Dectin-1 mediates the biological effects of beta-glucans

Abstract

The ability of fungal-derived beta-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-alpha, 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-alpha 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-alpha in response to fungi, a critical step required for the successful control of these pathogens.

Figure 1.

Zymosan-induced TNF-α production is β-glucan dependent and mediated by Dectin-1. (a) GluP specifically inhibits the production of TNF-α in response to unlabeled zymosan in thioglycollate-elicited Mφ. (b) RAW264.7 Mφ express low levels of endogenous Dectin-1 as measured by FACS^® staining using mAb 2A11. Increased levels of this receptor are obtained by stably transducing the Mφ with HA-tagged Dectin-1, including two cytoplasmic tail mutants. (c) RAW264.7 Mφ overexpressing Dectin-1 (RAW-Dectin; ▴) have an enhanced ability to bind FITC-labeled zymosan compared with the vector-transduced control (RAW-FB; ▪) and (d) show a dose-dependent increase in TNF-α production.

Figure 2.

Dectin-1 induces the production of TNF-α in response to live yeasts. RAW-Dectin Mφ display an enhanced ability to induce TNF-α in response to live (a) C. albicans and (b) S. cerevisiae. Live yeasts were added at a multiplicity of infection of 5:1. Note that the response to S. cerevisiae is significantly greater than to C. albicans and that these responses are β-glucan dependent.

Figure 3.

TLRs are required for TNF-α production in response to zymosan. (a) BMDM from TLR-2– or MyD88-deficient mice do not produce TNF-α after overnight incubation with unlabeled zymosan. The response to overnight incubation with 5 ng/ml LPS is shown as a control. (b–e) V5-Tagged TLR-2 (red) and HA-tagged Dectin-1 (green) colocalize within 5 min after the addition of zymosan. (b) Light micrograph showing RAW-Dectin cells binding zymosan. Anti-HA (c) and anti-V5 (d) staining showing colocalization (e) of Dectin-1 and TLR-2, respectively, at the zymosan phagocytic cup. The cell indicated with an arrow was not transfected with V5-tagged TLR-2 and is presented as a staining control. Colocalization of these receptors at the cell surface in contact with zymosan were also observed after 30-min incubation at 37°C with Cytochalasin D (unpublished data).

Figure 4.

The β-glucan–dependent TNF-α response to zymosan is mediated by Dectin-1. Although opsonization increases the amount of FITC-labeled zymosan bound by RAW-FB Mφ (a), TNF-α production is only marginally increased and is no longer β-glucan dependent (b). In contrast, Mφ overexpressing Dectin-1 (RAW-Dectin) still possess an enhanced β-glucan–dependent response to opsonized zymosan.

Figure 5.

The cytoplasmic tail of Dectin-1 is involved in mediating the TNF-α response to zymosan. The cell lines expressing the Dectin-1 cytoplasmic tail mutants display an enhanced ability to bind FITC-labeled zymosan (a), but they do not transduce an intracellular signal inducing the TNF-α response to this particle (b). This effect is not due to lack of internalization of the zymosan as signaling still occurs, and is even enhanced, when phagocytosis is inhibited with cytochalasin D (c; cytoD).