isolated from Ganoderma lucidum alone and in combinations with interferons. J Ethnopharmacol, 2000; 72:451–458.

24.

O'Shea

, Ma

, Lipsky

. Citokines and autoimmunity. Nat Rev Immunol, 2002; 2:37–45.

25.

Akira

. Roles of STAT3 defined by tissue-specific gene targeting. Oncogene, 2000; 19:2607–2611.

26.

Gao

, Zhou

, Wen

, Huang

, Xu

. Mechanism of the antiulcerogenic effect of Ganoderma lucidum polysaccharides on indomethacin-induced lesions in the rat. Life Sci, 2002; 72:731–745.

27.

Zhao

, Zeng

. Elevated plasma levels of tumor necrosis factor in chronic heart failure with cachexia. Int J Cardiol, 1997; 58:257–261.

28.

Sharma

, Anker

. Cytokines, apoptosis and cachexia: the potential for TNF antagonism. Int J Cardiol, 2002; 85:161–171.

29.

Tolosa

, Morlá

, Iglesias

, Busquets

, Lladó

, Olmos

. IFN-γ prevents TNF-α -induced apoptosis in C2C12 myotubes through down-regulation of TNF-R2 and increased NF-κ B activity. Cell Signal, 2005; 17:1333–1342.

30.

Gadina

, Hilton

, Johnston

, Morinobu

, Lighvani

, Zhou

, Visconti

, O'Shea

. Signaling by type I and II cytokine receptors: ten years after. Curr Opin Immunol, 2001; 13:363–373.

31.

Feldmann

, Brennan

, Maini

. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol, 1996; 14:397–440.

32.

van der Hem

, van der Vliet

, Bocken

CFM

, Kino

, Hoitsma

, Tax

WJM

. Ling zhi-8: studies of a new immunomodulating agent. Transplantation, 1995; 60:438–443.

33.

Rubel

, Dalla Santa

, Fernandes

, Lima Filho

JHC

, Figueiredo

, Di Bernardi

, Moreno

, Leifa

, Soccol

. High immunomodulatory and preventive effects against sarcoma 180 in mice fed with Ling Zhi or Reishi mushroom Ganoderma lucidum (W. Curt.: Fr.) P. Karst. (Aphyllophoromycetideae) mycelium. Int J Med Mushroom, 2008; 10:37–48.

34.

Napoli

, Ignarro

. Nitric oxide and atherosclerosis. Nitric Oxide, 2001; 5:88–97.

35.

Chesrown

, Monnier

, Visner

, Nick

. Regulation of inducible nitric oxide synthase mRNA levels by LPS, IFN-gamma, TGF-beta, and IL-10 in murine macrophage cell lines and rat peritoneal macrophages. Biochem Biophys Res Commun, 1994; 200:126–134.

36.

Khatri

, Johnson

, Magid

, Lessner

, Laude

, Dikalov

, Harrison

, Sung

, Rong

, Galis

. Vascular oxidant stress enhances progression and angiogenesis of experimental atheroma. Circulation, 2004; 109:520–525.

37.

Liu

, Ooi

VEC

, Chang

. Free radical scavenging activities of mushroom polysaccharide extracts. Life Sci, 1997; 60:763–771.

38.

Yang

, Huang

, Jozwik

, Lin

, Glasman

, Caohuy

, Srivastava

, Esposito

, Gillette

, Hartley

, Pollard

. Cardiac glycosides inhibit TNF-α/NF-κB signaling by blocking recruitment of TNF receptor-associated death domain to the TNF receptor. Proc Natl Acad Sci U S A, 2005; 102:9631–9636.

39.

Newman

, Yang

, Pawlus

, Block

. Cardiac glycosides as novel cancer therapeutic agents. Mol Interv, 2008; 8:36–49.

Medicinal Mushroom Ganoderma lucidum (Leyss: Fr) Karst. Triggers Immunomodulatory Effects and Reduces Nitric Oxide Synthesis in Mice

Abstract

Introduction

Materials and Methods

Mushroom culture

G. lucidum mycelium content

Diet preparation

Study design

Ex vivo experiment

Cytokines

NO

O2 •−

H2O2

Phagocytosis

Lysosomal volume

Statistical analysis

Results

G. lucidum mycelium composition

Effect on populations of spleen lymphocytes

Plasma concentrations of cytokines

Concentrations of cytokines ex vivo

Discussion

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

References

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