Factors Contributing to Sex Differences in Mice Inhaling Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a respiratory fungal pathogen and an allergen, commonly detected in flooded indoor environments and agricultural settings. Previous studies in Balb/c mice showed that repeated inhalation of live and dry A. fumigatus spores, without any adjuvant, elevated allergic immune response and airway remodeling. Sex-specific differences can influence host-pathogen interactions and allergic-asthma related outcomes. However, the effect of host sex on immune response, in the context of A. fumigatus exposure, remains unknown. In this study, we quantified the multivariate and univariate immune response of C57BL/6J mice to live, dry airborne A. fumigatus spores. Our results corroborate previous results in Balb/c mice that repeated inhalation of live A. fumigatus spores is sufficient to induce mucus production and inflammation by day 3 post last challenge, and antibody titers and collagen production by day 28 post-challenge. Principal Component Analysis (PCA) showed that females exhibited significantly higher levels of immune components than males did. Taken together, our data indicate that host-sex is an important factor in shaping the immune response against A. fumigatus, and must be considered when modeling disease in animals, in designing diagnostics and therapeutics for A. fumigatus-associated diseases or while drafting evidence-based guidelines for safe mold levels.

Keywords: IgE; airway remodeling; collagen; mucus; principal component analysis.

Figure 1

The study design. Six weeks old male or female C57BL/6J mice were anesthetized with an intraperitoneal injection of ketamine and xylazine, and laid in a supine position with their nostrils sticking into the ports of the inhalation chamber [11]. An 8-day-old culture of Aspergillus fumigatus on Saboraud Dextrose Agar was hooked to the air supply on the right end of the chamber and steady airflow blew off the dry, live A. fumigatus spores, which were inhaled by the mice. This challenge was repeated for 10 min., once per week for three weeks. Naïve (untreated) mice were maintained as controls. The mice were euthanized at days three or twenty-eight post third fungal challenge. Serum and bronchoalveolar lavage fluid (BALF) were collected and stored at -20 °C for antibody analysis. The bronchoalveolar lavage (BAL) was cytospun and stained with Quick-Dip stains for leukocyte analysis and whole left lungs were fixed, sectioned at 5 µm and stained with Sirius Red/Fast Green or Periodic Acid Schiff stains for collagen or mucus/goblet cell metaplasia, respectively. The figure was created with BioRender.com.

Figure 2

Principal Component Analysis to assess the effect of different factors on immune response mounted by male and female mice at days 0 (naïve), 3 or 28 post third A. fumigatus challenge. (A) Eigenvalues for 10 principal components showed that the first two components had eigenvalues greater than 1 and captured 74.792% of the variance. (B) Principal Component 1 versus 2 graph showed different clusters for day 0 (naïve), 3 and 28 mice. Male and female mice clustered differently on all timepoints (p value = 0.004), and the distance between the two sexes increased with time post-challenge (p < 0.001).

Figure 3

Antibody titers (mean ± SEM) in serum (A and B) or bronchoalveolar lavage fluid (C) obtained from male and female mice at days 0 (naïve), 3 or 28 post third A. fumigatus challenge. *, ***; p-value ≤ 0.05 and 0.001, respectively, indicate a comparison of mean ± SEM values between day 0 (naïve) and A. fumigatus challenged mice. The solid line and corresponding p-values indicate the pairwise comparisons between the sexes at each timepoint.

Figure 4

Leukocyte counts (mean ± SEM) observed in the bronchoalveolar lavage (BAL) wash obtained from male and female mice at days 0 (naïve), 3 or 28 post third A. fumigatus challenge. *, **, ***; p-value ≤ 0.05, 0.01 and 0.001, respectively, indicate a comparison of mean ± SEM values between day 0 (naïve) and A. fumigatus challenged mice. Average cells per high power field (hpf) are represented in (A) Neutrophils; (B) Eosinophils; (C) Lymphocytes; (D) Macrophages; (E) Total BAL cells

Figure 5

Collagen production observed in lung sections obtained from male and female mice at days 0 (naïve), 3 or 28 post third A. fumigatus challenge. The collagen deposition (pinkish red thread like structures indicated by black arrows) around five random terminal airways were scored for each mouse, by two blinded personnel, and the mean ± SEM values are reported in (G). The scale bars in (A–F) represented by red lines correspond to 60 µm. ***, ****; p-value ≤ 0.001 and 0.0001, respectively, indicate the comparison of day 0 (naïve) and A. fumigatus challenged mice.

Figure 6

Mucus production observed in lung sections obtained from male and female mice at days 0 (naïve), 3 or 28 post third A. fumigatus challenge. The mucus production and goblet cell metaplasia (pinkish-purple stain indicated by black arrows) around five random terminal airways were scored for each mouse, by two blinded personnel, and the mean ± SEM values are reported in (G). The scale bars in (A–F) represented by red lines correspond to 60 µm. ***, ****; p-value ≤ 0.001 and 0.0001, respectively, indicate the comparison of day 0 (naïve) and A. fumigatus challenged mice.