Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4+ T cells, CD11c+ cells, and Gr-1+ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.

Alternative macrophage activation is associated with exacerbated disease in murine models of pulmonary cryptococcosis. The present study evaluated the efficacy of interferon-␥ transgene expression by Cryptococcus neoformans strain H99␥ in abrogating alternative macrophage activation in infected mice. Macrophage recruitment into the lungs of mice after infection with C. neoformans strain H99␥ was comparable with that observed in mice challenged with wild-type C. neoformans. However, pulmonary infection in mice with C. neoformans strain H99␥ was associated with reduced pulmonary fungal burden, increased pulmonary Th1-type and interleukin-17 cytokine production, and classical macrophage activation as evidenced by increased inducible nitric oxide synthase expression, histological evidence of enhanced macrophage fungicidal activity, and resolution of inflammation. In contrast, progressive pulmonary infection, enhanced Th2-type cytokine production, and the induction of alternatively activated macrophages expressing arginase-1, found in inflammatory zone 1, Ym1, and macrophage mannose receptor were observed in the lungs of mice infected with wild-type C. neoformans. These alternatively activated macrophages were also shown to harbor highly encapsulated , replicating cryptococci. Our results demonstrate that pulmonary infection with C. neoformans strain H99␥ results in the induction of classically activated macrophages and promotes fungal clearance. These studies indicate that phenotype , as opposed to quantity , of infiltrating macrophages correlates with protection against pulmonary C . neoformans infection.

Experimental pulmonary Cryptococcus neoformans infection in BALB/c mice is associated with polarized Th2-type cytokine production, alternative macrophage activation, and severe bronchopneumonia. In contrast, pulmonary infection with a C. neoformans strain that secretes IFN-γ, H99γ, elicits Th1-type cytokine production and classical macrophage activation. Additionally, mice infected with H99γ resolve the acute infection and are subsequently protected against challenge with wild type C. neoformans. The present study characterizes macrophage activation during the protective response to wild type C. neoformans in mice previously immunized with H99γ. We observed increased pulmonary Th1-type cytokine production in lung homogenates and classical macrophage activation as evidenced by enhanced expression of inducible nitric oxide synthase (iNOS) in the lungs of H99γ immunized mice compared to mice given a non-protective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages from day 7 post-challenge H99γ immunized mice cultured in vitro were fungistatic against C. neoformans, whereas cryptococcal growth was uncontrolled within macrophages from HKCn immunized mice. Th2-type cytokine production and induction of alternatively activated macrophages were also observed in lungs of HKCn immunized mice during re-challenge. Gene expression arrays showed that classical macrophage activation during challenge infection in H99γ immunized mice was associated with induction of the transcription factor STAT1 and its downstream targets IRF-1, SOCS-1, CXCL9 and CXCL10. These studies demonstrate that protective responses to C. neoformans challenge in immunized mice include classical macrophage activation and enhanced macrophage fungistasis of C. neoformans yeasts. Finally, the classical activation phenotype of protective anti-cryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.