Different β-Glucans – Different Effects On Natural, Adaptive And Trained Immunity

Introduction

Directive Publications Vaclav Vetvicka Materials All β-glucan samples were either donated by the manufacturers or purchased from their distributors, as listed in Table 1. Glucans were tested as sold at retail, dosed by equal product mass, without independent verification of content. Ovalbumin, polymyxin B, cyclophosphamide, and Wright stain were purchased from Sigma (St. Louis, MO, USA). Table 1. Individual glucans. Glucan Source Manufacturer 5 Defenders mushroom Real Mushrooms, Calgary, Canada Turkey Tail mushroom Eos Corp., Hong Kong, China Host Defense Mushroom mushroom Fungi Perfecti, Olympia, WA, USA Maitake D mushroom Mushroom Wisdom, Rutherford, NJ, USA RYL Beta 550 yeast Youngevity, Scottsdale, AZ, USA ABBi16 yeast AB Biotek, Casteggio, Italy #300 yeast Transfer Point, Columbia, SC, USA 19 , 2026 Phagocytosis assay Phagocytosis of 2-hydroxyethyl methacrylate (HEMA) microspheres was performed as previously described.7 Briefly, 0.1 mL of peripheral blood collected from mice treated with various doses of β-glucan or phosphate-buffered saline (PBS) was incubated in vitro with 0.05 mL of HEMA microspheres (5 × 10⁸ particles/mL) at 37 °C for 60 min with intermittent shaking. Blood smears were prepared and stained with Wright stain. Cells containing three or more HEMA particles were considered phagocytosis-positive. All experiments were performed in triplicate, and at least 300 cells were evaluated per sample. IL-2 secretion Purified splenocytes (2 × 10⁶ cells/mL) isolated from mice treated with 100 μg of β-glucan or PBS were suspended in RPMI 1640 medium supplemented with 5% FCS and seeded into 24-well tissue culture plates. Cells were incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO 2 . Concanavalin A (1 μg/well; Sigma) served as the positive control. At the end of the incubation period, culture supernatants were collected, and IL-2 concentrations were determined using a Quantikine Mouse IL-2 ELISA kit (Abcam, Cambridge, UK). Antibody response The antibody response to ovalbumin was evaluated as previously described.5 Mice were immunized twice, 14 days apart, with 100 μg of ovalbumin. Blood samples were collected on Day 21, and serum was isolated for analysis. Experimental groups received daily intraperitoneal (i.p.) injections of β-glucan throughout the experimental period. Ovalbumin-specific antibody levels were determined by ELISA. Mice immunized with ovalbumin emulsified in Freund's adjuvant (Sigma) served as the positive control. Trained immunity To evaluate the induction of trained immunity, mice received a single intraperitoneal injection of 100 μg of the indicated β-glucan preparation. Seven days later, animals were inoculated orthotopically into the mammary fat pad with 1 × 10⁶ Ptas64 cells suspended in PBS. Three weeks after tumor implantation, mice were euthanized, tumors were excised, and tumor weights were determined as previously described. 8 RESULTS Commercial dietary supplements contain small amounts of biologically inert excipients that are not expected to possess immunological activity. Nevertheless, it is theoretically possible that unknown quantities of these excipients could affect the actual amount of active ingredient present in a capsule. For example, a capsule labeled as containing 10 mg of β-glucan may have a total weight exceeding 10 mg because of the presence of inactive fillers. To address this possibility, we previously compared the biological activity of a β-glucan preparation available both as a purified powder and as encapsulated capsules. The activities of equivalent doses prepared from both formulations were identical, even at the lowest dose tested. 7 We first evaluated phagocytosis, one of the primary effector functions of innate immunity. Phagocytosis is the process by which phagocytic cells engulf and subsequently digest microorganisms, foreign particles, and cellular debris. In order to find the optimal dose, we evaluated the effets of three different doses. β-Glucans enhance phagocytic activity through interactions with several pattern-recognition receptors, particularly complement receptor 3 (CR3) and Dectin-1. Phagocytic activity was assessed using synthetic 2-hydroxyethyl methacrylate (HEMA) microspheres following two weeks of treatment with three different doses of each

Vaclav Vetvicka Directive Publications β-glucan preparation. As summarized in Table 2, most β-glucan samples significantly enhanced phagocytosis in a dose- dependent manner. The greatest stimulatory activity was observed with Glucan #300. Table 2. Effects of various glucans on phagocytosis. Sample 50 (µg) 100 (µg) 200 (µg) 5 Defenders 38.4 ± 1.2* 40.5 ± 2.2* 42.8 ± 3.1* Turkey Tail 32.8 ± 2.1 36.2 ± 2.0 37.9 ± 1.5* Host Defense Mushroom 38.0 ± 2.9* 44.1 ± 3.3* 48.1 ± 3.7* Maitake D 38.8 ± 2.2* 44.8 ± 1.8* 52.1 ± 3.7* RYL Beta 550 40.2 ± 1.9* 46.6 ± 3.0* 48.9 ± 2.6* ABBi16 43.5 ± 2.5* 46.6 ± 3.2* 50.1 ± 3.8* #300 47.2 ± 2.9* 58.6 ± 4.0* 67.8 ± 3.9* Control values (PBS) were 31.9 ± 2.9. The dose means a single ip. injection in PBS/mouse. *Significant difference between tested groups and PBS control group at P ≤ 0.05 level. Results represent mean values from three experiments ± SD. We next examined IL-2 production by splenocytes isolated from control and β-glucan-treated mice. Following 72 h of in vitro incubation, IL-2 concentrations were measured in culture supernatants. Because unstimulated spleen cells produced no detectable IL-2, all β-glucan preparations significantly increased IL-2 secretion (Table 3). Once again, Glucan #300 exhibited the highest activity. However, none of the tested preparations induced IL-2 production to the level achieved by the Concanavalin A positive control. Table 3. Effect of samples on IL-2 production. Sample 100 (µg) 5 Defenders 181.5 ± 11.2* Turkey Tail 119.3 ± 18.6* Host Defense Mushroom 308.4 ± 21.8* Maitake D 380.4 ± 25.5* RYL Beta 550 155.2 ± 12.9* ABBi16 503.9 ± 21.8* #300 874.4 ± 32.1* Neg. Control 0 Pos. Control 994.8 ± 35.7* *Significant difference between tested groups and PBS control group at P ≤ 0.05 level. Results represent mean values from three experiments ± SD. The effects of β-glucan treatment on adaptive immunity were further evaluated by measuring the antibody response to ovalbumin. Mice were immunized twice with ovalbumin at two-week intervals, and serum samples were collected seven days after the second immunization. Most β-glucan preparations significantly enhanced antigen-specific antibody production (Table 4). The strongest responses were observed in mice treated with Maitake D and Glucan #300, whereas the Host Defense Mushroom preparation did not produce a statistically significant effect. Table 4. Effect of glucan supplementation on antibody formation. Glucan % of control (ovalbumin only) 5 Defenders 189.0 ± 14.9* Turkey Tail 208.9 ± 19.7* Host Defense Mushroom 111.6 ± 15.2 Maitake D 300.4 ± 34.7* RYL Beta 550 153.5 ± 17.9* ABBi16 253.3 ± 20.1* #300 344.1 ± 24.1* Ovalbumin + FA 461.7 ± 30.8* *Significant difference between tested groups and PBS control group at P ≤ 0.05 level. Results represent mean values from three experiments ± SD. 19 , 2026

Vaclav Vetvicka Directive Publications Finally, we investigated the ability of β-glucans to induce trained immunity capable of suppressing breast tumor growth. Mice received a single administration of the test samples prior to tumor challenge. As shown in Table 5, pretreatment with Turkey Tail, Maitake D, and Glucan #300 significantly inhibited subsequent breast tumor growth, demonstrating their ability to induce trained immunity with measurable antitumor activity. Table 5. Effets of glucan on tumor growth. Glucan mg of tumor weight 5 Defender 602.1 ± 51.1 Turkey Tail 484.6 ± 28.4* Host Defense Mushroom 625.4 ± 49.3 Maitake D 508.6 ± 37.7* RYL Beta 550 671.1 ± 30.9 ABBi16 638.4 ± 40.1 #300 348.9 ± 27.3* PBS 725.5 ± 38.9 *Significant difference between tested groups and PBS control group at P ≤ 0.05 level. Results represent mean values from three experiments ± SD. 19, 2026 DISCUSSION The immunomodulatory activity of β-glucans is determined by multiple structural characteristics rather than by a single property. The glucan preparations that consistently demonstrated strong activity across phagocytosis, IL-2 production, antibody formation, and trained immunity most likely possess an optimal combination of favorable structural features, whereas the less active preparations differ in one or more of these characteristics. The principal determinants of β-glucan biological activity are discussed below. Yeast-derived β-glucans from Saccharomyces cerevisiae consist of a β-(1,3)-linked backbone with β-(1,6)-linked side chains, a structural configuration that is efficiently recognized by innate immune receptors. In contrast, mushroom- derived β-glucans vary considerably among species, and many commercial mushroom preparations are produced from mycelia cultivated on grain substrates, resulting in relatively low and highly variable β-glucan content. Cereal β-glucans are composed primarily of mixed β-(1,3)/(1,4) linkages and interact only weakly with Dectin-1. Moreover, even among S. cerevisiae preparations, differences in yeast strain and cultivation conditions can substantially influence branching pattern, molecular weight, purity, and overall biological activity. Consequently, two products marketed simply as "yeast β-glucan" may exhibit markedly different immunomodulatory properties. Molecular weight is another major determinant of biological activity. In general, high-molecular-weight β-glucans are more potent immunomodulators because they activate complement more efficiently and are capable of inducing trained immunity. In contrast, low-molecular-weight fragments and short β-glucan oligosaccharides generally display reduced biological activity. Thus, molecular weight likely contributes substantially to the differences in activity observed among the preparations evaluated in the present study. The physical form of β-glucan also influences its biological effects. Particulate β-glucans, including whole-glucan particles, promote clustering of Dectin-1 receptors at the cell surface, forming a phagocytic synapse that enhances phagocytosis, oxidative burst, and cytokine production. These particulate forms are also those most consistently associated with the induction of trained immunity. In contrast, soluble β-glucans preferentially interact with complement receptor 3 (CR3) and generally induce a different, and often less pronounced, cytokine profile. The coordinated enhancement of phagocytosis, IL-2 production, and trained immunity observed for the most active preparations in the present study is consistent with the activity of intact, high-molecular- weight particulate β-glucans acting primarily through Dectin- 1-mediated signaling. 9 Trained immunity enhances host resistance by functionally reprogramming innate immune cells through long-lasting epigenetic and metabolic modifications. Macrophages and monocytes are the principal cellular targets of this process. β-Glucan-induced trained immunity has been shown to enhance antibody responses and improve vaccine efficacy, including experimental cancer vaccines. Notably, a single administration of β-glucan is sufficient to induce these long- lasting functional changes. 10 Consequently, β-glucans are increasingly recognized as promising inducers of trained immunity and have attracted considerable interest as potential adjuvants in cancer immunotherapy. 11 The present study demonstrated substantial differences in the biological activities of commercially available β-glucan preparations. Among the products evaluated, Glucan #300 consistently exhibited the greatest immunostimulatory

Vaclav Vetvicka Directive Publications activity across multiple assays. Several additional preparations displayed moderate immunological activity, whereas others produced only limited stimulation of immune function. Whether yeast-derived β-glucans are intrinsically superior to mushroom-derived β-glucans remains uncertain, as biological activity depends not only on the source but also on molecular weight, branching structure, purity, solubility, particle size, and manufacturing procedures. Further studies directly comparing well-characterized β-glucan preparations will be necessary to define the structural features responsible for optimal immunomodulatory activity.

References

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