• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. Aug 2009; 77(8): 3450–3457.
Published online Jun 1, 2009. doi:  10.1128/IAI.00297-09
PMCID: PMC2715691

Cytokine Signaling Regulates the Outcome of Intracellular Macrophage Parasitism by Cryptococcus neoformans[down-pointing small open triangle]

Abstract

The pathogenic yeast Cryptococcus neoformans and C. gattii commonly cause severe infections of the central nervous system in patients with impaired immunity but also increasingly in immunocompetent individuals. Cryptococcus is phagocytosed by macrophages but can then survive and proliferate within the phagosomes of these infected host cells. Moreover, Cryptococcus is able to escape into the extracellular environment via a recently discovered nonlytic mechanism (termed expulsion or extrusion). Although it is well established that the host's cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Here, we report a systematic analysis of the influence of Th1, Th2, and Th17 cytokines on the outcome of the interaction between macrophages and cryptococci. We show that Th1 and Th17 cytokines activate, whereas Th2 cytokines inhibit, anticryptococcal functions. Intracellular yeast proliferation and cryptococcal expulsion rates were significantly lower after treatment with the Th1 cytokines gamma interferon and tumor necrosis factor alpha and the Th17 cytokine interleukin-17 (IL-17). Interestingly, however, the Th2 cytokines IL-4 and IL-13 significantly increased intracellular yeast proliferation while reducing the occurrence of pathogen expulsion. These results help explain the observed poor prognosis associated with the Th2 cytokine profile (e.g., in human immunodeficiency virus-infected patients).

The two encapsulated yeast species Cryptococcus neoformans (serotypes A and D) and C. gattii (serotypes B and C), the causative agents of cryptococcosis, can cause life-threatening infections of the central nervous system (e.g., meningoencephalitis) (9).

Initial infection with Cryptococcus is believed to occur via the inhalation of airborne propagules and the subsequent colonization of the respiratory tract (9). In mouse and rat model systems, C. neoformans is internalized by alveolar macrophages shortly after inhalation (17, 22). Furthermore, C. neoformans phagocytosis by mouse, rat, guinea pig, and human macrophages in vitro has been demonstrated repeatedly (8, 16, 37, 49) and is triggered by direct recognition of the yeast or by receptor-mediated recognition via complement or antibodies (38). However, Cryptococcus seems to have developed a unique method to manipulate host macrophages. After phagocytosis, C. neoformans can survive and proliferate within these infected host cells, eventually leading to macrophage lysis (2, 15, 17, 18, 33, 50). Moreover, a novel expulsive mechanism by which the yeast can exit macrophages without killing the host cell, thus avoiding a local inflammatory response, has recently been described (3, 34).

Results from restriction fragment length polymorphism analyses suggest that initial infection with Cryptococcus often occurs in early childhood and can be followed by a long latent phase in immunocompetent individuals (21). However, C. neoformans is generally capable of disseminating to other organs within the human body and shows a predilection for the central nervous system, where it can lead to life-threatening meningitis and meningoencephalitis (27). Although the molecular basis of latency and expulsion is not known, the so-called Trojan Horse model suggests that replication in and eventual expulsion from macrophages may offer a potential explanation for how C. neoformans stays latent and spreads within the host without triggering immediate immune responses (11, 46). An improved understanding of the interaction between macrophages and Cryptococcus is therefore critical for the development of more effective therapies.

In healthy hosts, the cryptococcal infection is usually self-limiting, suggesting effective clearance or maintenance in a latent state by phagocytic cells. The outcome of cryptococcosis depends on the immune status of the infected individual and the cytokine pattern generated in response to the pathogen. Although it is well established that the host's cytokine profile dramatically affects the outcome of cryptococcal disease, the molecular basis for this effect is unclear. Both Th1 and Th2 cytokines are involved in protection against C. neoformans, but whereas Th1-associated cytokines are essential for natural immunity, Th2-associated immunity is not protective in mice (6, 24, 25). Increased expression of Th1 cytokines, such as tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), results in improved fungal control (19, 29, 36, 53), while IFN-γ knockout mice show increased fungal burdens (4). In 2007, Müller et al. (39) showed a significant role for the Th17 response and the proinflammatory cytokine interleukin-17 (IL-17) in modulating the survival of Cryptococcus-infected mice. In contrast, Th2 cytokines such as IL-4 and IL-13 reduce the host's ability to deal with C. neoformans in vivo (7, 14, 28, 39).

Despite these observations from animal models, little work on the in vitro effects of Th1, Th17, and Th2 cytokines on the interaction between macrophages and Cryptococcus has been done. Here, we report a systematic study of cytokine influence on macrophage-Cryptococcus interactions for a representative selection of Cryptococcus strains. Our results demonstrate that Th1- and Th17-stimulated macrophages are significantly better at phagocytosing cryptococci and at controlling the intracellular proliferation of this pathogen than Th2-stimulated cells. In contrast, Th2-activated macrophages show a significantly lower rate of cryptococcal expulsion than Th1- or Th17-activated cells. Together, these data help explain the susceptibility phenotype associated with Th2 cytokine profiles in vivo.

MATERIALS AND METHODS

Yeast strains and growth conditions.

C. neoformans serotype A strains H99 and ATCC 90112, both originally isolated from patient cerebrospinal fluids, and C. gattii serotype B strain R265, a clinical isolate from an outbreak on Vancouver Island, Canada, were cultured in liquid YPD medium [1% peptone, 1% yeast extract, 2% d-(+)-glucose] for 24 h at 25°C with shaking at 240 rpm prior to experimental use (34).

Mammalian cells and growth conditions.

The semiadherent macrophage-like cell line J774 and human primary monocyte-derived macrophages were used for experimental work. The J774 cell line is derived from a reticulum sarcoma that arose in a female BALB/c/NIH mouse (43). The cells were used between passages 5 and 20 after thawing and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml streptomycin, and 100 U/ml penicillin at 37°C and 5% CO2 (34). Human primary peripheral blood monocytes were isolated from buffy coats in samples supplied by the local blood transfusion unit from eight independent healthy volunteers. To separate and collect the mononuclear cells, each sample was diluted twofold and a 30-ml aliquot was centrifuged over a 20-ml Ficoll-Paque PLUS cushion at 400 × g for 30 min. The mononuclear layer was collected and washed multiple times with phosphate-buffered saline (PBS; pH 7.2) to remove platelets. Monocytes were isolated by adherence to plastic at a concentration of 4 × 106 to 6 × 106 cells/ml in RPMI 1640 medium supplemented with 2% FBS, 2 mM glutamine, 100 U/ml streptomycin, and 100 U/ml penicillin at 37°C and 5% CO2 for 1 h. Nonadherent lymphocytes were removed with warm PBS, and the adherent cells differentiated into macrophages in RPMI 1640 medium containing 100 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml streptomycin, and 100 U/ml penicillin (culture medium) at 37°C and 5% CO2. After incubation overnight, the cells were washed with warm PBS and detached with ice-cold PBS on ice for 30 min. The macrophages were collected, resuspended, and plated into 24-well plates at a concentration of 5 × 105 cells/well in RPMI 1640 medium containing 100 U/ml GM-CSF. The next day, the medium was replaced by GM-CSF-free RPMI 1640 medium, and the cells were cultured for another 4 days at 37°C and 5% CO2 before assays were commenced.

Infection of macrophages with Cryptococcus.

One milliliter of J774 cells (105 cells/ml) in culture medium were plated into each well of a 24-well tissue culture-treated plate 24 h prior to infection and kept at 37°C and 5% CO2. One hour before infection, J774 cells were switched into serum-free Dulbecco's modified Eagle's medium (supplemented as RPMI 1640 medium but without FBS) with, if applicable, the following recombinant mouse cytokines (ImmunoTools GmbH) at the indicated concentrations: 10 U/ml IFN-γ, 1 ng/ml TNF-α, 10 ng/ml IL-17, 10 ng/ml IL-4, and 10 ng/ml IL-13. The human primary macrophages were preincubated with recombinant human cytokines (ImmunoTools GmbH) in RPMI 1640 medium for 24 h prior to the experiment and in serum-free RPMI 1640 medium for 1 h before infection. At the same time, Cryptococcus cells from 24-h-old liquid cultures were washed three times with PBS, counted in a hemocytometer, and opsonized with 10 μg/ml of the monoclonal antibody 18B7 (a kind gift from Arturo Casadevall) or 10% human serum at 37°C for 1 h. Human sera from healthy volunteers were obtained from blood samples that were allowed to clot for 3 h at 37°C before the serum fraction was drawn off and used immediately. After preincubation, the opsonized yeast cells were directly added to the J774 cells or the peripheral blood macrophages at a ratio of 10 yeast cells per macrophage and phagocytosis was allowed to proceed for 2 h at 37°C in a 5% CO2 atmosphere. Afterwards, noninternalized yeast cells were removed by extensive washes with prewarmed PBS, and the effectiveness of washing was confirmed under a microscope (34).

Phagocytosis assay.

To assess the extent of Cryptococcus phagocytosis, J774 cells or human primary macrophages were grown on acid (1 M HCl)-washed 13-mm glass coverslips and infected as described above. Cells were fixed on the coverslips with 4% paraformaldehyde for 20 min at 4°C. The coverslips were washed three times in PBS and twice in distilled water before being mounted in Mowiol mounting medium (100 mM Tris-HCl, pH 8.5, 9% Mowiol, 25% glycerol) onto microscope glass slides. A total of at least 1,000 cells per sample coverslip were observed, and samples were scored according to the number of cells with internalized yeast cells. The extent of Cryptococcus phagocytosis was calculated as the percentage of cells with internalized Cryptococcus (referred to hereinafter as the percent phagocytosis). Three individual experiments for each condition were performed, and the data were tested for normality by using the Kolmogorov-Smirnov test, for homogeneity of variances by using the Levene statistic, and for statistically significant differences among the mean data by using a one-way analysis of variance. Multiple comparisons (using Tukey's honestly significant difference [HSD] test) were performed to identify statistically significant differences between pairs. A P value of <0.05 after controlling for multiplicity was considered to be statistically significant.

Proliferation assay.

The ability of Cryptococcus to proliferate within J774 cells and human primary macrophages was analyzed in proliferation assays. Following infection, fresh serum-free culture medium was added to the wells and the cells were further cultured at 37°C in a 5% CO2 atmosphere. When pretreated with cytokines, the cultures were maintained at the concentrations mentioned above. Samples were taken after infection and the removal of noninternalized yeast cells, at 0, 18, 24, 48, and 72 h. Intra- and extracellular yeast cells were counted separately with a hemocytometer after trypan blue staining. To determine the number of extracellular yeast cells, the extracellular medium was collected into a reaction tube, each well was washed with 200 μl of PBS, and the wash fluid was collected into the same tube to gather any remaining extracellular yeast. Cells with intracellular Cryptococcus cells remain attached to the bottom of the dish. To count the intracellular yeast cells, cells were lysed in 200 μl of distilled H2O at 37°C for 30 min. The cells were scraped off the bottom of the dish and collected, an additional 200 μl of PBS was used to wash each well, and the wash fluid was added to the same collection tube. The intracellular yeast cells were then counted, and the results were compared to the number at time zero. The maximal intracellular proliferation rate (IPR) was used as a measure of intracellular proliferative capacity and was calculated as the highest intracellular yeast count (typically at 18 or 24 h) divided by the initial intracellular yeast count at time zero. The IPR was calculated separately for each individual experiment and for each cytokine treatment (relative to values for cells incubated with the same cytokine to control for differential uptake patterns). Three individual experiments for each condition were performed, and the data were tested for normality by using the Kolmogorov-Smirnov test, for homogeneity of variances by using the Levene statistic, and for statistically significant differences among the mean data by using a one-way analysis of variance. Multiple comparisons (using Tukey's HSD test) were performed to identify statistically significant differences between pairs. A P value of <0.05 after controlling for multiplicity was considered to be statistically significant. The growth of yeast cells alone and that of J774 cells and human primary macrophages alone under identical conditions were also recorded to rule out direct effects of cytokine treatment on macrophage or yeast cell survival.

Live-cell imaging.

Following the infection of J774 cells and human primary macrophages with Cryptococcus as described above, fresh serum-free culture medium and the corresponding cytokine used for pretreatment were added to the wells before further culture at 37°C and 5% CO2 in a controlled chamber (OKOLAB). Cells were visualized on a Nikon Eclipse TE2000-U microscope with a 20× phase-contrast objective and a 1× optivar lens. Images were captured every 90 s for 20 h with a Nikon Digital Sight DS-Qi1MC camera and compiled into time-lapse movies using the software NIS-Elements AR 3.0. The numbers of expulsion events in three independent experiments for each cytokine were determined by visual examination with the naked eye. Differences between treatments were tested for statistical significance by using the χ2 test, and a P value of <0.05 was considered to be statistically significant.

RESULTS

Macrophage activation.

Macrophages were treated with different cytokines to mimic Th1, Th17, and Th2 activation. We chose to investigate the Th1 cytokines IFN-γ and TNF-α, the Th17 cytokine IL-17, and the Th2 cytokines IL-4 and IL-13, as there is already considerable information available describing the influence of these cytokines on cryptococcosis in vivo. To avoid artifacts due to overstimulation, we used the lowest cytokine concentrations previously demonstrated to induce Th1, Th2, or Th17 phenotypes, as follows: 10 U/ml IFN-γ (40), 1 ng/ml TNF-α (45), 10 ng/ml IL-17 (23), 10 ng/ml IL-4 (52), and 10 ng/ml IL-13 (52). To confirm that the cytokines were nonetheless functional at these concentrations, we monitored macrophage morphology and phagocytic capacity after cytokine exposure.

Untreated J774 and primary macrophages appeared mainly round or occasionally slightly spread out. However, following the exposure of J774 cells and primary macrophages to mouse and human recombinant cytokines, respectively, most cells were extensively elongated and spread out, demonstrating that cells are effectively activated by cytokines at the concentrations tested (Fig. (Fig.1A).1A). To detect any cytokine-evoked cytotoxic effects on macrophages, growth was monitored over 72 h. Neither J774 cells (data not shown) nor human monocyte-derive macrophages (Fig. (Fig.1B)1B) exhibited any cytokine-induced cytotoxicity.

FIG. 1.
Treatment with cytokines at the chosen concentrations results in morphological changes in both J774 and human primary monocyte-derived macrophages but does not influence macrophage survival. J774 and human primary monocyte-derived macrophages were treated ...

Given the well-established role of proinflammatory cytokines in enhancing phagocytosis (29), we used the ability of macrophage-like cells and human primary macrophages to phagocytose Cryptococcus when treated with Th1, Th17, and Th2 cytokines as a second parameter to show the efficacy of cytokine treatment. Untreated or cytokine-treated macrophages were infected with C. neoformans strain ATCC 90112 or H99 or C. gattii strain R265 and analyzed for uptake rates (percent phagocytosis). While Th1/Th17 cytokines induced an increase in cryptococcal phagocytosis, neither IL-4 nor IL-13 treatment significantly altered uptake rates (data not shown). Similar results were obtained for all three cryptococcal strains in both mouse and human macrophages, suggesting that Th1/Th17-dependent enhancement of phagocytosis is not strain specific.

Intracellular proliferation in J774 macrophages.

Cryptococcus is known to survive and proliferate within macrophages. We made use of a recently developed method for monitoring intracellular Cryptococcus proliferation in cell culture systems (our unpublished data). In vitro, the intracellular yeast cell number increases steadily for the first 18 to 24 h, before the number starts to decline due to host cell lysis. We used the maximal IPR as a measure of proliferative capacity that enables easy comparison of IPRs under different conditions (Fig. (Fig.2A2A).

FIG. 2.
Intracellular Cryptococcus proliferation is increased in J774 cells treated with Th2 cytokines but not altered in those treated with Th1/Th17 cytokines. Untreated J774 cells and cells treated with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), ...

To analyze if Th1, Th17, or Th2 cytokines influence intracellular cryptococcal proliferation, untreated or cytokine-pretreated J774 macrophages were infected with the C. neoformans strain ATCC 90112 or H99 or the C. gattii strain R265 and the yeast were monitored for intracellular proliferation over 72 h. For all three strains, treatment with the Th1 cytokine IFN-γ did not alter the IPR whereas the IPR in macrophages treated with the Th2 cytokine IL-4 was significantly increased (Fig. (Fig.2B).2B). To confirm that these findings were not specific to these two cytokines, we measured the IPR following J774 macrophage pretreatment with TNF-α (Th1 cytokine), IL-17 (Th17 cytokine), or IL-13 (Th2 cytokine). The three cryptococcal strains showed similar trends in this experimental analysis, and so, for simplicity, we present representative data for only one strain. In all cases, Th1/Th17 cytokines did not alter the IPR while Th2 activation increased it (Fig. (Fig.2C).2C). None of the cytokine treatments altered the timing of the maximal IPR, which always occurred at either 18 or 24 h postinfection.

Intracellular proliferation in primary human macrophages.

To rule out any cell line-specific effects and to test the clinical relevance of the results of this study, assays of intracellular proliferation in primary human monocyte-derived macrophages were conducted. Like J774 macrophages, human macrophages activated with the Th1 cytokine TNF-α or the Th17 cytokine IL-17 exhibited yeast IPRs much lower than those in cells treated with the Th2 cytokine IL-4 or IL-13. It is interesting that, in contrast to J774 cells, human primary macrophages that were untreated or treated with IFN-γ tended to have yeast IPRs higher than those in cells activated with TNF-α or IL-17 (Fig. (Fig.3),3), although future investigation of this phenomenon would require a larger donor pool in order to overcome significant donor-to-donor variability. Thus, the enhancement of the IPR that occurs following Th2 stimulation is conserved between mouse and human cells.

FIG. 3.
Intracellular Cryptococcus proliferation is increased in Th2 cytokine- but not altered in TNF-α and IL-17 cytokine-treated human primary macrophages. Human primary macrophages, untreated or treated with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), ...

We considered the possibility that mammalian cytokines may directly affect yeast growth. However, none of the recombinant mouse or human cytokines used in this study caused significant differences in the growth rate relative to that of untreated controls over 72 h, either in the presence or in the absence of macrophages (data not shown). Thus, cytokine signaling alters the capacity of macrophages to control intracellular cryptococci, rather than having an impact on extracellular killing or the yeast directly.

Influence of cytokine signaling on expulsion events in J774 macrophages.

Expulsion is believed to be a possible trafficking mechanism by which Cryptococcus disseminates within the infected individual without triggering local inflammation (34). Thus, the influence of the selected Th1, Th17, and Th2 cytokines on the occurrence of cryptococcal expulsion was analyzed using live-cell imaging. A total of 10,031 J774 macrophages (of which 1,208 showed internalized yeast cells) and 115 expulsion events were observed. The three Cryptococcus strains showed similar results (Fig. (Fig.4).4). However, expulsion is a very rare event (34), and to enable statistical analysis of the results for the different treatments, the data for all three strains were pooled and analyzed using a χ2 test. No statistically significant differences between untreated macrophages and cells activated with the Th1 cytokine IFN-γ (P > 0.5) or TNF-α (P > 0.2) or the Th17 cytokine IL-17 (P > 0.2) were found. Strikingly, however, the occurrence of expulsion in cultures treated with the Th2 cytokine IL-4 (P = 0.0001) or IL-13 (P = 0.0001) was significantly reduced (more than threefold) compared to that in the untreated cultures. Furthermore, a pairwise comparison of expulsion rates between Th1/Th17 and Th2 cytokine-treated cultures showed that the expulsion rate in each of the proinflammatory cytokine environments was significantly higher than that in each Th2 cytokine environment (P, <0.01 for all combinations).

FIG. 4.
The occurrence of cryptococcal expulsion from Th2 cytokine-treated J774 macrophages is reduced compared to that from untreated cells, but expulsion from TNF-α and IL-17 cytokine-treated J774 macrophages is not altered. J774 cells left untreated ...

Influence of cytokine signaling on expulsion events in human primary monocyte-derived macrophages.

To test whether the reduction of expulsion in response to Th2 cytokines is an artifact of the J774 cell line, human primary macrophages were activated with the Th1 cytokines IFN-γ and TNF-α, the Th17 cytokine IL-17, or the Th2 cytokines IL-4 and IL-13, infected with cryptococci, and then visualized and analyzed for the occurrence of expulsion. As in J774 macrophages, the three cryptococcal strains showed similar trends (Fig. (Fig.5),5), and we therefore pooled the data for all three strains to permit statistical analysis. As for J774 macrophages, expulsion rates did not differ significantly between untreated macrophages and cells activated with the Th1 cytokine IFN-γ (P > 0.12) or the Th17 cytokine IL-17 (P > 0.68), although expulsion events were slightly (P = 0.03) more common among TNF-α-treated cells. In contrast, however, the occurrence of expulsion in cultures treated with the Th2 cytokines IL-4 and IL-13 was significantly reduced compared to that in untreated samples (P, 0.0001 and 0.04, respectively). Thus, Th2 cytokines reduce the rates of expulsion from both J774 and human primary macrophages.

FIG. 5.
The occurrence of cryptococcal expulsion from Th2 cytokine-treated human primary macrophages is reduced compared to that from untreated cells, but expulsion from TNF-α and IL-17 cytokine-treated cells is not altered. Human primary macrophages ...

Complement-opsonized Cryptococcus IPR in and expulsion from cytokine-treated J774 macrophages.

While opsonizing antibodies to Cryptococcus may or may not be present during an infection, complement opsonization should occur in all healthy individuals. To determine the effects of Th1, Th17, and Th2 cytokines on complement-opsonized yeast, intracellular proliferation in and the occurrence of expulsion from cytokine-treated J774 cells infected with serum-opsonized C. neoformans strain ATCC 90112 cells were assessed. Like antibody-opsonized yeast, complement-opsonized yeast proliferated more readily in, but were expelled less frequently from, Th2-stimulated macrophages than Th1- or Th17-stimulated macrophages (Fig. (Fig.6).6). Thus, the effects of cytokine activation appear to be largely independent of the opsonin present on the cryptococcal surface.

FIG. 6.
Complement-mediated opsonization does not change the effect of cytokine stimulation on cryptococcal behavior. Human primary macrophages left untreated or treated with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), Th17 (10 ng/ml IL-17), or Th2 ...

DISCUSSION

The importance of a Th1 immune response for controlling the cryptococcal burden is evident from the high incidence of severe C. neoformans infections in human immunodeficiency virus (HIV)-infected patients (51) and from data obtained with numerous mouse models (19, 29, 36, 53). Here, we provide a molecular explanation for these in vivo observations. Our results strongly suggest the inhibition of anticryptococcal macrophage functions, such as phagocytosis and intracellular clearance, by Th2 cytokines, which would be likely to lead to higher fungal burdens in hosts showing a Th2-dominated response, as reported previously (7, 14, 39).

We selected three different Cryptococcus strains to represent the two most important serotypes. The C. neoformans serotype A strains ATCC 90112 and H99, both clinical isolates from patient cerebrospinal fluids, represent the serotype most commonly isolated from immunocompromised patients, which is responsible for 95% of all C. neoformans infections (26). The C. gattii serotype B strain R265 is a clinical isolate from the ongoing cryptococcosis outbreak in immunocompetent individuals in British Columbia, Canada, an outbreak that highlights the potential of Cryptococcus as an emerging pathogen (20, 35). Trends for the three strains were similar throughout the study, and thus, the influence of cytokine signaling on cryptococcal pathogenesis is likely to be common to both Cryptococcus species, despite the different disease etiologies associated with the two species.

We deliberately selected the lowest possible cytokine concentrations for this study in order to minimize the risk of artifacts. Given this situation, we recognize that there may be more dramatic effects on cryptococcal behavior at higher concentrations and, thus, that the Th1-Th17-Th2 balance may profoundly affect the path of disease progression in infected patients.

We have demonstrated that intracellular yeast proliferation is increased after Th2 activation of macrophages. These findings provide a molecular explanation for the protective effect of Th1 cytokines such as IFN-γ and TNF-α (29, 36, 53) and Th17 cytokines such as IL-17 (39) and the nonprotective effect of Th2 cytokines such as IL-4 and IL-13 (7, 14, 39) in mouse model systems.

Two additional interesting observations can be made based on the IPR data set. First, it is intriguing that IFN-γ suppresses the cryptococcal IPR in J774 cells far more effectively than that in human primary macrophages. This observation is in agreement with the data in previous reports demonstrating a reduction in the inhibition of cryptococcal growth in human alveolar and monocyte-derived macrophages after IFN-γ treatment (30, 44). Such findings may indicate differences in IFN-γ-related regulation of anticryptococcal functions between mouse and human macrophages. Second, in J774 cells, treatment with proinflammatory cytokines did not alter cryptococcal proliferation compared with that in untreated controls. This result is suggestive of the fact that J774 macrophages may already be partially activated by the presence of opsonized cryptococci and/or the tissue culture environment.

We have also demonstrated for the first time that Th2, but not Th1 or Th17, cytokine treatment leads to a significant reduction in the occurrence of cryptococcal expulsion, a recently described phenomenon that appears to be unique to Cryptococcus (3, 34). The relevance of expulsion for disease progression is, as yet, unclear. However, cryptococci expelled from circulating monocytes will likely be exposed to a far greater immune attack than those that remain intracellular. In addition, reduced expulsion may favor the dissemination of Cryptococcus to the central nervous system in parasitized macrophages via a Trojan Horse mechanism (10, 31). Thus, the suppression of cryptococcal expulsion by a dominant Th2 cytokine profile may benefit the pathogen and reduce the host's likelihood of surviving an infection. It is important that reduced expulsion alone is insufficient to account for the increased IPR observed in Th2-stimulated macrophages; even in Th1-stimulated cells, expulsion is a very rare effect (34) that cannot account for the observed differences in IPRs. Thus, cytokine signaling operates in at least two ways, first, to modify intracellular proliferation and, second, to influence the likelihood of expulsion.

How might cytokine signaling have an impact on intracellular cryptococcal behavior? The most likely scenario is that cytokine activation triggers changes in the composition of the phagosome that favor either expulsion (Th1/Th17) or proliferation (Th2). Recent data suggest that one such change may be the availability of metal ions. Many cryptococcal virulence factors such as laccase, superoxide dismutase, catalase, and urease depend upon metal ions and thus cation homeostasis. Interestingly, the anti-inflammatory cytokines IL-4 and IL-13 have been suggested to enhance iron uptake and storage by macrophages by suppressing the activation of iron regulatory proteins 1 and 2, leading to translational repression of the iron storage protein ferretin or transcriptional activation of the membrane receptor for iron uptake (52). Thus, greater metal ion availability may increase the activity of cryptococcal virulence factors and may lead to increased intracellular proliferation in Th2-stimulated cells. In this regard, it is intriguing that urease, a cryptococcal virulence factor (13), promotes a nonprotective Th2 immune response within the lung (42), suggesting that Cryptococcus may actively bias the host's cytokine profile to its own advantage.

The incidence of cryptococcosis increases throughout the course of HIV infection and correlates with the loss of a Th1 response in HIV-infected patients (1) and with a Th2-type cytokine profile in transplant recipients (48). Our data indicate that the loss of Th1 cytokines significantly reduces the ability of macrophages to deal with Cryptococcus and prevents efficient cryptococcal clearance in HIV-infected patients and, therefore, that proinflammatory cytokines may be a useful therapeutic agent for the treatment of cryptococcosis. IFN-γ has been successfully applied to enhance chemotherapy of systemic cryptococcosis in BALB/c mice (32) and in SCID mice (12) and, thus, has been suggested for use in a potential therapeutic regimen for humans. However, there seem to be differences in IFN-γ-related signaling in mice and humans. IFN-γ has clearly been demonstrated to have a protective role in mouse model systems of cryptococcosis (5, 24) and to increase fungicidal activity of murine macrophages (19). However, among human cells, IFN-γ treatment reduces the capacity of human alveolar macrophages (44) and human monocyte-derived macrophages (30) to inhibit cryptococcal growth, a finding that is supported by our data. Although IFN-γ levels at the site of infection have been negatively correlated with cryptococcal CFU (47) and IFN-γ therapy has proved to be successful for one patient (41), these in vitro data suggest that IFN-γ treatment may not always be an appropriate therapeutic approach.

Taken together, our data suggest that the poor prognosis associated with Th2 cytokine profiles in cryptococcal infection may result in part from a combination of increased intracellular proliferation and reduced expulsion of the pathogen. These effects are independent of the cryptococcal strain or the phagocytic opsonin and may therefore represent a general phenomenon associated with intracellular pathogens.

Acknowledgments

We thank Arturo Casadevall for providing the 18B7 antibody used in this study.

This work was financially supported by grants from the Medical Research Council (G0601171) and the Darwin Trust of Edinburgh.

Notes

Editor: A. Casadevall

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 June 2009.

REFERENCES

1. Altfeld, M., M. M. Addo, K. A. Kreuzer, J. K. Rockstroh, F. L. Dumoulin, K. Schliefer, L. Leifeld, T. Sauerbruch, and U. Spengler. 2000. T(H)1 to T(H)2 shift of cytokines in peripheral blood of HIV-infected patients is detectable by reverse transcriptase polymerase chain reaction but not by enzyme-linked immunosorbent assay under nonstimulated conditions. J. Acquir. Immune Defic. Syndr. 23287-294. [PubMed]
2. Alvarez, M., and A. Casadevall. 2007. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages. BMC Immunol. 816. [PMC free article] [PubMed]
3. Alvarez, M., and A. Casadevall. 2006. Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages. Curr. Biol. 162161-2165. [PubMed]
4. Arora, S., Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and G. B. Huffnagle. 2005. Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis. J. Immunol. 1746346-6356. [PubMed]
5. Bava, A. J., J. Afeltra, R. Negroni, and R. A. Diez. 1995. Interferon gamma increases survival in murine experimental cryptococcosis. Rev. Inst. Med. Trop. Sao Paulo 37391-396. [PubMed]
6. Beenhouwer, D. O., S. Shapiro, M. Feldmesser, A. Casadevall, and M. D. Scharff. 2001. Both Th1 and Th2 cytokines affect the ability of monoclonal antibodies to protect mice against Cryptococcus neoformans. Infect. Immun. 696445-6455. [PMC free article] [PubMed]
7. Blackstock, R., and J. W. Murphy. 2004. Role of interleukin-4 in resistance to Cryptococcus neoformans infection. Am. J. Respir. Cell Mol. Biol. 30109-117. [PubMed]
8. Bulmer, G. S., and J. R. Tacker. 1975. Phagocytosis of Cryptococcus neoformans by alveolar macrophages. Infect. Immun. 1173-79. [PMC free article] [PubMed]
9. Casadevall, A., and J. R. Perfect. 1998. Cryptococcus neoformans, 1st ed., vol. 1. American Society for Microbiology, Washington, DC.
10. Charlier, C., K. Nielsen, S. Daou, M. Brigitte, F. Chretien, and F. Dromer. 2009. Evidence of a role for monocytes in dissemination and brain invasion by Cryptococcus neoformans. Infect. Immun. 77120-127. [PMC free article] [PubMed]
11. Chretien, F., O. Lortholary, I. Kansau, S. Neuville, F. Gray, and F. Dromer. 2002. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia. J. Infect. Dis. 186522-530. [PubMed]
12. Clemons, K. V., J. E. Lutz, and D. A. Stevens. 2001. Efficacy of recombinant gamma interferon for treatment of systemic cryptococcosis in SCID mice. Antimicrob. Agents Chemother. 45686-689. [PMC free article] [PubMed]
13. Cox, G. M., J. Mukherjee, G. T. Cole, A. Casadevall, and J. R. Perfect. 2000. Urease as a virulence factor in experimental cryptococcosis. Infect. Immun. 68443-448. [PMC free article] [PubMed]
14. Decken, K., G. Kohler, K. Palmer-Lehmann, A. Wunderlin, F. Mattner, J. Magram, M. K. Gately, and G. Alber. 1998. Interleukin-12 is essential for a protective Th1 response in mice infected with Cryptococcus neoformans. Infect. Immun. 664994-5000. [PMC free article] [PubMed]
15. Del Poeta, M. 2004. Role of phagocytosis in the virulence of Cryptococcus neoformans. Eukaryot. Cell 31067-1075. [PMC free article] [PubMed]
16. Diamond, R. D., and J. E. Bennett. 1973. Growth of Cryptococcus neoformans within human macrophages in vitro. Infect. Immun. 7231-236. [PMC free article] [PubMed]
17. Feldmesser, M., Y. Kress, P. Novikoff, and A. Casadevall. 2000. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 684225-4237. [PMC free article] [PubMed]
18. Feldmesser, M., S. Tucker, and A. Casadevall. 2001. Intracellular parasitism of macrophages by Cryptococcus neoformans. Trends Microbiol. 9273-278. [PubMed]
19. Flesch, I. E., G. Schwamberger, and S. H. Kaufmann. 1989. Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans. J. Immunol. 1423219-3224. [PubMed]
20. Fraser, J. A., R. L. Subaran, C. B. Nichols, and J. Heitman. 2003. Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot. Cell 21036-1045. [PMC free article] [PubMed]
21. Garcia-Hermoso, D., G. Janbon, and F. Dromer. 1999. Epidemiological evidence for dormant Cryptococcus neoformans infection. J. Clin. Microbiol. 373204-3209. [PMC free article] [PubMed]
22. Goldman, D. L., S. C. Lee, A. J. Mednick, L. Montella, and A. Casadevall. 2000. Persistent Cryptococcus neoformans pulmonary infection in the rat is associated with intracellular parasitism, decreased inducible nitric oxide synthase expression, and altered antibody responsiveness to cryptococcal polysaccharide. Infect. Immun. 68832-838. [PMC free article] [PubMed]
23. Higgins, S. C., A. G. Jarnicki, E. C. Lavelle, and K. H. Mills. 2006. TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J. Immunol. 1777980-7989. [PubMed]
24. Hoag, K. A., M. F. Lipscomb, A. A. Izzo, and N. E. Street. 1997. IL-12 and IFN-gamma are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice. Am. J. Respir. Cell Mol. Biol. 17733-739. [PubMed]
25. Huffnagle, G. B. 1996. Role of cytokines in T cell immunity to a pulmonary Cryptococcus neoformans infection. Biol. Signals 5215-222. [PubMed]
26. Hull, C. M., and J. Heitman. 2002. Genetics of Cryptococcus neoformans. Annu. Rev. Genet. 36557-615. [PubMed]
27. Idnurm, A., Y. S. Bahn, K. Nielsen, X. Lin, J. A. Fraser, and J. Heitman. 2005. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat. Rev. Microbiol. 3753-764. [PubMed]
28. Kawakami, K., M. Hossain Qureshi, T. Zhang, Y. Koguchi, Q. Xie, M. Kurimoto, and A. Saito. 1999. Interleukin-4 weakens host resistance to pulmonary and disseminated cryptococcal infection caused by combined treatment with interferon-gamma-inducing cytokines. Cell. Immunol. 19755-61. [PubMed]
29. Kawakami, K., S. Kohno, J. Kadota, M. Tohyama, K. Teruya, N. Kudeken, A. Saito, and K. Hara. 1995. T cell-dependent activation of macrophages and enhancement of their phagocytic activity in the lungs of mice inoculated with heat-killed Cryptococcus neoformans: involvement of IFN-gamma and its protective effect against cryptococcal infection. Microbiol. Immunol. 39135-143. [PubMed]
30. Levitz, S. M., and T. P. Farrell. 1990. Growth inhibition of Cryptococcus neoformans by cultured human monocytes: role of the capsule, opsonins, the culture surface, and cytokines. Infect. Immun. 581201-1209. [PMC free article] [PubMed]
31. Luberto, C., B. Martinez-Marino, D. Taraskiewicz, B. Bolanos, P. Chitano, D. L. Toffaletti, G. M. Cox, J. R. Perfect, Y. A. Hannun, E. Balish, and M. Del Poeta. 2003. Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans. J. Clin. Investig. 1121080-1094. [PMC free article] [PubMed]
32. Lutz, J. E., K. V. Clemons, and D. A. Stevens. 2000. Enhancement of antifungal chemotherapy by interferon-gamma in experimental systemic cryptococcosis. J. Antimicrob. Chemother. 46437-442. [PubMed]
33. Ma, H., J. E. Croudace, D. A. Lammas, and R. C. May. 2007. Direct cell-to-cell spread of a pathogenic yeast. BMC Immunol. 815. [PMC free article] [PubMed]
34. Ma, H., J. E. Croudace, D. A. Lammas, and R. C. May. 2006. Expulsion of live pathogenic yeast by macrophages. Curr. Biol. 162156-2160. [PubMed]
35. MacDougall, L., S. E. Kidd, E. Galanis, S. Mak, M. J. Leslie, P. R. Cieslak, J. W. Kronstad, M. G. Morshed, and K. H. Bartlett. 2007. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerg. Infect. Dis. 1342-50. [PMC free article] [PubMed]
36. Milam, J. E., A. C. Herring-Palmer, R. Pandrangi, R. A. McDonald, G. B. Huffnagle, and G. B. Toews. 2007. Modulation of the pulmonary type 2 T-cell response to Cryptococcus neoformans by intratracheal delivery of a tumor necrosis factor alpha-expressing adenoviral vector. Infect. Immun. 754951-4958. [PMC free article] [PubMed]
37. Mitchell, T. G., and L. Friedman. 1972. In vitro phagocytosis and intracellular fate of variously encapsulated strains of Cryptococcus neoformans. Infect. Immun. 5491-498. [PMC free article] [PubMed]
38. Mitchell, T. G., and J. R. Perfect. 1995. Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clin. Microbiol. Rev. 8515-548. [PMC free article] [PubMed]
39. Müller, U., W. Stenzel, G. Kohler, C. Werner, T. Polte, G. Hansen, N. Schutze, R. K. Straubinger, M. Blessing, A. N. McKenzie, F. Brombacher, and G. Alber. 2007. IL-13 induces disease-promoting type 2 cytokines, alternatively activated macrophages and allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans. J. Immunol. 1795367-5377. [PubMed]
40. Murray, H. W., G. L. Spitalny, and C. F. Nathan. 1985. Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma. J. Immunol. 1341619-1622. [PubMed]
41. Netea, M. G., A. E. Brouwer, E. H. Hoogendoorn, J. W. Van der Meer, M. Koolen, P. E. Verweij, and B. J. Kullberg. 2004. Two patients with cryptococcal meningitis and idiopathic CD4 lymphopenia: defective cytokine production and reversal by recombinant interferon-gamma therapy. Clin. Infect. Dis. 39e83-87. [PubMed]
42. Osterholzer, J. J., R. Surana, J. E. Milam, G. T. Montano, G. H. Chen, J. Sonstein, J. L. Curtis, G. B. Huffnagle, G. B. Toews, and M. A. Olszewski. 2009. Cryptococcal urease promotes the accumulation of immature dendritic cells and a non-protective T2 immune response within the lung. Am. J. Pathol. 174932-943. [PMC free article] [PubMed]
43. Ralph, P., J. Prichard, and M. Cohn. 1975. Reticulum cell sarcoma: an effector cell in antibody-dependent cell-mediated immunity. J. Immunol. 114898-905. [PubMed]
44. Reardon, C. C., S. J. Kim, R. P. Wagner, and H. Kornfeld. 1996. Interferon-gamma reduces the capacity of human alveolar macrophages to inhibit growth of Cryptococcus neoformans in vitro. Am. J. Respir. Cell Mol. Biol. 15711-715. [PubMed]
45. Roilides, E., A. Dimitriadou-Georgiadou, T. Sein, I. Kadiltsoglou, and T. J. Walsh. 1998. Tumor necrosis factor alpha enhances antifungal activities of polymorphonuclear and mononuclear phagocytes against Aspergillus fumigatus. Infect. Immun. 665999-6003. [PMC free article] [PubMed]
46. Santangelo, R., H. Zoellner, T. Sorrell, C. Wilson, C. Donald, J. Djordjevic, Y. Shounan, and L. Wright. 2004. Role of extracellular phospholipases and mononuclear phagocytes in dissemination of cryptococcosis in a murine model. Infect. Immun. 722229-2239. [PMC free article] [PubMed]
47. Siddiqui, A. A., A. E. Brouwer, V. Wuthiekanun, S. Jaffar, R. Shattock, D. Irving, J. Sheldon, W. Chierakul, S. Peacock, N. Day, N. J. White, and T. S. Harrison. 2005. IFN-gamma at the site of infection determines rate of clearance of infection in cryptococcal meningitis. J. Immunol. 1741746-1750. [PubMed]
48. Singh, N., B. D. Alexander, O. Lortholary, F. Dromer, K. L. Gupta, G. T. John, R. del Busto, G. B. Klintmalm, J. Somani, G. M. Lyon, K. Pursell, V. Stosor, P. Munoz, A. P. Limaye, A. C. Kalil, T. L. Pruett, J. Garcia-Diaz, A. Humar, S. Houston, A. A. House, D. Wray, S. Orloff, L. A. Dowdy, R. A. Fisher, J. Heitman, M. M. Wagener, and S. Husain. 2008. Pulmonary cryptococcosis in solid organ transplant recipients: clinical relevance of serum cryptococcal antigen. Clin. Infect. Dis. 46e12-18. [PMC free article] [PubMed]
49. Swenson, F. J., and T. R. Kozel. 1978. Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated macrophages. Infect. Immun. 21714-720. [PMC free article] [PubMed]
50. Tucker, S. C., and A. Casadevall. 2002. Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad. Sci. USA 993165-3170. [PMC free article] [PubMed]
51. Wadhwa, A., R. Kaur, and P. Bhalla. 2008. Profile of central nervous system disease in HIV/AIDS patients with special reference to cryptococcal infections. Neurologist 14247-251. [PubMed]
52. Weiss, G., C. Bogdan, and M. W. Hentze. 1997. Pathways for the regulation of macrophage iron metabolism by the anti-inflammatory cytokines IL-4 and IL-13. J. Immunol. 158420-425. [PubMed]
53. Wormley, F. L., Jr., J. R. Perfect, C. Steele, and G. M. Cox. 2007. Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain. Infect. Immun. 751453-1462. [PMC free article] [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...