Immunomodulatory Properties of Polysaccharides from Lentinula edodes

Lentinula edodes (Berk.) Pegler, also known as shiitake mushroom, is a popular edible macrofungus and a source of numerous bioactive substances with multiple beneficial health effects. L. edodes-derived polysaccharides are the most valuable compounds, with anticancer, antioxidant, antimicrobial, and immunomodulatory properties. It has been demonstrated that their biological activity depends on the extraction method, which affects monosaccharide composition, molecular weight, branching degrees, and helical conformation. In this review, we discuss the immunomodulatory properties of various polysaccharides from L. edodes in animal models and in humans.


Introduction
Lentinula edodes (Berk.) Pegler, also known as shiitake mushroom, is the most popular edible macrofungus, belonging to the Basidiomycota [1]. L. edodes grows naturally in the humid and warm regions of North and South America, Asia, and Australia and is also widely cultivated all over the world [2]. Numerous studies have demonstrated that L. edodes fruiting bodies and mycelium are a source of various nutrients and bioactive substances with multiple beneficial health effects [2,3]. L. edodes contains about 68-78% of carbohydrates (mono-, di-, tri-, and polysaccharides), exogenous amino acids (including arginine, histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, valine, and tryptophan), lipids (5-8% of dry matter), vitamins (including B1, B2, B12, C, D, E), minerals (including Ca, K, Mg, Mn, P, Zn, and Na), fiber, and numerous substances with antibacterial, antiviral, and antifungal properties [3,4]. The main compounds of L. edodes are presented in Figure 1. L. edodes-derived polysaccharides are widely studied and the most valuable compounds with proven anticancer, antioxidant, antiaging, antimicrobial, and immunomodulatory properties [5,6]. Studies on the mechanism of the immunomodulatory action of fungal polysaccharides suggested that it is a complex interaction between immunological, metabolic, and epigenetic changes [7]. As detailed below, β-glucans are the main polysaccharides found in the fungal cell wall and are responsible for most of the biological effects. Glucan molecules are considered to be pathogen-associated molecular patterns (PAMPs) and various pattern-recognition receptors (PRRs) can recognize them (please see below for details) [7,8].
It is worth emphasizing that besides polysaccharides, other L. edodes compounds are biologically active [5,6]. Findings from recent studies indicated that exogenous amino acids regulate cytokines and nitric oxide (NO) synthesis, antibodies production, and lymphocytes proliferation [3]. In addition, evidence of B and D vitamins role in the immune response has accumulated in past years. It has been demonstrated that B vitamins regulate the function of T helper (Th) cells, natural killer (NK) cells, and macrophages, including cells proliferation, cytokines synthesis, and NO production. Vitamin D also regulates the function of all immune cells, including T and B lymphocytes, monocytes/macrophages, and dendritic vitamins regulate the function of T helper (Th) cells, natural killer (NK) cells, and macrophages, including cells proliferation, cytokines synthesis, and NO production. Vitamin D also regulates the function of all immune cells, including T and B lymphocytes, monocytes/macrophages, and dendritic cells. Minerals present in L. edodes, particularly Mg and Zn, are essential for the innate and adaptive immune response. They regulate the function of macrophages, neutrophils, T and B lymphocytes, as well as NK cells [4].
It has been demonstrated that cultivation and extraction method are essential steps in obtaining polysaccharides because they affect their monosaccharide composition, different molecular weight, branching degrees, helical conformation, as well as biological activity [7,8]. In this review, we discuss immunomodulatory properties of polysaccharides from L. edodes in animal models and in humans.

Surface Receptors and Signaling Pathways
Over the past three decades, numerous studies have shown that L. edodes-derived polysaccharides exhibit immunomodulatory properties. It has been confirmed that they can modulate both innate and adaptive immune responses, and thus should be considered as biological response modifiers (BRMs) [9,10]. L. edodes biologically active polysaccharides mostly belong to the group of β-glucans. Glucans are PAMPs, therefore numerous cell surface receptors on monocytes/macrophages, neutrophils, natural killer (NK) cells, dendritic cells (DCs), as well as T and B cells recognize them [11,12]. So far, several polysaccharide-binding receptors have been identified, including complement receptor 3 (CR3), dectin-1, and lactosylceramide (LaCer). CR3 is expressed on neutrophils, monocytes, macrophages, and NK cells and recognizes mainly β-1,3-glucans [11]. The interaction of polysaccharides with CR3 leads to enhanced spleen tyrosine kinase (Syk) phosphorylation, augmented phosphatidylinositol3 kinase (PI3K) activation, and In this review, we discuss immunomodulatory properties of polysaccharides from L. edodes in animal models and in humans.

Immunomodulatory Properties of Polysaccharides from L. edodes-Studies Performed in Animals
Numerous studies have been conducted to determine the immunomodulatory effects of polysaccharides isolated from mycelium or fruiting bodies of L. edodes in animal models. Due to the fact that fungal polysaccharides from varoius sources have different composition, molecular weight, and conformation, they can bind to multiple receptor types with different affinity and activate various signalling pathways. Therefore, it is very likely that future research will contribute to the discovery of new receptors for muschroomderived polysaccharides.

Immunomodulatory Properties of Polysaccharides from L. edodes-Studies Performed in Animals
Numerous studies have been conducted to determine the immunomodulatory effects of polysaccharides isolated from mycelium or fruiting bodies of L. edodes in animal models.
Chen et al. [8] investigated immunomodulatory properties of three polysaccharide fractions (named F1, F2, and F3) isolated from L. edodes water extracts in BALB/c mice. All three fractions were composed of glucose (Glc), galactose (Gal), and mannose (Man) in different proportions. F1 was mainly composed of 38.0% Glc, 12.2% Gal, and 12.3% Man. F2 mainly consisted of 28% Glc, 4.6% Gal, and 4.4% Man; F3 contained 13.6% Glc, 2.7% Gal, and 2.5% Man. Moreover, all three fractions had different molecular weights (136 kDa, 14-61 kDa, and 14-35 kDa, respectively). The study demonstrated that F1, F2, and F3 polysaccharides increased thymus index and footpad thickness in mice. In addition, all three fractions increased proliferation of Concanavalin A-and LPS-stimulated splenocytes. The group also revealed that F2 and F3 upregulated serum immunoglobulin(Ig)G and IgM levels and enhanced NK cells cytotoxicity. These results suggest that F1, F2, and F3 polysaccharides isolated from L. edodes had immunoenhancing activity. The fraction with the highest molecular weight (F1) improved cellular immunity, while F2 and F3 improved both cellular and humoral immunity.
In a study by Pan et al. [17], the effect of supplementation of L. edodes-derived 1,3/1,6-β glucan on C57BL/6J mice gut microbiota was evaluated. It was reported that both shortterm (7 days) and long-term (15 weeks) supplementation of β-glucan (at the dose of 1.5 mg per mouse/day) inhibited macrophage accumulation in the colon of mice fed by a highfat diet and downregulated the expression of proinflammatory cytokines, including IL-6, TNF-α and IL-1β.
The immunomodulatory properties of bioprocessed polysaccharide (BPP) isolated from L. edodes liquid mycelial culture supplemented with black rice bran were studied by Kim and colleagues [18]. BPP is a β-glucan composed of glucose, galactose, rhamnose, fucose, mannose, and xylose. It was found that BPP upregulated spleen lymphocyte proliferation, as well as increased serum IFN-α levels in mice infected with Salmonella Typhimurium. In addition, BPP administration augumented Th1 cytokines production (including IL-1β, IL-2, IL-6, and IL-12) in mice splenocytes but did not affect Th2 cytokines production (IL-4, IL-5, and IL-10). The same polysaccharide, isolated from mycelial culture supplemented with tumeric reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum and upregulated the expression of IL-1β, IL-10, TNF-α and inducible nitric oxide synthase in response to various Salmonella infections [19].
In another study, the biological activity of five novel polysaccharides (described as SLNT1, SLNT2, JLNT1, JLNT2, and JLNT3) isolated from the dried fruit body of L. edodes was evaluated in H22-bearing mice [20]. All polysaccharides consisted of glucose and had similar structures of (1→3)-linked-β-D-glucan and triple-helix conformation. Biological activity analyzes demonstrated that supplementation of two out of five tested polysaccharides with the highest molecular weights (SLNT1 and JLNT1) suppressed H22 tumor growth. Moreover, these two polysaccharides stimulated IL-2 and TNF-α production and induced the tumor cell apoptosis.
In another study, the impact of lentinan, a (1→3)-β-glucan with (1→6) branches, encapsulated into CaCO 3 microparticles on lymphocyte activation, as well as humoral and cellular immunity in BALB/c mice, was analyzed [22]. Animals were immunized with ovalbumin (OVA) and injected with lentinan (0.2 mL at a concentration of 10 µg/mL). It was demonstrated that lentinan-CaCO 3 -OVA enhanced lymphocyte proliferation, increased activation of B cells, and upregulated the ratio of CD4 + to CD8 + T cells in spleen lymphocytes. In addition, increased secretion of immunoglobulins IgG and IL-2, IL-4, IFN-γ, and TNF-α was observed.
In another similar research, lentinan was covalently attached to multiwalled carbon nanotubes (MWCNTs) and its immunoregulatory capacities were analyzed in BALB/c mice [23]. Hydrophobic MWCNTs in this study were used as an adjuvant in a vaccine delivery system for intensification of the immune response. The study demonstrated that lentinan attached to MWCNTs (L-MWCNTs) had higher solubility, lower cytotoxicity, and higher biostability than soluble lentinan. L-MWCNTs enhanced production of IL-4, IL-6, TNF-α, and IFN-γ. Moreover L-MWCNTs upregulated the percentage of CD4 + and CD8 + T cells in the spleen and enhanced the production of anti-OVA IgG antibodies. The observed enhancement of the immunomodulatory effects of lentinan was associated with the fact that nanotubes rapidly entered immune cells, contained larger amounts of antigens, and potentiated cellular and humoral immunity.
Wang and colleagues [24] investigated the effect of lentinan on the immune cells of BALB/c mice with sepsis. Animals received 40, 100 or 250 mg/kg of lentinan daily (by intraperitoneal injection). It was found that the drug (at all doses tested) significantly decreased IL-10 production and FoxP3 expression by CD4 + CD25 + Tregs. Moreover, it attenuated LPS-stimulated Erk-FoxO1 activation. The obtained results led to the hypothesis that lentinan could be used as an immunoregulatory drug preventing sepsis in patients with burns, however, further studies in this field are needed.
The immunomodulatory effects of orally administered lentinan were also studied in rainbow trout (Oncorhynchus mykiss) with LPS-induced inflammation [25]. It has been demonstrated that lentinan decreased the expression of TNF-and IFN-related genes involved in acute inflammatory reactions.
In another study, the impact of lentinan supplemenation on immune response in Brown Norway rats was studied by McCormack et al. [26]. Lentinan (12 mg/kg) was orally administered for 20 weeks and the number of immune cells, as well as serum levels of GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, and TNF-α, was determined. It was found that lentinan supplementation increased the number of circulating monocytes and CD8 + T cells and decreased the ratio of CD4/CD8 cells. Moreover, this drug reduced the production of IL-4, IL-6, IL-10, and GM-CSF.
Zheng et al. [27] isolated and purified a polysaccharide, named L-II from the fruiting body of L. edodes, and evaluated its effects on the cellular immune response in Sarcoma-180bearing mice. The polysaccharide L-II consisted of d-glucopyranose and had molecular weight of 2.03 × 10 5 Da. Animals were treated with L-II in a doses 1, 5, and 10 mg/kg body weight. It was demonstrated that L-II in all tested concentrations increased the thymus and spleen weight, delayed-type hypersensitivity response, as well as the phagocytosis of macrophages. Moreover, this polysaccharide elevated serum TNF-α and IFN-γ but not IL-2. In addition, L-II increased nitrogen oxide production and catalase activity in macrophages.
Jeff et al. [28], likewise, isolated and purified two mannogalactoglucan-type polysaccharides (WPLE-N-2 and WPLE-A0.5-2), from the fruiting bodies of L. edodes, and evaluated their effects on the cellular immune response of Sarcoma-180-bearing mice. Both polysaccharides were α-glucans composed of glucose, galactose, and mannose. Animals were treated with 100 mg/kg body weight of the polysaccharides for 10 days. The group observed a significant tumor regression in the polysaccharides' groups. Moreover, polysaccharides upregulated NO production in peritoneal macrophages and macrophage phagocytosis and enhanced concanavalin and LPS-induced splenocytes proliferation.
Park and colleagues [29] investigated whether a standardized mycelial extract of L. edodes, containing αand β-glucans, amino acids, and minerals (named AHCC ® ) promotes the therapeutic effect of immunotherapy in cancers in mice. The group used a combination of oral AHCC ® and dual immune checkpoint blockade (DICB), including PD-1/CTLA-4 blockade. It was demonstrated that the treatment reduced tumor growth and increased granzyme B and Ki-67 expression by tumor-infiltrating CD8 + T cells in MC38 colon-cancerbearing mice compared with a combination of water and DICB.
All of the results, which are summarized in Table 1, suggest that polysaccharides isolated from L. edodes have immunomodulatory properties. However, different studies gave opposite results. Reasons for such divergences may be different animal models used in studies, different chemical structure and molecular weight of analyzed polysaccharides, or even lack of precise analysis of which of the components of the extracts/powdered L. edodes is responsible for the observed immunomodulatory activity. Therefore, future investigation in this field is needed. Wang et al. [20] Polysaccharide (homogenous β-glucan composed of rhamnose, arabinose, galactose, and glucose) isolated from L. edodes spent compost (ASMCP) Kunming strain mice with LPS-induced kidney injury 1. Reduced the serum levels of TNF-α, IL-6 and IL-1β. 2. Reduced serum levels of urea nitrogen, creatinine, and uric acid. 3. Increased activity of superoxide dismutase, catalase, and malondialdehyde.

Immunomodulatory Properties of Polysaccharides from L. edodes-Studies Performed in Humans
The first studies on the biological activity of L. edodes polysaccharides in humans were carried out in the 1980s.
The immune activating effects of lentinan were firstly described in healthy humans by Aoki [30] and Tani et al. [31]. It was found that this drug in vitro enhanced the proliferation of peripheral blood mononuclear cells (PBMCs) and increased the production of lymphokine-activated killer cel and NK cells activity. Similar results were obtained in gastric cancer patients when lentinan was administered intravenously (2 mg of lentinan for 7 days) with classic chemiotherapy [31,32]. Moreover, it was found that this drug significantly increased IL-1α, IL-1β, and TNF-α production by PBMCs of these patients, and increased the ratio of activated T cells and cytotoxic T cells in the spleen [30,32]. In another study, conducted in patients with gastric cancer the effect of lentinan on lymphocyte subsets of peripheral blood, lymph nodes, and tumor tissues was evaluated [33]. A total of 2 mg of lentinan was intravenously administered twice to twelve patients. No changes in the lymphocyte subsets of peripheral blood were found, however, the number of CD4 + T cells in lymph nodes was elevated. In addition, the number of tumor-inflitrating CD4 + , Leu11, and LeuM3 cells was significantly increased.
The effect of orally administered lentinan on the immune response in healthy elderly humans was investigated in the placebo-controlled clinical study in 2011 [34]. Forty-two participants were randomly assigned to two groups given either 2.5 mg/day Lentinex ® or placebo. It was found that the number of total CD3 + T cells and CD4 + T cells was lower in the placebo group than in the Lentinex ® group, however, no changes in the number of CD8 + T cells were detected. In addition, it was demonstrated that the number of B cells in the placebo group was nonsignificantly decreased and nonsignificantly increased in the Lentinex ® group. Interestingly, the difference between groups after six weeks after the end of the supplementation was significant. The number of CD56 + NK cells increased significantly in both groups, but there was no difference between the groups. Changes in humoral response were also described. The complement C3 decreased significantly in both groups, but no difference between the groups was observed. No statistically significant changes in IgG, IgA, and IgM levels were found between the studied groups.
In another randomized, placebo-controlled clinical trial, the hypocholesterolemic, microbiota-modulatory, and immunomodulatory effect of L. edodes extract was analyzed in hypercholesterolemic patients [35]. Fifty-two participants were assigned to two groups given either a β-D-glucan-enriched (BGE) mixture (10.4 g/day) obtained from L. edodes fruiting bodies ensuring a 3.5 g/day of fungal β-D-glucans or a placebo incorporated in three different food products. No significant differences in lipid-or cholesterol-related parameters were found compared with the placebo group. In addition, the BGE showed no immunomodulatory effect (including changes in IL-1β, IL-6, and TNF-α production). However, BGE modulated the colonic microbiota.
The immunomodulating properties of L. edodes mushroom have also been investigated in healthy young adults in another randomized study [36]. Fifty-two participants were randomized to consume either one (5 g) or two (10 g) servings of dried, prepared L. edodes daily for 4 weeks. It was demonstrated that mushrooms consuming increased ex vivo proliferation of γδ-T cells and NK-T cells, as well as the expression of activation receptors on these cells. In addition, an increased level of secretory IgA in saliva and decreased level of C-reactive protein in serum was observed. The concentration of IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-17, IFN-γ, TNF-α, macrophage inflammatory protein-1α/chemokine C-C ligand 3 (MIP-1α/CCL3), and MIP-1β in PBMCs culture supernatants was also analyzed. It was found that mushroom intake upregulated IL-4, IL-10, TNF-α, and IL-1α levels and downregulated MIP-1α/CCL3. No changes were observed for IL-6, IL-1β, MIP-1β, IL-17, and IFN-γ. It was concluded that regular L. edodes consumption resulted in improved immunity, however, it was not analyzed which component of the mushroom exerted immunomodulatory effects.
Wang et al. [37] evaluated the influence of lentinan on cytokines profile and T cell subsets in 73 patients with nonsmall cell lung cancer (NSCLC) treated with vinorelbin and cisplatin. As expected, all patients with NSCLC had a higher subset ratio of CD3 + CD8 + T cells. It was found that the combined therapy of lentinan-vinorelbin-cisplatin resulted in a significant increase in CD3 + CD56 + NKT cells, CD3 + CD8 + , and CD3 + CD4 + subsets. Moreover, therapy with lentinan decreased the number of CD4 + CD25 + Tregs, increased the production of IL-10 and TGF-β1, and upregulated IFN-γ, TNF-α, and IL-12. It was concluded that lentinan-based chemo-immunotherapy could be a promising strategy in NSCLC treatment.
Our group also investigated the immunomodulatory properties of polysaccharides from L. edodes in healthy human immune cells. We isolated from the mycelial cultures a selenium(Se)-enriched analog of lentinan (named Se-L) and, as a reference, the non-Seenriched fraction (named L) and evaluated its effects on the proliferation of human PBMCs stimulated with various T and B-cells mitogens and influence on superoxide anions (O 2 − ) production by granulocytes [38]. Our study demonstrated that none of the polysaccharides has a significant effect on the generation of reactive oxygen species by granulocytes. However, we found that both fractions significantly decreased the proliferation of PMBCs stimulated with anti-CD3 antibody and phytohemagglutinin (PHA) in a dose-dependent manner. Structural studies of the obtained fraction revealed that it is a protein-containing mixture of high molar mass polysaccharides (MW 3.9 × 10 6 g/mol and 2.6 × 10 5 g/mol) αand β-glucans composed of glucose or mannose. At this early stage of the research, it was not determined which of the components of the fraction was responsible for the immunosuppressive activity. Therefore, in our next study, we separated homogenous (Se)-enriched fraction (named Se-Le-30), defined four-polysaccharide components of this fraction, and determined their structures and immunomodulatory properties in human immune cells [39,40]. We found that Se-Le-30 downregulated the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated and decreased the production of TNF-α by CD3 + T cells. This result suggests that Se-Le-30 fraction is a selective T cells immunosuppressant, which presumably acts through the modulation of signaling via the TCR-CD3 receptor complex. Later analyses demonstrated that all four polysaccharide components of Se-Le-30 also inhibited the proliferation of anti-CD3-stimulated PBMCs, but the inhibitory effect was weaker than that of the Se-Le-30 fraction. In continuation of our study, we extracted from a postculture medium of L. edodes exopolysaccharides fraction (Se-containing and nonselenated) composed mainly of a highly branched 1-6-αmannoprotein of molecular weight 4.5 × 10 6 Da and also evaluated its effect on human PBMCs and granulocytes. The analysis demonstrated that similarly to polysaccharides isolated from L. edodes mycelium, exopolysaccharides have no influence on the production of reactive oxygen species by granulocytes but reduce PMBCs proliferation induced by the anti-CD3 antibody. However, the observed effect was much weaker than that for the fractions isolated from mycelium [41].
Most of the results (summarized in Table 2) strongly suggest that polysaccharides from L. edodes have immunomodulatory properties in humans, however, various polysaccharide fractions may significantly differ in biological activity. These differences result both from the polysaccharides source (fruiting bodies or mycelium) and extraction method, which affects monosaccharide composition, molecular weight, branching degrees, and helical conformation [7,8]. Lentinan, a (1→3)-β-glucan with (1→6) branches, extracted from fruiting bodies of L. edodes, functions as an effective immunostimulatory agent. In contrast, polysaccharides with different structure isolated from L. edodes mycelium have selective immunosuppressive activity. Table 2. Main studies of immunomodulatory effects of L. edodes polysaccharides in humans.

Sample
Participants Immunomodulatory Effects Reference