Abstract
Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity1. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin2,3, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.
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Acknowledgements
We thank the staff of the University of Aberdeen animal facility for their support and care for our animals, C. G. Park for providing recombinant langerin, and S. Filler and R. Cramer for advice. Funding was provided by the Wellcome Trust (102705, 097377, 093378, 099197, 108430, 101873), the Medical Research Council Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1). K.J.K.-C is supported by the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health; V.A. by an ANR-DST COMASPIN grant (ANR-13-ISV3-0004); B.H. by German Science Foundation (www.dfg.de) grant no. HE 7565/1-1; J.-P.L., I.V. and V.A. by the ANR and FRM DEQ2015-331722; A.C. by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), and by the Fundação para a Ciência e Tecnologia (FCT) (IF/00735/2014 and SFRH/BPD/96176/2013).
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G.D.B., J.-P.L. and J.A.W. conceived and designed the study and guided the interpretation of the results. A.E.C., M.H.T.S. and V.A. performed the majority of the experiments and data analysis. S.B., D.M.R., P.A., S.E.H., I.M.D., B.K., A.P., J.A.W., C.C., M.d.G.T.S., C.A.W., R.Y. and B.H. conducted experiments and data analysis. Y.L. and T.F. performed the glycan microarray experiments and data analysis. M.J., M.G.N., F.L.v.d.V., J.F.L., A.Cam. and A.Car. provided the human patient data and analysis. A.A.B., K.J.K.-C., I.V., M.P., C.Z., M.Z. and N.A.R.G. provided critical conceptual input and reagents. G.D.B. drafted the manuscript. All authors discussed the results, edited and approved the draft and final versions of the manuscript.
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Extended data figures and tables
Extended Data Figure 1 MelLec recognizes ligands on selected fungi and fungal morphotypes.
a, b, Cartoon representations of the structures of Fc-MelLec (a) and the full-length receptor (b). Lollipop structures represent predicted glycosylation sites. c, Fc-MelLec or Fc-CLEC12B (Fc-control) staining of C. albicans hyphae, generated in RPMI with 10% fetal bovine serum for 90 min. Fungal particles were analysed by flow cytometry. Experiment was repeated independently twice, with similar results. d, Representative light microscopy images and immunofluorescence micrographs using Fc-MelLec or anti-galactomannan (GM, control) as probes to detect ligands on A. fumigatus mycelium. Experiments were repeated three times independently, with similar results. e, Representative light microscopy images and immunofluorescence micrographs showing the surface distribution of MelLec ligands on C. cladosporioides using Fc-MelLec as a probe. The lower panels show fungal cells after treatment with 1 M NaOH. Experiments were repeated three times independently, with similar results. f, IL-2 production by MelLec-expressing BWZ reporter cells after stimulation by anti-MelLec antibody (αMelLec) crosslinking or with ΔrodA (1:1, 5:1, 10:1) or ΔrodAΔpksP (5:1, 10:1) A. fumigatus conidia, as indicated. Values shown are mean ± s.d. Experiment was repeated three times independently, with similar results.
Extended Data Figure 2 Glycan microarray analyses of mouse Fc-MelLec and human langerin.
These 496 lipid-linked probes are arranged according to their backbone sequences as annotated in the coloured panels below the figure. GAGs, glycosaminoglycans; Lac, lactose; LacNAc, N-acetyllactosamine; LNnT, lacto-N-neotetraose; LNT, lacto-N-tetraose; Misc., miscellaneous; PolyLac, polylactosamine. The signals are means of fluorescence intensities of duplicate spots, printed at 5 fM per spot level with error bars representing half of the difference between the two values. The signals shown together with the probe sequences are in Supplementary Table 1. NS, not significant.
Extended Data Figure 3 MelLec recognizes DHN-melanin.
a, Representative immunofluorescence micrographs using Fc-MelLec as a probe showing the surface distribution of MelLec-ligands on wild-type A. fumigatus and various melanin-deficient mutants. Lower panels show conidia treated with 1 M NaOH. b, Representative immunofluorescence micrographs and light microscopy images of ΔrodAΔpksP A. fumigatus conidia stained with Fc-MelLec or ConA-FITC. c, Representative histograms showing the presence or absence of MelLec or Dectin-1 ligands on ΔrodA or ΔrodAΔpksP A. fumigatus conidia. Fungal particles were stained with Fc-MelLec (red) or Fc-Dectin-1 (green) and analysed by flow cytometry. Grey histograms indicate secondary-only control. d, Representative histogram showing the presence of Dectin-1 ligands on ΔpksP A. fumigatus conidia. Fungal particles were stained with Fc-Dectin-1 (green) or Fc-CLEC12B (Fc-control; blue) and analysed by flow cytometry. e, Representative histogram showing the presence of MelLec ligands on melanin ghosts of A. fumigatus conidia. Fungal particles were stained with Fc-MelLec (red) or Fc-CLEC12B (Fc-control; blue) and analysed by flow cytometry. In a–e, experiments were repeated three times independently, with similar results. f, Flow-cytometric analysis of melanized (red) and non-melanized (grey) Cryptococcus neoformans yeast and melanin ghosts, and B16 melanoma cells47, stained with Fc-MelLec. The experiment was repeated independently twice, with similar results.
Extended Data Figure 4 MelLec recognizes naphthalene-diol.
a, Detection of MelLec ligands in ghosts of melanin mutants of A. fumigatus by ELISA, as indicated. Values show mean ± s.d. b, Representative immunofluorescence and light micrograph images of Fc-MelLec ligands on NaOH-treated A. fumigatus B5233 conidia after pretreatment with ghosts of ΔpksP or Δayg1 conidia, as indicated. c, Representative immunofluorescence micrographs and light microscopy images of MelLec ligands on NaOH-treated wild-type A. fumigatus conidia after pretreatment with or without YWA1. In a–c, experiments were repeated at least three times independently, with similar results. d, Detection of Fc-MelLec or Fc-Dectin-1 ligands on ΔrodA conidia after pretreatment with (blue) or without (red) YWA1. The experiment was repeated independently twice, with similar results. e, f, Detection of 1,8-DHN (e) and 1,2-DHN and 1,4-DHN (f) by Fc-MelLec and Fc-control using ELISA. Values show mean ± s.d. g, Detection of 1,8-DHN, naphthalene and 1-naphthol by Fc-MelLec using ELISA. Values show mean ± s.d. In e–g, experiments were repeated at least three times independently, with similar results.
Extended Data Figure 5 MelLec is expressed at the cell surface.
a, RT–PCR detection of MelLec expression in various tissues. The expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in these samples, also used for the characterization of MCL36, is shown as a control. The experiment was performed once; for gel source data, see Supplementary Fig. 1. b, Flow-cytometric analysis of surface expression of haemagglutinin-tagged mouse (m) and human (h) MelLec on the surface of NIH3T3 fibroblasts (black open histograms). NIH3T3 cells transfected with vector only served as controls (grey filled histograms). c, Western-blot analysis of lysates of haemagglutinin-tagged MelLec expressing NIH3T3 cells under reducing and non-reducing conditions and with (+) and without (-) N-glycosidase. Haemagglutinin-tagged CLEC12A (ref. 31) expressing NIH3T3 cells served as controls (for blot source data, see Supplementary Fig. 1). d, Relative binding of FITC-labelled ΔrodA A. fumigatus conidia to NIH3T3 cells transduced with vector only, Dectin-1 or MelLec, as determined by flow cytometry. Values shown are mean ± s.d., analysed by one-way ANOVA. e, Screening of hybridoma supernatants on MelLec-expressing (red) and parental (black) NIH3T3 cells. In b–e, experiments were repeated at least three times independently, with similar results. *P ≤ 0.05; NS, not significant.
Extended Data Figure 6 Mouse MelLec is not expressed by myeloid cells.
a, b, Flow-cytometric analysis of MelLec expression on various ex vivo and in vitro derived myeloid cells (a) and peripheral blood, bone marrow, lymph nodes and spleen (b). Experiments were repeated at least twice independently, with similar results. c, Flow-cytometric analysis of MelLec expression on CD61+ platelets. Experiment was repeated at least three times independently, with similar results. BM, bone marrow; DC, dendritic cell; LPS, lipopolysaccharide; mø, macrophage.
Extended Data Figure 7 MelLec expression in tissues and generation of Clec1a−/− mice.
a, Immunofluorescence microscopy of MelLec-expressing versus control NIH3T3 cells labelled with anti-MelLec antibody (green). Nuclei are stained with DAPI (blue). Experiments were repeated at least three times independently, with similar results. b, Exemplar flow-cytometric gating strategy for identification of live cells from tissue. c, Flow-cytometric analysis of MelLec expression on live CD45−CD31+ EpCAM− and EpCAM+ populations in the liver. d–j, Flow cytometric analysis of MelLec expression on live CD45−CD31+ cells in the heart (d), kidney (e) and small intestine (f), and on live CD45−EpCAM+ cells in the heart (g), kidney (h), small intestine (i) and epidermis (j). In b–j, experiments were repeated at least twice independently, with similar results. Black lines, isotype controls. k, Schematic of the wild-type Clec1a locus, gene targeting vector, PCR primer sites and correctly targeted recombinant allele. l, PCR analysis of gene-targeted mice (for gel source data, see Supplementary Fig. 1). +/+, wild-type, +/− heterozygous and −/− homozygous for the targeted allele. m, Immunofluorescence microscopy of naive lung tissue from Clec1a−/− mice (labelling of wild-type lung is shown in Fig. 3b). n, Analysis of MelLec expression in disaggregated lung tissue from wild-type (wt) or Clec1a−/− mice by flow cytometry. In l–n, experiments were repeated at least three times independently, with similar results.
Extended Data Figure 8 Clec1a−/− mice show early inflammatory defects upon challenge with A. fumigatus.
a, Survival (left) and weight measurements (right) of immunocompetent mice after i.t. infection with 107 A. fumigatus conidia (n = 4 mice per group). Values shown are mean ± s.d. b, Exemplar flow-cytometric gating strategy for the identification of CD45+ cells from bronchoalveolar lavage. c, Representative FACS profiles of pulmonary CD11b+ Ly6Ghigh neutrophils in wild-type and Clec1a−/− mice 4 h after challenge with 107 A. fumigatus conidia (wild-type n = 29 mice; Clec1a−/− n = 26 mice). d, e, Pulmonary CD11b+ Ly6Ghigh neutrophils (wild-type n = 29 mice, Clec1a−/− n = 26 mice) (d) and cytokines (n = 25 mice per group) (e) in mice 4 h after challenge with 107 A. fumigatus conidia. f, Pulmonary CD11b+ Ly6Ghigh neutrophils in mice 4 h after challenge with melanin ghosts (160 μg) of A. fumigatus (wild-type n = 15 mice; Clec1a−/− n = 10 mice). Samples with blood contamination were excluded. g, Pulmonary CD11b+ Ly6Ghigh neutrophils in mice 24 h after challenge with 107 A. fumigatus conidia (wild-type n = 33 mice, Clec1a−/− n = 30 mice). h, Cellular inflammatory profiles of mice 24 h after challenge with 107 A. fumigatus conidia (wild-type n = 33 mice, Clec1a−/− n = 30 mice). Alveolar macrophages were defined as CD11c+Siglec-F+, inflammatory macrophages as CD11b+ F4/80+ and eosinophils as CD11b+Siglec-F+. In d–h, values shown are mean ± s.e.m. of pooled data from at least two independent experiments, analysed by two-sided Mann–Whitney U test. i, Expression of MelLec on pulmonary CD45− CD31+ cells isolated from uninfected mice (black) and mice 24 h after infection with A. fumigatus conidia (red). Grey line, isotype control (n = 3 mice per group). j, Pulmonary CD11b+ Ly6Ghigh neutrophils in mice 4 h after challenge with 107 ΔpksP A. fumigatus conidia (wild-type n = 7 mice, Clec1a−/− n = 8 mice). Values shown are mean ± s.e.m. of pooled data from two independent experiments, analysed by two-sided Mann–Whitney U test. k, Representative FACS profiles of pulmonary CD11b+ Ly6Ghigh neutrophils in wild-type and Clec1a−/− mice 4 h after challenge with 107 ΔpksP A. fumigatus conidia (wild-type n = 7 mice, Clec1a−/− n = 8 mice). *P ≤ 0.05; NS, not significant.
Extended Data Figure 9 Clec1a−/− mice show alterations in antifungal immunity during systemic infection.
a, Survival of corticosteroid-treated mice after i.t. infection with 105 A. fumigatus conidia (n = 15 mice per group). Pooled data from two independent experiments, analysed by log-rank test. b, Fungal burdens in various mouse tissues, as indicated, 4 days after i.v. infection with 106 A. fumigatus conidia (n = 12 mice per group). Values shown are mean ± s.e.m. of pooled data from two independent experiments, analysed by two-sided Mann–Whitney U test. c, Tissue section of kidney from day-4-infected Clec1a−/− mouse stained with Grocott’s methenamine silver stain and haematoxylin (n = 3 mice per group). d, Immunofluorescence microscopy of brain from day-2-infected Clec1a−/− mouse (n = 3 mice per group). Fungi are stained with calcofluor white (blue), leukocytes with Gr1 (green) and DNA with propidium iodide (red). e, Fungal burdens in various mouse tissues, as indicated, four days after i.v. infection with 106 ΔpksP A. fumigatus conidia (n = 4 mice per group). Values shown are mean ± s.d., analysed by two-sided Mann–Whitney U test. *P ≤ 0.05; NS, not significant.
Extended Data Figure 10 A single nucleotide polymorphism in human MelLec influences anti-Aspergillus inflammatory responses.
a, Representative histograms showing the presence or absence of human MelLec ligands on ΔrodA or ΔrodAΔpksP A. fumigatus conidia, as determined by flow cytometry. Fc-CLEC12B was used as a control (Fc-control). The experiment was repeated at least three times independently, with similar results. b, Survival of irradiated wild-type mice reconstituted with wild-type or Clec1a−/− bone marrow (BM), as indicated, after i.v. infection with 106 A. fumigatus conidia (n = 16 mice per group). Pooled data from two independent experiments, analysed by log-rank test. c, Inflammatory cytokine production in monocyte-derived macrophages isolated from genotyped individuals, after stimulation with lipopolysaccharide (wild-type n = 14 individuals, SNP allele n = 5 individuals). Values shown are mean ± s.d., analysed by two-sided Mann–Whitney U test. d, Inflammatory cytokine production in peripheral blood mononuclear cells isolated from genotyped Dutch individuals, after stimulation with heat-killed A. fumigatus conidia (wild-type n = 72 individuals, SNP allele n = 17 individuals). Boxes represent the median values and interquartile ranges; whiskers represent minimum and maximum values, analysed by two-sided Mann–Whitney U test. e, Inflammatory cytokine production in transduced RAW264.7 macrophages expressing wild-type or SNP allele after stimulation with ΔrodA or ΔrodAΔpksP A. fumigatus conidia, as indicated. Values shown are mean ± s.d., analysed by one-way ANOVA and repeated at least three times independently, with similar results. *P ≤ 0.05; NS, not significant.
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Stappers, M., Clark, A., Aimanianda, V. et al. Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus. Nature 555, 382–386 (2018). https://doi.org/10.1038/nature25974
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DOI: https://doi.org/10.1038/nature25974
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