Dectin-1 and IL-17A suppress murine asthma induced by Aspergillus versicolor but not Cladosporium cladosporioides due to differences in β-glucan surface exposure

Abstract

There is considerable evidence supporting a role for mold exposure in the pathogenesis and expression of childhood asthma. Aspergillus versicolor and Cladosporium cladosporioides are common molds that have been implicated in asthma. In a model of mold-induced asthma, mice were repeatedly exposed to either A. versicolor or C. cladosporioides spores. The two molds induced distinct phenotypes, and this effect was observed in both BALB/c and C57BL/6 strains. C. cladosporioides induced robust airway hyperresponsiveness (AHR), eosinophilia, and a predominately Th2 response, whereas A. versicolor induced a strong Th17 response and neutrophilic inflammation, but very mild AHR. Neutralization of IL-17A resulted in strong AHR and eosinophilic inflammation following A. versicolor exposure. In Dectin-1-deficient mice, A. versicolor exposure resulted in markedly attenuated IL-17A and robust AHR compared with wild-type mice. In contrast, C. cladosporioides induced AHR and eosinophilic inflammation independent of IL-17A and Dectin-1. A. versicolor, but not C. cladosporioides, spores had increased exposure of β-glucans on their surface and were able to bind Dectin-1. Thus, the host response to C. cladosporioides was IL-17A- and Dectin-1-independent, whereas Dectin-1- and IL-17A-dependent pathways were protective against the development of asthma after exposure to A. versicolor.

Figure 1. A. versicolor and C. cladsoporioides induce distinct inflammatory phenotypes

(A) BALB/c mice were exposed IT to 10⁶ A. versicolor (A. ver) or C. cladosporioides (C. clad) spores 3 times a week for 3 weeks and 48 hours after the last challenge, AHR was measured by Flexivent; (B) BALF was analyzed for total and differential counts; (C) total serum IgE. Data are representative of 3 independent experiments are expressed as mean and SEM. N=6-8 mice per group. p<0.05, **p<.01, and ***p<0.001 as compared to saline. #p<0.05, ##p<0.01, and ###p<0.001 as compared to A. versicolor.

Figure 2. Similar Distribution of A. versicolor and C. cladosporioides in the lungs of exposed mice

(A) Silver stained sections from mice 24 hours following a single exposure to 10⁶ spores at 400x magnification. Arrows indicate pulmonary antigen presenting cells containing mold spores. Scale bars are 50um (B) Quantification of spores per high power field from silver stained sections. Data are representative of 2 independent experiments and are expressed as mean and SEM. N=3-4 mice per group.

Figure 3. A. versicolor induces Th17 cells while C. cladsoporioides induces Th2 cells

(A) Contour plots and (B) quantification of intracellular cytokine staining for IL-13 or IL-17A in CD4+ lung cells. (C) Relative levels of mRNA for IL-17A and IL-4 in whole lung. Data are representative of 3 independent experiments are expressed as mean and SEM. N=6-8 mice per group. *p<0.05, **p<0.01, and ***p<0.001 as compared to saline. ##p<0.01 and ###p<0.001 as compared to A. versicolor.

Figure 4. A. versicolor induces allergic airway disease when IL-17A is blocked

(A) BALB/c mice were exposed to A. versicolor or C. cladosporioides spores and treated with either isotype control antibody or anti-IL-17A and 48 hours after the last challenge AHR. (B) Comparison of AHR data at 100mg/ml of methacholine. (C) BALF total and differential counts. (D) Relative levels of mRNA for IL-5 in whole lung. Data are representative of 2 independent experiments and expressed as mean and SEM. N=5-6 mice per group. *p<0.05 and **p<0.01, and ***p<0.001 as compared to saline. #p<0.05, ##p<0.01, and ###p<0.001 as compared to A. versicolor or C. cladosporioides isotype antibody treated mice.

Figure 5. Increased Th2 cells and decreased Th17 cells in Dectin-1 -/- mice exposed to A. versicolor

(A) Contour plots and (B,C) quantification of intracellular cytokine staining for IL-17A or IL-13 in CD4+ cells isolated from the lungs of A. versicolor exposed mice (B) or C. cladosporioides exposed mice (C). Data are representative of 3 independent experiments and expressed as mean and SEM. N=4-6 mice per group. *p<0.05, **p<0.01, and ***p<0.001 as compared to saline. ##p<0.01 and ###p<0.001 as compared to A. versicolor wild type mice.

Figure 6. Dectin-1 protects against allergic inflammation induced by A. versicolor

(A, C) Wild type C57Bl/6 or Dectin-1 -/- mice were exposed to A. versicolor (A) or C. cladosporioides (C) spores and AHR was assessed 48 hours later. (B, D) Total and differential counts of BALF for A. verisoclor (B) or C. cladosporioides (D) exposed mice. Data are representative of 3 independent experiments and expressed as mean and SEM. N=4-6 mice per group. *p<0.05, **p<.01, and ***p<0.001 as compared to saline. #p<0.05, ##p<0.01, and ###p<0.001 compared to exposed Dectin-1 -/- mice.

Figure 7. A. versicolor spores have increased beta-glucan surface availability and Dectin-1 binding compared to C. cladosporioides spores

(A) Immunofluorescence images of beta-glucan staining taken at 1000x for A. versicolor and C. cladosporioides spores. Scale bar is 10um. (B) Dectin-1 binding assay for A. versicolor and C. cladosporioides spores. Data are representative of 2 independent experiments and are expressed as mean and SEM. N=3 samples per group.

Figure 8. Working model

Exposure of beta-glucans (red) on the surface of A. versicolor promote signaling through Dectin-1 to induce a Th17 response. In the absence of Dectin-1, A. versicolor signals through an alternate pathway to induce Th2 cells and asthma. Beta-glucans (red) in C. cladosporioides are not readily available on the surface (black), thus preventing signaling through a Dectin-1, and promoting the use of an alternate pathway to induce a Th2 response and asthma.