Extracellular vesicles from Cryptococcus neoformans modulate macrophage functions

Abstract

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-alpha), interleukin-10 (IL-10), and transforming growth factor beta (TGF-beta). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.

FIG. 1.

Incorporation of vesicular components by macrophages. DiI-stained vesicles (red fluorescence) were incubated with RAW 264.7 macrophages, followed by staining of the nucleus with DAPI (blue fluorescence). (A) A plasma membrane was stained with FITC-labeled cholera toxin subunit B (green fluorescence). (B) Macrophages were incubated with serum samples from patients with cryptococcosis followed by Alexa Fluor 488 anti-human IgG antibody. The merged image at the right (third panel) demonstrates that DiI-stained compartments colocalize, at least partially, with antigenic vesicular components (arrowheads). Scale bars, 10 μm. (C) Macrophage viability is apparently not affected by the vesicles, as demonstrated by the measurement of LDH activity in supernatants of macrophages treated with vesicles (at a 2 μg/ml sterol concentration) or incubated in regular medium. As a positive control for enzyme activity, the macrophages were incubated with Triton X-100. (A_i − A_f)₃₄₀ nm indicates the difference between the initial and final absorbance readings.

FIG. 2.

Vesicle-stimulated macrophages produce nitric oxide. (A) Dose-dependent production of NO was observed for vesicle fractions from both acapsular and encapsulated fungi. The asterisks indicate P < 0.05. Vesicle concentrations are expressed as a function of the sterol content in their membranes. (B) Macrophages were incubated overnight with vesicles from different strains at a final concentration of 0.4 μg/ml of sterol. GXM and L-NIL were added in final concentrations of 30 μg/ml and 3 μM, respectively. The levels of nitric oxide production in the culture medium (no vesicles) were below the detection limit of the technique. Bars represent averages of three measurements; brackets denote standard deviations. Data shown are representative of the results of three independent experiments.

FIG. 3.

Profile of cytokine production by macrophages in response to stimulation with vesicle fractions. Macrophages were stimulated with vesicles isolated from culture supernatants from encapsulated (HEC3393) and acapsular (CAP67) C. neoformans cells. The sterol concentration corresponded to 0.4 μg/ml in all fractions. The basal production of each cytokine in the medium alone is also shown (first bars). TNF-α, TGF-β, and IL-10 concentrations in the supernatant were determined by capture ELISA. Statistical significance values are highlighted; P < 0.05. Bars represent averages of three measurements; brackets denote standard deviations. Data shown are representative of the results of three independent experiments.

FIG. 4.

Macrophages stimulated with vesicles exhibit enhanced phagocytosis. (A) Macrophages were treated overnight with vesicles derived from CAP67 or HEC3393 at a sterol concentration of 1 μM. Gray peaks represent uninfected macrophages; open histograms show the population analysis after interaction with FITC-C. neoformans within the M1 region, and the corresponding macrophage fluorescence increase is indicated numerically. These data are representative of the results of three individual experiments. The percentages of positive fluorescence (M1) are indicated. Asterisks denote P > 0.05. (B) Analysis by fluorescence microscopy of the material prepared for flow cytometry. Infected cells were visualized using differential interference contrast microscopy and fluorescence microscopy. Scale bars, 10 μM.

FIG. 5.

Fungicidal activity of vesicle-stimulated macrophages. Macrophages treated with vesicles isolated from encapsulated or acapsular C. neoformans were incubated with the encapsulated C. neoformans strain for different periods for CFU determination of numbers of intracellular C. neoformans. CFU values were multiplied using the dilution factor to generate absolute cell numbers. Bars represent averages of three measurements; brackets represent standard deviations. Statistically significant differences are highlighted.