Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus

Abstract

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the TLR family are not absolutely required for host defense against A. fumigatus in nonimmunosuppressed hosts, suggesting that other pattern recognition receptors are involved. We show in this study that naive mice (i.e., not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1(-/-)) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1(-/-) mice demonstrated impaired IL-1alpha, IL-1beta, TNF-alpha, CCL3/MIP-1alpha, CCL4/MIP-1beta, and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1(-/-) mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1(-/-) mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent, and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Figure 1. Enhanced susceptibility of Dectin-1 deficient mice after challenge with A. fumigatus

Wild-type and Dectin-1^−/− mice were challenged intratracheally with (A) 5 × 10⁷ conidia in a volume of 50 ul or (B) 7 × 10⁷ conidia in a volume of 50 ul and survival was monitored for 5 days. The Figure illustrates cumulative results from two independent studies per inoculum dose. *** represents a P value of < 0.001 (log rank Mantel-Cox test). (C) Real-time PCR analysis of A. fumigatus 18S rRNA levels in the lungs of wild-type and Dectin-1^−/− mice challenged intratracheally with 7 × 10⁷ conidia and collected at 24 h and 48 h post-challenge. The Figures illustrates cumulative data from three independent studies (n = 5 mice/group for each study). Data are expressed as mean A. fumigatus 18S rRNA levels + SEM. * represents a P value of < 0.05 (Unpaired two-tailed Student’s t test).

Figure 2. Defective A. fumigatus recognition by Dectin-1 deficient neutrophils

(A) Neutrophil levels in BAL fluid were assessed by flow cytometry of individual mice 24 h after intratracheal challenge with 7 × 10⁷ conidia. The Figure illustrates data from one of two independent studies (n = 5 mice/group). Data are expressed as neutrophils/ml of BAL fluid + SEM. ** P < 0.01 (Unpaired two-tailed Student’s t test). (B) Neutrophils were isolated from the peritoneum of wild-type and Dectin-1^−/− mice challenged with 3% thioglycollate for 14 h and co-cultured with live A. fumigatus swollen conidia for 2 h. ROS production was assessed using dihydrorhodamine 123 conversion. The Figure illustrates cumulative data in triplicate wells from one of two independent studies. Data are expressed as mean fluorescent intensity ± SEM. ** and *** represent a P value of < 0.01 and 0.001, respectively (Unpaired two-tailed Student’s t test). (C) Peritoneal neutrophils were isolated and co-cultured with live A. fumigatus swollen conidia at a cell to organism ratio of 1:1 for 2-4 h. Controls included live A. fumigatus swollen conidia cultured in medium alone for 2-4 h. Thereafter, RNA was isolated from wild-type, Dectin-1^−/− and A. fumigatus cultures and A. fumigatus 18S rRNA levels were quantified by real-time PCR. The Figure illustrates cumulative results from two independent studies each containing triplicate wells for wild-type, Dectin-1^−/− and A. fumigatus cultures. Data are expressed as mean A. fumigatus 18S rRNA levels + SEM. * represents a P value of < 0.05 comparing wild-type to Aspergillus; # represents a P value of < 0.05 comparing wild-type to Dectin-1^−/− (Unpaired two-tailed Student’s t test).

Figure 3. Histological evidence for A. fumigatus invasion and lung damage in Dectin-1 deficient mice

Representative H&E-stained (left) and GMS-stained (right) lung sections from (A) wild-type mice and (B) Dectin-1^−/− mice challenged intratracheally with 7 × 10⁷ conidia for 48 h. Original magnification of 200X. Scale bar in A, left image represents 100 μm.

Figure 4. Requirement for Dectin-1 in the inflammatory response to A. fumigatus in vivo and in vitro

(A) Wild-type and Dectin-1^−/− mice were challenged intratracheally with 7 × 10⁷ conidia and 24 h after exposure, IL-α, IL-1β, TNF-α, CCL3/MIP-α, CCL4/MIP-1β and CXCL1/KC levels were quantified in lung homogenates by Bio-Plex. The Figure illustrates cumulative data from four independent studies (n = 5 mice/group for each study). Data are expressed as mean pg/ml + SEM. *, ** and *** represent a P value of < 0.05, 0.01 and 0.001, respectively (Unpaired two-tailed Student’s t test). (B) Alveolar macrophages were isolated via BAL from wild-type or Dectin-1^−/− mice and cultured with live A. fumigatus conidia for 24 h at a macrophage to conidium ratio of 1:1. Cytokine/chemokine concentrations in co-culture supernatants were determined by Bio-Plex. Unstimulated levels have been subtracted from each column. The Figure illustrates cumulative results from four independent studies. Data are expressed as mean pg/ml + SEM. * and ** represents a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test).

Figure 5. Compromised lung function in A. fumigatus-challenged Dectin-1 deficient mice

Wild-type and Dectin-1^−/− mice were challenged intratracheally with 4 × 10⁷ conidia and 72 h after exposure, respiratory mechanics were analyzed via mechanical ventilation using the flexiVent system. Pulmonary function measurements were (A) airway resistance (R_N) and (B) whole lung resistance (R). The Figure illustrates cumulative data from two independent studies (n = 5 mice/group for each study. Data are expressed as mean airway or whole lung resistance ± SEM. * and *** P < 0.05 and < 0.001, respectively (Unpaired two-tailed Student’s t test).

Figure 6. IL-17 levels in the lungs of Dectin-1 deficient mice exposed to A. fumigatus

Wild-type and Dectin-1^−/− mice were challenged intratracheally with 7 × 10⁷ conidia and 24 h after exposure, (A) IL-17, (B) IL-12p40 and (C) IL-23 levels were quantified in lung homogenates. The Figure illustrates cumulative data from three independent studies (n = 5 mice per group per time point). Data are expressed as mean pg/ml + SEM. * and ** represent a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test).

Figure 7. Neutralization of IL-17 impairs early A. fumigatus lung clearance

Wild-type and Dectin-1^−/− mice were challenged intratracheally with 7 × 10⁷ conidia. Six hours after challenge, mice were administered 50 μg of rat anti-mouse IL-17 or rat IgG2A antibodies intratracheally. IL-17 levels were quantified in lung homogenates (A) 24 h and (C) 48 h after challenge by Bio-Plex. The Figure illustrates cumulative data from two independent studies (n = 5 mice per group per time point). Data are expressed as mean pg/ml + SEM. * and ** represents a P value of < 0.05 and 0.01, respectively (Unpaired two-tailed Student’s t test). Real-time PCR analysis of A. fumigatus 18S rRNA levels in the lungs of wild-type and Dectin-1^−/− mice administered anti-IL-17 or isotype control antibodies (B) 24 h and (D) 48 h after challenge. The Figure illustrates cumulative data from two independent studies (n = 5 mice per group per time point). Data are expressed as mean A. fumigatus 18S rRNA + SEM. * represents a P value of < 0.05 (Unpaired two-tailed Student’s t test).