Persisting Cryptococcus yeast species Vishniacozyma victoriae and Cryptococcus neoformans elicit unique airway inflammation in mice following repeated exposure

Abstract

Background: Allergic airway disease (AAD) is a growing concern in industrialized nations and can be influenced by fungal exposures. Basidiomycota yeast species such as Cryptococcus neoformans are known to exacerbate allergic airway disease; however, recent indoor assessments have identified other Basidiomycota yeasts, including Vishniacozyma victoriae (syn. Cryptococcus victoriae), to be prevalent and potentially associated with asthma. Until now, the murine pulmonary immune response to repeated V. victoriae exposure was previously unexplored.

Objective: This study aimed to compare the immunological impact of repeated pulmonary exposure to Cryptococcus yeasts.

Methods: Mice were repeatedly exposed to an immunogenic dose of C. neoformans or V. victoriae via oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were collected to examine airway remodeling, inflammation, mucous production, cellular influx, and cytokine responses at 1 day and 21 days post final exposure. The responses to C. neoformans and V. victoriae were analyzed and compared.

Results: Following repeated exposure, both C. neoformans and V. victoriae cells were still detectable in the lungs 21 days post final exposure. Repeated C. neoformans exposure initiated myeloid and lymphoid cellular infiltration into the lung that worsened over time, as well as an IL-4 and IL-5 response compared to PBS-exposed controls. In contrast, repeated V. victoriae exposure induced a strong CD4⁺ T cell-driven lymphoid response that started to resolve by 21 days post final exposure.

Discussion: C. neoformans remained in the lungs and exacerbated the pulmonary immune responses as expected following repeated exposure. The persistence of V. victoriae in the lung and strong lymphoid response following repeated exposure were unexpected given its lack of reported involvement in AAD. Given the abundance in indoor environments and industrial utilization of V. victoriae, these results highlight the importance to investigate the impact of frequently detected fungal organisms on the pulmonary response following inhalational exposure. Moreover, it is important to continue to address the knowledge gap involving Basidiomycota yeasts and their impact on AAD.

Keywords: Cryptococcus neoformans; Vishniacozyma victoriae; allergic disease; exposure; fungi; inflammation; yeast.

Figure 1

Exposure Paradigms. Mice were exposed to (A) set doses of either C. neoformans, V. victoriae, or PBS control via oropharyngeal aspiration. (B) Deposition was confirmed by exposing mice one time and then collecting tissue for analysis approximately 1 hour after the exposure. (C) For the repeated exposure study, mice were repeatedly exposed to the same dose of yeast or PBS control every other day for a total of six exposures, with tissue collection occurring either 1 day or 21 days after the final exposure.

Figure 2

C. neoformans-induced inflammation worsens over time, whereas V. victoriae-induced inflammation resolves. (A–C) Representative micrographs of stained lung sections from mice one day (left) or 21 days (right) following repeated exposure to PBS (top), 10⁴ C. neoformans (middle), or 10⁶ V. victoriae cells (bottom). (A) H&E stain, 100x magnification. (B) PAS-stain, 200x magnification. (C) GMS-stain, 400x magnification. (D) Inflammation, (E) Perivascular Mononuclear Inflammation, (F) Peribronchial Mononuclear Inflammation and (G) Goblet Cells histology scores of lungs following repeated exposure to PBS (black), 10⁴ C. neoformans cells (yellow) or 10⁶ V. victoriae cells (blue) at 1 day post final exposure (circles, crossed bars, left) or 21 days post final exposure (squares, solid bars, right). n=3 per group, *P<0.05, **P < 0.01. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test comparing each exposure together for the same timepoint.

Figure 3

Inflammation, airway changes, and yeast infiltration in lungs of mice after repeated exposure to 10⁴ V. victoriae cells. Representative micrographs of stained sections of lung from mice 1 day (top) and 21 days (bottom) following repeated exposure 10⁴ V. victoriae cells. Goblet cells are by arrowheads, and yeast cells by arrows. (A, D) H&E stain, 100x magnification. (B, E) PAS-stain, 200x magnification. (C, F) GMS-stain, 400x magnification.

Figure 4

C. neoformans exposure has a stronger impact on myeloid populations in the BALF over time compared to V. victoriae. Cell quantifications (numbers) in BALF following repeated exposure to PBS (black), 10⁴ C. neoformans cells (yellow) or 10⁶ V. victoriae cells (blue) at 1 day post final exposure (circles, crossed bars, left) or 21 days post final exposure (squares, solid bars, right). (A) CD45⁺ Cells, (B) Neutrophils, (C) Ly6C^hi/med Monocytes, (D) CD11b⁺ Dendritic Cells, and (E) CD103⁺ Dendritic Cells. n=3-7 per group, *P<0.05, **P < 0.01, ***P < 0.001. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test comparing each exposure together for the same timepoint.

Figure 5

Lymphoid populations are elevated in the BALF 21 days following repeated yeast exposure. Cell populations in BALF following repeated exposure to PBS (black), 10⁴ C. neoformans cells (yellow) or 10⁶ V. victoriae cells (blue) at 1 day post final exposure (circles, crossed bars, left) or 21 days post final exposure (squares, solid bars, right). Cells = cell number. (A) Lymphocytes, (B) B Cells, (C) NK Cells, (D) T Cells, (E) CD4⁺ T Cells, and (F) CD8⁺ T Cells. n=3-7 per group, *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test comparing each exposure together for the same timepoint.

Figure 6

Repeated C. neoformans exposure continues to increase cell populations in the BALF at 21 days post final exposure. Mean cell quantifications in BALF following repeated exposure to PBS, 10⁴ C. neoformans cells or 10⁶ V. victoriae cells at 1 day post final exposure compared to 21 days post final exposure. n=3-7 per group. Values used for this heatmap were obtained by calculating the average quantities at 21 days post final exposure and subtracting the average quantities at 1 day post final exposure. A positive Z score (pink) indicates that the quantification of cells increased at 21 days post final exposure compared to 1 day post final exposure. Negative Z scores (blue) indicate that the cell quantifications decreased by 21 days post final exposure compared to 1 day post final exposure. Asterisks overlaying the heatmap indicate P values comparing the quantifications of each time point for the same exposure. *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were determined by ordinary one-way ANOVA with Sidak’s multiple comparisons test comparing the same exposure at 1 day post final exposure and 21 days post final exposure.

Figure 7

C. neoformans increases IL-4 and IL-5 in lung one day following repeated exposure. Cytokine concentrations in lung homogenate were determined via Luminex after repeated exposure to PBS (black), 10⁴ C. neoformans cells (yellow) or 10⁶ V. victoriae cells (blue) at 1 day post final exposure (circles, crossed bars, left) or 21 days post final exposure (squares, solid bars, right). (A) IL-4, (B) IL-5, (C) IL-13, (D) Eotaxin (CCL11), and (E) IL-33. n=3 per group, *P < 0.05, **P < 0.01. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test comparing each exposure together for the same timepoint.