A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry <i>Aspergillus fumigatus</i> Conidia

<div><p>Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from <i>Aspergillus fumigatus</i> wild-type (WT) or a melanin-deficient (<i>Δalb1</i>) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of <i>A. fumigatus</i> WT spores. Germination was associated with specific IgG to intracellular proteins while <i>Δalb1</i> spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.</p></div

Total cell counts in the BALF.

<p>Following 8 dry conidial exposures, mice were sacrificed at the indicated time points to determine the kinetics of the cellular influx to the lung. Total cell numbers were obtained through acridine orange staining and quantified using an automated cell counter. Each cell population was quantified by multiplying the frequency of each by the total cell counts. Data are presented as the average ± standard error of measure. (Control n = 30 mice/time point, Exposed n = 7–10 mice/group/time point). ****P≤0.0001, ***P≤0.001, **P≤0.01, *P≤0.05.</p

Histopathology of sections derived from <i>Aspergillus fumigatus</i> WT exposed mice.

<p>A) Representative histopathology sections from WT exposed mice sacrificed at the indicated time points. Top panel-H&E stained sections at 100× magnification, Bottom panel-PAS stained sections at 100× magnification. B) GMS stained sections at 400× magnification. Black arrow heads indicate swollen conidia (24 hr), while red arrow heads indicate conidia germination and emergence of hyphal tubes (48 hr). C) Quantification of conidia and germination (swollen conidia+germ tube formation) over time. Values were obtained by quantifying the number of conidia visualized by counting 100 random fields of view covering both lung fields at a magnification of 400×. Conidia were considered swollen when the size was >2× that of resting conidia. Data are presented as the average ± standard error of measure. ****P≤0.0001, ***P≤0.001, **P≤0.01.</p

Intracellular cytokine flow cytometry analysis of the BALF. IL-17⁺ CD4⁺ and CD8⁺ T cells were quantified by multiplying the frequency of each individual cell population by the total cell counts.

<p>Mice were sacrificed at the indicated time points after the 8th exposure. Data are presented as the average ± standard error of measure. n = 7–10 mice/group per time point. ****P≤0.0001, **P≤0.01.</p

Preliminary acoustical generator exposure chamber experiments.

<p>A) Illustration showing the acoustical generator inhalation exposure system. Supply air is HEPA filtered and directed into the acoustical generator. The acoustical generator is then sent a signal to vibrate at a designated frequency resulting in the formation of fungal aerosols from inoculated rice grains. The fungal aerosol is then directed into a multi-animal, nose-only chamber. After passing through the animal's breathing zone the air is filtered before being sent into the exhaust system. A real-time particle counter attached to the computer calculates the concentration of fungal particles being deposited into the airways, and the DataRAM reports that number to the computer, which can be altered during the exposure to obtain the desired deposition concentration. B) The <i>Aspergillus fumigatus</i> aerosol particle size distribution produced by the acoustical generator. The size of single or aggregate <i>A. fumigatus</i> conidia is 2–5 µm. C) Field emission scanning electron microscopy image of <i>A. fumigatus</i> fungal conidia deposited on a polycarbonate filter collected from one of the sampling ports of the nose-only exposure chamber.</p

Proteomic analysis of immunoreactive conidia protein bands identified in sIgG Western blot.

<p>Proteomic analysis of immunoreactive conidia protein bands identified in sIgG Western blot.</p

Specific IgG from <i>A. fumigatus</i> WT or <i>Δalb1</i> exposed mice.

<p>Western blot analysis of specific IgG in serum from mice exposed to acoustically aerosolized conidia from (A) WT or (B) <i>Δalb1</i> strains. Lanes 1-molecular weight markers, 2-ASF WT conidial extract, 3-<i>Δalb1</i> conidial extract, 4-ASF WT hyphal extract, 5-Δalb1 hyphal extract (10 µg protein/lane). Numbered bands were excised and identified using LC/MS analyses. The asterisks (*) denote weak binding of WT–exposed serum antibody to hydrophobin. C) ELISA analysis of IgG specific for <i>A. fumigatus</i> WT and <i>Δalb1</i> conidial proteins or hyphal extracts. Results are representative of the mean OD₄₀₅ values for each mouse sera diluted 1∶200 ± the standard deviation of duplicate ELISA wells coated with 3 µg/mL protein.</p