Over the last 40-yr, the majority of research on glucans has focused on β-(1→3)-glucans. Recent studies indicate that β-(1→6)-glucans may be even more potent immune modulators than β-(1→3)-glucans. Mechanisms by which β-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble β-(1→6)-glucans with macrophage cell lines and primary peritoneal macrophages and the cellular and molecular consequences of this interaction. Our results indicate the existence of a specific β-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known β-(1→3)-glucans receptors are not responsible for β-(1→6)-glucan recognition and interaction. The receptor-ligand uptake/interaction has an apparent dissociation constant (KD) of ∼4-μM, and was associated with phosphorylation of ERK and JNK but not Iκ-α or p38. Our results indicate that macrophage interaction with β-(1→6)-glucans may lead to modulation of genes associated with anti-fungal immunity and recruitment/activation of neutrophils. In summary, we show that macrophages specifically bind and internalize β-(1→6)-glucans followed by activation of intracellular signaling and modulation of anti-fungal immune response-related gene regulation. Thus, we conclude that the interaction between innate immunity and β-(1→6)-glucans may play an important role in shaping the anti-fungal immune response.

The ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.

Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IκBα is a crucial step in the NFκB activation pathway. We investigated IκBα phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IκBα by 48-192%. Pre-or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IκBα levels in CLP mice. Phospho-IκBα in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKβ kinase activity, IκBα phosphorylation and degradation, and NFκB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 μg/mL) and/or glucan phosphate (1 μg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKβ kinase activity, phosphorylation and degradation of IκBα, and NFκB nuclear binding activity. The data indicate that one mechanism by which (1→3)-β-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKKβ kinase activity with subsequent decreases in IκBα phosphorylation and NFκB activation.

The immunostimulatory property of glucan isolates from three marine yeasts (Debaryomyces hansenii S8, Debaryomyces hansenii S169 and Candida tropicalis S186) and one Baker's yeast (Saccharomyces cerevisiae S36) as examined for potential application as immunostimulants in Penaeus monodon postlarvae against White Spot Syndrome Virus (WSSV) infection. Structural characterization of the glucan component in the isolates by proton nuclear magnetic resonance (NMR) indicated similar structures containing (1-3)-linked anhydroglucose repeat units (AGRUs) in the backbone with (1-6)-linked AGRUs in side chains that are (1-6)-linked to the backbone AGRUs. Glucan from C. tropicalis (S186) with the highest molecular weight and the lowest level of branching supported maximum survival (69%) followed by the other two marine yeast (S169 and S8) glucans of 27% and 23% respectively while glucan from Baker's yeast, S. cerevisiae S36 with the lowest molecular weight and the highest level of branching exhibited poor survival (4%) in P. monodon post challenge WSSV. The present study showed that the glucan isolate from marine yeast with a higher molecular weight and a lower degree of branching acts as better immunostimulants in P. monodon postlarvae than did the glucan isolate from S. cerevisiae.

OBJECTIVE:: To examine peripheral leukocyte Dectin-1 regulation in clinically relevant models of fungal and polymicrobial sepsis. DESIGN:: Prospective animal study. SETTING:: University medical school research laboratory. SUBJECTS:: Age, weight, and sex matched ICR/HSD mice. INTERVENTIONS:: Mice were infected with Candida albicans (1 × 10, intravenously) or were subjected to cecal ligation and puncture to induce polymicrobial sepsis. MEASUREMENTS:: Blood, spleen, and peritoneal exudate were harvested and leukocytes were isolated. Leukocytes were evaluated for membrane-associated Dectin-1 expression and cell phenotype by flow cytometry. MAIN RESULTS:: In C. albicans infection, Dectin-1-positive blood and splenic leukocytes were increased from 23.5% to 58.9% over the course of infection. The increased percentage of Dectin-1-expressing cells was primarily attributable to neutrophilia. However, the amount of Dectin-1 expressed by blood and splenic neutrophils in C. albicans-infected mice was decreased by a range of 49.0% to 53.3%. C. albicans infection also resulted in an infiltration of Dectin-1-positive macrophages and neutrophils into the kidney. In contrast, polymicrobial sepsis decreased blood leukocyte Dectin-1-expressing cells by up to 51.4%. This reduction was due to a decrease in Dectin-1-positive neutrophils in the periphery. However, the percentage of Dectin-1-expressing cells in the peritoneal cavity increased by 774% with cecal ligation and puncture. Treatment of isolated neutrophils with three soluble glucans, mannan, lipopolysaccharide, or a variety of cytokines revealed that glucans, alone or in combination, were the only treatment that resulted in a decrease in Dectin-1-positive neutrophils. CONCLUSIONS:: We conclude that peripheral leukocyte Dectin-1 expression is differentially regulated in fungal vs. polymicrobial sepsis. These data demonstrate that leukocyte Dectin-1 levels are modulated in response to infections of fungal and nonfungal origin.

A comparative study was performed to evaluate the immunostimulatory effect of yeast and yeast-derived glucan in white prawn Fenneropenaeus indicus (sub-adults of ∼20 gm). Feed with a whole cell biomass of marine yeast Candida sake S165 (CSY) at a concentration of 10% (w/w) and another feed with 0.2% glucan of C. sake S165 (CSG) were used in the study. Fenneropenaeus indicus were fed with these diets for 40 days and subsequently challenged with the white spot syndrome virus (WSSV). Haematological parameters such as the total haemocyte count, phenoloxidase activity, superoxide anion (O2-) level, haemolymph peroxidase level and post-challenge survival against WSSV infection were determined to assess the immune status. In the present experiment, a higher immunity index and post-challenge survival were recorded in shrimps fed with the whole cell yeast diet. The better immunostimulatory performance of the whole cell yeast diet compared with the glucan diet could be attributed to the cellular constituents of yeast including the cell wall glucan, nucleotides, carotenoid pigments and vitamins. Here we observed that whole cell yeast performed better as an immunostimulant than the extracted cell wall glucans. Therefore, the use of yeast biomass in diets, rather than the yeast cell wall extract, glucan, would confer better protection against microbial infection besides reducing the cost of shrimp production.

Previous studies have demonstrated that bacterial lipopolysaccharide (LPS) and heat killed Staphylococcus aureus (SA) activation of inflammatory cells depended in part upon activation of heterotrimeric Gi proteins. It has also been shown that (1→3) beta-D-glucan can suppress inflammatory cell activation by microbial products although the cellular mechanism of the glucan effect remains to be clearly defined. We hypothesized that Gi proteins function as a common convergent signaling pathway for both LPS and SA leading to monocyte mediator production. Additionally, we hypothesized that soluble glucan suppresses LPS and SA induced cytokine production via Gi protein coupled signaling. Human THP-1 promonocytic cells were pretreated with pertussis toxin (PTx, 100ng/ml or 1 microgram/ml) 6 hours prior to stimulation with LPS (10 microgram/ml) and SA (10 microgram/ml) and/or soluble glucan (10 microgram/ml). Both LPS and SA significantly (p<0.05) induced cytokine production IL-6 >TNF alpha >IL-1 beta >GM-CSF >IL-10 >IFN gamma. The induction of these cytokines was significantly (p<0.05) suppressed by PTx. Glucan treatment alone had no effect on cytokine production but suppressed (P<0.05) LPS and SA induced cytokines. PTx further augmented (p<0.05) the inhibitory effect of glucan on the LPS and SA induced cytokine expression. The data support the hypothesis that Gi proteins function as a common signaling protein for both LPS and SA induction of pro-and anti-inflammatory cytokines and that soluble glucan effectively suppresses cytokine production to the microbial stimuli. In contrast, the effect of soluble glucan on inhibiting cellular activation by LPS and SA is Gi protein independent.

The use of 4-acetoxy-2,2-dimethylbutanoyl protecting group for the C2-hydroxyl allows the selective formation of β-glycosides without producing α-glycosides. This very bulky protecting group can be removed under mild conditions.

Glucan is a natural product immunomodulator that has been reported to enhance early wound repair. The mechanism of glucan-stimulated wound repair was thought to be indirect via macrophage release of wound growth factors. However, recent data indicate that there are glucan-specific receptors on human fibroblasts that can modulate cellular function following interaction with the glucan ligand. In this study we examined the effect of glucan on activation of the transcription factors activator protein-1 (AP-1) and specificity protein-1 (Sp1) in normal human dermal fibroblasts. AP-1 and Sp1 are involved in the regulation of cytokine and procollagen genes. In addition, we evaluated the effect of glucan on wound growth factor and vascular endothelial growth factor (VEGF) mRNA expression in primary cultures of normal human dermal fibroblasts. Glucan (1 μg/ml) stimulated fibroblast AP-1 and Sp1 activation in a time-dependent manner, although the temporal kinetics varied between the two transcription factors. AP-1 binding activity was increased (p<0.05) at early time intervals (1, 2, 4, 8 and 12 h), while Sp1 nuclear binding activity was increased (p<0.05) at later time intervals (12, 24, 36 and 48 h). Glucan (1 μg/ml) stimulated fibroblast expression of neurotrophin 3 (NT-3), platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), fibroblast growth factor acidic (aFGF), fibroblast growth factor basic (bFGF), transforming growth factor alpha (TGFα), transforming growth factor beta (TGFβ) and VEGF mRNA at 8 h.

Polymeric carbohydrates have been reported to modulate inflammatory responses in vitro and in vivo. Previous reports suggest that certain carbohydrate polymers, such as (1→3)-β-D-glucans, may possess free radical scavenging activity. If glucans are free radical scavengers then it might explain, in part, the ability of these ligands to modulate inflammatory responses. The present study examined the free radical scavenging activity of a variety of carbohydrate polymers and the effect of the polymers on free radical levels in a murine macrophage cell line. All of the carbohydrates exhibited concentration dependent antioxidant effects (EC50 range = 807 to 43 μg/ml). However, the antioxidant activity for the carbohydrates was modest in comparison with PDTC (EC50 = 0.13 μg/ml) and the carbohydrate concentration required for antioxidant activity was high (×̄ EC50 = 283 μg/ml). The antioxidant ability of the polymers was greater (p < .05) than their monosaccharide constituents, i.e., dextrose EC50 = 807 vs. glucan sulfate EC50 = 43 μg/ml. Coincubation of glucans with murine J774a.1 cells increased free radical levels when compared to controls. Therefore, the weak free radical scavenging activity of glucan polymers cannot explain their modulatory effect on inflammatory responses in tissue culture and/or disease models of inflammation.