Eosinophil deficiency compromises lung defense against Aspergillus fumigatus

Abstract

Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated with A. fumigatus exposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance of A. fumigatus from the lung. Acute A. fumigatus challenge in normal mice induced the recruitment of CD11b+ Siglec F+ Ly-6G(lo) Ly-6C(neg) CCR3+ eosinophils to the lungs, which was accompanied by an increase in lung Epx (eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impaired A. fumigatus clearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression of Epx (eosinophil peroxidase) and Prg2 (major basic protein) as well as lower interleukin 1β (IL-1β), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity against A. fumigtaus in vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response after A. fumigatus exposure.

FIG 1

Eosinophils are recruited to lung after A. fumigatus challenge. (A) BALB/c wild-type mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia (AF), and 24 h postexposure, whole lungs were collected, total RNA was isolated and transcribed to cDNA, and quantitative real-time PCR was performed for Epx. Gene expression was normalized to Gapdh, and fold changes between naive mice (set at 1) and A. fumigatus-exposed BALB/c mice were determined using the 2^−ΔΔCT method. Cumulative data from three independent studies (n = 3 to 5 mice per group per study) are illustrated. *, P value of <0.05 (paired two-tailed Student t test). (B) BALB/c wild-type mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia, and 24 or 48 h postexposure, lung cells were isolated via bronchoalveolar lavage, Fc blocked, stained with a LIVE/DEAD staining kit, and thereafter stained with fluorochrome-conjugated CD11c, CD11b, Gr-1, and Siglec F. Cumulative data from three independent studies (n = 2 or 3 mice per group per time point per study) are illustrated. Data are expressed as absolute numbers of live cells in lung lavage fluid. * and ***, P values of <0.05 and <0.0001, respectively (unpaired two-tailed Student t test).

FIG 2

Enhanced susceptibility of eosinophil-deficient mice after challenge with A. fumigatus. (A) BALB/c WT and dblGATA1-deficient (dblGATA1) mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia, and 24 and 48 h after exposure, lung fungal burden was assessed by real-time PCR analysis of A. fumigatus 18S rRNA levels. Cumulative data from two independent studies (n = 4 or 5 mice per group per time point per study) are illustrated. Data are expressed as means + standard errors of the means. * and ***, P values of <0.05 and <0.0001, respectively (unpaired two-tailed Student t test). (B) Representative GMS-stained lung sections of two individual WT (top) and dblGATA1-deficient (bottom) mice 48 h post-A. fumigatus challenge. (C to E) WT and dblGATA1-deficient mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia, and lung cells were isolated via bronchoalveolar lavage, Fc blocked, stained with a LIVE/DEAD staining kit, and thereafter stained with fluorochrome-conjugated antibodies differentiating neutrophils (C), dendritic cells (D), and monocytes/macrophages (E). Cumulative data from three independent studies (n = 3 or 4 mice per group per study) are illustrated. Data are expressed as absolute numbers of live cells in lung lavage fluid.

FIG 3

Eosinophil-deficient mice challenged with A. fumigatus demonstrate reductions in specific eosinophil antimicrobial factors. BALB/c WT and dblGATA1-deficient (dblGATA1) mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia, and 24 and 48 h after exposure, whole lungs were collected, total RNA was isolated and transcribed to cDNA, and quantitative real-time PCR was performed for Epx (A), Prg2 (B), Ear1 (C), and Ear2 (D). Gene expression was normalized to Gapdh, and fold changes between WT mice (set at 1) and dblGATA1-deficient mice were determined using the 2^−ΔΔCT method. Cumulative data from two independent studies (n = 4 or 5 mice per group per time point per study) are illustrated. *, **, and ***, P values of <0.05, <0.01, and <0.0001, respectively (paired two-tailed Student t test).

FIG 4

Eosinophil-deficient mice challenged with A. fumigatus demonstrate differences in lung proinflammatory cytokine and chemokine levels. BALB/c WT and dblGATA1-deficient (dblGATA1) mice were challenged intratracheally with 7 × 10⁷ A. fumigatus conidia, and 24 and 48 h after exposure, levels of IL-1β (A), IL-6 (B), IL-17A (C), G-CSF (D), (E) GM-CSF (E), and CXCL1 (F) were quantified in lung homogenates by Bio-Plex. Cumulative data from two or three independent studies (n = 4 to 6 mice per group per time point per study) are illustrated. Data are expressed as mean concentrations + standard errors of the means. * and ***, P values of <0.05 and 0.001, respectively (unpaired two-tailed Student t test).

FIG 5

Eosinophils recognize and respond to A. fumigatus. Bone marrow cells were isolated from naive BALB/c mice and cultured with 100 ng/ml of stem cell factor and 100 ng/ml of FLT3-L. After 4 days, cells were replated in medium supplemented with 10 ng/ml of IL-5 for an additional 6 days. (A) Representative Giemsa-stained cytospin of bone marrow-derived eosinophils at day 10 of culture showing characteristic nuclear structure and secretory granules. (B) Bone marrow-derived eosinophils (1 × 10⁵ cells) were cultured in the presence of A. fumigatus conidia (3 × 10⁵) in a total volume of 100 μl for 24 h (AF + Eos). Controls included A. fumigatus conidia (3 × 10⁵) cultured alone in a volume of 100 μl for 24 h (AF alone). Cumulative data from four independent studies are illustrated. Each dot represents a single well. ***, P value of <0.0001 (unpaired two-tailed Student t test). (C) Bone marrow-derived eosinophils (1 × 10⁶ cells; 100 μl) were cultured in the top chamber of a 0.4-μm-pore-containing Transwell membrane in the presence of A. fumigatus conidia (3 × 10⁶; 300 μl) for 24 h [Eos (Top)/AF (Bottom)]. Controls included 100 μl of medium in the top chamber in the presence of A. fumigatus conidia (3 × 10⁶; 300 μl) cultured alone in the bottom chamber for 24 h [Media (Top)/AF (Bottom)]. Cumulative data from three independent studies are illustrated. Each dot represents a single well. ***, P value of <0.0001 (unpaired two-tailed Student t test). (D) Bone marrow-derived eosinophils (1 × 10⁶ cells) were lysed in 100 μl of PhosphoSafe extraction buffer (10 min at room temperature, followed by centrifugation at 12,000 × g for 10 min). Lysates (100 μl) were added in triplicate to 3 × 10⁵ A. fumigatus conidia in a volume of 100 μl for 24 h. Controls included PhosphoSafe extraction buffer (100 μl) added in triplicate to 3 × 10⁵ A. fumigatus conidia in a volume of 50 μl for 24 h. Cumulative data from three independent studies are illustrated. Each dot represents a single well. ***, P value of <0.0001 (unpaired two-tailed Student t test). (E and F) Bone marrow-derived eosinophils were cultured as for panel B, and supernatants were collected after 24 h and clarified by centrifugation. IL-1β, CCL2, and CCL4 (E) and IL-4, IL-13, IL-9, and CCL11 (F) were quantified by Bio-Plex. Controls included bone marrow-derived eosinophils cultured in medium alone (gray columns). Cumulative data from two independent studies are illustrated. ** and ***, P values of <0.01 and <0.0001, respectively (unpaired two-tailed Student t test). Un, unstimulated.