C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia - PubMed (original) (raw)

. 2009 Feb 10;106(6):1897-902.

doi: 10.1073/pnas.0805177106. Epub 2009 Jan 26.

Makoto Matsumoto, Osamu Takeuchi, Tetsuhiro Matsuzawa, Eri Ishikawa, Machie Sakuma, Hiroaki Tateno, Jun Uno, Jun Hirabayashi, Yuzuru Mikami, Kiyoshi Takeda, Shizuo Akira, Takashi Saito

Affiliations

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

Sho Yamasaki et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRgamma-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRgamma, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to alpha-mannose but not mannan. Thus, Mincle may recognize specific geometry of alpha-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle(-/-) mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle(-/-) mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Mincle recognizes Malassezia species and delivers activation signal. (A) Screening of pathogenic fungus for Mincle-ligand activity. Reporter cell line expressing Mincle, FcRγ, and NFAT-GFP was established and left unstimulated or stimulated with plate-coated anti-Mincle mAb (10 μg/ml) for 18 h (inset). Mincle-expressing NFAT-GFP reporter cells were cocultured with indicated pathogenic fungi (open, 1%; closed, 10%) for 18 h, or stimulated with plate-coated anti-Mincle as a positive control (gray). NFAT-GFP induction was analyzed by flow cytometry. Data are representative of 3 independent experiments. (B) Microscopic analysis. Mincle-expressing reporter cells were cocultured with M. pachydermatis (arrowhead) and analyzed with fluorescence microscopy (IX-81; Olympus). (Scale bars: 5 μm.) (C) Dose-dependent activation by M. pachydermatis. Reporter cells expressing Mincle and FcRγ (closed) and only FcRγ (open) were cocultured with indicated amount (cells/ml) of M. Pachydermatis for 18 h. (D) Anti-Mincle mAb blocked _Malassezia_-induced NFAT activation. Reporter cells were cultured with M. pachydermatis in the presence of rat IgG or anti-Mincle mAbs and GFP expression was determined by flow cytometry. (E) Critical role of Mannose-binding motif of Mincle for Malassezia recognition. MincleWT (WT), MincleE169Q/N171D (EPN -> QPD) were expressed together with FcRγ in NFAT-GFP reporter cells. Cells were treated and analyzed as in (D). (F) Mincle specifically recognizes α-mannose. Mincle-Ig (1 μg/ml) precomplexed with Cy3-anti-human IgG (0.5 μg/ml) in TBS containing 1 mM CaCl2 or 10 mM EDTA was applied on the glycoconjugated microarray. After incubation at 20 °C for 3 h, binding was detected by an evanescent-field fluorescence-assisted scanner. Data were analyzed with the Array Pro analyzer ver. 4.5.

Fig. 2.

Fig. 2.

Generation of Mincle-deficient mice. (A) Genomic Mincle structure and targeting constructs with neomycin resistance (Neo) insertion. The Mincle exons are shown as black box. (B) Genomic PCR analysis of Mincle-targeted allele. Genomic DNA isolated from +/+, +/− and −/− mice were amplified with primer pairs for wild-type allele (+) or targeted allele (−) as shown in arrowheads in (A). (C) Surface expression of Mincle. BMMφ from WT (Mincle+/+) and Mincle-deficient (Mincle−/−) mice were stimulated with 1 ng/ml LPS for 18 h and stained with anti-rIgG1-biotin (thin line) or anti-Mincle-biotin (thick line) and Streptavidine-APC. (D) Western blot analysis. Thioglycolate-elicited peritoneal macrophages from Mincle+/+ and Mincle−/− mice were left unstimulated (−) or stimulated with 1, 3, 10 × 106 of M. pachydermatis for 18 h. Cells were lysed and blotted with anti-Mincle and anti-actin mAbs as a control.

Fig. 3.

Fig. 3.

Mincle deficiency failed to respond to Malassezia. (A) _Malassezia_-induced cytokine production. Mincle+/+ and Mincle−/− BMMφ were stimulated with 1, 3, 10 × 106 of M. pachydermatis, 100 μg/ml zymosan or 1 ng/ml LPS for 18 h. *, P < 0.05. (B) _Malassezia_-induced cytokine transcription. BMMφ was stimulated with 10 × 106 M. pachydermatis or 100 μg/ml zymosan for 4 h, and mRNA expression was determined by real time PCR. Relative mRNA levels were expressed as fold induction. *, P < 0.05. (C) Mincle+/− (WT) and Mincle−/− (KO) mice were injected with 4 × 107 M. pachydermatis i.p. After 18 h, the peritoneal cavity was washed out with 5 ml of saline and cytokine concentration was determined by ELISA. Each symbol represents an individual mouse. Data are representative of two independent experiments. *, P < 0.05. (D) Mincle+/+ or Mincle+/− (WT) and Mincle−/− (KO) mice were injected with 2.5 × 107 M. pachydermatis i.p. At 20 h after injection, peritoneal cells were stained with CD11b and Gr1 and analyzed by flowcytometry. Total number of peritoneal cells (Left) and CD11b+Gr1+ cells (Right) were indicated. Data are representative of two independent experiments. *, P < 0.05.

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