The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory.

Immunological memory is thought to be mediated exclusively by lymphocytes. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection, which suggests the presence of innate immunological memory. Here we identified an important role for the stress-response transcription factor ATF7 in innate immunological memory. ATF7 suppressed a group of genes encoding factors involved in innate immunity in macrophages by recruiting the histone H3K9 dimethyltransferase G9a. Treatment with lipopolysaccharide, which mimics bacterial infection, induced phosphorylation of ATF7 via the kinase p38, which led to the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks. A partially disrupted chromatin structure and increased basal expression of target genes were maintained for long periods, which enhanced resistance to pathogens. ATF7 might therefore be important in controlling memory in cells of the innate immune system.

Dectin-1 Reactivity to Paramylon Derived from Euglena gracilis EOD-1.

Euglena gracilis is a microalga that has recently attracted attention because of its bioactivities. Paramylon (PM), a major ß-1,3-glucan, constitutes 70-80% of the cells of the E. gracilis EOD-1 strain. Dectin-1 is a pattern recognition receptor that recognizes ß-glucan. However, it is unclear whether PM binds to dectin-1. In this study, we investigated the reactivity of EOD1PM with dectin-1 by analyzing the binding of soluble murine and human dectin-1-Fc fusion protein (m dectin-1 Fc, h dectin-1 Fc) to EOD1PM using flow cytometry and enzyme-linked immunosorbent assay (ELISA). m Dectin-1 Fc bound to EOD1PM particles when m dectin-1-Fc is added. Furthermore, the binding specificity was examined in a competitive reaction following addition of a soluble antigen. It was found that the binding of m dectin-1-Fc to EOD1PM was not inhibited by the addition of dextran or ovalbumin but by the addition of solubilized EOD1PM or Candida cell wall- solubilized ß-glucan. In addition, the h dectin-1-Fc fusion protein was found to specifically bind to EOD1PM. These results suggest that dectin-1 recognizes and binds to the ß-glucan structure of EOD1PM. Dectin-1 is expressed in leukocytes as a ß-glucan receptor and is involved in the expression of various biological activities; therefore, the dectin-1 pathway may be involved in the biological activity of EOD1PM.

Development of a Highly Sensitive ß-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant ß-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-ß-D-glucan recognition protein (S-BGRP) and a (1→6)-ß-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-ß-D-glucan from fungi. S-BGRP and (1→6)-ß-glucanase mutant proteins reacted with ß-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived ß-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-ß-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of ß-glucan levels by the SSMC method using recombinant ß-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test.

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.

Latent 1,3-ß-D-glucan acts as an adjuvant for allergen-specific IgE production induced by Japanese cedar pollen exposure.

BACKGROUND: The pollen grains of several plant species contain 1,3-ß-D-glucan (BG). BG activates dendritic cells (DCs) and subsequently regulates the innate immune responses. Within Japan, the most common disease associated with type-I hypersensitivity is Japanese cedar pollinosis. However, the role of BG in Japanese cedar pollen (JCP) remains unclear. This study examined the localization and immunological effects of BG in JCP. METHODS: The localization of BG in JCP grain was determined by immunohistochemical staining using a soluble dectin-1 protein probe and a BG recognition protein (BGRP). The content of BG extracted from JCP was measured by a BGRP-based ELISA-like assay. The cytokine production by bone marrow-derived DCs (BMDCs) obtained from wild-type and BG receptor (dectin-1) knock-out mice was examined in vitro. The mice were intranasally administered JCP grains and the specific serum Ig levels were then quantified. RESULTS: BG was detected in the exine and cell wall of the generative cell and tube cell of the JCP grain. Moreover, BG in the exine stimulated production of TNF-α and IL-6 in the BMDCs via a dectin-1-dependent mechanism. Meanwhile, JCP-specific IgE and IgG were detected in the serum of wild-type mice that had been intranasally administered with JCP grains. These mice also exhibited significantly enhanced sneezing behavior. However, dectin-1 knock-out mice exhibited significantly lower JCP-specific IgE and IgG levels compared to wild-type mice. CONCLUSIONS: Latent BG in JCP can act as an adjuvant to induce JCP-specific antibody production via dectin-1.

Coronary Vasculitis Induced in Mice by the Cell Wall Mannoprotein of Candida krusei.

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances, such as the hot water extract of C. albicans (CADS) and Candida water-soluble fraction (CAWS), induced coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the hot water extract of C. krusei, inherently resistant to fluconazole, induces vasculitis in mice. Three strains of C. krusei, NBRC1395, NBRC1162, and NBRC10737, were cultured in natural (Y) and chemically defined (C) media and cell wall mannoprotein (MN) fractions were prepared by autoclaving cells (CKY1395MN, CKC1395MN, CKY1162MN, CKC1162MN, CKY10737MN, and CKC10737MN). All MN fractions reacted strongly with Concanavalin A (Con A) and dectin-2 and induced anaphylactoid shock in ICR mice. MNs induced severe coronary vasculitis in DBA/2 mice, resulting in cardiac hypertrophy. MNs also induced coronary vasculitis in C57Bl/6 mice. These results suggest that the MNs of non-albicans Candida, such as C. krusei, induce similar toxicity to those of C. albicans.

N-Terminal (1→3)-ß-d-Glucan Recognition Proteins from Insects Recognize the Difference in Ultra-Structures of (1→3)-ß-d-Glucan.

Recognition of (1→3)-ß-d-glucans (BGs) by invertebrate ß-1,3-d-glucan recognition protein (BGRP) plays a significant role in the activation of Toll pathway and prophenoloxidase systems in insect host defense against fungal invasion. To examine the structure diversity of BGRPs for the recognition of physiochemically different BGs, the binding specificity of BGRPs cloned from four different insects to structure different BGs was characterized using ELISA. Recombinant BGRPs expressed as Fc-fusion proteins of human IgG1 bound to the solid phase of BGs. Based on the binding specificities, the BGRPs were categorized into two groups with different ultrastructures and binding characters; one group specifically binds BGs with triple-helical conformation, while the other group recognizes BGs with disordered conformations like single-helical or partially opened triple helix. The BGRPs from the silkworm and the Indian meal moth bound to the BGs with a triple-helical structure, whereas BGRPs from the red flour beetle and yellow mealworm beetle showed no binding to triple-helical BGs, but bound to alkaline-treated BGs that have a partially opened triple-helical conformation. This evidence suggests that the insect BGRPs can distinguish between different conformations of BGs and are equipped for determining the diversity of BG structures.

Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1.

Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Effects of Outdoor-Grown Royal Sun Medicinal Mushroom Agaricus brasiliensis KA21 (Agaricomycetes) Fruiting Body on Canine Malassezia Dermatitis.

Brazil-grown outdoor-cultivated Agaricus brasiliensis KA21 fruiting body (KA21) significantly increases the production of serum anti-beta-glucan antibody. Therefore, KA21 ingestion may be useful for the prevention and alleviation of fungal infections. This study aimed to determine the effects of KA21 in fungal infections in animals. KA21 was administered to nine dogs infected with Malassezia. Notably, the anti-beta-glucan antibody titer remained unchanged or tended to decrease in the oral steroid arm, whereas in the non-steroid arm, antibody titer increased in almost all animals after KA21 ingestion. Dogs showing improved clinical symptoms exhibited increased anti-beta-glucan antibody titers. The results of this study suggest that KA21 ingestion may alleviate the symptoms of Malassezia and other fungal infections and that continuous ingestion may help prolong recurrence-free intervals. Additionally, the ingestion of KA21 during oral steroid dosage reduction or discontinuation may enable smoother steroid withdrawal.

ß-mannosyl linkages inhibit CAWS arteritis by negatively regulating dectin-2-dependent signaling in spleen and dendritic cells.

AIMS: CAWS, Candida albicans water-soluble fraction, is an extracellular mannoprotein produced by C. albicans NBRC1385. It is a ligand of dectin-2, the C-type lectin receptor for innate immunity, and has strong potency for induction of vasculitis in DBA/2 mice. The structure of this mannoprotein is known to be modulated by the culture conditions. To clarify the structure required for vasculitis, CAWSs were prepared in the two culture conditions with or without pH control, and biological properties were compared. METHODS: CAWSs prepared by the standard protocol and pH controlled at 7.0 were designated as CAWS and CAWS727, respectively. The antigenicity was detected by the anti-Candida mannan IgG. These chemical structures were assessed by nuclear magnetic resonance analysis and the lectin array system. The in vitro activity of CAWSs was tested by tumor necrosis factor-α (TNF-α) induction using bone marrow-derived dendritic cells and spleen cell cultures. RESULTS: The antigenicity of CAWS727 was similar to CAWS but the nuclear magnetic resonance analysis showed a higher ratio of ß-mannosyl linkages were detected in CAWS727. The lectin array showed relative affinities of CAWS727 to α-mannosyl specific lectins were weaker than those of CAWS. CAWS induced severe vasculitis in DBA/2 mice while CAWS727 did not. CAWS significantly induced TNF-α but CAWS727 did slightly. In addition, CAWS-induced TNF-α production was inhibited by mixing with CAWS727 in a concentration dependent manner. CONCLUSION: The α-mannosyl linkages of Candida mannan is a key molecule for the immunotoxicity. CAWS727, which conatins ß-mannosyl linkages, competitively bound to lectin receptors, and resulted in reductions in the inflammatory response.

Analysis of the titer and reactivity of antibody/ies against fungal cell wall ß-glucans in human sera.

The aim of this work was to characterize an antibody response to ß-glucan (BG), a major component of the fungal cell wall, at each isotype in human sera. The titer and reactivity of the anti-human BG antibody were examined using enzyme-linked immunosorbant assay plates coated with Candida soluble cell wall ß-glucan as a standard antigen. The antibody was detected using anti-human immunoglobulin (Ig) G, IgM, and IgA. Its major class was IgG in all subjects. The antibody titer varied significantly. The anti-BG antibody showed greater reactivity to ß-glucans derived from pathogenic fungi than monoglycosyl-branched ß-glucans derived from mycelia culture medium. In addition, it was suggested that the anti-BG IgM antibody was bound relatively strongly to the ß1,3-glucan backbone and the anti-BG IgG antibody to ß1,6-glucan. The anti-BG antibody plays a variety of roles, according to class, in the host's response to fungi. We propose a new index of human response to ß-glucan that effects the understanding of the response to ß-glucan in humans.

Effect of Agaricus brasiliensis-derived cold water extract on Toll-like receptor 2-dependent cytokine production in vitro.

Agaricus brasiliensis (Agaricus blazei Murrill) is well known as a medicinal mushroom. Fruit body of A. brasiliensis is rich in ß-glucan and has shown benefits for various diseases. Both hot and cold water extraction are traditional methods for intake of this mushroom extract. In the present study, we prepared cold water extract of the fruit body of A. brasiliensis (ACWS). The 1,3-ß-glucan segment of this fraction was too small and did not interact with the 1,3-ß-glucan receptor, dectin-1. However, ACWS could induce production of various cytokines including IL-6 from murine splenocytes. Therefore, we aimed to identify the receptor that modulates IL-6 production using ACWS. We focused our attention on Toll-like receptors (TLRs) and examined them as follows. (i) The interaction between TLRs and ACWS was screened using HEK293 cells transfected with TLR plasmid. (ii) IL-6 production from splenocytes induced by ACWS was inhibited by treatment of anti-TLR antibodies. (iii) Direct binding activity between TLR protein and ACWS was assessed by ELISA-like assay. ACWS was found to activate HEK293 cells via TLR2, 4 and 5. However, only anti-TLR2 monoclonal antibody suppressed IL-6 production from splenocytes. In addition, ACWS has the ability to bind directly to TLR2 protein. Accordingly, we suggest that fruit body of A. brasiliensis has some water-soluble TLR ligand complexes, and TLR2 on splenocytes strongly induces IL-6 production.

Disruption of actin cytoskeleton enhanced cytokine synthesis of splenocytes stimulated with beta-glucan from the cauliflower medicinal mushroom, Sparassis crispa Wulf.:Fr. (higher Basidiomycetes) in vitro.

Beta-glucan (BG) is a representative pathogen-associated molecular pattern (PAMP) produced by pathogenic fungi. SCG is a BG obtained from Sparassis crispa, which stimulates splenocytes in DBA/2 mice to produce cytokines, such as GM-CSF, IFN-γ, and TNF-α. In the present study, we analyzed the molecular mechanism of SCG-mediated cytokine synthesis using cytocharasin D (CytD), an inhibitor of actin polymerization. It was found that GM-CSF and TNF-α synthesis of splenocytes stimulated with SCG, but not with lipopolysaccharide, was significantly enhanced in the presence of CytD. CRDO, partially hydrolyzed linear 1,3-BG curdlan, stimulated splenocytes of DBA/2 mice slightly to produce cytokines. CRDO, acting as an antagonist in the presence of SCG, changed to a strong agonist in the presence of CytD. CytD also enhanced cytokine synthesis of bone marrow-derived dendritic cells. Taken together, cytokine productivity of BG was significantly dependent on molecular weight, and CytD treatment is useful to enhance the sensitivity for analyzing the immunostimulating activity of BG in vitro.

Induction of IFN-γ by a highly branched 1,3-ß-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways.

We have previously elucidated the precise structure of a unique type of 1,3-ß-D-glucan, AP-FBG (Aureobasidium pullulans-fermented ß-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-ß-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.

Vasculitis and anaphylactoid shock in mice induced by the polysaccharide fraction secreted into culture supernatants by the fungus Candida metapsilosis.

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not ß-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.

Granulocyte macrophage colony-stimulating factor is required for cytokine induction by a highly 6-branched 1,3-ß-D-glucan from Aureobasidium pullulans in mouse-derived splenocytes.

We have previously obtained and elucidated the precise structure of a highly branched 1,3-ß-D-glucan (with 6-monoglucopyranosyl side chains), Aureobasidium pullulans-fermented ß-D-glucan (AP-FBG), from the fungus A. pullulans. However, the mechanism(s) of the effects of AP-FBG on in vitro mouse primary cells have not been analyzed in detail. Herein, we report that the induction of cytokines by AP-FBG was dependent on the existence of a granulocyte macrophage colony-stimulating factor (GM-CSF); this is similar way to be a typical 1,3-ß-D-glucan from Sparassis crispa (SCG), which is a 1,3-ß-D-glucopyranosyl backbone with single 1,6-ß-D-glucopyranosyl side branching units every three residues. In other words, the production of cytokines in DBA/2-mouse-derived splenocytes by AP-FBG was completely hampered by an anti-GM-CSF neutralizing monoclonal antibody. Furthermore, the addition of exogenous GM-CSF to C57BL/6-derived splenocytes, which are less sensitive to AP-FBG, induced the production of cytokines by AP-FBG. Therefore, GM-CSF is indispensable for the induction of cytokines by AP-FBG in mouse-derived splenocytes. This finding has provided a new insight into our understanding of the actions of ß-D-glucan but will also aid in the design and development of more effective ß-D-glucan agents.

Characterization of blood beta-1,3-glucan and anti-beta-glucan antibody in hemodialysis patients using culinary-medicinal Royal Sun Agaricus, Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).

Beta-glucan is a major component of fungal cell walls and shows various immunopharmacological activities including antitumor activity. Previously, we detected anti-beta-glucan antibody in human sera. Anti-beta-glucan antibody participates in the immune response to fungal cell wall beta-glucan. Patients on dialysis are at high risk of infection including fungal infections. We examined the plasma beta-glucan level and the titer of anti-beta-glucan antibody in dialysis patients. We measured plasma beta-1,3-glucan concentrations with the limulus G test and anti-beta-glucan antibody titers by ELISA with Candida beta-glucan-coated plates. We also examined the influence of the period of dialysis and the kind of dialysis membrane. The patients were positive for beta-1,3-glucan in their plasma. The anti-beta-glucan antibody titer was lower in the dialysis patients than in healthy volunteers. Long-term dialysis patients showed lower anti-beta-glucan antibody titers than short-term dialysis patients. No significant difference was found between the kinds of dialysis membrane. The titer of anti-beta-glucan antibody as recognition molecule of beta-glucan was low in dialysis patients compared with healthy volunteers. This is likely to be one factor explaining the sensitivity to infection of the dialysis patients. An appropriate application of culinary-medicinal mushroom such as Agaricus brasiliensis has potential for the prevention of fungal infection in dialysis patients.

Binding Specificity of a New Artificial ß-Glucan Recognition Protein and Its Application to ß-Glucan Detection in Mushroom Extracts.

ß-1,3-D-glucan (BG) activates innate immunity and enhances immune responses. Fungi, such as mushrooms, produce a relatively large amount of BG, the structure and molecular weight of which varies depending on the species of fungi. This study was conducted to develop a detection probe for quantifying or detecting BG from fungi using BG-binding proteins. The binding properties of a new ß-glucan recognition protein (BGRP) against various BGs were compared. With reference to the amino acid sequences of BGRP in insects, an artificial BGRP (supBGRP) was designed with higher production efficiency using gene recombination technology. SupBGRP was produced in Escherichia coli with high efficiency, and its reactivity with BG from fungi was the highest among the BG-binding proteins examined. SupBGRP exhibited high reactivity with 1,6-branched BG and will be useful for the quantification and detection of fungal BG.

Open-Label Study of the Influence of Food Containing the Royal Sun Mushroom, Agaricus brasiliensis KA21 (Agaricomycetes), on ß-Glucan-Specific Antibody Production in Healthy Human Volunteers.

The edible mushroom Agaricus brasiliensis contains a large amount ß-glucan, which is mainly composed of a ß-1,6-glucan structure. In this study, we investigated the effect of A. brasiliensis strain KA21 on the anti-ß-glucan antibody titer in healthy humans and the role of antibodies as an immunomodulator. Twenty-two healthy volunteers were fed the dried fruiting body of A. brasiliensis (900 or 1500 mg/day) for 12 weeks. The anti-ß-glucan antibody titer in the serum was determined by enzyme-linked immunosorbent assay. Immunoglobulin G (IgG) against ß-glucan was significantly upregulated after intake of A. brasiliensis. Murine experiments demonstrated improvement of anti-ß-glucan antibody production after intraperitoneal injection of Agaricus-derived ß-glucan. To understand the role of antibody against ß-glucan in exclusion of pathogenic fungi, we examined the interaction between HL-60 cells and antibody-treated heat-killed Candida albicans. Flow cytometry analysis indicated the upregulation of Candida-positive HL-60 cells after treatment with human IgG, whereas the competitive assay demonstrated that the main epitope of Candida-reacted IgG was the ß-1,6-glucan structure. Binding between HL-60 and IgG-opsonized C. albicans was suppressed by anti-Fcγ receptor 1 (FcγRI) neutralizing antibody. Finally, using FcγRI-expressed cells with the nuclear factor of activated T-cell reporter assay, we demonstrated that higher titers of anti-ß-glucan IgG can induce stronger Fc receptor-mediated cell activation through the formation of an antibody-ß-glucan complex. In conclusion, oral ingestion of A. brasiliensis KA21 promotes anti-ß-glucan antibody production and may contribute to preventing fungal infection through the activation of immune cells by forming antibody-ß-glucan complexes via an FcγR-dependent pathway.