Much effort has focused on identifying microbial ligands and the receptors that drive the differentiation of DCs into producers of either IL-12 or IL-23 [8,9,23]. In fungi, activation of members of the Toll-like receptor (TLR) family, mainly TLR2 and TLR4, in DCs has been linked to the induction of IL-12 leading to protective TH-1 responses against Candida and Aspergillus [24,25]. On the other hand, activation of dectin-1, a C-type lectin expressed on DCs that recognizes the polysaccharide b-glucan, triggers the induction of IL-23 through the syk-CARD-9 signaling pathway [26], and drives robust TH-17 response [26]. Interestingly, while the yeast form of Candida albicans induces IL-12, the invasive, tissue-infiltrating form of the fungus (hyphae) selectively induces IL-23 and drives TH-17 responses [26]. Therefore, it appears that tissue invasion by fungi triggers activation of the IL-23/TH-17 pathway that has a central role in the initiation of an acute inflammatory response via neutrophil recruitment and the induction of epithelial antimicrobial peptides [6,7]. The important role of dectin-1-syk-CARD-9 signaling in induction of TH-17 responses and protective mucosal immunity against Candida has been convincingly shown both in mice [27,28] and recently in humans [29,30]. In view of the complexity and the structural differences in the fungal cell wall of fungi [31,32], whether activation of dectin-1 signaling is a major pathway for TH-17 induction in response to airborne opportunistic fungal pathogens that currently cause the majority of fungal infections in immunocompromised patients, and whether this ability is related to specific growth stages and/or the inherent pathogenic behavior of each fungus in currently unknown. Here we studied human DC responses against the two predominant airborne opportunistic fungal pathogens, Aspergillus and Rhizopus, and the pathogenic fungus Histoplasma. Importantly, we found that opportunistic fungal pathogens (Aspergillus and Rhizopus) but not primary pathogenic fungi (Histoplasma) prime dendritic cells (DCs) to produce high levels of IL-23, which drives TH-17 responses. The induction of these IL-23 producing DCs depended on activation of dectin-1 and is mediated by b-glucan exposed in the cell wall of the invasive form of opportunistic fungal pathogens. By contrast, the yeast form of the pathogenic fungus Histoplasma, which lacks cell wall exposure of b-glucan, failed to induce IL-23 producing DCs. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma, not only abrogated the pathogenicity of this fungus, but also triggered the induction of IL- 23 producing DCs. Thus, b-glucan exposure in the cell wall of fungi is essential for the generation of IL-23 producing DCs and TH-17 immune responses and may represent a key factor that regulates protective immunity to opportunistic but not primary pathogenic fungi. Results The invasive stage of growth of opportunistic fungal pathogens but not that of primary pathogenic fungi drives the induction of IL-23 producing DCs To test the ability of airborne fungi that cause human infections to drive DC-mediated TH-17 responses, we used Aspergillus and Rhizopus as model opportunistic fungal pathogens and Histoplasma, a pathogenic fungus that causes infections in immunocompetent individuals [6]. Previous studies suggested that DCs are capable of discriminating between the different stages of fungal growth (spores vs. hyphae) to prime the development of TH-1 and TH-2 responses [24,25]. We therefore tested if the induction of TH-17 responses is also dependent on specific stage of growth of fungi by human DCs. Human DCs were infected with invasive stage IL-23 Regulates Virulence (hyphae) or the resting stage (spores) of Aspergillus or Rhizopus, and cytokine secretion was analyzed after overnight culture. We found that DC infected with the invasive stage but not the resting stage of both fungi secreted high amounts of IL-23 (Figure 1A) and only low amounts of IL-12 (Figure 1B). Importantly, IL-23 production by DCs has been recognized to be a key driver of TH-17 responses [8,9,19,23]. IL-23 producing DCs induced by the invasive stage of Aspergillus or Rhizopus also secreted high levels of IL-1, IL-6, and TNF-a (Figure S1). Next, we evaluated DCs infected with the pathogenic fungus Histoplasma, one of the few fungi that cause invasive infections in immunocompetent individuals [6,32]. Histoplasma is a dimorphic fungus that adapts a particular stage of growth (yeast) at mammalian temperatures [6,32]. This growth adaptation is considered a virulence trait of Histoplasma that allows the fungus to escape immune recognition and establish persistent infection within host macrophages [32]. Interestingly, in contrast to opportunistic fungi, we found that the yeast form of Histoplasma failed to elicit IL-23 producing DCs (Figure 1A). Together, these data demonstrate that the invasive form of opportunistic fungal pathogens Aspergillus or Rhizopus but not of the pathogenic fungus Histoplasma drives the induction of IL-23 producing DCs. The invasive form of opportunistic fungal pathogens but not that of primary pathogenic fungi exposes b-glucan in the cell wall surface b-glucan has been shown to be a key driver of IL-23 production in DCs [23,26]. Previous studies have shown that Aspergillus exposes b-glucan on the cell wall surface during the invasive stage of growth (hyphae) [33–35]. Thus, we sought to determine whether like Aspergillus also Rhizopus but not Histoplasma exposes b- glucan in the cell wall of the invasive stage of fungal growth. Immunofluorescene studies using a b-glucan specific monoclonal antibody confirmed the exposure of b-glucan in hyphae of Aspergillus (Figure 2). Interestingly, abundant staining of b-glucan was also found on the surface of hyphae of Rhizopus (Figure 2), which is in contrast to previous reports that failed to identify b- glucan on the cell wall of Rhizopus and instead suggested that chitin and chitosan comprise the main polysaccharides of this fungus [36]. Importantly, there was no evidence of b-glucan staining in the spores of both opportunistic fungal pathogens and in the yeast form of the pathogenic fungus Histoplasma (Figure 2). These data indicate that b-glucan exposure selectively occurs in the invasive stage of growth of opportunistic fungal pathogens but not in primary pathogenic fungi, a finding that correlates with the ability of these fungi to induce IL-23 producing DC. Induction of IL-23 producing DCs by opportunistic fungal pathogens is dectin-1 dependent and is mediated by b-glucan Next, we sought to demonstrate that the selective exposure of b- glucan in the hyphae of opportunistic fungal pathogens drives the induction of IL-23 producing DCs. We used laminarin, a competitive inhibitor of b-glucan for its binding to dectin-1, the receptor for b-glucan expressed on DCs [27,37]. Increasing concentrations of laminarin abrogated the induction of IL-23 production by DCs infected with hyphae of Aspergillus or Rhizopus (Figure 3A). As a control, we stimulated DCs with the TLR4 agonist LPS, and showed that laminarin had no effect on the production of IL-23 by DCs (Figure S2). Furthermore, pre- incubation of DCs with a neutralizing anti-dectin-1 antibody resulted in complete inhibition of IL-23 production in DCs infected with hyphae of both opportunistic fungi, while it had no PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12955