Glucans are immunomodulatory carbohydrates found in the cell walls of fungi and certain bacteria. We examined the pharmacokinetics of three water-soluble glucans (glucan phosphate, laminarin, and scleroglucan) after oral administration of 1 mg/kg doses in rats. Maximum plasma concentrations for glucan phosphate occurred at 4 h. In contrast, laminarin and scleroglucan showed two plasma peaks between 0.5 and 12 h. At 24 h, 27 Ϯ 3% of the glucan phosphate and 20 Ϯ 7% of the laminarin remained in the serum. Scleroglucan was rapidly absorbed and eliminated. The liver did not significantly contribute to the clearance of plasma glucan. Biological effects were further studied in mice. Following oral administration of 1 mg, glucans were bound and internalized by intestinal epithelial cells and gut-associated lymphoid tissue (GALT) cells. Internalization of glucan by intestinal epithelial cells was not Dectindependent. GALT expression of Dectin-1 and toll-like receptor (TLR) 2, but not TLR4, increased following oral administration of glucan. Oral glucan increased systemic levels of interleukin (IL)-12 (151 Ϯ 15%) in mice. Oral glucan administration also increased survival in mice challenged with Staphylococcus aureus or Candida albicans. These data demonstrate that orally administered water-soluble glucans translocate from the gastrointestinal (GI) tract into the systemic circulation. The glucans are bound by GI epithelial and GALT cells, and they modulate the expression of pattern recognition receptors in the GALT, increase IL-12 expression, and induce protection against infectious challenge.

Glucans are structurally diverse fungal biopolymers that stimulate innate immunity and are fungal pathogen-associated molecular patterns. Dectin-1 is a C-type lectin-like pattern recognition receptor that binds glucans and induces innate immune responses to fungal pathogens. We examined the effect of glucan structure on recognition and binding by murine recombinant Dectin-1 with a library of natural product and synthetic (133)-␤/(136)-␤-glucans as well as nonglucan polymers. Dectin-1 is highly specific for glucans with a pure (133)-␤-linked backbone structure. Although Dectin-1 is highly specific for (133)-␤-D-glucans, it does not recognize all glucans equally. Dectin-1 differentially interacted with (133)-␤-D-glucans over a very wide range of binding affinities (2.6 mM-2.2 pM). One of the most striking observations that emerged from this study was the remarkable high-affinity interaction of Dectin-1 with certain glucans (2.2 pM). These data also demonstrated that synthetic glucan ligands interact with Dectin-1 and that binding affinity increased in synthetic glucans containing a single glucose side-chain branch. We also observed differential recognition of glucans derived from saprophytes and pathogens. We found that glucan derived from a saprophytic yeast was recognized with higher affinity than glucan derived from the pathogen Candida albicans. Structural analysis demonstrated that glucan backbone chain length and (136)-␤ side-chain branching strongly influenced Dectin-1 binding affinity. These data demonstrate: 1) the specificity of Dectin-1 for glucans; 2) that Dectin-1 differentiates between glucan ligands based on structural determinants; and 3) that Dectin-1 can recognize and interact with both natural product and synthetic glucan ligands.

Glucans are (1-3)-beta-D-linked polymers of glucose that are produced as fungal cell wall constituents and are also released into the extracellular milieu. Glucans modulate immune function via macrophage participation. The first step in macrophage activation by (1-3)-beta-D-glucans is thought to be the binding of the polymer to specific macrophage receptors. We examined the binding/uptake of a variety of water soluble (1-3)-beta-D-glucans and control polymers with different physicochemical properties to investigate the relationship between polymer structure and receptor binding in the CR3- human promonocytic cell line, U937. We observed that the U937 receptors were specific for (1-->3)-beta-D-glucan binding, since mannan, dextran, or barley glucan did not bind. Scleroglucan exhibited the highest binding affinity with an IC(50)of 23 nM, three orders of magnitude greater than the other (1-->3)-beta-D-glucan polymers examined. The rank order competitive binding affinities for the glucan polymers were scleroglucan>>>schizophyllan > laminarin > glucan phosphate > glucan sulfate. Scleroglucan also exhibited a triple helical solution structure (nu = 1.82, beta = 0.8). There were two different binding/uptake sites on U937 cells. Glucan phosphate and schizophyllan interacted nonselectively with the two sites. Scleroglucan and glucan sulfate interacted preferentially with one site, while laminarin interacted preferentially with the other site. These data indicate that U937 cells have at least two non-CR3 receptor(s) which specifically interact with (1-->3)-beta-D-glucans and that the triple helical solution conformation, molecular weight and charge of the glucan polymer may be important determinants in receptor ligand interaction.

Comparative LDH secretion, Ext_data_figure2.ep a, Assessment of Candida induced cell death of PBMCs after 24 hours Extended Data Fig. 2.Extended Data Fig. 3 Relative C. auris induced ROS production and heatsensitivity of the cell wall components responsible for the C. auris induced cytokine production. Ext_data_figure3.ep sa, Neutrophil ROS release after 1-hour stimulation without (RPMI; negative control) or with heat-killed C. albicans, C. auris strains or zymosan (positive control), depicted in relative light units (RLU) either as time-course (left) or as area under the curve (AUC, right), n=9. b, PBMC ROS release after 1-hour stimulation without (RPMI; negative control) or with heat-killed C. albicans, C. auris strains or zymosan (positive control), depicted in RLU either as time-course (left) or as AUC (right), n=6. c, TNF-α, IL-6, IL-1β, and IL-1Ra levels in the supernatant of PBMCs after stimulation without (RPMI; negative control) or with heat-killed C. albicans and C. auris from all five geographical clades for 24 hours, n=8. d, PBMC production of cytokines IFN-γ (n=10; n=7 for C. auris 10051895), IL-10 (n=6), IL-17 (n=6), and IL-22 (n=14; n=6 for C. auris 10051893; n=11 for C. auris 10051895) after stimulation without (RPMI; negative control) or with heat-killed C. albicans and C. auris for 7 days. Graphs represent mean ± SEM, data are pooled from at least two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p = 0.001, a-b Time curves (left panels) were assessed for statistical differences between C. auris strains and C. albicans by a two-way ANOVA, Area Under curve (AUC) means (right panels) were compared using the two-sided Wilcoxon signed rank test, c-d twosided Wilcoxon matched pairs signed-rank test comparing respective C. auris strains with C. albicans as control or reference species. Data used to make this figure can be found in Source Data Extended Data Fig. 3.Extended Data Fig. 4 Transcriptional changes induced by purified cell wall components and their respective exposure on C. albicans and C. auris Ext_data_figure4.ep s . a, Heatmap displaying the Log 2 Fold change (color scale) of the top 50 DEG of C. albicans live, for both Candida species and their cell wall components, β-glucan and mannan, at 4 hour (left panel) and 24 hours (right panel). b, Flow cytometry plot based on forward scatter component (FSC) and side scatter component (SSC), demonstrating C. surface. auris strains are slightly smaller and of higher complexity than C. albicans. c, Flow cytometry-based comparison of cell wall components of C. albicans and C. auris strains. Mean fluorescent intensity (MFI) of thimerosal-fixed Candida cells stained for Fc-Dectin-1, a marker for β-glucan (left), and ConA, a marker for mannans (right). Graphs represent mean ± SEM of the 3 means, each performed with three replicates in three independent measurements, * p < 0.05, Kruskall Wallis test with two-sided Dunn's multiple comparison test was performed comparing the respective C. auris strains with the two C. albicans refere...

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