Differential cytokine production and Toll-like receptor signaling pathways by Candida albicans blastoconidia and hyphae

Abstract

Toll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicans by TLRs. In contrast to Candida blastoconidia, which stimulated large amounts of gamma interferon (IFN-gamma), the tissue-invasive Candida hyphae did not stimulate any IFN-gamma by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-gamma was TLR4 dependent, as shown by the significantly decreased IFN-gamma production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor alpha (TNF-alpha) than macrophages of control mice did when stimulated with Candida blastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-alpha in response to both Candida blastoconidia and hyphae. Candida hyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candida stimulation, whereas Candida recognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candida blastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.

FIG. 1.

Production of TNF-α by PBMC from four healthy volunteers after stimulation with various concentrations of heat-killed C. albicans blastoconidia and hyphae. Data are represented as means ± standard errors of the means from two separate experiments.

FIG. 2.

Production of TNF-α (A) and IFN-γ (B) by PBMC after stimulation with heat-killed C. albicans blastoconidia (10⁷ CFU/ml) and hyphae (10⁷ CFU/ml). Data are represented as means ± standard errors of the means from two separate experiments with cells of six volunteers. *, P < 0.05, t test.

FIG. 3.

Induction of TNF-α (A), IFN-γ (B), and IL-10 (C) production by PBMC from five healthy volunteers after preincubation with anti-TLR4 antibody or isotype control antibody and stimulation with heat-killed C. albicans blastoconidia (10⁷ CFU/ml) or hyphae (10⁷ CFU/ml). TNF-α was measured after 24 h, and IFN-γ and IL-10 were measured after 48 h. Data are represented as means ± standard errors of the means from two separate experiments. *, P < 0.05, t test.

FIG. 4.

IFN-γ and IL-10 production by PBMC from five healthy volunteers after preincubation with RPMI as a control or glucan phosphate (50 μg/ml) and stimulation for 48 h with heat-killed C. albicans blastoconidia (10⁷ CFU/ml). Data are represented as means ± standard errors of the means. *, P < 0.05, t test.

FIG. 5.

TNF-α (A) and IL-10 (B) production by peritoneal macrophages from TLR2^+/+ mice and TLR2^−/− mice in response to heat-killed C. albicans blastoconidia (10⁷ CFU/ml) or hyphae (10⁷ CFU/ml), LTA (0.1 μg/ml), and E. coli LPS (0.1 μg/ml). Data are represented as means ± standard errors of the means from three separate experiments with three to nine mice per strain. *, P < 0.05, t test.

FIG. 6.

TNF-α (A) and IL-10 (B) production by peritoneal macrophages from TLR4^+/+ mice and TLR4^−/− ScCr mice in response to heat-killed C. albicans blastoconidia (10⁷ CFU/ml) or hyphae (10⁷ CFU/ml), LTA (0.1 μg/ml), and E. coli LPS (0.1 μg/ml). IFN-γ (C) production by splenic lymphocytes from TLR4^+/+ mice and TLR4^−/− mice in response to heat-killed C. albicans blastoconidia (10⁷ CFU/ml) or hyphae (10⁷ CFU/ml) during a 48-h stimulation. Data are represented as means ± standard errors of the means from two different experiments with three to nine mice per strain. *, P < 0.05, t test.

FIG. 7.

Loss of TLR4-mediated signals during yeast-to-hypha transition of C. albicans. Recognition of C. albicans blastoconidia involves both TLR2 and TLR4. The yeast-to-hypha transition results in the loss of proinflammatory TLR4-mediated signals and a TLR2-mediated anti-inflammatory cytokine profile, used by C. albicans as an escape mechanism from the host defense.