Figure 4. Enforced b-glucan surface exposure in the non- pathogenic Histoplasma Dags1 mutant drives IL-23 producing DCs. (A) Representative confocal microscopy images of b-glucan surface staining in yeast cells in the Dags1 mutant of Histoplasma and the isogenic complementary strain Ags1 + (left panel). Overlay images indicate the pattern of surface exposure on yeast cell surface (DIC/b- glucan immunostaining; right panel). (B) IL-23 production by DCs stimulated with the isogenic complementary strain Ags1+ of Histoplas- ma and the Dags1 mutant untreated or following overnight enzymatic digestion with b-glucanase (10 U/ml). IL-23 was selectively produced following stimulation of DCs with Dags1 mutant. Data are expressed as mean 6 SEM values of four independent experiments. *, P,0.001, paired Student’s t test. doi:10.1371/journal.pone.0012955.g004 DCs stimulated with curdlan or fungal hyphae following the addition of polymyxin-B (PMX-B) (Figure S5), thus excluding LPS contamination in DC cultures. IL-23 Regulates Virulence The addition of neutralizing antibodies against IL-23 inhibited IL-17 production by na¨ıve and memory CD4+ T cells polarized with Aspergillus-stimulated DCs close to the baseline levels (Figure 5C). Importantly, neutralizing antibodies against IL-23 did not affect IFN-c production by CD4+ T cells polarized with Aspergillus-stimulated DCs (Figure 5C). Therefore, IL-23 producing DCs induced by hyphae of opportunistic fungi drive TH-17 responses. Discussion The emergence of life-threatening infections caused by airborne opportunistic fungal pathogens in an expanding population of non-neutropenic patients with defects in T-cell function, such as transplant recipients, patients with autoimmune diseases receiving biologic therapies or corticosteroids, and individuals with AIDS [1,2], illustrates the pivotal role of adaptive immunity in antifungal host defense. Recent studies in mice demonstrate that the newly identified TH-17/IL-23 axis regulates epithelial host defense against a growing list of fungi [10–14]. In view of the considerable differences in generation and function of TH-17 cells between mice and humans [8,9], understanding the regulatory mechanisms for induction of antifungal TH-17 responses in humans is important for development of targeted therapeutic strategies. Although purified b-glucan acts as a potent adjuvant for the induction of IL-23 producing DCs and TH-17 priming [26], the structural complexity and differential cellular composition of various fungi makes it difficult to predict whether, which, and how common molecular patterns in cell wall of different fungi link innate sensing by DCs with TH-17 pathway induction. Herein, we identified a common innate sensing pathway in human DCs that is triggered by opportunistic fungi. We found that this pathway was dependent on activation of dectin-1 signaling by b-glucan exposure in the hyphae of opportunistic fungal pathogens, and resulted in robust induction of IL-23 producing DCs that primed TH-17 antifungal responses. Importantly, we also demonstrated that pathogenic fungi evade TH-17 induction by concealing immunos- timulatory b-glucans with other polysaccharide layers and thus avoiding activation of dectin-1 signaling. Our initial studies identified that the signature cytokine of the TH-17 pathway, IL-23, was selectively produced by human DCs infected with hyphae of Aspergillus or Rhizopus, by not by DCs infected with dormant spores of opportunistic fungi or the yeast form of the pathogenic fungus Histoplasma. Importantly, we found that generation of these IL-23 producing DCs correlated with selective b-glucan surface exposure in the hyphae of Aspergillus and Rhizopus. This was an unexpected finding based on biochemical studies that failed to identify b-glucan in the cell wall of Rhizopus (36), and clinical evidence of lack of detectable amounts of b- glucan release in human infections caused by this fungus [4,5]. Our results corroborate with recent studies that identified b-glucan synthetase, the enzyme responsible for synthesis of b-glucan, in Rhizopus [40]. Next, by using specific dectin-1 inhibitors and b- glucan enzymatic digestion, we confirmed that induction of IL-23 producing DCs is dependent on activation of dectin-1 signaling and is mediated by b-glucan exposure in the hyphae of opportunistic fungi. Importantly, surface exposure of b-glucan in the Dags1 mutant of Histoplasma that lacks the upper layer of a-glucan, not only abrogated its virulence, but also triggered the induction of IL- 23 producing DCs via activation of dectin-1 signaling. Our study is the first to explore the interaction of DCs with fungi of the class Zygomycetes and the regulatory mechanisms of TH-17 induction against airborne opportunistic fungi in humans. PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12955