Sarah E. Hardison

Pulmonary Infection with an Interferon-γ-Producing Cryptococcus neoformans Strain Results in Classical Macrophage Activation and Protection

Alternative macrophage activation is associated with exacerbated disease in murine models of pulmonary cryptococcosis. The present study evaluated the efficacy of interferon-gamma transgene expression by Cryptococcus neoformans strain H99gamma in abrogating alternative macrophage activation in infected mice. Macrophage recruitment into the lungs of mice after infection with C. neoformans strain H99gamma was comparable with that observed in mice challenged with wild-type C. neoformans. However, pulmonary infection in mice with C. neoformans strain H99gamma 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 H99gamma 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.

Role of IL-17A on Resolution of Pulmonary C. neoformans Infection

The current studies evaluated the role of interleukin (IL)-17A in the induction of protective immunity against pulmonary cryptococcosis in mice. Protection against pulmonary infection with C. neoformans strain H99γ was associated with increased IL-17A production. Signaling through the IFN-γ receptor (R) was required for increased IL-17A production, however, a Th17-type cytokine profile was not observed. Neutrophils were found to be the predominant leukocytic source of IL-17A, rather than T cells, suggesting that the IL-17A produced was not part of a T cell-mediated Th17-type immune response. Depletion of IL-17A in mice during pulmonary infection with C. neoformans strain H99γ resulted in an initial increase in pulmonary fungal burden, but had no effect on cryptococcal burden at later time points. Also, depletion of IL-17A did not affect the local production of other cytokines. IL-17RA−/− mice infected with C. neoformans strain H99γ survived the primary infection as well as a secondary challenge with wild-type cryptococci. However, dissemination of the wild-type strain to the brain was noted in the surviving IL-17RA−/− mice. Altogether, our results suggested that IL-17A may be important for optimal protective immune responsiveness during pulmonary C. neoformans infection, but protective Th1-type immune responses are sufficient for protection against cryptococcal infection.

Protective immunity against pulmonary cryptococcosis is associated with STAT1-mediated classical macrophage activation.

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 NO synthase in the lungs of H99γ-immunized mice compared with mice given a nonprotective immunization with heat-killed C. neoformans (HKCn). Furthermore, macrophages isolated from H99γ-immunized mice on day 7 postchallenge and 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 rechallenge. 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 IFN regulatory factor-1, suppressor of cytokine signaling-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 anticryptococcal macrophages is likely mediated via STAT1 signal transduction pathways.

Interleukin-17 Is Not Required for Classical Macrophage Activation in a Pulmonary Mouse Model of Cryptococcus neoformans Infection

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that causes disease in individuals with suppressed cell-mediated immunity. Recent studies in our laboratory have shown that increases in pulmonary Th1-type and interleukin-17A (IL-17A) cytokine production, classical macrophage activation, and sterilizing immunity are elicited in response to infection with a gamma interferon (IFN-γ)-producing C. neoformans strain, H99γ. IL-17A-treated macrophages, compared to IL-4-treated macrophages, have been demonstrated to exhibit increased microbicidal activity in vitro, a characteristic consistent with classical macrophage activation. The purpose of these studies is to determine the role of IL-17A in the induction of classically activated macrophages following infection with C. neoformans. Immunohistochemistry and real-time PCR were used to characterize the macrophage activation phenotype in lung tissues of mice treated with isotype control or anti-IL-17A antibodies and given an experimental pulmonary infection with C. neoformans strain H99γ. The pulmonary fungal burden was resolved, albeit more slowly, in mice depleted of IL-17A compared to the fungal burden in isotype control-treated mice. Nonetheless, no difference in classical macrophage activation was observed in IL-17A-depleted mice. Similarly, classical macrophage activation was evident in mice deficient in IL-17A or the IL-17 receptor A, which mediates IL-17A signaling, following pulmonary infection with wild-type C. neoformans strain H99 or H99γ. These studies suggest that IL-17A may play a role in the early immune response to C. neoformans but is not required for classical macrophage activation in mice experimentally infected with C. neoformans.

Fatal Disseminated Cryptococcus gattii Infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1’s virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.

Induction of Protective Immunity Against Cryptococcosis

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen that can cause life-threatening infections of the central nervous system in immune compromised individuals resulting in high morbidity and mortality. Consequently, several studies have endeavored to understand those mechanisms that mediate resistance and susceptibility to Cryptococcus infection. In this review, we will examine the contributions of various components of the innate and adaptive immune response toward protection against cryptococcosis. We will focus our discussion on studies presented at the 8th International Conference on Cryptococcus and Cryptococcosis (ICCC). Remarkable progress has been made toward our understanding of host immunity and susceptibility to cryptococcal infection and the potential for vaccine development.

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.

Candida albicans VPS4 contributes differentially to epithelial and mucosal pathogenesis

We have previously demonstrated that the C. albicans pre-vacuolar protein sorting gene VPS4 is required for extracellular secretion of the secreted aspartyl proteases Sap2p and Saps4–6p. Furthermore, the vps4Δ null mutant has been shown to be markedly hypovirulent in a murine tail vein model of disseminated candidiasis. In these experiments, we sought to further define the role of the pre-vacuolar secretion pathway mediated by the pre-vacuolar sorting gene VPS4 in the pathogenesis of epithelial and mucosal infection using a broad range of virulence models. The C. albicans vps4Δ mutant demonstrates reduced tolerance of cell wall stresses compared to its isogenic, complemented control strain. In an in vitro oral epithelial model (OEM) of tissue invasion, the vps4Δ mutant caused reduced tissue damage compared to controls. Further, the vps4Δ mutant was defective in macrophage killing in vitro, and was attenuated in virulence in an in vivo Caenorhabditis elegans model representative of intestinal epithelial infection. In contrast, the vps4Δ mutant caused a similar degree of tissue damage in an in vitro uroepithelial model of Candida infection compared with controls. Furthermore, in an in vivo murine model of vaginal candidiasis there was no reduction in fungal colony burden and no differences in vaginal histopathology compared to wild-type and complemented controls. These results suggest that VPS4 contributes to several key aspects of oral epithelial but not uroepithelial infection, and in contrast to systemic infection, plays no major role in the pathogenesis of Candida vaginitis. By using a wide range of virulence models, we demonstrate that C. albicans VPS4 contributes to virulence according to the specific tissue that is infected. Thus, in order to gain a full understanding of C. albicans virulence in relation to a particular gene or pathway of interest, a selected range of infection models may need to be utilized.

Induction of Broad-Spectrum Protective Immunity against Disparate Cryptococcus Serotypes

Cryptococcosis is a fungal disease caused by multiple Cryptococcus serotypes; particularly C. neoformans (serotypes A and D) and C. gattii (serotypes B and C). To date, there is no clinically available vaccine to prevent cryptococcosis. Mice given an experimental pulmonary vaccination with a C. neoformans serotype A strain engineered to produce interferon-γ, denoted H99γ, are protected against a subsequent otherwise lethal experimental infection with C. neoformans serotype A. Thus, we determined the efficacy of immunization with C. neoformans strain H99γ to elicit broad-spectrum protection in BALB/c mice against multiple disparate Cryptococcus serotypes. We observed significantly increased survival rates and significantly decreased pulmonary fungal burden in H99γ immunized mice challenged with Cryptococcus serotypes A, B, or D compared to heat-killed H99γ (HKH99γ) immunized mice. Results indicated that prolonged protection against Cryptococcus serotypes B or D in H99γ immunized mice was CD4+ T cell dependent and associated with the induction of predominantly Th1-type cytokine responses. Interestingly, immunization with H99γ did not elicit greater protection against challenge with the Cryptococcus serotype C tested either due to low overall virulence of this strain or enhanced capacity of this strain to evade host immunity. Altogether, these studies provide “proof-of-concept” for the development of a cryptococcal vaccine that provides cross-protection against multiple disparate serotypes of Cryptococcus.