Aspergillus fumigatus triggers inflammatory responses by stage-specific beta-glucan display

Abstract

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-alpha/MIP-2 secretion by alveolar macrophages. Fungal beta-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal beta-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed beta-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-alpha/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.

Figure 1. Live but Not Heat-Killed A. fumigatus Conidia Induce Inflammatory Responses

(A and B) In vivo cellular recruitment into lung airways. (A) Absolute number of BAL cells, total neutrophils (Ly6G^hi, CD11c⁻) in BAL, and total macrophages (CD11c⁺, Ly6G^int) in BAL for C57BL/6 mice 24 h after intratracheal instillation of PBS-Tw (vehicle), 10⁷ heat-killed, or 10⁷ live conidia. The bar graphs show the average cell numbers + standard deviation from four mice per group. One of three representative experiments is shown. (B) Flow cytometric analysis of live BAL cells stained for Ly6G and CD11c. Gates representing neutrophils (Ly6G^hi, CD11c⁻) and AMØs (CD11c⁺, Ly6G^int) are shown and the frequencies of these cell populations are indicated in representative BAL samples. (C–E) Ex vivo TNF-α/MIP-2 secretion by BMMØs (C), BMDCs (D), and AMØs (E) stimulated with live or heat-killed A. fumigatus conidia, PBS-Tw, or LPS (100 ng/ml) for 18 h in medium containing 0.5 μg/ml voriconazole. TNF-α/MIP-2 concentrations in the culture supernatants were determined by ELISA. In (C), the values for TNF-α/MIP-2 secretion induced by LPS were reduced by a factor of ten. The bar graphs represent the average cytokine production ± standard deviation by cells in 3–4 wells per condition.

Figure 2. Germinating A. fumigatus Conidia Are Highly Inflammatory

(A) Absolute number of BAL cells, total neutrophils in BAL, and total macrophages in BAL for C57BL/6 mice 24 h after intratracheal instillation with either heat-killed swollen conidia or live conidia. The bar graphs show the average cell numbers + standard deviation from four mice per group. (B) Flow cytometric analysis of live BAL cells stained for Ly6G and CD11c as in Figure 1B. (C) A. fumigatus conidia were allowed to initiate germination for 3, 5, or 7 h, heat-killed, and added to BMMØ cultures for 18 h. TNF-α/MIP-2 concentrations in the supernatants were determined by ELISA. The bar graphs represent the average ± standard deviation of three wells per condition. One of four experiments is shown.

Figure 3. Germinating but Not Resting A. fumigatus Conidia Display β-Glucans on the Cell Surface

(A) The mAb 744 detects β-glucans. Decreasing laminarin concentrations were coated onto ELISA plates in the presence or absence of laminarinase. 10 μg/ml mAb 744 was added to the wells and detected using an alkaline phosphatase conjugated anti-mouse IgM. (B–D) A. fumigatus conidia were incubated in RPMI for 7 h, stained with mAb 744 (B), mAb 744 depleted by zymosan (C), or IgM control antibody (D), followed by Alexa Fluor 594-anti-mouse IgM, and examined by confocal microscopy. Representative DIC/epifluorescence confocal images of resting conidia (open arrowheads), swollen conidia (black arrowheads), and early germlings (white arrowheads) are shown in overlay. Scale bar = 10 μm.

Figure 4. Dectin-1 Is Recruited to AMØ Phagosomes Containing Swollen Conidia with Surface-Exposed β-Glucans

(A–H) AMØs were harvested from C57BL/6 mice 45 min after intratracheal instillation of 10⁷ heat-killed swollen conidia (A–D) or heat-killed resting conidia (E–H), processed at 4 °C, and immunostained for ingested conidial β-glucans with mAb 744, followed by a FITC-coupled anti-mouse IgM (B and F) and for Dectin-1 (C and G) with goat anti-Dectin polyclonal antibodies, followed by Alexa Fluor 594-coupled anti-goat IgG. AMØs were examined by DIC microscopy (A and E) and epifluorescence confocal microscopy (B–D and F–H). (D and H) Merged images of β-glucan and Dectin-1 immunofluorescence. Scale bar = 10 μm.

Figure 5. Dectin-1- and MyD88-Mediated Signals Are Induced by A. fumigatus Conidia

(A–D) WT (white bars, [A–C]), MyD88^−/− (dark grey bars, [B–D]), and TLR-2^−/− (light grey bars, [B]) BMMØs (A–C) or AMØs (D) were stimulated with conidia for 18 h in medium containing 0.5 μg/ml voriconazole, and TNF-α/MIP-2 secretion was measured by ELISA. (A,C,D) BMMØs (A and C) and AMØs (D) were incubated with 2A11 (anti-Dectin-1 mAb) or an isotype control antibody in the presence of 2 μM cytochalasin D. (A and B) ELISA results from test conditions were averaged among three to five experiments and expressed as a percentage ± standard deviation of the averaged value obtained for the control condition. (C and D) show representative experiments. All experiments were performed with three to six replicates per condition.