Cryptococcus neoformans variants generated by phenotypic switching differ in virulence through effects on macrophage activation

Abstract

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense, serve as a site for fungal replication, and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1, PD-L2, and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition, macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue, which were reduced by AM depletion. In conclusion, our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection, which ultimately results in death.

FIG. 1.

(A and B) Differences in survival of SM- and MC-infected SCID (A) and TgE26 (B) mice. Mice (5 to 10 mice per group) were infected i.t. as described in Materials and Methods Levels of survival were compared by using a log rank test. (C) Macrophage-specific staining with MAC3 (Mac) of SM- and MC-infected SCID mice (day14; magnification, ×20) and TgE26 mice (day 21; magnification, ×10) demonstrated that there was enhanced recruitment of macrophages in MC-infected SCID and TgE26 lung tissue compared to SM-infected lung tissue. HE, hematoxylin and eosin stain.

FIG. 2.

Activation of AMs isolated from SM- and MC-infected C57BL/6J mice (five mice per group) confirmed that there was alternative activation. (A) RT-PCR demonstrated that at day 7 (d-7) there were significantly higher levels of arginase (ARG) mRNA (A) in AMs derived from MC-infected mice than in AMs isolated from SM-infected mice. The data were obtained by comparison with the abundance of actin. (B and C) Immunohistochemical analysis with arginase-specific Abs of lungs (day 14) (B) and in vitro production of arginase from the supernatant of isolated AMs from SM- and MC-infected mice (C) both confirmed that there was increased arginase production in MC-infected AMs. There was a significant difference between samples obtained from SM-infected mice and samples obtained from MC-infected mice (P < 0.05) as determined by a t test. The error bars indicate standard deviations.

FIG. 3.

(A to C) mRNA levels in AMs isolated from SM- and MC-infected C57BL/6J mice (five mice per group) as measured by RT-PCR showed that there were significant differences in PD-L1 (A), PD-L2 (B), and MHC-II (C) levels at 7 days (d-7) and 14 days postinfection. The data were obtained by comparison with the abundance of actin. (D) Flow cytometric analysis of AMs isolated from sham-, SM-, and MC-infected mice. The preparations were stained with PD-L1, PD-L2, and MHC-II Abs as described in Material and Methods. SM7 and MC7, SM- and MC-infected mice at 7 days postinfection, respectively; SM14 and MC14, SM- and MC-infected mice at 14 days postinfection, respectively. *, significant difference between SM and MC samples (P < 0.05) as determined by a t test (n = 5). The error bars indicate standard deviations.

FIG. 4.

T-cell proliferation in the presence of AMs derived from SM- and MC-infected C57Bl/6J mice at 7 days (d-7) and 14 days postinfection. Proliferation was measured in quadruplicate by using a ViaLight high-sensitivity cell proliferation kit as described in Materials and Methods. The following preparations were used in this experiment: negative controls consisting of macrophages and T cells (isolated from noninfected mice) alone (donor T-cells alone and AM alone); ConA-stimulated T-cells in the presence of SM and MC macrophages (AM + ConA stimulated T-cells); cultures with Ag-specific T-cell proliferation induced by heat-inactivated SM and MC cells in the presence of the corresponding AMs from SM- and MC-infected mice (AM + Ag stimulated T-cells); and cultures with T-cell proliferation with the Ags (heat-inactivated SM and MC cells) reversed (AM + Reverse Ag stimulated T-cells). *, significant difference between SM and MC samples (P < 0.05) as determined by a t test. The error bars indicate standard deviations. AM, alveolar macrophage.

FIG. 5.

Differences in CD4⁺ and CD4⁺ IL-17A⁺ T-cell recruitment between SM- and MC-infected C57BL/6J mice. (A) Flow cytometric analysis of lung leukocytes with Abs specific for CD4⁺ cells and intracellular staining for CD4⁺ IL-17A⁺ cells (five mice per group) at 7 days (d-7) and 14 days postinfection. (B) Cytokine IL-17 production as measured by ELISA was higher in MC-infected lungs (five mice per group). *, significant difference between SM and MC samples (P < 0.05) as determined by a t test. The error bars indicate standard deviations.

FIG. 6.

(A) H&E staining of lung tissue from mice at the time of death, showing differences in the inflammatory response between SM- and MC-infected C57BL/6J mice. There was less inflammatory response in AM-depleted MC-infected mice than in MC-infected wild-type mice. In contrast to the findings for SM-infected mice, there was no difference in the inflammatory response between AM-depleted mice and wild-type control mice. (B) Cytokine IL-17 production as measured by ELISA was lower in lungs of MC-infected AM-depleted mice at 14 days postinfection (d-14) (five mice per group). (C) Flow cytometric analysis of lung leukocytes showing lower levels of CD4⁺ IL-17A⁺ cells in AM-depleted MC-infected mice than in MC-infected control mice. *, significant difference between SM and MC samples (P < 0.05) as determined by a t test. The error bars indicate standard deviations.