Recognition and blocking of innate immunity cells by Candida albicans chitin

Abstract

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.

Fig. 1.

Characterization of the chitin isolated from C. albicans. Chitin preparations were isolated and acid hydrolyzed as described in Materials and Methods. Samples containing 20-μl aliquots of hydrolyzed sample from C. albicans (panel B) or from shrimp shells (Sigma; panel C) were analyzed by HPAEC-PAD using a CarboPac PA200 analytical column. For panel A, high-purity standards (1 μg each; Sigma) were mixed and separated by HPAEC-PAD as indicated. GlcNAc, N-acetylglucosamine; Glc, glucose; Man, mannose.

Fig. 2.

Chitin blocks the recognition of heat-killed and live C. albicans. Human PBMCs were preincubated at 37°C for the indicated times with 10 μg/ml chitin and then stimulated with 1 × 10⁵ yeasts/ml of either live (panel A) or heat-killed (HK; panel B) C. albicans NGY152. For controls, PBMCs were also stimulated with medium or 10 μg/ml chitin. TNF-α, IL-6, IL-10, IFN-γ, and IL-1β were measured after 24 h of incubation. A similar experiment was conducted with S. Typhimurium as the stimulus (panel C). The results are pooled duplicate data from three separate experiments with a total of 6 volunteers (means ± SDs). * and **, P values of <0.01 and <0.05, respectively, for results obtained with chitin-treated cells compared to those obtained with untreated cells.

Fig. 3.

Caspofungin-treated C. albicans cells have high chitin contents at the cell wall (A) and stimulate reduced levels of cytokines (B). C. albicans NGY152 was grown for 6 h in Sabouraud medium supplemented with 0.032 μg/ml caspofungin and was stained with the fluorescent lectin WGA-FITC (panel A) or used to stimulate cytokine production by human PBMCs (panel B). For panel A, all pictures were taken at the same exposure time, and WGA-FITC is able to bind the accessible chitin only at the cell surface. The scale bars represent 10 μm. In panel B, the results are pooled duplicate data from three separate experiments with a total of 6 volunteers (means ± SDs). *, P value of <0.05 for results obtained with caspofungin-treated cells compared to those obtained with control cells. IL-1β concentrations stimulated by live control cells and live caspofungin-treated cells were 0.17 ± 0.01 ng/ml and 0.06 ± 0.03 ng/ml, respectively, and the IL-10 concentrations under these conditions were 0.17 ± 0.08 ng/ml and 0.05 ± 0.01 ng/ml, respectively. Control cells were untreated C. albicans NGY152. HK, heat-treated C. albicans cells.

Fig. 4.

Staining of chitin in C. albicans wild-type (CAF2-1) and chs3Δ null mutant cells by using calcofluor white (A) and the fluorescent lectin WGA-FITC (B). The former stain detects chitin in all cells, while WGA is able to bind to accessible chitin only at the cell surface. The scale bars represent 10 μm. HK, heat-treated C. albicans cells.

Fig. 5.

Cytokine production in human PBMCs stimulated by live or heat-killed (HK) yeast cells of a C. albicans wild-type strain (CAF2-1) (WT) and a chs3Δ null mutant. After 24 h of incubation, supernatants were removed and used to measure the levels of TNF-α (panel A), IL-6 (panel B), IL-10 (panel C), IL-1α (panel D), IL-1β (panel E), and IFN-γ (panel F). The results are pooled duplicate data from three separate experiments with a total of 6 volunteers (means ± SDs). * and **, P values of <0.05 and <0.01, respectively, for results obtained with chs3Δ null mutant cells compared to those obtained with wild-type cells.

Fig. 6.

Chitin does not block the recognition of specific agonists of TLR2 and TLR4. Human PBMCs were preincubated with 10 μg/ml chitin for 60 min and then stimulated with live C. albicans NGY152, 1 μg/ml Pam₃CSK₄, or 10 ng/ml LPS from E. coli. After 24 h of incubation, supernatants were assayed for TNF-α (panel A), IL-6 (panel B), IL-1β (panel C), and IL-10 (panel D). The results are pooled duplicate data from two separate experiments with a total of 8 volunteers (means ± SDs). *, P value of <0.01 for results obtained with chitin-treated cells compared to those obtained with untreated cells.

Fig. 7.

Blocking effect of chitin on the C. albicans recognition is not TLR2 or TLR4 dependent. Peritoneal macrophages from C57BL/6J (WT), TLR2^−/−, and TLR4^−/− mice were preincubated at 37°C for 60 min with 10 μg/ml chitin and then stimulated with heat-killed C. albicans NGY152 at a concentration of 1 × 10⁵ yeasts/ml. After 24 h of incubation, supernatants were removed and assayed for TNF-α (panel A), IL-6 (panel B), IL-1α (panel C), and IL-1β (panel D). The results are pooled duplicated data from two separate experiments with a total of 9 animals per group (means ± SDs). * and **, P values of <0.05 and <0.01, respectively, for results obtained with chitin-treated cells compared to those obtained with untreated cells.

Fig. 8.

Dectin-1 is required for the blocking effect of chitin on C. albicans recognition. (A) FACS analysis of chitin or β-glucan preparations incubated as described in the text. (B) Peritoneal macrophages from C57BL/6J (WT) and dectin-1^−/− mouse PBMCs were preincubated at 37°C for 60 min with 10 μg/ml chitin and then stimulated with heat-killed C. albicans NGY152 at a concentration of 1 × 10⁵ yeasts/ml. After 24 h of incubation, supernatants were removed and assayed for TNF-α production. The results are pooled duplicated data from a total of 9 animals per group (means ± SDs). *, a P value of <0.05 for results obtained with chitin-treated cells compared to those obtained with untreated cells.