Dectin-1/CLEC7A is a pattern recognition receptor that recognizes β-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. β-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate β-glucans are thought to be poor activators of innate immunity. We show that β-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large β-glucan-stimulated DC generate significantly more IL-1β, IL-6, and IL-23 compared to those stimulated with the smaller β-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to β-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1β production determined by β-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that β-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

β-Glucans are known for their ability to trigger both protective and damaging immune responses. Here we have explored the role of the beta-glucan receptor Dectin-1 in archetypical models of protective and non-protective immunomodulation induced by beta-glucan rich ligands. In the first model, we explored the role of Dectin-1 in the ability of soluble purified β-glucans to mediate protection against systemic Staphylococcus aureus infection in mice. In the second model, we explored the role of Dectin-1 in zymosan induced multiple organ dysfunction syndrome. In both cases, these β-glucan rich compounds had marked effects invivo which were unaltered by Dectin-1 deficiency, suggesting that this receptor has a redundant role in these murine models.

β-Glucans are known for their ability to trigger both protective and damaging immune responses. Here we have explored the role of the beta-glucan receptor Dectin-1 in archetypical models of protective and non-protective immunomodulation induced by beta-glucan rich ligands. In the first model, we explored the role of Dectin-1 in the ability of soluble purified β-glucans to mediate protection against systemic Staphylococcus aureus infection in mice. In the second model, we explored the role of Dectin-1 in zymosan induced multiple organ dysfunction syndrome. In both cases, these β-glucan rich compounds had marked effects invivo which were unaltered by Dectin-1 deficiency, suggesting that this receptor has a redundant role in these murine models.

Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 °C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1→3)-β-d-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1→3)-β-d-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1→3, 1→6)-β-d-glucan were present in the recovered precipitate, but not linear (1→3)-β-d-glucan. Surprisingly, NMR analysis indicated that (1→6)-β-d-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1→6)-β-d-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1→6)-β-d-glucan as the major carbohydrate component along with trace amounts of mannan and (1→3, 1→6)-β-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1→6)-β-d-glucan from other fungi.

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.

Effect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a filamentous fungi Acremonium diospyri following alkali-acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1→3)-β-d-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus. On the other hand, ASG, which contained 93% (1→3)-α-glucan, did not induce significant immune response in shrimp. Here we report that the difference in immunostimulatory potential between AIG and ASG is due to the difference in the percentage of (1→3)-β-d-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1→3)-β-d-glucan as the major fraction.

β-Glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian β-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble β-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

Objectives: Hormones and cytokines are known to act as regulatory messengers between the neuroendocrine and immune systems. The innate immune system identifies infectious agents by means of pattern-recognition receptors. These receptors recognize pathogen-specific macromolecules called pathogen-associated molecular patterns. Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity via interaction with membrane receptors on immune-competent cells. Glucans are also released into the systemic circulation of patients with fungal infections. Recent evidence confirms the existence of glucan-specific receptors on cells outside the immune system. We hypothesized that glucans may directly interact with pituitary cells as an early signaling event in fungal infections. Methods: We characterized the receptor-mediated interaction of glucan derived from Candida albicans with pituitary cells using surface plasmon resonance. Prolactin levels were assayed by commercial ELISA. TLR2, TLR4 and CD14 mRNA levels were assessed by RT-PCR. Results: A single glucan-specific binding site was identified on rodent somatomammotroph (KD = 3.9) μM) and human folliculostellate cell (KD = 3.6) μM) membranes. Coincubation of glucan with somatomammotroph cells for 72 h significantly (p < 0.01) increased prolactin accumulation by 56-62% over that observed in cells treated with media alone. Glucan also increased TLR4 and CD14 gene expression in human folliculostellate cells. Conclusions: Pituitary cells directly recognize and respond to fungal cell wall glucans resulting in stimulation of pituitary cell TLR4 and CD14 gene expression. In addition, glucan stimulates secretion of prolactin, a hormone that plays an important role in the response to infection.

Rodents are a natural host for the dimorphic pathogenic fungi Coccidioides immitis and Coccidioides posadasii, and mice are a good model for human infection. Humans and rodents both express Dectin-1 and Toll-like receptor 2 (TLR2) on myeloid cells, and those receptors collaborate to maximize the cytokine/chemokine responses to spherules (the tissue form of the fungi) and to formalin-killed spherules (FKS). We showed that Dectin-1 is necessary for resistance to pulmonary coccidioidomycosis, but the importance of TLR2 in vivo is uncertain. Myeloid differentiation factor 88 (MyD88) is the adapter protein for TLR2 and -4, interleukin-1R1 (IL-1R1), and IL-18R1. MyD88/TRIF -/- and MyD88 -/- mice were equally susceptible to C. immitis infection, in contrast to C57BL/6 (B6) controls. Of the four surface receptors, only IL-1R1 was required for resistance to C. immitis, partially explaining the susceptibility of MyD88 -/- mice. We also found that FKS stimulated production of IL-1Ra by bone marrow-derived dendritic cells (BMDCs), independent of MyD88 and Dectin-1. There also was a very high concentration of IL-1Ra in the lungs of infected B6 mice, supporting the potential importance of this regulatory IL-1 family protein in the largely ineffective response of B6 mice to coccidioidomycosis. These results suggest that IL-1R1 signaling is important for defense against C. immitis infection.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12794号 / 論農博第2790号 / 新制||農||1019(附属図書館) / 学位論文||H25||N4806(農学部図書室) / 30813 / (主査)教授 栗原 達夫, 教授 植田 和光, 教授 平竹 潤 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM