Th2 but not Th1 immune bias results in altered lung functions in a murine model of pulmonary Cryptococcus neoformans infection

Abstract

Changes in airway dynamics have been reported in the rat model of pulmonary cryptococcosis. However, it is not known if Cryptococcus neoformans-induced changes in lung functions are related to the immunophenotype that develops in response to cryptococcal infection in the lungs. In this study we performed a parallel analysis of the immunophenotype and airway resistance (standard resistance of the airways [SRAW]) in BALB/c mice infected with highly virulent C. neoformans strain H99 and moderately virulent strain 52D. H99 infection evoked a Th2 response and was associated with increased SRAW, while the SRAW for 52D infection, which resulted in a predominantly Th1-skewed response, did not differ from the SRAW for uninfected mice. We found that an altered SRAW in mice did not positively or negatively correlate with the pulmonary fungal burden, the magnitude of inflammatory response, the numbers of T cells, eosinophils or eosinophil subsets, neutrophils, or monocytes/macrophages, or the levels of cytokines (interleukin-4 [IL-4], IL-10, gamma interferon, or IL-13) produced by lung leukocytes. However, the level of a systemic Th2 marker, serum immunoglobulin E (IgE), correlated significantly with SRAW, indicating that the changes in lung functions were proportional to the level of Th2 skewing in this model. These data also imply that IgE may contribute to the altered SRAW observed in H99-infected mice. Lung histological analysis revealed severe allergic bronchopulmonary mycosis pathology in H99-infected mice and evidence of protective responses in 52D-infected mice with well-marginalized lesions. Taken together, the data show that C. neoformans can significantly affect airflow physiology, particularly in the context of a Th2 immune response with possible involvement of IgE as an important factor.

FIG. 1.

Pulmonary and cerebral growth and clearance of C. neoformans in BALB/c mice. Mice were inoculated intratracheally with 10⁴ cells of either C. neoformans 52D or H99. (A) The pulmonary fungal burden was evaluated at weekly intervals. 52D-infected mice, n = 12 (week 1), n = 12 (week 2), n = 19 (week 3), and n = 10 (week 4); H99-infected mice, n = 9 (week 1), n = 15 (week 2), n = 17 (week 3), and n = 7 (week 4). (B) Cerebral fungal burden was evaluated at week 3 postinfection. 52D-infected mice, n = 14; H99-infected mice, n = 12. Data, pooled from three separate matched experiments, are shown as mean and SEM numbers of CFU per organ. *, P < 0.05, and ***, P < 0.001, between the H99 and 52D groups at the matching time points; +, P < 0.05 within the 52D-infected group compared to week 1; ‡, P < 0.001 within the H99-infected group compared to the fungal burden 1 week earlier.

FIG. 2.

Pulmonary inflammatory response in mice infected with either C. neoformans 52D or H99. Lungs were collected from uninfected and H99- or 52D-infected BALB/c mice at week 3 postinfection. (A) Total numbers of recruited pulmonary leukocytes were enumerated from individual mice following enzymatic dispersion of whole lungs. Uninfected mice, n = 4; H99-infected mice, n = 12; 52D-infected mice, n = 28. (B) Numbers of lymphocyte subsets were determined by staining samples of leukocyte suspensions from infected mice with fluorochrome-labeled antibodies specific for CD4⁺, CD8⁺, and CD19⁺ lymphocytes and analyzed by flow cytometry. Uninfected mice, n = 4; H99-infected mice, n = 11; 52D-infected mice, n = 10. (C) Differential myeloid subset recruitment was determined by microscopic enumeration of cytospun-leukocyte isolates. Uninfected mice, n = 4; H99-infected mice, n = 12; 52D-infected mice, n = 8. Data, pooled from three separate matched experiments (A) and two separate matched experiments (B and C), are expressed as means and SEM. Neut, neutrophils; Eos, eosinophils; Mono/Mac, monocytes/macrophages. NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 3.

Cytokine production from C. neoformans-infected BALB/c mice. Lung leukocytes were isolated from infected and uninfected mice at week 3 postinfection. (A) Isolated leukocytes (5 × 10⁶ cells/ml) were cultured for 24 h, and supernatant was collected from the cell culture and analyzed by ELISA for the cytokines IL-4, IL-10, IFN-γ, and IL-13. Uninfected mice, n = 7; H99-infected mice, n = 30; 52D-infected mice, n = 27. (B) Total RNA from 5 × 10⁶ isolated lung leukocytes was prepared, and first-strand cDNA was synthesized. The mRNA levels for cytokine IL-12p35 and TNF-α in each sample were compared to the GAPDH mRNA expression level and are expressed as percentages of the GAPDH level (relative expression). Uninfected mice, n = 6; H99-infected mice, n = 22; 52D-infected mice, n = 22. Data, pooled from four separate matched experiments, are expressed as means and SEM. NS, not significant; **, P < 0.01; ***, P < 0.001.

FIG. 4.

Production of serum IgE from C. neoformans-infected BALB/c mice. Blood was collected from uninfected and infected BALB/c mice at week 3 postinfection. Sera were evaluated to determine the concentration of total IgE using the ELISA. Uninfected mice, n = 7; H99-infected mice, n = 17; 52D-infected mice, n = 30. Data, pooled from three separate matched experiments, are expressed as means and SEM. ***, P < 0.001.

FIG. 5.

Morphological patterns of pulmonary inflammation and pathological lesions in C. neoformans-infected lungs: photomicrographs of lung histology for BALB/c mice infected with either C. neoformans 52D or H99. Lungs were collected from infected mice at week 3 postinfection, fixed, processed for histology, and either stained with PAS stain and photographed with a ×20 objective (A to C) or stained with hematoxylin and eosin and photographed with a ×40 objective (D and F) or a ×100 objective (E). (A) Lung of uninfected control mouse. (B) Lung of a C. neoformans H99-infected mouse. Note the uncontrolled and widespread dissemination of cryptococcal cells in the absence of inflammation. (C) Lung of a C. neoformans 52D-infected mouse. Note the tight leukocyte infiltrate forming a granuloma around the yeast cells and clear segregation between uninfected and infected tissue. Note the presence of PAS stain-positive goblet cells and mucus accumulation in both groups of mice. (D and E) Lungs of a C. neoformans H99-infected mouse, showing pulmonary eosinophilia (Eos) (D) and evidence of alternative activation macrophages (E). YM crystals in the alveolar macrophages are indicated by arrows. (F) Lung of a C. neoformans 52D-infected mouse showing containment of infection within a tight mononuclear infiltrate (Th1 granuloma).

FIG. 6.

Eosinophil phenotypes of C. neoformans-infected BALB/c mice. Eosinophils from lung digest samples isolated from C. neoformans-infected BALB/c mice were analyzed by flow cytometry at week 3 postinfection. (A) Eosinophil populations were identified by CCR3 expression and the side and forward scatter characteristics (CCR3⁺ SSC^hi FSC^low). The overlaid histograms show the frequency of CD48⁺ CD44⁺ eosinophils in either H99-infected mice (black histograms) or 52D-infected mice (gray histograms) compared with the results for the isotype control (white histograms). The percentages of CD48⁺ and CD44⁺ are indicated above the gate, and the percentages of autofluorescent cells are indicated below the gate. Note that there was no significant difference in the expression frequency of either surface marker in either strain when the value for the isotype control was subtracted. (B) Total numbers of activated CD44⁺ and CD48⁺ eosinophils. Uninfected mice, n = 1; H99-infected mice, n = 5; 52D-infected mice, n = 5. Data are shown as means and SEM. ***, P < 0.001.

FIG. 7.

Effect of C. neoformans 52D and H99 infection on lung functions. At 3 weeks postinfection, measurements of lung functions in mice were made using a dual-chamber plethysomograph and Buxco system at baseline and after administration of increasing doses of aerosolized MCh (0, 2.5, 5, 10, 20, 40, and 80 mg/ml) as described in Materials and Methods. (A) Baseline airway resistance. Uninfected mice, n = 8; H99-infected mice, n = 15; 52D-infected mice, n = 18. (B) Airway responsiveness of mice to MCh. Uninfected mice, n = 12; H99-infected mice, n = 12; 52D-infected mice, n = 20. Data, pooled from three separate matched experiments, are expressed as means and SEM. *, P < 0.05 in comparison with the uninfected control.

FIG. 8.

Correlations between the serum IgE level and airway resistance in infected mice at week 3 postinfection. (A) Correlation for H99-infected mice at an MCh concentration of 5 mg/ml. (B) Correlation for 52D-infected mice at an MCh concentration of 5 mg/ml. H99-infected mice, n = 13; 52D-infected mice, n = 15. Data were pooled from three parallel experiments.