Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans

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 was 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).

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 with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), Th17 (10 ng/ml IL-17), or Th2 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines and then monitored to assess cytokine efficacy at the chosen cytokine concentrations. (A) Representative images of cytokine-treated J774 and human primary macrophage cells. Note the polarized and spread-out patterns of activated, but not control, cells. (B) Cytokine treatment does not significantly influence human primary macrophage survival. The data are presented as the means of results from at least three individual experiments.

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), Th17 (10 ng/ml IL-17), or Th2 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines were infected with C. neoformans strain ATCC 90112 or H99 or C. gattii strain R265 and then further incubated, under the same conditions used during infection, to monitor intracellular yeast proliferation. (A) Schematic graph illustrating how the IPR was derived. (B) The IPR is not altered under the influence of the Th1 cytokine IFN-γ but increases under the influence of the Th2 cytokine IL-4 for all three selected strains. (C) The IPR of ATCC 90112 is unaltered following treatment with the Th1 cytokines IFN-γ and TNF-α and the Th17 cytokine IL-17 but increases significantly following treatment with the Th2 cytokines IL-4 and IL-13. The data are presented as the means of results from at least three individual experiments ±2 standard errors of the means. The means were tested for statistical significance by using Tukey's HSD test, assuming equal variances. Statistically significant differences between the test results and the control values are indicated by asterisks (*, P < 0.05; **, P ≤ 0.01).

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), Th17 (10 ng/ml IL-17), or Th2 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines, were infected with C. neoformans strain ATCC 90112 or H99 or C. gattii strain R265 and then further incubated under the same conditions used during infection to monitor intracellular yeast proliferation. The IPR is low following treatment with the Th1 cytokine TNF-α or the Th17 cytokine IL-17 but higher following treatment with the Th2 cytokine IL-4 or IL-13, although due to high donor variability and the multiplicity of testing, not all comparisons are significant at the 5% level. The data are presented as the means of results from at least three individual experiments ±2 standard errors of the means. Statistically significant differences among the indicated results and those for TNF-α and IL-17 are denoted by asterisks (*, P < 0.05; ***, P ≤ 0.001).

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 or treated with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), Th17 (10 ng/ml IL-17), or Th2 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines were infected with C. neoformans strain ATCC 90112 or H99 or C. gattii strain R265 and then further incubated, under the same conditions used during infection, while being monitored for cryptococcal expulsion. The occurrence of expulsion following the treatment of cells with the Th2 cytokine IL-4 or IL-13 is significantly reduced compared to that from untreated or Th1/Th17-treated cells. The presented data were obtained from at least three individual experiments and analyzed for statistical significance by χ² tests. Statistically significant differences are indicated by asterisks (**, P ≤ 0.01).

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 either untreated or treated with Th1 (10 U/ml IFN-γ or 1 ng/ml TNF-α), Th17 (10 ng/ml IL-17), or Th2 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines were infected with C. neoformans strain ATCC 90112 or H99 or C. gattii strain R265 and then further incubated, under the same conditions used during infection, while being monitored by live-cell imaging for cryptococcal expulsion. The occurrence of expulsion following the treatment of cells with the Th2 cytokine IL-4 or IL-13 is significantly reduced compared to that from untreated or Th1/Th17-treated cells. The presented data were obtained from at least three individual experiments and analyzed for statistical significance by χ² tests. Statistically significant differences are indicated by asterisks (**, P ≤ 0.01).

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 (10 ng/ml IL-4 or 10 ng/ml IL-13) cytokines were infected with C. neoformans strain ATCC 90112, opsonized in fresh human sera, and then further incubated, under the same conditions used during infection, to monitor intracellular yeast proliferation or to monitor cryptococcal expulsion by live-cell imaging. (A) The IPR following treatment with the Th1 cytokine TNF-α or the Th17 cytokine IL-17 is significantly lower than that following treatment with either of the Th2 cytokines. The data are presented as the means of results from at least three individual experiments ±2 standard errors of the means. (B) The occurrence of expulsion following treatment with the Th2 cytokine IL-4 or IL-13 is reduced compared to that from untreated cells or cells treated with IFN-γ. Data were obtained from at least three individual experiments. Statistically significant differences are indicated by asterisks (*, P < 0.05).