Pulmonary granuloma formation during latent Cryptococcus neoformans infection in C3HeB/FeJ mice involves progression through three immunological phases

Abstract

Cryptococcus neoformans is a fungal pathogen that can cause lethal disease in immunocompromised patients. Immunocompetent host immune responses, such as formation of pulmonary granulomas, control the infection and prevent disseminated disease. Little is known about the immunological conditions establishing the latent infection granuloma in the lungs. To investigate this, we performed an analysis of pulmonary immune cell populations, cytokine changes, and granuloma formation during infection with a latent disease-causing clinical isolate in C3HeB/FeJ mice over 360 days. We found that latently infected mice progress through three phases of granuloma formation where different immune profiles dominate: an early phase characterized by eosinophilia, high IL-4/IL-13, and C. neoformans proliferation in the lungs; an intermediate phase characterized by multinucleated giant cell formation, high IL-1α/IFNγ, granuloma expansion, and increased blood antigen levels; and a late phase characterized by a significant expansion of T cells, granuloma condensation, and decreases in lung fungal burden and blood antigen levels. These findings highlight a complex series of immune changes that occur during the establishment of granulomas that control C. neoformans in the lungs and lay the foundation for studies to identify critical beneficial immune responses to Cryptococcus infections.IMPORTANCECryptococcus neoformans is a fungal pathogen that disseminates from the lungs to the brain to cause fatal disease. Latent C. neoformans infection in the lungs is controlled by organized collections of immune cells called granulomas. The formation and structure of Cryptococcus granulomas are poorly understood due to inconsistent human pathology results and disagreement between necrotic granuloma-forming rat models and non-necrotic granuloma-forming mouse models. To overcome this, we investigated granuloma formation during latent C. neoformans infection in the C3HeB/FeJ mouse strain which forms necrotic lung granulomas in response to other pathogens. We found that latent C. neoformans granuloma formation progresses through phases that we described as early, intermediate, and late with different immune response profiles and granulomatous characteristics. Ultimately, we show that C3HeB/FeJ mice latently infected with C. neoformans form non-necrotic granulomas and could provide a novel mouse model to investigate host immune response profiles.

Keywords: C3HeB/FeJ; Cryptococcus neoformans; adaptive immunity; cryptococcosis; granuloma; host-pathogen interactions; innate immunity; latent infection; mycology; pathogenesis; pulmonary infection; tuberculosis.

Fig 1

Disease outcomes of latent cryptococcal infection in C3HeB/FeJ mice. (A) Kaplan-Meier survival curves for C3HeB/FeJ and WT-B6 mice infected with latent UgCl223 or KN99α. Tissue fungal burdens for (B) lungs and (C) brain. Dashed line indicates the mean CFUs for KN99α infection at 20 DPI. (D) CrAg LFA maximum positive titers for sacrificed and survival cohort mice. Dashed line indicates the mean LFA titer for KN99α infection at 20 DPI. (E) LFA titers for survival cohort mice over time with lines connecting titers collected from the same mouse. Panels B–D were analyzed via one-way ANOVA with Tukey’s post hoc. * = P value < 0.05, *** = P value < 0.001.

Fig 2

C3HeB/FeJ mice form non-necrotic pulmonary granulomas composed primarily of CD68⁺ macrophages, neutrophils, and CD4⁺ T cells. Lungs were collected from infected C3HeB/FeJ mice at predetermined time points over 360 days and stained with hematoxylin and eosin. Number of mice lungs imaged per time point (N) = 5. Representative images of whole lung and a 5× magnified region for each time point are shown.

Fig 3

SP140KO mice do not replicate the immune response seen in C3HeB/FeJ mice during cryptococcal infection. Lungs of UgCl223-infected C3HeB/FeJ, wild-type C57Bl/6 (WT-B6), and SP140^−/− C57Bl/6J (SP140^−/− B6) were collected at 30 DPI and analyzed by flow cytometry to enumerate 11 different CD45⁺ cell types. The top-left graph depicts total CD45⁺ cell counts while the rest of the graphs depict that cell type’s proportion of all CD45⁺ cells. Groups were analyzed via one-way ANOVA with Tukey’s post hoc. Only biologically relevant pairwise comparisons are shown (i.e., comparisons within the same mouse background or comparisons of different mouse backgrounds of same infection status). * = P value < 0.05, ** = P value < 0.01, **** = P value < 0.0001.

Fig 4

Pulmonary cytokine levels change during latent infection. Pearson-correlation-clustered heatmap of pulmonary cytokine profiles from C3HeB/FeJ mice latently infected with UgCl223 at designated time points between 0 and 360 days post-infection. Cytokine levels from lung serum were measured using a ProcartaPlex multiplex immunoassay kit. Early, intermediate, and late indicate phases of granuloma formation. Number of lung serum samples (N) = 3–5 for each column.

Fig 5

Pulmonary immune cell populations change during latent infection. (A) Proportion of pulmonary CD45⁺ cells in C3HeB/FeJ mice infected with latent UgCl223 or lethal KN99α strains. Representative time points from each phase are displayed. (B) Total cell counts for CD45⁺, T cell, B cell, eosinophil, neutrophil, and interstitial macrophages (IMac). Dashed lines represent cell counts for KN99α at 14 DPI. Tissues were collected from UgCl223 infected mice at designated time points or KN99α mice at intermediate (14 DPI) or late (21 DPI) time points of the lethal infection. Ordinary one-way ANOVA comparing means to uninfected. * = P value ≤ 0.05.

Fig 6

Pulmonary CD4⁺ and CD8⁺ T cell populations experience differential expansion. Total cell counts for CD4⁺ and CD8⁺ effector, regulatory (Treg), and tissue-resident memory (TRM) cells. Lungs were collected from UgCl223 infected mice at designated time points, homogenized, and analyzed using a T cell-specific flow cytometry panel. Ordinary one-way ANOVA comparing means to 0 DPI. * = P value < 0.05, ** = P value < 0.01, *** = P value < 0.001, **** = P value < 0.0001.

Fig 7

C3HeB/FeJ mice form non-necrotic pulmonary granulomas composed primarily of CD68⁺ phagocytes, Ly6G⁺ neutrophils, and CD4⁺ T cells. Lungs were collected from infected C3HeB/FeJ mice and stained with fluorescent markers targeting F4/80 (red), CD4 (green), and CD68 (cyan) or EPX (red), Ly6G (green), and CD68 (cyan). Representative images at each phase of granuloma formation were taken at 20× magnification while insets were taken at 5× magnification.

Fig 8

Phases of the pulmonary immune response during latent cryptococcal infection. The histological, pathobiological, and immunological temporal patterns of pulmonary immune responses during latent cryptococcal infection reveal distinct immunological phases. The early phase is characterized by rapidly replicating C. neoformans cells, eosinophilia, and IL-4, IL-13, and pro-inflammatory cytokine production. The intermediate phase is characterized by mouse mortality, elevated antigenemia, pro-inflammatory cytokines, and significant increases in interstitial macrophages and CD11b⁻ dendritic cells. The late phase is marked by significant reduction in lung fungal burden and antigenemia, RANTES and IL-10 cytokine abundance, significant increases in both lymphocyte and myeloid cell populations, and mature non-necrotic granuloma formation.