C-type lectin Langerin is a beta-glucan receptor on human Langerhans cells that recognizes opportunistic and pathogenic fungi

Abstract

Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and beta-glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and beta-glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections.

Fig. 1

Langerin recognizes Candida species, M. furfur and Saccharomyces species. (A and B) Yeast strains were coated onto ELISA plates (10⁷/ml) and binding to recombinant Langerin was determined. Polysaccharide mannan was used to determine the specificity of the binding. (C and D) Polysaccharide mannan (C) and C. albicans (D) were coated onto ELISA plates at different concentrations. Both mannan and blocking antibody against Langerin (10E2) (de Witte et al., 2007) were used to determine specificity. (E) Binding of life and heat-treated C. albicans to Langerin. Data represent results from at least three independently performed ELISAs and was performed with two different batches of yeast strains. Error bars represent standard deviation of triplicates.

Fig. 2

Titration of a selection of fungi. Yeast strains from Candida species (A and B), Cryptococcus neoformans (C), Cryptococcus gattii (D), and Saccharomyces cerevisiae (E) were coated onto ELISA plates at different concentrations. Mannan was used to determine specificity. Data are representative of at least three independent experiments. Error bars represent standard deviation of triplicates.

Fig. 3

Langerin is a receptor for β-glucans. (A) Langerin specifically interacts with mannose, fucose and GlcNAc structures. Carbohydrate binding to Langerin was determined by recombinant Langerin ELISA. Specificity was determined by mannan, anti-Langerin (10E2) (de Witte et al., 2007) or isotype control antibodies. (B–F) Langerin is a receptor for β-glucans present on curdlan, laminarin and zymosan. Mannan (C), zymosan (D), laminarin (E) and curdlan (F) were coated onto ELISA plates in different concentrations. Binding to Langerin was inhibited by addition of mannan or laminarin. Data represent results from at least three independent experiments. Error bars represent standard deviation of triplicates.

Fig. 4

Candida species and zymosan interact with cellular Langerin. (A) Langerin and dectin-1 expression on Jurkat cell-line transduced with Langerin. Thin line represents isotype control, filled histogram represents specific staining. (B) Langerin expressing cell-line was incubated with different concentrations of FITC-labelled fungi and binding was measured by flow-cytometric analysis. Pre-incubation with mannan was used to determine specificity. Data represent results from at least three independent experiments. Error bars represent standard deviation of triplicates.

Fig. 5

Langerin is the primary receptor for fungi on LCs. (A) CD1a, Langerin, dectin-1 and DC-SIGN expression on primary immature LCs. Thin line represents isotype control, filled histogram represents specific staining. (B) Binding of different fungi to human LCs was investigated. FITC-labelled fungi were incubated with LCs at MOI 10. Specificity was determined by mannan, blocking antibodies against Langerin and dectin-1, or isotype controls. Binding was measured by flow cytometry. Data represent results from at least three different donors. Error bars represent standard deviation of triplicates.

Fig. 6

Electron microscopic analysis of Candida albicans interaction with LCs. (A) Electron microscopic picture of LCs which has internalized C. albicans (C). Arrow is directed towards C. albicans. (B–D) Gold labelling of Langerin demonstrates Langerin localization at the binding site of LCs and C. albicans. Arrows are directed towards Langerin gold labelling. Picture (B) is a magnification of (A), and (D) is a magnification of (C).