Phenotypic switching in Cryptococcus neoformans contributes to virulence by changing the immunological host response.

Cryptococcus neoformans is an encapsulated yeast that causes disease primarily in patients with impaired immunity (13, 50). C. neoformans can undergo microevolution during chronic cryptococcosis (16). One mechanism for rapid microevolution is phenotypic switching, a phenomenon that generates switch variants, which exhibit differences in virulence relative to the parent strain (19, 22). We previously described phenotypic switching in C. neoformans strain RC-2, a serotype D strain (20). In that strain, the parent smooth colony (SM) variant switches to a mucoid colony (MC) variant at a rate of 0.5 × 10−4. Phenotypic switching also occurs in vivo during chronic infection and is associated with a lethal outcome in mice (20). In addition, the MC variant is selected for in mice that are infected with a switching strain and treated with antifungal drugs or with a capsule-specific monoclonal antibody (MAb) (18). Hypervirulence of the MC variant has been consistently documented in different mouse strains, including the more resistant BALB/c mice, as well as the highly sensitive C57BL/6 mice, which can develop a chronic pulmonary infection. Although the host immune response mounted against the SM and MC switch variants has not been fully characterized, lower interleukin 10 (IL-10) levels were observed in MC-infected than in SM-infected mice (20). IL-10 is a multifunctional cytokine, as reviewed in detail elsewhere (42), and is produced by many different cells, including alternatively and classically activated macrophages, dendritic cells, B lymphocytes, and CD4+ regulatory T cells. IL-10 is a major regulator of innate immunity and interferes with the production of inflammatory mediators by polymorphonuclear neutrophils, monocytes, and macrophages. IL-10-deficient (IL-10−/−) mice develop chronic inflammatory bowel disease as a result of an inappropriate innate immune response to intestinal bacterial antigens. IL-10 is also classified as a Th2-type cytokine that downregulates Th1-type-associated cytokines (42), like IL-12 and IL-18, in antigen-presenting cells, as well as reducing expression of major histocompatibility complex class II molecules and costimulatory molecules. Therefore, IL-10−/− mice are often more resistant to intracellular pathogens. IL-10 directly affects the function of CD4+ T cells by inhibiting the expression of IL-2, tumor necrosis factor (TNF), IL-5, and chemokine receptor CXCR4. IL-10 also affects the humoral response, because it regulates isotype switching in B cells (32). Several investigators have examined the role of IL-10 in C. neoformans infection. Blackstock et al. demonstrated that IL-10−/− mice infected intratracheally (i.t.) with C. neoformans strain NU-2 survive longer than the wild-type control (3). Beenhouwer et al. showed similar findings in mice that were intravenously (i.v.) infected with C. neoformans strain 20467 (2). Hernandez et al. highlighted the complexity of the role of IL-10 in chronic cryptococcosis and showed a difference between IL-4−/− and IL-10−/− mice, with augmented levels of IL-12 and tumor necrosis factor alpha (TNF-α) in the lungs of IL-10−/− mice, as well as a reduction of Th2-type cytokines (24). Because IL-10 is a multifunctional cytokine that could affect the pathogenesis of chronic cryptococcosis on different levels, we pursued the observation that SM- and MC-infected mice consistently exhibited differences in IL-10 in lung tissue. We hypothesized that phenotypic switch variants may be differently affected by IL-10 deficiency, as they elicit strikingly different inflammatory responses. Hence, the objective of this study was to compare the effects and outcomes of infection with the isogenic C. neoformans SM and MC switch variants in IL-10−/− mice. We concluded from our data that the contribution of IL-10 to the pathogenesis of cryptococcosis is complex and also dependent on the infecting strain.