Purpose
To compare three fractions of a medicinal mushroom blend (MMB), MyCommunity, on immune-activation, inflammation-regulation, and induction of biomarkers involved in regenerative functions.
Methods
A seventeen-species MMB was sequentially extracted: first, saline solution at ambient temperature, followed by re-extraction of the solids in ethanol, and finally resuspension of the homogenized ethanol-insoluble solids in cell-culture media. Fractions were tested on peripheral blood mononuclear cells from three healthy donors. Immunostaining, flow-cytometry, and Luminex protein-arrays measured immune-cell activation and cytokine response. Dose-responses for induction of the CD69 early activation marker and individual cytokine and growth-factor responses for each donor were evaluated. The CD69 and the combined cytokine and growth-factor results were subjected to Non-metric Multidimensional Scaling (NMDS) and multivariate ordination to aid interpretation of the aggregate immune response and pairwise permutational MANOVA on a distance-matrix to evaluate statistical differences between treatments on pooled data from all donors.
Results
Differential effects were induced by water-soluble, ethanol-soluble, and insoluble immunomodulatory compounds of the MMB. The aqueous and ethanol fractions upregulated expression of CD69 on all tested cell types. Monocyte-activation was correlated with the ethanol fraction, while NKT and non-NK non-T cell-activation was more closely correlated with the aqueous fraction. The solid fraction was the most potent inducer of Tumor Necrosis Factor-α, as well as the anti-viral cytokines interferon-γ, MCP-1 (CCL-2), MIP-1α (CCL-3), and MIP-1β (CCL-4), and induced G-CSF and b-FGF—growth-factors involved in regenerative functions—and the anti-inflammatory cytokine IL-1ra.
Conclusion
The aqueous, ethanol, and insoluble compounds within MMB induced differential immune-activating, anti-inflammatory, and regenerative effects. This in vitro data suggests that, upon consumption, MMB may induce a concerted series of immunomodulatory events based on the differential solubility and bioavailability of the active constituents. These differential responses support both immune-activation and resolution of the host defense-induced inflammatory reactions, thus assisting a post-response return to homeostasis.
Introduction
Mushrooms have been embraced for centuries due to their nutritional and medicinal properties. They have been historically used in the treatment of infectious disease, gastrointestinal disorders and asthmatic conditions, as well as to support overall wellbeing.1 Fungi now occupy their own kingdom, but they were once considered plants due to their resemblance and root-like structures. One of many characteristics that separate fungal from plant organisms is the cell wall structure. The cell walls of fungi contain chitin,2 a modified form of the polysaccharide cellulose. Chitin is comprised of β-(1→4)-linked N-acetylglucosamine monomers, whereas cellulose is comprised of β-(1→4)-linked glucose units. Chitin degrades into a mixture of shorter-chained polysaccharides along with monosaccharide products.3,4 This degradation can occur with a variety of processing techniques that implement heat and drying.
Mushroom polysaccharides possess documented immunomodulatory properties, specifically through the activation of natural killer cells, macrophages, and neutrophils, as well as induction of innate immune cytokines and interleukins.5 β-glucans are another class of polymers present in the cell walls of fungi. The generic term β-glucan refers to the polymeric form of glucose residues connected by β-(1→3), β-(1→4), and β-(1→6)-linkages. The type of β-glucans isolated from fungi consist mainly of a linear backbone of β-(1→3) glucose monomers and side branches comprised β-(1→3) and β-(1→6)-linked oligosaccharides.6
The most widely studied β-glucans are comprised of (1→3)-β, and (1,6)-β linkages, which exhibit immunostimulatory and antitumor properties.7,8 These polysaccharides are ligands for the dectin-1 and toll-like receptor 2 (TLR-2) receptor systems expressed on macrophages and dendritic cells, inducing NK cells, neutrophils, T-cells, B-cells, as well as TNF-a, IL-4, and IL-6 signaling.9 The Complement Receptor-3 (CD11b/CD18) in context of extracellular matrix is also involved in immune responses to fungal β-glucans.10 Research by Quayle et al demonstrates that other structural components in the fungal cell wall matrix affect pattern recognition receptor activity.11 The TLR-2 activity of polysaccharide-K (PSK) was reduced by 81% upon treatment with lipoprotein lipase, revealing the biological activity of a previously unreported lipid in the PSK complex. Mushroom β-glucans may contain other functional groups that contribute additional biological effects, particularly regarding antioxidant activity.12 Mushroom polysaccharide and β-glucan products are common dietary supplements, often available as whole mushrooms, mycelial powders, dried or liquid extracts from fruitbodies or from mycelium.
Specialized protein-bound polysaccharide products have been developed as adjuvant immunotherapies in oncological clinical settings. Polysaccharide-K (PSK, Krestin®, Kureha) and polysaccharide peptide (PSP) are produced from the cultured mycelium of the turkey tail mushroom (Trametes versicolor) and are used as antineoplastic agents and immunostimulants in China and Japan.13–18 Lentinan is a polysaccharide extracted from the mycelium and fruiting body of the shiitake mushroom (Lentinula edodes),19 and has demonstrated efficacy as a biological response modifier and chemotherapy adjuvant in gastrointestinal and lung cancers.20 Grifolan is another polysaccharide extracted from maitake (Grifola frondosa) which enhances production of IL-6, IL-1, and TNF-a.21
Another class of immunologically active compounds from mushrooms, which are typically much smaller in size compared to cell-wall polymers, are secondary metabolites. These are compounds that may not be absolutely required for the growth of the organism but assist in its survival by offering protection and communication among other important but not metabolically essential functions. When consumed, secondary metabolites are associated with myriad biological effects, including antioxidant, anti-viral, anti-inflammatory, neuroregenerative, and hepatoprotective effects. Classes of fungal metabolites with medical significance include sterols, terpenes, and phenols, notable examples of which include ganoderic acid in Ganoderma lucidum,22 erinacines in Hericium erinaceus,23–25 and betulin in Inonotus obliquus,26 and cordycepin in Cordyceps militaris.27
A growing body of international scientific and medical research continues to help define the precise biochemical pathways leading to improved physiological outcomes. Cordycepin, 3ʹ-deoxyadenosine, has shown very potent anti-inflammatory effects in a spectrum of in vitro and animal models, specifically via effects on adipose-derived mesenchymal stem cells, where higher doses helped maintain the stem-ness of the cells and lower doses supported osteogenic differentiation.28 Isolated compounds from reishi (Ganoderma lucidum) were tested on the MCF-7 breast cancer cell line and its non-transformed counterpart MCF10A and showed selective killing of the transformed cells, both actively growing, and quiescent slow-cycling cancer stem cells.29 Polysaccharides from reishi30 and maitake31 have also been shown to promote and enhance the survival/renewal abilities of primitive hematopoietic stem/progenitor cells. For maitake, this was specifically linked to β-glucan-mediated increases in the production of granulocyte-colony stimulating factor.32 Notably, this research demonstrated that this effect supported stem cell transplantation in a NOD/SCID mouse model. To the best of our knowledge, non-β-glucan fractions of maitake were not previously evaluated in stem cell related studies.
While much prior research on medicinal mushrooms has focused on the solid, β-glucan-rich fraction, and β-glucan-mediated responses are clearly important, focusing on this compound class in isolation clearly does not reflect the overall bioactivity of a complex blend when consumed for immune support. Emerging evidence suggests that a blend of mushrooms may provide additive or synergistic effects on the host immune response. Preclinical work on a 7-mushroom blend (Ganoderma resinaceum, Cordyceps sinensis, Trametes versicolor, Hericium erinaceus, Inonotus obliquus, Grifola frondosa, Agaricus brasiliensis f. blazei, Phellinus linteus) discovered greater NK cell upregulation with a blend over any one isolated species.33 Findings from another study suggest synergistic radical scavenging activity with a combination of Boletus edulis and Marasmius oreades mushroom extracts.34
The purpose of this study was to investigate the differential immunological effects of aqueous, ethanol, and solid fractions of MMB in vitro, to determine the differences in biological activity between the soluble and insoluble fractions of a complex medicinal mushroom blend (MMB), a blend of 17 mushroom species (Table 1) that is used by consumers for seasonal immune wellness, and by clinicians to enhance innate immunity. The study of a complex blend rather than isolated single compounds was of importance. The experimental model included effects on multiple immune cell types in vitro, because of parallels to events in the gut mucosal tissue where dendritic cells and macrophages sample antigens in the gut lumen, and present to lymphocytes in the gut-associated mucosal tissue, leading to local immune cell activation, and cytokine secretion that has systemic effects.
Reagents
Phosphate-buffered saline, Roswell Park Memorial Institute 1640 medium, penicillin–streptomycin 100×, interleukin-2 (IL-2), and lipopolysaccharide (LPS) from Salmonella enterica were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Lympholyte-Poly was obtained from Thermo-Fisher Scientific (Waltham, MA, USA). CD69 fluorescein isothiocyanate, CD56 phycoerythrin, CD3 peridinin chlorophyll protein, and heparin Vacutainer tubes were purchased from Becton-Dickinson (Franklin Lakes, NJ, USA). Bio-Plex Pro™ human cytokine arrays were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA).
Medicinal Mushroom Blend
The medicinal mushroom blend MyCommunity is sold internationally under the brand Host Defense® MushroomsTM and was obtained from the manufacturer—Fungi Perfecti, LLC, Olympia, WA. It is a certified organic freeze-dried blend of 17 medicinal mushroom species (mycelium/fruiting bodies) for immune system health: Royal sun blazei (Agaricus brasiliensis f. blazei), cordyceps (Cordyceps militaris), enokitake (Flammulina velutipes), amadou (Fomes fomentarius), agarikon (Fomitopsis officinalis), artist conk (Ganoderma applanatum), reishi (Ganoderma lucidum s.l.), Oregon ganoderma (Ganoderma oregonense s.l.), maitake (Grifola frondosa), lion’s mane (Hericium erinaceus), chaga (Inonotus obliquus), shiitake (Lentinula edodes), mesima (Phellinus linteus), birch polypore (Piptoporus betulinus), pearl oyster (Pleurotus ostreatus), split gill polypore (Schizophyllum commune), and turkey tail
Testing for Endotoxins
The MMB powder was tested for endotoxin levels at Associates of Cape Cod Inc., East Falmouth MA, using the quantitative kinetic turbidimetric method for the detection of Gram-negative bacterial endotoxin, and reported in Endotoxin Units (EU).
Testing for Beta-Glucans
The whole MMB powder, as well as the freeze-dried solids of the aqueous and ethanol MMB fractions, were each tested for beta-glucan content by the Megazyme® assay at Venture Laboratories (Lexington, KY). This analysis involves total glucan hydrolysis by sulfuric acid and alpha-glucan hydrolysis by various enzymes. Total glucan and α-glucan content are measured spectrophotometrically, and β-glucans are estimated mathematically by the difference of these two values. All materials submitted for analysis were from the same lot number of material used in the immunological testing.
Preparation of Mushroom Fractions for Immune Bioassays
The powder was handled in the following manner, using pyrogen-free disposables: A 100 mg/mL suspension was prepared in phosphate-buffered saline (PBS) and allowed to rehydrate and extract aqueous compounds for 1 hr at 20°C under gentle agitation. The suspension was centrifuged at 400 g for 10 mins, and the aqueous fraction harvested. Ethanol (95%) was added to the pellet and vortexed, and extraction of non-aqueous, ethanol-soluble compounds allowed for 1 hr at 20°C under gentle agitation. The suspension was centrifuged at 400 g for 10 mins, and the ethanol fraction harvested. The remaining solid pellet was resuspended in PBS. The aqueous and ethanol fractions were filtered through a 0.22-micron syringe filter before adding to cell cultures. The solid fraction was passed through homogenization spin columns (QIAshredder, Qiagen, Hercules, CA), but were not filtered through a 0.22-micron filter. From each fraction, serial dilutions were made in pyrogen-free physiological saline.
Immune Cell Activation
Peripheral venous blood was drawn from three healthy human donors upon written informed consent, as approved by the Sky Lakes Medical Center Institutional Review Board, Federalwide Assurance 2603. The blood was drawn into heparin vacutainer vials, and the peripheral blood mononuclear cells (PBMC) isolated using Lympholyte Poly by centrifugation for 35 mins at 400 g. The PBMC were washed twice in PBS, counted, and the density adjusted to establish cultures with a cell density at 106/mL, using Roswell Park Memorial Institute 1640 medium containing penicillin–streptomycin and fetal bovine serum.
The highly inflammatory lipopolysaccharide (LPS) from Salmonella enterica was used as a positive control for immune-cell activation. Serial dilutions of products or LPS (10 ng/mL) were added to cultures at a volume of 20 μL, and cultures were then incubated at 37°C, 5% CO2 for 24 hrs. In parallel, IL-2 was used as a positive control for natural killer (NK)-cell activation, at a concentration of 100 IU/mL. Untreated negative control cultures consisted of PBMC exposed to phosphate-buffered saline in the absence of test products. All treatments, including each dose of test product and each positive and negative control, were tested in triplicate. After 24 hrs, cells were transferred to V-bottom microtiter plates, washed in PBS containing bovine serum albumin and sodium azide, and stained for 10 mins with fluorochrome-labeled anti-CD3, anti-CD56, and anti-CD69 monoclonal antibodies at the recommended concentration. PBMC were then fixed in formalin. The fluorescence intensities for CD3, CD56, and CD69 were measured by flow cytometry, using an Attune acoustic-focusing flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). Data analysis utilized gating on forward and side scatter to evaluate CD69 expression on lymphocyte versus monocyte/macrophage subsets. The lymphocyte subpopulation was further analyzed for CD69 expression on CD3-CD56+ NK cells, CD3+CD56+ NKT cells, CD3+CD56- T cells, and non-NK non-T lymphocytes.
Production of Cytokines, Chemokines, and Growth Factors
After 24 hrs of incubation, after transfer to V-bottom microtiter plates and before cells were stained for flow-cytometry analysis, the culture supernatants were harvested from the PBMC cultures described above. Levels of cytokines, chemokines, and growth factors were quantified using Bio-Plex protein arrays (Bio-Rad Laboratories Inc., Hercules CA, USA) and utilizing xMAP technology (Luminex, Austin, TX, USA).
Statistical Analysis
ata organization, exploration, and analysis were conducted using the statistical computing language R (version 3.5.2) implemented in the RStudio (version 1.0.143) software environment.35,36 Most data handling was conducted using the base package, with additional functions from the dplyr package (version 0.7.8) used in the reorganization of the raw data.37 Statistical analysis was implemented using the R ‘stats’ (version 3.5.2) and “car” (version 3.0–2) packages,38 as well as the packages “vegan” (version 2.5–4), and “vegan3d” (version 1.1–2) for multivariate ordination plots and Non-metric Multidimensional Scaling (NMDS).39,40 Multiple comparison test after Kruskal–Wallis used the “pgirmess” package (version 1.6.9), and the “RVAideMemoire” package (version 0.9–72) was used for pairwise permutational MANOVA with Bonferroni correction.