IL-23 Regulates Virulence Figure 2. The invasive form of opportunistic fungal pathogens but not that of pathogenic fungi exposes b-glucan in the cell wall surface. Representative confocal microscopy images of b-glucan surface staining of resting (spores) and invasive (hyphae) stages of growth of the opportunistic fungi Aspergillus or Rhizopus and the invasive (yeast) form of the pathogenic fungus Histoplasma. Live fungal cells were fixed with 1% paraformaldehyde and stained with a mouse b-(1–3)-glucan-specific monoclonal antibody followed by staining with a secondary goat anti-mouse Alexa488 antibody (green). The antibody failed to detect surface b-glucan on the spores of opportunistic fungi and in the invasive form of Histoplasma but detected high amounts of the polysaccharide in germinating hyphae of both Aspergillus and Rhizopus (left panels). The overlay images (DIC/b-glucan immunostaining; right panels) indicate the pattern of surface b-glucan staining in the different stages of growth of fungi. Data shown are representative of 3 independent experiments. doi:10.1371/journal.pone.0012955.g002 TH-17 responses by human DCs are typically driven by IL-23, but may also require, in particular at the initial stages of differentiation other pro inflammatory cytokines, including IL1b or IL1b plus IL-6 [19–23]. We found that IL-23 producing DCs also secreted large amounts of IL-1b, which was dependent on activation of dectin-1 as it was completely abolished in the presence of dectin-1 neutralizing antibody (Figure S3A). In addition, IL-6 was also produced by IL-23 producing DCs, although its secretion was only partially dependent on activation of dectin-1 (Figure S3B). Notably, there was no significant reduction in IL-6 production by Figure 3. Induction of IL-23 producing DCs by opportunistic fungal pathogens is dectin-1 dependent and is mediated by b-glucan. (A) IL-23 production by human monocyte-derived DCs pre incubated for 1 h with increasing concentrations of the dectin-1 inhibitor laminarin (0, 0.01, 0.1, and 1 mg/ml) and subsequently stimulated with hyphae of either Aspergillus (open cycles) or Rhizopus (closed cycles), at a 1:1 ratio. (B) IL-23 production by DCs stimulated with hyphae of either Aspergillus (black bars) or Rhizopus (white bars), or b-glucan (scattered bars) or LPS (gray bars) alone or following 1 h pre incubation with an anti-dectin-1 blocking antibody (10 mg/ml). Data are expressed as mean 6 SEM values for DCs derived from three different donors. (C) Representative confocal microscopy images of b-glucan surface staining in hyphae of Aspergillus or Rhizopus untreated or following overnight enzymatic digestion with b-glucanase (10 U/ml). (D) IL-23 production by DCs stimulated with hyphae of Aspergillus (white bars) or Rhizopus (black bars) untreated or following overnight enzymatic digestion with b-glucanase. Data shown are representative of 3 independent experiments. Error bars represent SD. *, P,0.001, paired Student’s t test. doi:10.1371/journal.pone.0012955.g003 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12955