Use of fungal derived polysaccharide-conjugated particles to probe Dectin-1 responses in innate immunity.

The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of β-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of "fungal like particles" was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified β-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the β-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the β-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating β-1,3-glucan beads with laminarinase, a specific β-1,3-gluconase, the reactivity of the anti-β-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly β-1,3-glucan. TNF-α was also measured by stimulating bone-marrow derived macrophages with the β-1,3-glucan beads, and showed a dose dependent response compared to soluble β-glucan, insoluble β-1,3-glucan, uncoated beads, and soluble β-1,3-glucan mixed with uncoated beads. Finally, β-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. β-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. These data describe a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.