Characterizing the Mechanisms of Nonopsonic Uptake of Cryptococci by Macrophages

Abstract

The pathogenic fungus Cryptococcus enters the human host via inhalation into the lung and is able to reside in a niche environment that is serum- (opsonin) limiting. Little is known about the mechanism by which nonopsonic phagocytosis occurs via phagocytes in such situations. Using a combination of soluble inhibitors of phagocytic receptors and macrophages derived from knockout mice and human volunteers, we show that uptake of nonopsonized Cryptococcus neoformans and C. gattii via the mannose receptor is dependent on macrophage activation by cytokines. However, although uptake of C. neoformans is via both dectin-1 and dectin-2, C. gattii uptake occurs largely via dectin-1. Interestingly, dectin inhibitors also blocked phagocytosis of unopsonized Cryptococci in wax moth (Galleria mellonella) larvae and partially protected the larvae from infection by both fungi, supporting a key role for host phagocytes in augmenting early disease establishment. Finally, we demonstrated that internalization of nonopsonized Cryptococci is not accompanied by the nuclear translocation of NF-κB or its concomitant production of proinflammatory cytokines such as TNF-α. Thus, nonopsonized Cryptococci are recognized by mammalian phagocytes in a manner that minimizes proinflammatory cytokine production and potentially facilitates fungal pathogenesis.

FIGURE 1.

Mannose receptor is important but dispensable during uptake of Cryptococcus particles. Mouse macrophage cell line J774.A1 (A) or differentiated BMMs (B) (WT or MR KO) or differentiated primary human macrophages (C) were challenged with either C. neoformans H99 (Cn, black bars) or C. gattii R265 (Cg, white bars) for 60 min, processed for immunofluorescence, and scored for phagocytosis as described in Materials and Methods. Where indicated, J774.A1 and primary human macrophages were pretreated with 100 μg/ml mannan (MAN) for 30 min before the addition of Cryptococcus particles. Phagocytosis indices were related to the values obtained from the negative controls. Number in bars indicate the total number of phagocytes counted. Results are expressed as the mean ± SD of at least three independent experiments.

FIGURE 2.

Uptake of Cryptococcus particles is Syk-dependent. Mouse macrophage cell line J774.A1 (A) or differentiated primary human macrophages (B) were challenged with unopsonized C. neoformans H99 (Cn, black bars) or C. gattii R265 (Cg, white bars) for 60 min, processed for immunofluorescence, and scored for phagocytosis as described in Materials and Methods. Phagocytosis indices were related to the values obtained from the negative controls. Number in bars indicate the total number of phagocytes counted. Results are expressed as the mean ± SD of at least three independent experiments.

FIGURE 3.

Activated Syk is essential for the uptake of Cryptococcus particles. Mouse macrophage cell line J774.A1 was challenged with either (IgG-opsonized or unopsonized, U/O) C. neoformans H99 or C. gattii R265 for 15 min (B), processed for immunofluorescence, and analyzed by confocal microscopy of localized phospho-Syk (B and C) as described in Materials and Methods. (A) Schematic diagram J774.A1 macrophage with intracellular actin cytoskeleton (red) and yeast particles (blue). To confirm phospho-Syk localization, the bottom of the cells was observed first [(A), grey dashed line and (B), bottom panels], before moving to the middle of the cells [(A), purple dashed line, (B), top panels]. Pixel intensities for 20 cells per sample were determined [(C), right] and normalized to the intensity at the center of the cell [(C), left]. (A and C) The green triangles denote phospho-Syk. The black line denotes the outline of a cell as imagined from the side (i.e., its z-axis). Results are expressed as the mean ± SD of at least three independent experiments. Scale bar, 20 μm.

FIGURE 4.

Dectins are required for uptake of Cryptococcus particles. Mouse macrophage cell line J774.A1 (A), differentiated primary human macrophages (B), or differentiated BMMs (C) (WT, Dectin-1 KO, or Dectin-2 KO) were challenged with either C. neoformans H99 (Cn, black bars) or C. gattii R265 (Cg, white bars) for 60 min, processed for immunofluorescence, and scored for phagocytosis as described in Materials and Methods. Where indicated, J774.A1 were pretreated with 100 μg/ml glucan-6-phosphate (G6P) for 30 min before the addition of Cryptococcus particles. Phagocytosis indices were related to the values obtained from the negative controls. Number in bars indicate the total number of phagocytes counted. Results are expressed as the mean ± SD of at least three independent experiments. *p < 0.05. ns, not significant (p ≥ 0.05).

FIGURE 5.

Administration of polysaccharides blocks uptake of Cryptococcus particles to hemocytes in the G. mellonella larvae model. Larvae were inoculated with 60 μg of blocking sugars 1 h prior to infection for 2 h with 10⁶ C. neoformans H99 (black bars) or C. gattii R265 (white bars). Uptake of yeast of hemocytes was determined under light microscopy. Results are expressed as the mean ± SD of at least three independent experiments. *p < 0.05 (related to PBS control). ns, not significant (p ≥ 0.05).

FIGURE 6.

Glucan administration protects G. mellonella larvae from infection by C. neoformans or C. gattii. Larvae were inoculated with 60 μg of blocking sugars 24 h prior to infection for a further 24 h with 10⁶ C. neoformans H99 (black bars) or C. gattii R265 (white bars). Fungal load was determined by serially diluting homogenized larvae and plating aliquots onto erythromycin containing agar plates. Yeast cell density were related to the values obtained from the negative (PBS) controls and expressed as cfu × 10⁵/larva. Results are expressed as the mean ± SD of at least three independent experiments. *p < 0.05 (related to PBS control). ns, not significant (p ≥ 0.05).

FIGURE 7.

Uptake of Cryptococcus did not affect NF-κB nuclear translocation. J774.A1 macrophages were challenged with a variety of opsonized or unopsonized pathogenic fungi, SRBCs, or soluble agonists (LPS or PMA), processed for immunofluorescences, analyzed by microscopy (A), and scored for p65 nuclear translocation (B), as described in Materials and Methods. (A) Representative images of PMA- (top) or LPS- (bottom) stimulated J774.A1 macrophages and stained to highlight either actin or p65. Actin was stained using rhodamine-phalloidin; p65 was stained using the anti–65 kDa subunit (p65) NFκB mAb with an anti-rabbit Alexa Fluor–488. Scale bar, 20 μm.

FIGURE 8.

Uptake of Cryptococcus did not affect proinflammatory cytokine response. J774.A1 macrophages (black bars) or differentiated primary human macrophages (white bars) were challenged with a variety of unopsonized pathogenic fungi (C. neoformans, Cn; C. gattii. Cg; Candida albicans, Ca) or LPS, and subsequent supernatants were analyzed by ELISA, as described in Materials and Methods. Results are expressed as the mean ± SD of at least three independent experiments. *p < 0.05.