Abstract
Abstract The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an α-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that α-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked α-d-Glcp residues substituted at position 6 with α-d-Glcp branches. Soluble α-glucan, but not β-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when α-glucan from conidial surface was removed by enzymatic treatment with α-amyloglucosidase, thus indicating an essential role of α-glucan in P. boydii internalization by macrophages. α-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with α-amyloglucosidase. Finally, α-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and α-glucan could be a target for intervention during fungal infections.
Highlights
Pseudallescheria boydii is a saprophytic fungus widespread in soil and polluted water and has recently emerged as an agent of localized as well as disseminated infections in both immunocompromised and immunocompetent hosts
The results demonstrate that TLR2 and TLR4 participate in the recognition of pathogenic fungi, the molecules that trigger the activation of the toll-like receptors (TLRs)-associated signaling pathway leading to the induction of the innate immune response are largely unknown
We described the structure of a highly purified ␣-glucan, obtained from P. boydii, that mediates P. boydii conidial phagocytosis and triggers macrophage activation in a mechanism involving CD14, TLR2, and MyD88
Summary
Mice—C57/BL6, BALB/c, and C57BL/10 mice were obtained from the Fundacao Oswaldo Cruz Breeding Unit (Rio de Janeiro, Brazil). After incubation at 37 °C in 5% of CO2 for 60 min in RPMI 1640 medium, the cells were rinsed with HBSS for removal of non-internalized conidia. Adherent cells were stimulated for 4 h, in RPMI medium, with the ␣-glucan, LPS, or Pam3Cys, at concentrations indicated in the figure legends After this period the supernatant was recovered for TNF determination by ELISA according to the manufacturer’s instructions. After 5 days, fresh medium was added to culture and with 7 days of culture, the cells were collected and bone marrow-derived dendritic cells were separated by Optiprep gradient (Sigma) by centrifugation at 600 ϫ g for 30 min at 24 °C. The level of significance was set at p Ͻ 0.05
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