Optimization of Aspergillus versicolor Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure

Abstract

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.

Keywords: Aspergillus; Aspergillus versicolor; aerosolization; allergen; murine nose-only exposure; repeated indoor fungal exposure.

Figure 1

Culture viability, morphology, and aerosolization of Aspergillus versicolor conidia. (A) A. versicolor was grown on sterile autoclave rice. Aliquots were tested for viability before and after heat inactivation. Asterisks represent statistical significance determined by t-test. **** p < 0.0001. n = 14/treatment. (B,C) A. versicolor conidia were aerosolized and examined via SEM. (D–F) Mass concentration of aerosolized spores. HIC—heat inactivated conidia.

Figure 2

Components of viable and heat-inactivated A. versicolor spores. (A) Whole protein lysates of live A. versicolor collected from malt extract agar (lane 2), rice (lane 3), and heat-inactivated conidia liberated from rice (lane 4). (B) Absolute values of secondary metabolites associated with A. versicolor, quantified from live (green) and inactivated (blue) conidia. Sterigmatocystin, aspercolorin, averantin, averufin, nidurufin, norsolorinic acid, seco-sterigmatocystin, versicolorin A, versicolorin C, versiconal acetate, and versiconol were quantified from aliquots of equivalent concentrations of cells (values are represented as ng/vial of 5 × 10⁵ spores). (C) Sterigmatocystin quantified from filters on the receiving end of the AGS. The negative control for this figure was a filter from an AOC pod. (D) Endotoxin quantified on rice and from filters on the receiving end of the AGS. The negative control for the rice grain section was sterile rice, and for the filter section was a filter from an AOC pod. Asterisks represent statistical significance, as determined by two-way ANOVA, comparing cell means within rows. * p < 0.05, *** p < 0.001, **** p < 0.0001; n = 3 per column.

Figure 3

Deposition of viable and heat-inactivated spores, via AGS, into the airways of mice. A single dose of A. versicolor was administered to live, awake mice via an acoustical generator system. Immediately following exposure, mice were euthanized, and the lungs were assessed using histopathology. (A) Conidia were quantified in the nose and lung of each mouse. Asterisks represent statistical significance determined by one-way ANOVA. * p < 0.05 n = 5/group (B) Representative GMS images of each exposure group focused on the nasal passage, alveoli, and bronchiole.

Figure 4

Aspergillus versicolor induces lymphoproliferation. A modified local lymph node assay was performed to identify lymphoproliferative doses of A. versicolor. A. fumigatus was used as a positive control, as it is known to be lymphoproliferative from prior studies. Values over the bars indicate the calculated SI value. Asterisks represent statistical significance, determined by one-way ANOVA. Asterisks over the bars indicate a comparison to the air-only control group. *** p < 0.001, **** p < 0.0001; n = 9–10 /group.

Figure 5

Four-week, repeated exposure murine dosimetry study. (A) Schematic of exposure paradigm. After a week, including two acclimations to the exposure pods, mice were exposed twice weekly for a total of eight exposures. One day after the following exposure, mice were euthanized, and samples were collected. (B) Conidia were quantified during each exposure and compiled for each group. Red, inverted triangles indicate aborted exposures that were later replaced. n = 8–9/group (C) Mouse body weight was recorded weekly to ensure animals were continuing to grow at the time of euthanasia. n = 5/group.

Figure 6

Flow cytometric analysis of cells infiltrating the lung following repeated exposure to A. versicolor. Flow cytometry was performed on cells from the bronchoalveolar lavage or the lung one-day post-final exposure to increasing doses of A. versicolor. Lymphocytes (A,E), alveolar macrophages (B,F), neutrophils (C,G) and eosinophils (D,H) were quantified as percent of single cells. Asterisks represent statistical significance as determined by one-way ANOVA with multiple comparisons. * p < 0.05, ** p < 0.01; n = 5/group.

Figure 7

Serum immunoglobulin quantification following a 4-week dosimetry study. Total concentrations of IgM ((A), n = 8/group), IgG ((B), n = 5/group), IgA ((C), n = 5/group), and IgE ((D), n = 5/group) were quantified in serum. Statistical significance was determined by one-way ANOVA with multiple comparisons, but no significant differences were observed.