Inhibition of monocytic interleukin-12 production by Candida albicans via selective activation of ERK mitogen-activated protein kinase

Abstract

Our previous data demonstrated that live Candida albicans inhibits interleukin-12 (IL-12) production by human monocytes. Here we explored whether C. albicans inhibits IL-12 via a released factor and whether the inhibition is mediated via mitogen-activated protein kinase (MAPK) regulation. Supernatant fluids were obtained from cultured C. albicans (SC5314) as well as cultured Saccharomyces cerevisiae after 20 h of incubation. At 2 h postincubation of monocytes with heat-killed C. albicans (HKCA) (2:1) to stimulate IL-12, concentrated fungal supernatant fluids were added and incubated for an additional 20 h. The present data show that, unlike supernatant fluids obtained from S. cerevisiae, the C. albicans supernatant fluids significantly suppressed IL-12 production induced by HKCA. This suggested that the inhibition is Candida specific. A preliminary biochemical analysis revealed that this secretory IL-12 inhibitory factor is glycoprotein in nature. The inhibitory activity had no effect on the phagocytosis of yeasts. Supernatant fluids from C. albicans markedly induced the phosphorylation of ERK44/42 MAPK, but not p38 and SAPK, 1 min after they were added to monocytes. To test if the induction of ERK44/42 MAPK was central to the IL-12 inhibition, we used gamma interferon (IFN-gamma) (1 ng/ml) plus lipopolysaccharide (LPS) (100 ng/ml) to stimulate IL-12 production by monocytes. The inhibition of ERK MAPK by the specific inhibitor PD 98059 significantly reduced phospho-ERK44/42 MAPK levels induced by C. albicans supernatant fluids in the IFN-gamma-plus-LPS-driven monocytes. Concomitantly, PD 98059 reversed the IL-12 inhibitory activity of the C. albicans supernatant (P < 0.01). These data indicate that C. albicans can inhibit IL-12 production by secreting an ERK44/42 MAPK-stimulating factor and thus can attenuate effective immune responses.

FIG. 1.

IL-12 production after treatment with supernatant fluids from C. albicans. (A) Supernatant fluids from C. albicans inhibited HKCA-induced IL-12 production by monocytes. Monocytes were incubated with HKCA (2:1) for 2 h, followed by the addition of concentrated supernatant fluids (>30 kDa) from C. albicans (equivalent to 1:0.3 monocytes to live C. albicans spores incubated overnight) or from S. cerevisiae. After overnight incubation, culture supernatant fluids were collected for IL-12 p70 quantitation. Monocytes only, monocytes plus HKCA, and monocytes, HKCA, and medium supplemented with FBS after filtration with a 30-kDa cutoff served as controls. CA-sup, supernatant fluids from C. albicans; SC-sup, supernatant fluids from S. cerevisiae. P values were determined by a paired t test (n = 3). (B) C. albicans culture supernatant fluid from FBS-free medium inhibits IL-12 p70 produced by monocytes in a dose-dependent manner. Supernatant fluids from C. albicans cultured in RPMI medium without FBS were collected. Protein concentrations were measured after concentration through Centricon 30-kDa cutoff filters. The amount of protein from the supernatant fluid was added as indicated to a monocyte (MN) culture (10⁶/ml ) with the addition of IFN-γ (100 pg/ml) and LPS (10 ng/ml). Monocyte cultures with or without IFN-γ plus LPS were used as controls. CA, C. albicans protein. P values were determined by a paired t test (n = 3).

FIG. 1.

IL-12 production after treatment with supernatant fluids from C. albicans. (A) Supernatant fluids from C. albicans inhibited HKCA-induced IL-12 production by monocytes. Monocytes were incubated with HKCA (2:1) for 2 h, followed by the addition of concentrated supernatant fluids (>30 kDa) from C. albicans (equivalent to 1:0.3 monocytes to live C. albicans spores incubated overnight) or from S. cerevisiae. After overnight incubation, culture supernatant fluids were collected for IL-12 p70 quantitation. Monocytes only, monocytes plus HKCA, and monocytes, HKCA, and medium supplemented with FBS after filtration with a 30-kDa cutoff served as controls. CA-sup, supernatant fluids from C. albicans; SC-sup, supernatant fluids from S. cerevisiae. P values were determined by a paired t test (n = 3). (B) C. albicans culture supernatant fluid from FBS-free medium inhibits IL-12 p70 produced by monocytes in a dose-dependent manner. Supernatant fluids from C. albicans cultured in RPMI medium without FBS were collected. Protein concentrations were measured after concentration through Centricon 30-kDa cutoff filters. The amount of protein from the supernatant fluid was added as indicated to a monocyte (MN) culture (10⁶/ml ) with the addition of IFN-γ (100 pg/ml) and LPS (10 ng/ml). Monocyte cultures with or without IFN-γ plus LPS were used as controls. CA, C. albicans protein. P values were determined by a paired t test (n = 3).

FIG. 2.

C. albicans SIIF is glycoprotein in nature. Equal amounts of C. albicans supernatant fluid (25 μl of SIIF) were added to different groups of monocytes, and their IL-12 levels were determined. Group 1 represents normal monocytes, while group 2 represents monocytes stimulated with supernatant fluids and IFN-γ plus LPS. The stimulated monocytes were incubated with untreated supernatant fluids (group 3), proteinase K-treated supernatant fluids (group 4), inactive proteinase K-treated supernatant fluids (group 5), or alkali-treated supernatant fluids (group 6). Data shown are means ± standard deviations of duplicate experiments.

FIG. 3.

The >30-kDa supernatant of C. albicans does not inhibit phagocytosis of killed blastospores by monocytes. Monocytes were first labeled with PE-conjugated monoclonal antibody CD14. Ethanol-killed C. albicans blastospores were labeled with FITC and opsonized by suspension in human AB serum. Then opsonized FITC-blastospores were incubated with PE-monocytes at a ratio of 5:1. For discrimination between ingested, membrane-bound, and free C. albicans, EtBr was added to the samples prior to the flow cytometric analysis. Thus, internalized blastospores remained green, while adherent and nonphagocytosed blastospores stained red. C. albicans phagocytosis was determined by gating the phagocytes and calculating the percentage of phagocyte-associated green fluorescent cells (refer to Materials and Methods for further details). The numbers above each box indicate the percentages of killed C. albicans cells that were phagocytosed by the monocytes. Cytochalasin D is an inhibitor of phagocytosis and served as a control. The supernatant was obtained from C. albicans. Data are representative of three experiments.

FIG. 4.

Effect of C. albicans supernatant fluid on phosphorylation of ERK, p38, and SAPK MAPK in monocytes. Monocytes (MN) were stimulated with or without LPS (1 μg/ml) as indicated, followed by the addition of >30-kDa C. albicans supernatants (equivalent to 1:0.3 monocytes to live C. albicans cells) for 1, 5, and 15 min, and then the monocytes were lysed with lysis buffer. The lysates were resolved by Nu-PAGE followed by Western blotting. RDs of phosphorylated ERK/p38/SAPK relative to the total amount of ERK/p38/SAPK are expressed in bar charts. Data are representative of three experiments. Mo, monocytes.

FIG. 5.

C. albicans-induced phosphorylation of ERK MAPK in monocytes is blocked by the specific inhibitor of ERK phosphorylation, PD 98059. Monocytes (MN) were incubated with >30-kDa C. albicans supernatants (equivalent to 1:0.3 monocytes to live C. albicans cells) for 1 min, with or without IFN-γ (1 ng/ml) and LPS (100 ng/ml) (to stimulate IL-12, connecting with the following experiments), and then were lysed with lysis buffer. The lysates were resolved by Nu-PAGE, followed by Western blotting with antibodies for phospho-ERK MAPK and total ERK MAPK. White plus sign, positive standard for phospho-ERK MAPK; I+L, LPS plus IFN-γ; Sup, >30-kDa supernatant of SC5314; ERKi, PD 98059 inhibitor of phosphorylated ERK MAPK.

FIG. 6.

Blocking of ERK phosphorylation in C. albicans-inhibited monocytes restores IL-12 production. Monocytes (MN) pretreated with or without PD 98059 (20 μM) for 45 min were stimulated with IFN-γ (1 ng/ml) and LPS (100 ng/ml), followed by incubation with the >30-kDa C. albicans supernatant (equivalent to 1:0.3 monocytes to live C. albicans cells) for 20 h. Supernatants were collected and IL-12 p70 was determined by ELISA. The P value is indicated (n = 4 tests). I+L, IFN-γ plus LPS; Sup, >30-kDa supernatant of SC5314; ERKi, PD 98059 inhibitor of phosphorylated ERK MAPK.