Antibody efficacy in murine pulmonary Cryptococcus neoformans infection: a role for nitric oxide

We investigated the pathogenesis of pulmonary Cryptococcus neoformans infection and passive Ab efficacy in mice deficient in inducible NO synthase (NOS2(-/-)) and the parental strain. Parental mice lived significantly longer than NOS2(-/-) mice after intratracheal infection, despite having a higher lung fungal burden. Administration of Ab reduced lung CFU in both NOS2(-/-) and parental mice, but prolonged survival and increased the inflammatory response only in parental mice. Ab administration was associated with increased serum nitrite and reduced polysaccharide levels in parental mice. Eosinophils were present in greater numbers in the lung of infected NOS2(-/-) mice than parental mice, irrespective of Ab administration. C. neoformans infection in NOS2(-/-) mice resulted in significantly higher levels of IFN-gamma, monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha than parental mice. Ab administration had different effects on infected NOS2(-/-) and parental mice with respect to IFN-gamma, monocoyte chemoattractant protein-1, and macrophage-inflammatory protein-1alpha levels. Ab administration increased lung levels of IFN-gamma in parental mice and reduced levels in NOS2(-/-) mice. The results indicate that NO is involved in the regulation of cytokine expression in response to cryptococcal pneumonia and is necessary for Ab efficacy against C. neoformans in mice. Our findings indicate a complex relationship between Ab efficacy against C. neoformans and cytokine expression, underscoring the interdependency of cellular and humoral defense mechanisms.     

中枢神经系统隐球菌感染动物模型的研究

目的 构建中枢神经系统隐球菌感染的动物模型。方法 给予小鼠脑内接种隐球菌构建中枢神经系统隐球菌感染的动物模型。小鼠被随机地分为实验组和对照组,给予实验组小鼠脑内接种隐球菌菌悬液,对照组小鼠脑内接种生理盐水。结果 从组织病理方面观察到,实验组小鼠脑组织中的蛛网膜下腔、软脑膜表面、脑实质内、侧脑室脉络丛组织内均可见隐球菌菌体,脑膜轻度增生,侧脑室轻度扩大,脉络丛血管轻度扩张充血。对照组小鼠脑组织可见侧脑室轻度扩大,蛛网膜下腔血管、脑实质内血管、脉络丛血管均有轻度扩张充血,而蛛网膜下腔、软脑膜表面、脑实质内及侧脑室和室旁均未见隐球菌浸润。从组织病理观察结果两组具有一定的对比性。结论 小鼠脑内接种隐球菌构建其中枢神经系统隐球菌感染的模型,为研究人中枢神经系统隐球菌病提供了一个工具。     

Contribution of interferon-γ in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans

Abstract In the present study, the role of interferon-γ (IFN-γ) in the host resistance against Cryptococcus neoformans was examined using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with live yeast cells, and the histological changes in the lungs and the number of microorganisms in the lung and brain were compared in mice treated and untreated with anti-IFN-γ monoclonal antibody (mAb) to define the contribution of endogenously synthesized IFN-γ in the natural course of infection. Administration of this mAb reduced the accumulation of inflammatory cells in the alveolar septa, peribronchial and perivascular areas, and promoted the expansive growth of microorganisms in the alveoli and destruction of alveolar structure. The neutralization of endogenous IFN-γ by mAb increased the number of microorganisms in the lung and brain, and significantly shortened the survival time of infected mice. On the other hand, administration of IFN-γ decreased the number of microorganisms in these organs, and significantly extended their survival time. Considered together, our results suggest that endogenous IFN-γ protects mice from infection with C. neoformans by inducing a cellular inflammatory response, potentiating the clearance of microorganism from the lungs and preventing its dissemination into the central nervous system.     

CCR2 expression determines T1 versus T2 polarization during pulmonary Cryptococcus neoformans infection

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. The objective of this study was to determine the role of CCR2 in leukocyte recruitment and development of T1-type cell-mediated immunity during pulmonary C. neoformans infection. Intratracheal inoculation of C. neoformans into CCR2 knockout (CCR2-/-) mice produced a prolonged pulmonary infection (5000-fold CFU at 6 wk compared with CCR2+/+ mice) and significant dissemination to the spleen and brain (160- and 800-fold greater). In addition, CCR2 deficiency resulted in significantly reduced recruitment of macrophages (weeks 1-3) and CD8+ T cells (weeks 1-2) into the lungs. The immune response in CCR2-/- mice was characterized by chronic pulmonary eosinophilia, crystal deposition in the lungs, pulmonary leukocyte production of IL-4 and IL-5 but not IFN-gamma, lack of anticryptococcal delayed-type hypersensitivity, and high levels of serum IgE. These results demonstrate that expression of CCR2 is required for the development of a T1-type response to C. neoformans infection and lack of CCR2 results in a switch to a T2-type response. Thus, CCR2 plays a critical role in promoting the development of T1- over T2-type immune responses in the lung following cryptococcus infection.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Cryptococcus neoformans capsular enlargement and cellular gigantism during Galleria mellonella infection

We have studied infection of Cryptococcus neoformans in the non-vertebrate host Galleria mellonella with particular interest in the morphological response of the yeast. Inoculation of C. neoformans in caterpillars induced a capsule-independent increase in haemocyte density 2 h after infection. C. neoformans manifested a significant increase in capsule size after inoculation into the caterpillar. The magnitude of capsule increase depended on the temperature, being more pronounced at 37°C than at 30°C, which correlated with an increased virulence of the fungus and reduced phagocytosis at 37°C. Capsule enlargement impaired phagocytosis by haemocytes. Incubation of the yeast in G. mellonella extracts also resulted in capsule enlargement, with the polar lipidic fraction having a prominent role in this effect. During infection, the capsule decreased in permeability. A low proportion of the cells (<5%) recovered from caterpillars measured more than 30 μm and were considered giant cells. Giant cells recovered from mice were able to kill the caterpillars in a manner similar to regular cells obtained from in vivo or grown in vitro, establishing their capacity to cause disease. Our results indicate that the morphological transitions exhibited by C. neoformans in mammals also occur in a non-vertebrate host system. The similarities in morphological transitions observed in different animal hosts and in their triggers are consistent with the hypothesis that the cell body and capsular responses represent an adaptation of environmental survival strategies to pathogenesis.     

Efficacy of voriconazole in experimental Cryptococcus neoformans infection

Voriconazole is a third generation triazole with improved activity against many fungal pathogens. We examined the efficacy of voriconazole in a murine infection model and evaluated the drug’s effect on cellular characteristics and serum polysaccharide levels. The antifungal reduced serum polysaccharide and significantly prolonged the survival of lethally infected animals.     

Chlamydospore formation during hyphal growth in Cryptococcus neoformans

Cryptococcus neoformans, a basidiomycetous fungal pathogen, infects hosts through inhalation and can cause fatal meningoencephalitis in individuals if untreated. This fungus undergoes a dimorphic transition from yeast to filamentous growth during mating and monokaryotic fruiting, which leads to the production of hyphae and airborne infectious basidiospores. Here we characterized a novel morphological feature associated with the filamentous stages of the life cycle of C. neoformans which resembles resting or survival structures known as chlamydospores in other fungi. The C. neoformans chlamydospore-like structure is rich in glycogen, suggesting that it might have a role as an energy store. However, characterization of mutants with decreased or increased levels of glycogen production showed that glycogen levels have little effect on filamentous growth, sporulation, or chlamydospore formation. These results suggest that the formation of chlamydospores is independent of glycogen accumulation level. We also show that chlamydospore formation does not require successful sporulation and that the presence of chlamydospores is not sufficient for sporulation. Although the biological functions of chlamydospores remain to be established for this pathogenic fungus, their formation appears to be an integral part of the filamentation process, suggesting that they could be necessary to support sexual sporulation under adverse conditions and thereby facilitate the production of infectious basidiospores or long-term survival propagules in harsh environments.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

2 cases of mycotic infection caused by Cryptococcus neoformans

The authors present two cases of Cryptococcus neoformans disease in two young persons. Its appears, in the first case, a diffuse ganglionnary and cutaneous lesion with an evolutive meningo-encephalic localization, and in the second case it appears only a meningitic lesion. These two observations are characterized by the apparition of the infection in a immunodepression patient and by an unfavorable evolution in spite of the treatment.     

Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (～50%), but at a much lower frequency during vegetative growth (～0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ～90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.     

Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells

Pathogenic yeast Cryptococcus neoformans causes devastating cryptococcal meningoencephalitis. Our previous studies demonstrated that C. neoformans hyaluronic acid was required for invasion into human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In this report, we demonstrate that C. neoformans hyaluronic acid interacts with CD44 on HBMEC. Our results suggest that HBMEC CD44 is a primary receptor during C. neoformans infection, based on the following observations. First, anti-CD44 neutralizing antibody treatment was able to significantly reduce C. neoformans association with HBMEC. Second, C. neoformans association was considerably impaired using either CD44-knock-down HBMEC or C. neoformans hyaluronic acid-deficient strains. Third, overexpression of CD44 in HBMEC increased their association activity towards C. neoformans. Fourth, confocal microscopic images showed that CD44 was enriched at and around the C. neoformans association sites. Fifth, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and actin translocated to the host membrane rafts. Our results highlight the interactions between C. neoformans hyaluronic acid and host CD44 and the dynamic results of these interactions, which may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts.     

Surface localization of glucosylceramide during Cryptococcus neoformans infection allows targeting as a potential antifungal

Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO(2)). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.     

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.     

Immunologic homeostasis during infection: coexistence of strong pulmonary cell-mediated immunity to secondary Cryptococcus neoformans infection while the primary infection still persists at low levels in the lungs

Maintenance of immunity to persistent pathogens is poorly understood. In this study, we used a murine model of persistent pulmonary fungal infection to study the ongoing cell-mediated immune response. CBA/J mice with low-level persistent Cryptococcus neoformans infection had CD4+ T cells of effector memory phenotype present in their lungs. Although unable to eliminate the primary infection to sterility, these mice displayed hallmarks of immunologic memory in response to rechallenge with C. neoformans: 1) the secondary cryptococcal challenge was controlled much more rapidly, 2) the inflammatory response developed and resolved more rapidly, 3) CD4+ T and CD8+ T cell responses were higher in magnitude, and 4) effector cytokine production by T cells was greatly enhanced. Depletion of CD4+ T cells at the time of secondary challenge adversely affected clearance of C. neoformans from the lungs. These results demonstrate that persistent low-level infection with C. neoformans does not impair the cell-mediated response to the fungus. Although they are relatively free of overt disease, these mice can respond with a rapid secondary immune response if the burden of C. neoformans increases. These data support the concept that immunologically healthy individuals can maintain low numbers of cryptococci that can become a nidus for re-activation disease during immunodeficient states such as AIDS.