Figure 1. The invasive stage of growth of opportunistic fungal pathogens but not that of primary pathogenic fungi drives the induction of IL-23 producing DCs. IL-23 (A) and IL-12 (B) produced by human monocyte-derived DCs (16106 cells per ml) following overnight stimulation with purified b-glucan (curdlan, 100 mg/ml), LPS (100 ng/ml), resting (spores) or invasive (hyphae) stages of growth of each opportunistic fungal pathogen (Aspergillus and Rhizopus) and the invasive form (yeast) of the pathogenic fungus Histoplasma, at a 1:1 ratio. The results are representative of 6 independent experiments. , indicates that the measured value was below the detection limit of the assay (,20 pg/ml). Error bars represent SD. *, P,0.001, paired Student’s t test. doi:10.1371/journal.pone.0012955.g001 IL-23 Regulates Virulence effect on IL-23 production by DCs stimulated with LPS (Figure 3B). Because b-glucan is not the only ligand of dectin-1 [37,38], we further investigated the role of b-glucan surface exposure by specific enzymatic digestion of b-glucan in the fungal cell walls using b- glucanase. The ability of b-glucanase to digest b-glucan in the cell wall of hyphae of Aspergillus and Rhizopus was confirmed by immunofluor- escene studies (Figure 3C). DCs infected with b-glucanase-treated hyphae were unable to produce any IL-23 (Figure 3D), indicating that hyphae of opportunistic fungal pathogens induce IL-23 producing DCs through b-glucan surface exposure. Enforced b-glucan surface exposure in the non-pathogenic Histoplasma Dags1 mutant drives IL-23 producing DCs In contrast to opportunistic fungal pathogens, in Histoplasma cell wall immunostimulatory b-glucans are covered in the yeast (infectious) form of the fungus by a layer of a-(1–3)-glucan [32,39]. Concealing of b-glucan by non-stimulatory a-(1–3)-glucan mole- cules is regarded as a virulence mechanism, because it allows Histoplasma to avoid immune recognition and survive within the host cells. As a proof of principle, b-glucan surface exposure in the Dags1 mutant of Histoplasma capsulatum that lacks a-(1–3)-glucan, renders it non-pathogenic in mice and results in increased release of TNFa in infected mouse macrophages through dectin-1 activation [39]. We hypothesized that similar to the induction of IL-23 release by b-glucan exposure in the hyphae of opportunistic fungal pathogens, unmasking of b-glucan in the Dags1 mutant of Histoplasma should trigger the release of IL-23 in human DCs. We initially confirmed by immunofluorescene studies that the yeast form of the Dags1 mutant of Histoplasma had surface exposure of b-glucan as opposite to its isogenic complementary Ags1+ strain (Figure 4A). Importantly, infection of human DCs with the yeast form of the Dags1 mutant of Histoplasma resulted in the production of IL-23 (Figure 4B); in contrast, there was no IL-23 production in DCs infected with the isogenic complementary Ags1+ strain of Histoplasma (Figure 4B). Additionally, IL-23 production by human DCs infected with the Dags1 mutant was inhibited following blocking of dectin-1 receptor or enzymatic digestion of b-glucan (Figure 4B). These results further support the role of b-glucan in driving IL-23 producing DCs and indicate that the exposure of cell wall b-glucans may restrict the pathogenic potential of fungi via the induction of the IL-23-TH-17 response pathway. IL-23 producing DCs induced by opportunistic fungal pathogens drive TH-17 responses Because IL-23 producing DCs represent the key drivers of TH- 17 responses (8, 9, 19, 23), we next sought to determine whether human DCs stimulated with opportunistic fungal pathogens are capable of driving TH-17 responses. Purified human na¨ıve and memory CD4+ T cells were activated polyclonally in the presence of supernatants of DCs previously stimulated with hyphae or spores of Aspergillus. DCs stimulated with hyphae but not spores of Aspergillus induced a predominant increase in IL-17 production versus IFN-c, indicating that the invasive form of this fungus can trigger DC-mediated TH-17 responses (Figure 5A). This finding was confirmed by intracellular cytokine staining of primed CD4+ T cells (Figure 5B), as there was a significant increase in IL-17+/ IFN-c2 single positive CD4+ T cells (mean increase from 4.6% to 9,7%, n = 5 experiments; P = 0.004) and IL-17+/IFN-c+ double positive CD4+ T cells (mean increase from 1,1% to 9,7%; n = 5 experiments; P = 0.002), (Figure S4). PLoS ONE | www.plosone.org 3 September 2010 | Volume 5 | Issue 9 | e12955