Generation of IL-23 producing dendritic cells (DCs) by airborne fungi regulates fungal pathogenicity via the induction of T(H)-17 responses

Abstract

Interleukin-17 (IL-17) producing T helper cells (T(H)-17) comprise a newly recognized T cell subset with an emerging role in adaptive immunity to a variety of fungi. Whether different airborne fungi trigger a common signaling pathway for T(H)-17 induction, and whether this ability is related to the inherent pathogenic behavior of each fungus is currently unknown. Here we show that, as opposed to primary pathogenic fungi (Histoplasma capsulatum), opportunistic fungal pathogens (Aspergillus and Rhizopus) trigger a common innate sensing pathway in human dendritic cells (DCs) that results in robust production of IL-23 and drives T(H)-17 responses. This response requires activation of dectin-1 by the fungal cell wall polysaccharide b-glucan that is selectively exposed during the invasive growth of opportunistic fungi. Notably, unmasking of b-glucan in the cell wall of a mutant of Histoplasma not only abrogates the pathogenicity of this fungus, but also triggers the induction of IL-23 producing DCs. Thus, b-glucan exposure in the fungal cell wall is essential for the induction of IL-23/T(H)-17 axis and may represent a key factor that regulates protective immunity to opportunistic but not pathogenic fungi.

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 (1×10⁶ cells per ml) following overnight stimulation with purified b-glucan (curdlan, 100 µg/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.

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.

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 µg/ml). Data are expressed as mean ± 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.

Figure 4. Enforced b-glucan surface exposure in the non-pathogenic Histoplasma Δags1 mutant drives IL-23 producing DCs.

(A) Representative confocal microscopy images of b-glucan surface staining in yeast cells in the Δags1 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 Histoplasma and the Δags1 mutant untreated or following overnight enzymatic digestion with b-glucanase (10 U/ml). IL-23 was selectively produced following stimulation of DCs with Δags1 mutant. Data are expressed as mean ± SEM values of four independent experiments. *, P<0.001, paired Student's t test.

Figure 5. IL-23 producing DCs induced by opportunistic fungi drive Th17 responses.

(A) IL-17 and IFN-γ production determined by ELISA in memory T cell cultures primed for 5 d in plates coated with anti-CD3 and anti-CD28, in the presence of supernatants (sups) of unstimulated DCs, or sups of DCs stimulated with either Aspergillus spores, or hyphae (above plots), and then re-stimulated for 24 h with anti-CD3 and anti-CD28. Data are expressed as mean ± SEM values for DCs derived from six different donors. (B) Intracellular cytokine staining for IL-17 and IFN-γ in memory CD4⁺ T cells primed and expanded as described in (A), and re-stimulated for 5 h with PMA and ionomycin. (C) ELISA of IL-17 and IFN-γ in 24-hour culture supernatants (sups) of naive and memory CD4⁺ T cells primed for 5 d in plates coated with anti-CD3 and anti-CD28, in the presence of sups of unstimulated DCs or Aspergillus spores-stimulated DCs or Aspergillus-hyphae stimulated DCs alone or in the presence of neutralizing anti-IL-23 antibodies. Data shown are representative of 5 independent experiments. Error bars represent SD. *, P<0.001, paired Student's t test.