In agreement to our findings, a recent study reported that dormant spores of Aspergillus were immunologically inert and did not induce DC or alveolar macrophage activation, because of the presence of a surface ‘rodlet layer’, composed of the hydrophobic RodA protein that masks underlying immunostimulatory molecules [41]. Previous studies suggested that coordinated activation of TLR-2 and TLR-4 by Aspergillus spores triggers a TH-1 protective response, whereas Aspergillus hyphae trigger TLR-2 activation and a TH-2 response [6,24]. Nonetheless, studies using different TLR and MyD88 knockout mice failed to convincingly show increased susceptibility to Aspergillus infection in the absence of immunosupression [7]. In contrast, dectin-1 knockout mice, display increased susceptibility to invasive aspergillosis without the need for administration of immunosuppressive agents, significantly attenuated proinflammatory response and decreased IL-17 production in the lung [14]. Notably, in agreement to our finding, macrophages from dectin-1 knockout mice fail to produce proinflammatory cytokines in response to Aspergillus infection [14]. Nonetheless, we cannot preclude that other immunostimu- latory molecules on the fungal cell wall are selectively exposed during invasive fungal growth to trigger PRRs in human DCs [42–44]. Likewise, collaborative activation of dectin-1 with surface TLRs and/or other PRR may be implicated in innate sensing of different opportunistic fungi [45,46]. The induction of IL-23 producing DCs by different microbial ligands is a key driver of TH-17 responses [8,9,19,23]. The ability of dectin-1 when compared to other PRRs for preferentially induction of IL-23 over IL-12, may be related to activation of a noncanonical NF-kappaB signaling pathway mediated by the serine-threonine kinase Raf-1 [47]. Indeed, supernatants from IL- 23 producing DCs generated by stimulation with Aspergillus hyphae or b-glucan induced the production of IL-17 in naive human CD4+ T cells and a robust expansion of IL-17 producing memory human CD4+ T cells stimulated polyclonally. Notably, IL-17 production was significantly reduced by an antibody neutralizing IL-23, as also suggested by other studies [19,21–23]. Overall, our studies demonstrate the important role of dectin-1 signaling as a major innate sensing pathway for induction on TH- 17 antifungal responses in humans. It is plausible that dysregu- lation of this pathway in patients with underlying immunodefi- ciency leads to unrestricted growth of opportunistic fungi and the development of life-threatening infections. Therefore, targeting this pathway for immunotherapy is an appealing strategy for prevention and treatment of patients at increased risk for development of opportunistic fungal infections. Future studies on the function of dectin-1/TH-17 axis in immunocompromised individuals might also provide a tool for risk stratification for development of fungal infections. Materials and Methods Reagents Highly purified Escherichia coli LPS, laminarin from Laminaria digitata, and b-1,3-D-glucanase from Helix pomatia were obtained from Sigma-Aldrich (St. Louis, MO). Purified particulate b-glucan (curdlan) was from Waiko (Tokyo, Japan). b-(1-3)-glucan-specific monoclonal antibody was from Biosupplies (Parkville, Australia). Blocking monoclonal antibodies for dectin-1 (MAB1859, clone 259931; 10 mg/ml) and appropriate isotype control antibodies were from R&D Biosystems. Neutralizing antibodies for the IL-23 p19 subunit (AF1716; 10 mg/ml), and IL-4 (34019; 10 mg/ml) were from R&D Biosystems. Neutralizing antibodies for IFN-c (B27; 10 mg/ml) were from BD Biosciences. IL-23 Regulates Virulence Microorganisms and culture conditions The Aspergillus fumigatus strain Af293 and the Rhizopus oryzae clinical isolate 557969 were grown on YAG-chloramphenicol agar plates for 3 days at 37uC. Fungal spores in the presence of sterile 0.1% Tween 20 in PBS were harvested by gentle shaking, washed, filtered, counted by a hemacytometer, and suspended at a concentration of 108 spores/ml. Germinating hyphae of Aspergillus and Rhizopus were obtained following growth in liquid RPMI 1640 media in a shaking incubator (220 rpm, 37uC), for 12 h and 8 h, respectively. Typically, .80% of conidia of each fungus were visibly swollen after 5 h. Enzymatic digestion of b-glucan in the hyphae of Aspergillus and Rhizopus was performed by using b-1-3-D-glucanase from Helix pomatia (Sigma). Hyphae of both fungi were incubated overnight in a shaking bath with 10 U/ml of b-glucanase at a temperature of 55uC and pH 5, 0. Inactivation of enzyme was achieved by 10 min incubation at 100uC followed by three washes in PBS. Verification of b-glucan digestion was performed by immuno- staining with a b-glucan monoclonal antibody. H. capsulatum Dags1 mutant and its isogenic complementary strain AGS1 (+) were derived from the clinical isolate G186A (ATCC 26029) and have been described in ref 39. Histoplasma yeasts were grown at 37uC with 95% air/5% CO2 in HMM supplemented with 100 mg/ml uracil. Dispersed Histoplasma yeasts were obtained by growth of liquid cultures to late exponential phase, removal of large yeast clumps by low-speed centrifugation (60 s at 1006g), three washes with Ham’s F-12 (Invitrogen, Carlsbad, CA), and counting with a hemacytometer. Inactivation of fungi was done by heat exposure (30 min, 65 C) or exposure to 1% paraformaldehyde (4uC, overnight). In initial DC stimulation experiments, amphotericin B (1 mg/ml) was added to the cell cultures immediate after infection with fungi to prevent germination or restrict overgrowth of germinating hyphae. Because there were no appreciable differences in cytokine production as compared to infection of DCs with inactivated fungi, all following studies were performed with inactivated fungal cells. Generation of monocyte-derived DCs This study was approved by the Institutional Review Board for human research at the M. D. Anderson Cancer Center in Houston. Separation of peripheral blood mononuclear cells (PBMCs) was performed by density gradient centrifugation using ficoll hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) from healthy donor blood obtained from the Gulf Coast Regional Blood Center, Houston, Texas. CD14+ cells were enriched from the PBMCs using CD14 microbeads and columns (Miltenyi Biotec, Auburn, CA). In order to generate DCs, CD14+ cells were cultured in the presence of IL-4 (100 ng/mL; R&D Systems, Minneapolis, MN) and granulocyte-macrophage colony-stimulat- ing factor (GM-CSF; 100 ng/mL R&D Systems, Minneapolis, MN) for 5 days at 26106 cells/ml in RPMI 1640 supplemented with2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS (v/ v). On day 5 the cells were 70 to 90% CD1a+ and 95% CD142. Then the culture medium containing IL-4 and GM-CSF was replaced with culture medium alone 2 h before infection with fungi or other treatments. Purification of naive and memory CD4+ T lymphocytes from adult blood Untouched na¨ıve and memory CD4+ T lymphocytes were purified from PBMCs by immunomagnetic depletion with the PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12955