Live Candida albicans suppresses production of reactive oxygen species in phagocytes

Abstract

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.

FIG. 1.

Live C. albicans suppresses production of ROS by J774 cells. (A) ROS production by unstimulated J774 cells was measured by luminol-enhanced chemiluminescence. Data represent mean RLU of three identical samples collected with 1-second integrations over 3 hours; error bars represent the standard deviations (SD) of the three measurements. J774 cells were treated with C. albicans at a yeast/phagocyte ratio of 5:1. (B) Bar heights indicate the mean area under the curve (AUC) of the data presented in panel A; error bars are the SD of the three AUC results. Data for J774 cells stimulated with PMA were acquired as for panel A, with the exception that PMA was added with the luminol mixture. (C) ROS production was measured using CM-H₂DCFDA. Bar heights represent the mean fluorescence intensity after 150 min of incubation, after background fluorescence was subtracted (error bars represent the SD). (D) Metabolic activities of live, UV yeast, and HK yeast were measured as the ability to metabolize the tetrazolium dye XTT. Bar heights indicate mean absorbances; error bars indicate SD. All experiments were performed at least three times (with the exception of the CM-H₂DCFDA experiment, which was performed twice), with the same trend observed each time. A representative experiment of each type is shown. Asterisks indicate results significantly different from those obtained with HK yeast (Student's t test; P < 0.005).

FIG. 2.

Live C. albicans suppresses ROS production in a dose-dependent manner. ROS production by J774 cells was measured in response to live or HK yeast at the indicated yeast/phagocyte ratio. Each experiment was performed with triplicate samples; the bar heights indicates the mean values and error bars represent the standard deviations. A representative experiment is presented; the experiment was repeated three times with the same trend observed each time. Live yeast resulted in significantly less ROS production than HK yeast at each yeast/phagocyte ratio (two-way ANOVA, P < 0.001 overall; for all individual yeast/phagocyte ratios, post hoc Student's t test comparisons yielded P values <0.001).

FIG. 3.

Live C. albicans suppresses production of ROS in primary phagocytes. ROS production by murine BMDM derived from BALB/c and C57BL/6 mice (A), murine elicited peritoneal PMN (B), hPBMC (C), or hPMN (D) was determined as for Fig. 1. Experiments using murine phagocytes were performed at least three times, with the same trend observed each time. A representative experiment is shown. Experiments using human phagocytes were performed using four different donors, as indicated. Donors for hPBMC and hPMN were not the same. For each donor, measurements were made in triplicate (with the exception of hPBMC donor B, for which results were measured in duplicate). Asterisks in panels A and B indicate results significantly different from those obtained with HK yeast (Student's t test; P < 0.001). For panels C and D, a one-way ANOVA of the entire data set indicated that there was significantly lower ROS production with live yeast for both hPBMC and hPMN (P < 0.001 for PBMC; P = 0.015 for PMN). Asterisks indicate that subsequent Student's t test comparisons for individual donors demonstrated a significant difference between HK and live yeast for all hPBMC donors (P < 0.01) but for none of the hPMN donors.

FIG. 4.

Candida species suppress ROS production but S. cerevisiae does not. ROS production by J774 cells in response to HK or live yeast was measured as for Fig. 1. A representative experiment is presented; the experiment was repeated three times, with the same trend observed each time. Each experiment was performed with triplicate samples; the bar heights indicates the mean values and error bars represent the standard deviations. Results were analyzed using a two-way ANOVA; ROS production in response to live S. cerevisiae was significantly different than with either of the Candida species (P < 0.001 for all comparisons). Live yeast induced less ROS than HK for both Candida species (P < 0.001 for both comparisons).

FIG. 5.

Live C. albicans and S. cerevisiae scavenge ROS at similar levels. Superoxide was produced in a cell-free system by the action of xanthine oxidase on hypoxanthine (as described in reference 24). The amount of ROS signal was evaluated using luminol-enhanced chemiluminescence. A decrease in ROS signal relative to that observed with buffer alone indicates ROS is being scavenged. As a positive control for ROS scavenging, SOD was added to some samples. The mean signal in SOD-containing samples was 2.68 × 10⁴; this value was too low for the bar to be visible on the graph. The experiment was performed three times; a representative experiment is shown. There was no difference in ROS signal between live C. albicans and S. cerevisiae (Student's t test, P = 0.21).

FIG. 6.

Cell wall ghosts prepared from either HK or live C. albicans yeast induce ROS production. Cell wall ghosts were isolated from live or HK yeast using a nondenaturing protocol. J774 cells were exposed to intact organisms or cell wall preparations at a concentration equivalent to the indicated Candida/phagocyte ratio. ROS production was measured as described for Fig. 1. A representative experiment is presented; the experiment was repeated three times with similar results. Bar heights indicate the means of three replicate samples; error bars indicate the standard deviations.

FIG. 7.

Exposure of 1,3-β-glucan by treatment with caspofungin does not prevent suppression of ROS production. (A) Bright-field (top panel) and epifluorescence (bottom panel) photomicrographs of yeast stained with anti-1,3-β-glucan antibody. Yeast were boiled for 10 min, HK, or treated during overnight growth with 1.25 ng/ml caspofungin or vehicle alone (0.1% dimethyl sulfoxide), as indicated. All epifluorescence images were captured using the same exposure time. (B) ROS production by J774 cells measured by luminol-enhanced chemiluminescence as for Fig. 1. A representative experiment is presented; the experiment was repeated three times with similar results. Bar lengths represent the means of triplicate values; error bars indicate the standard deviations. There was no significant difference in ROS production among untreated, caspofungin-treated, and vehicle-treated yeast as analyzed by one-way ANOVA (P > 0.80 for all comparisons). There were significant differences between HK yeast and all other groups (one-way ANOVA, P < 0.005); the comparison between yeast killed at 100°C versus 65°C was significant upon post hoc analysis (Student's t test, P = 0.046).

FIG. 8.

Secreted factors do not appear to suppress ROS production. (A) A schematic of the experiment is shown. Medium was harvested from J774 cells that had been exposed to HK yeast, live yeast, or no stimulus (stimulus A) under the same conditions used for an ROS assay. The medium was harvested, filtered, and then used to pretreat a second set of J774 cells; these cells were exposed to HK or live yeast (stimulus B) and ROS production was measured. (B) Results of the above experiment, presented as means ± standard deviations from a representative experiment. The experiment was repeated three times, with similar results. As analyzed by a two-way ANOVA, significantly more ROS was produced when stimulus A was HK compared with buffer (*, P < 0.05).