Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling

Abstract

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Keywords: Stachybotrys chartarum fungal fragmentation; fungal exposure; pulmonary arterial remodeling.

Figure 1.

Fragmentation of Stachybotrys chartarum. Aerodynamic particle size distributions of (A) viable and (B) heat-inactivated S. chartarum conidia (HIC) collected during exposures. Data are a representation of the particle size distribution observed over multiple exposures and have been normalized to 1,000 particle counts for comparison. S. chartarum = Stachybotrys chartarum.

Figure 2.

Pulmonary arterial remodeling. Representative photomicrographs of hematoxylin and eosin–stained sections of lung from mice exposed to viable (A) strain A and (B) strain B. The mice were killed at 24 hours after 4 or 13 weeks of exposure. Lung from an air-exposed mouse is shown for reference. (C) Representative photomicrographs showing the range of changes seen in pulmonary arteries in lungs of mice exposed to viable fungal conidia. The blue arrows indicate different inflammatory cells (i.e., eosinophils and macrophages). Scale bars: 200 μm and 100 μm. PA = pulmonary arteries (indicated by black arrows); TB = terminal bronchiole.

Figure 2.

Pulmonary arterial remodeling. Representative photomicrographs of hematoxylin and eosin–stained sections of lung from mice exposed to viable (A) strain A and (B) strain B. The mice were killed at 24 hours after 4 or 13 weeks of exposure. Lung from an air-exposed mouse is shown for reference. (C) Representative photomicrographs showing the range of changes seen in pulmonary arteries in lungs of mice exposed to viable fungal conidia. The blue arrows indicate different inflammatory cells (i.e., eosinophils and macrophages). Scale bars: 200 μm and 100 μm. PA = pulmonary arteries (indicated by black arrows); TB = terminal bronchiole.

Figure 3.

Cell populations of BAL fluid (BALF) after exposure to strain A and strain B. (A–F) BALF collected at 24 and 48 hours after 4- and 13-week exposures to strain A (A–C) or strain B (D–F) consisted of eosinophils (A and D), neutrophils (B and E), and macrophages (C and F). Values are expressed as mean ± SEM; n = 7 per group. *P ≤ 0.05 for exposed group versus air-only control group. ns = not significant.

Figure 4.

Cell populations of BAL fluid after exposure to strain A HIC and strain B HIC. (A–F) BALF collected at 24 and 48 hours after a 4- and 13-week exposure to strain A HIC (A–C) or strain B HIC (D–F) consisted of eosinophils (A and D), neutrophils (B and E), and macrophages (C and F). Values are expressed as mean ± SEM; n = 7 per group. *P ≤ 0.05 for exposed group versus air-only control group.

Figure 5.

Heat map of genes involved in the T-helper cell type 1 (Th1) and Th2 immune response pathways. Altered genes involved in the Th1 and Th2 response pathways after 4 weeks and 13 weeks of exposure to strain A and to strain B. Genes are color coded (red and green for up- and downregulation, respectively) and are arranged by Euclidean distance; n = 3–5 per group.

Figure 6.

Heat map of genes involved in the Th17 immune response pathway. Altered genes involved in the Th17 response pathways after 4-week and 13-week exposures to strain A and to strain B. Genes are color coded (red and green for up- and downregulation, respectively) and are arranged by Euclidean distance; n = 3–5 per group.