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Cryptococcal meningitis is one of the most common life-threatening diseases caused by Cryptococcus infection. Increasing evidence indicates that type 2 immunity is associated with disease progression by promoting fungal growth and dissemination. However, factors that govern this pathogenic response during infection are still elusive. In this study, we investigated the role of IL-25, one of the type 2–inducing cytokines produced by epithelial cells, in contributing to the pathogenesis of cryptococcosis. We found that pulmonary but not systemic infection with a high-virulence strain of C. neoformans significantly induced pulmonary IL-25 expression in the lungs but not brains. In response to pulmonary infection, mice deficient in the surface IL-17 receptor B, a component of the IL-25R, exhibited improved survival with a decreased brain fungal burden. The absence of IL-25R signaling diminished the type 2 and enhanced the type 1 immune response that directed macrophage polarization toward M1 macrophages. Interestingly, Cryptococcus-mediated IL-25 signaling suppressed the expression of cytokines and chemokines associated with protection in the brain, including Ifng, Il1b, Ip10, and Nos2, without affecting brain cellular inflammation and microglia cell activation. Il17rb−/− mice receiving cryptococcal-specific CD4+ T cells from wild-type had a shorter survival time with higher fungal burden within the brain and an elevated expression of M2 macrophage markers than those receiving cryptococcal-specific CD4+ T cells from Il17rb−/− mice. Taken together, our data indicated that IL-25 signaling subverts the induction of protective immunity and amplifies the type 2 immune response that may favor the development of cryptococcal disease and the fungal dissemination to the CNS.

Cryptococcosis is an infectious disease caused by two fungal species, Cryptococcus neoformans and Cryptococcus gattii. While C. neoformans affects mainly immunocompromised patients, C. gattii infects both immunocompetent and immunocompromised individuals. Laccase is an important virulence factor that contributes to the virulence of C. neoformans by promoting pulmonary growth and dissemination to the brain. The presence of laccase in C. neoformans can shift the host immune response toward a nonprotective Th2-type response. However, the role of laccase in the immune response against C. gattii remains unclear. In this study, we characterized laccase activity in C. neoformans and C. gattii isolates from Thailand and investigated whether C. gattii that is deficient in laccase might modulate immune responses during infection. C. gattii was found to have higher laccase activity than C. neoformans, indicating the importance of laccase in the pathogenesis of C. gattii infection. The expression of laccase promoted intracellular proliferation in macrophages and inhibited in vitro fungal clearance. Mice infected with a lac1Δ mutant strain of C. gattii had reduced lung burdens at the early but not the late stage of infection. Without affecting type-1 and type-2 responses, the deficiency of laccase in C. gattii induced cryptococcus-specific interleukin-17 (IL-17) cytokine, neutrophil accumulation, and expression of the neutrophil-associated cytokine gene Csf3 and chemokine genes Cxcl1, Cxcl2, and Cxcl5 in vivo, as well as enhanced neutrophil-mediated phagocytosis and killing in vitro. Thus, our data suggest that laccase constitutes an important virulence factor of C. gattii that plays roles in attenuating Th17-type immunity, neutrophil recruitment, and function during the early stage of infection.

Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.