Figure 1.
Effect of cryptococcal laccase on fungal clearance.
Mice were inoculated intratracheally with 104 C. neoformans strain H99 or lac1Δ. Lungs were harvested at weeks 1, 2, and 3 post-infection for analysis of fungal burden (A); brains were harvested at weeks 2, and 3 post-infection for analysis of fungal burden (B). Note the dramatically decreased fungal burden in the lungs and brains of lac1Δ infected mice compared with H99 infected mice. Data, mean CFU±SEM log10, were pooled from 2–3 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.
Figure 2.
Effect of cryptococcal laccase on the recruitment of total lung leukocytes and leukocyte subsets.
Lung leukocytes were isolated from mice infected with H99 or lac1Δ, antibody, stained and analyzed using flow cytometric analysis as per Materials and Methods. The total number of CD45+ leukocytes was determined by multiplying the frequency of CD45+ by total number cells at 2 and 3 wpi (A); Representative FACS plots show the staining strategy to isolate lung leukocyte subsets within gated populations of CD45+ leukocytess. Macrophage, Eosinophil, Neutrophil, and Lymphocyte subsets were identified based on the expression of Gr-1 vs CD11c. Total numbers of Macrophages, Eosinophils, Neutrophils, and Lymphocytse were determined by multiplying the frequency of each subset by the total number of CD45+ leukocytes at 3 wpi (B). Bars represent data (mean ± SEM) from 2–3 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.
Figure 3.
Effect of cryptococcal laccase on the activation of pulmonary macrophages.
Lung leukocytes were isolated from uninfected mice or mice infected with C. neoformans strain H99 or lac1Δ at 3 wpi. (A) Pulmonary macrophages were purified as described in methods. RNA was extracted, converted to cDNA, and evaluated by qPCR to quantify alternative (Arg1) versus classical (iNOS) macrophage activation gene expression. The data were normalized to GAPDH mRNA levels and expressed as percentage of GAPDH (% GAPDH). (B) Pulmonary macrophages (CD11c+/F4/80+) were gated from CD45+ lung leukocytes by flow cytometry analysis. The activation phenotype of pulmonary macrophages was evaluated by the surface expression of MHC class II (MHC II) and Mac2. Specific antibody staining is depicted as solid lines and isotype-control staining as shaded histograms. The bar graph presents mean fluorescence intensity (MFI) of positive cells derived from these histograms. Note that H99 infection was associated with increased expression of Arg1, compared with macrophages obtained from lac1Δ-infected mice. In contrast, H99 infection was associated with increased expression of Mac2 and decreased expression of MHC II in the pulmonary macrophages, compared with lac1Δ -infected mice. Bars represent data (mean ± SEM) from 2–3 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.
Figure 4.
Effect of cryptococcal laccase on morphological patterns of pulmonary inflammation and pathological lesions in C. neoformans infected lungs.
Lungs collected from H99-infected (A, C,) and lac1Δ-infected (B, D,) mice were perfused with buffered formalin, fixed, and processed for histology at week 3 postinfection. Representative photomicrographs of H&E + mucicarmine-stained slides taken at 40× (A, B) and 100× (C, D) objective power. Note that sections obtained from wt-infected lungs frequently revealed macrophages harboring intracellular cryptococci encircled by large capsular halos (black arrows) as compared to lung section obtained from Lac1Δ-infected lungs which revealed numerous macrophages with intracellular inclusions consistent with degraded cryptococcal organisms (blue arrows). Also, the extended macrophages in H99-infected mice are heavily laden with YM1/2 crystals (red arrows) which were not observed in lac1Δ-infected lungs. Lastly, note that H99 infection leads to the accumulation of eosinophils (yellow arrow) in the lung, compared to the accumulation of lymphocytes (green arrow) in lac1Δ-infected lungs.
Figure 5.
Effect of cryptococcal laccase on lung leukocyte cytokine production.
Lung leukocytes were isolated from the lungs of mice infected with C. neoformans strain H99 or lac1Δ at 3 wpi and cultured for 24 h at 5×106 cells/ml. Cytokine levels were evaluated by ELISA in cell culture supernatants. Bars represent mean cytokine concentration ±SEM (pg/ml). Bars represent mean ± SEM from 2 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.
Figure 6.
Effect of cryptococcal laccase on the recruitment and differentiation of CD4+ and CD8+ T-cells.
Leukocytes were isolated from the lungs of mice infected with C. neoformans strain H99 or lac1Δ at 3 wpi. The populations of CD4+ and CD8+ T lymphocytes were identified and enumerated using flow cytometry (A); Representative plots show intracellular IFN-γ and IL-17A staining in the lung CD4+ and CD8+ T cells. The bar graphs presents the percentage of IFN-γ and IL17A positive cells within lung CD4+ and CD8+ T cells (B). Bars represent data (mean ± SEM) from 2 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant; NS, no significant difference between wt and mutant.
Figure 7.
Effect of adoptively transferer CD4+ T cells on fungal clearance.
Lung associated lymph nodes (LALN) were collected from uninfected (week 0) and H99 and lac1Δ infected mice at 2 wpi. The total cells from LALN were isolated and portion used to extract mRNA. The expression of selected “polarizing” cytokines was analyzed by qRT-PCR. Cytokine gene expression was normalized to GAPDH mRNA levels. These values were expressed as fold induction over baseline values expressed in uninfected mice (A); Remaining cells were used for magnetic bead purification of CD4+ T cells, and analyzed by flow cytometry to confirm the purity (B); Purified CD4+ T cells from H99 and lac1Δ infected mice at 2 wpi were adoptively transferred (i.v.) to naïve (i)) and H99 infected (at day 6 post infection) (ii)) mice. The naïve mice were challenged by 104 H99 on the next day after the transfer. Mice that received PBS alone were used as controls. Brains were harvested as described in methods and results and fungal burdens evaluated (C). Bars represent data (mean ± SEM) from 2 separate matched experiments, N = 6 and above for each of the analyzed parameters; *p<0.05 in comparison between wt and mutant.