Cryptococcus neoformans Evades Pulmonary Immunity by Modulating Xylose Precursor Transport

Abstract

Cryptococcus neoformans is a fungal pathogen that kills almost 200,000 people each year and is distinguished by abundant and unique surface glycan structures that are rich in xylose. A mutant strain of C. neoformans that cannot transport xylose precursors into the secretory compartment is severely attenuated in virulence in mice yet surprisingly is not cleared. We found that this strain failed to induce the nonprotective T helper cell type 2 (Th2) responses characteristic of wild-type infection, instead promoting sustained interleukin 12p40 (IL-12p40) induction and increased IL-17A (IL-17) production. It also stimulated dendritic cells to release high levels of proinflammatory cytokines, a behavior we linked to xylose expression. We further discovered that inducible bronchus-associated lymphoid tissue (iBALT) forms in response to infection with either wild-type cryptococci or the mutant strain with reduced surface xylose; although iBALT formation is slowed in the latter case, the tissue is better organized. Finally, our temporal studies suggest that lymphoid structures in the lung restrict the spread of mutant fungi for at least 18 weeks after infection, which is in contrast to ineffective control of the pathogen after infection with wild-type cells. These studies demonstrate the role of xylose in modulation of host response to a fungal pathogen and show that cryptococcal infection triggers iBALT formation.

Keywords: Cryptococcus neoformans; encapsulated pathogens; fungal pathogenesis; iBALT; inducible bronchus-associated lymphoid tissue; pathogenic fungus; pulmonary immunity; xylose.

FIG 1

Fungal burden and pulmonary cytokine production in WT- and uxt1Δ uxt2Δ mutant-infected mice. All panels show the combined results of two independent experiments (5 mice per group per experiment). (A to C) Tissue homogenates of infected A/JCr mice were plated for CFU at the indicated dpi (open symbols, WT; red symbols, uxt1Δ uxt2Δ strain; dashed line, initial inoculum). Values from individual mice are plotted along with the mean ± standard error of the mean (SEM). (D to I) Cytokine levels in lung homogenates at the indicated dpi (dashed line, naive; gray bars, WT; red bars, uxt1Δ uxt2Δ strain). *, P < 0.05; **, P < 0.01; ***, P < 0.005 for Student's t test comparing WT to mutant infection.

FIG 2

Accumulation of T and B cells in the lungs of mice infected with uxt1Δ uxt2Δ mutant cells is required to prevent disease progression. CD19⁺ B cells (A), CD4⁺ T cells (B), and CD8⁺ T cells (C) were enumerated in the lungs of infected mice by flow cytometry (gray, WT; red, uxt1Δ uxt2Δ mutant). Plotted are the combined mean ± SEM values from two independent experiments (5 mice per group per experiment). *, P < 0.05; **, P < 0.01; ***, P < 0.005 by Student's t test. (D) Survival of mice after intranasal inoculation with 5 × 10⁴ cells of the WT (n = 5) or uxt1Δ uxt2Δ strain (n = 10). Results shown are combined from two independent experiments. **, P < 0.01; ***, P < 0.005 by the log rank test.

FIG 3

Cytokine production by dendritic cells stimulated with cryptococcal antigens. (A) CD11c⁺ DC populations in the lungs of infected mice were quantified by flow analysis. (B to E) DCs were coincubated for 24 h with heat-killed cells of the strains indicated or subjected to no treatment (no tx), and levels of IL-1β (B), IL-6 (C), TNF-α (D), and IL-12p40 (E) in the supernatant were quantified by ELISA. Shown are the mean ± SD (n = 3) values for one representative experiment from five independent experiments that yielded similar results. **, P < 0.01 by one-way ANOVA with Tukey’s post hoc test.

FIG 4

Mutant cryptococci are primarily restricted to lymphoid structures. Micrographs show Movat-stained lung sections from A/JCr mice infected with the WT or uxt1Δ uxt2Δ strain as above. Scale bars, 100 μm (top row) and 10 μm (bottom row). White arrows highlight fungi (stained turquoise). Images shown are representative of two independent studies (4 to 5 mice per group per experiment).

FIG 5

iBALT complexity in the context of C. neoformans infection. (A and B) Representative immunofluorescent staining of B cells (B220⁺), T cells (CD3⁺), plasma cells (IgG⁺), and CXCL13 in the lungs of infected A/JCr mice. Scale bar = 100 μm. (C to E) Morphometric analysis of iBALT structures in A/JCr mice infected with the WT (gray bars) and uxt1Δ uxt2Δ strain (red bars). (F) Quantification of immunofluorescent staining of CXCL13. Plots show the combined mean ± SEM of two independent studies (4 to 5 mice per group per experiment); colors as in panels C to E. *, P < 0.05; ***, P < 0.005 by Student's t test.