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Tyrosine phosphatase SHP-2 mediates C-type lectin receptor–induced activation of the kinase Syk and anti-fungal TH17 responses

Nature Immunology volume 16, pages 642–652 (2015)Cite this article

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Abstract

Fungal infection stimulates the canonical C-type lectin receptor (CLR) signaling pathway via activation of the tyrosine kinase Syk. Here we identify a crucial role for the tyrosine phosphatase SHP-2 in mediating CLR-induced activation of Syk. Ablation of the gene encoding SHP-2 (Ptpn11; called 'Shp-2' here) in dendritic cells (DCs) and macrophages impaired Syk-mediated signaling and abrogated the expression of genes encoding pro-inflammatory molecules following fungal stimulation. Mechanistically, SHP-2 operated as a scaffold, facilitating the recruitment of Syk to the CLR dectin-1 or the adaptor FcRγ, through its N-SH2 domain and a previously unrecognized carboxy-terminal immunoreceptor tyrosine-based activation motif (ITAM). We found that DC-derived SHP-2 was crucial for the induction of interleukin 1β (IL-1β), IL-6 and IL-23 and anti-fungal responses of the TH17 subset of helper T cells in controlling infection with Candida albicans. Together our data reveal a mechanism by which SHP-2 mediates the activation of Syk in response to fungal infection.

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Figure 1: SHP-2 is tyrosine-phosphorylated and regulates the expression of genes encoding pro-inflammatory products upon activation of dectin-1.
Figure 2: SHP-2 is required for CLR- and C. albicans–induced gene expression.
Figure 3: SHP-2 mediates the activation of Syk elicited by dectin-1- and C. albicans–induced signaling.
Figure 4: SHP-2 mediates translocation of Syk to the membrane and colocalization of Syk with dectin-1 and FcRγ.
Figure 5: SHP-2 recruits Syk to dectin-1 upon infection with C. albicans.
Figure 6: SHP-2-mediated CLR signaling in DCs is indispensable for anti-fungal TH17 responses.
Figure 7: SHP-2-mediated CLR signaling in macrophages and neutrophils is critical for anti-fungal innate immune responses.

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Acknowledgements

We thank J.V. Ravetch (The Rockefeller University) for FcRγ-deficient mice; Y. Iwakura (The University of Tokyo) for dectin-1-deficient mice; S. Yamasaki (Kyushu University) for Mincle-deficient mice; B. Qian for technical assistance in histology; and Y. Xu and J. Yan for technical assistance in confocal microscopy. Support by National Science and Technology Major Projects (2014CB541902 to H.X.; and 81270627 to H.H.Z.), the National Natural Science Foundation of China (31070779 and 31170862 to H.X.; 31270917 to M.D.; and 31100623 to A.Z.), the US National Institutes of Health (R01HL096125 to G.-S.F.; P01 HL103453 to X.Li; and R01EY018612 to E.P.), the Chinese Academy of Sciences “100-talent” program (H.X.), the National Program for Returned Overseas Talents (H.X.) and the Science and Technology Commission of Shanghai Municipality (Pujiang program 12PJ1406100 for H.H.Z.).

Author information

Authors and Affiliations

  1. Key Laboratory of Molecular Virology and Immunology, Vaccine Center, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China

    Zihou Deng, Shixin Ma, Aiping Zang, Yiyuan Fang, Tiantian Li, Huanjing Shi, Mei Liu, Min Du, Guangxun Meng, Changbin Chen, Yan Zhang, Xiaoming Zhang & Hui Xiao

  2. Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA

    Hao Zhou & Xiaoxia Li

  3. Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Ohio, USA

    Patricia R Taylor & Eric Pearlman

  4. State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Helen He Zhu

  5. State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Jiangye Chen

  6. Shanghai Institute of Immunology and Hongqiao International Institute of Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Fubin Li

  7. Department of Immunology, Tongji University School of Medicine, Shanghai, China

    Xin-Ming Jia

  8. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Xin Lin

  9. Department of Pathology and Division of Biological Sciences, University of California San Diego, La Jolla, California, USA

    Gen-Sheng Feng

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  1. Zihou Deng
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Contributions

Z.D. and H.X. designed the research; Z.D., S.M., H.Z., A.Z., Y.F., T.L., H.S., M.L. and M.D. performed experiments; P.R.T., H.H.Z., J.C., G.M., F.L., C.C., Y.Z., X.-M.J., X. Lin, X.Z., E.P., X.Li and G.-S.F. provided materials and technical support; Z.D., H.Z., X.Li and H.X. analyzed the data; and Z.D., E.P., X.Li, G.-S.F. and H.X. wrote the paper.

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Correspondence to Hui Xiao.

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Integrated supplementary information

Supplementary Figure 1 SHP-2 is tyrosine-phosphorylated and regulates pro-inflammatory gene expression after stimulation with dectin-1 ligands.

(a) Wild-type BMDMs primed with IL-4 (10ng/ml) overnight were either untreated or treated by Zymd (100μg/ml) for various times and Immunoblotting was conducted by respective antibodies as indicated. (b,c) BMDCs were untreated or pretreated by laminarin for 30 min, followed by 100μg/ml Zymd treatment (b); WT and dectin-1-/- BMDCs differentiated by GM-CSF (20ng/ml) and IL-4 (10ng/ml) were stimulated by Zymd (100μg/ml) (c). Immunoblot analysis was conducted with anti-p-SHP2 and anti-p-SYK. (d) BMDCs from WT and dectin-1 deficient mice (dectin-1-KO) were stimulated by Zymd (100μg/ml) for 24 h, supernatants were collected for ELISA. (e) Peritoneal macrophages were isolated from wild-type mice intraperitoneally injected with 4% Thioglycolate broth in PBS for 4 days, and stimulated by Zmyd (100μg/ml) for various times. (f) BMDMs with or without IL-4 (10ng/ml) priming overnight were either untreated or treated by Zymd (100μg/ml). (g) BMDCs differentiated by GM-CSF (20ng/ml) w/ or w/o IL-4 (10ng/ml) were stimulated with Pam3 (100ng/ml), Zymd (100μg/ml), or LPS (100ng/ml) for 24 hours and secreted TNF was measured by ELISA. (h) Cell lysates from BMDMs, BMDCs, spleen B cells and T cells generated from Shp-2fl/fl, DC-Shp-2-/- and MN-Shp-2-/- mice were probed by anti-SHP2 and anti-GAPDH. (i) Cell lysates from BMDCs generated from Shp-2fl/fl and DC-Shp-2-/- mice were probed by indicated antibodies. (j) Cell lysates from BMDMs generated from Shp-2fl/fl and MN-Shp-2-/- mice were probed by indicated antibodies. Data are presented as mean ± SEM from three samples of one representative experiment of three. Note: * p<0.05, ** p<0.01, *** p<0.001.

Supplementary Figure 2 SHP-2 plays a critical role in dectin-1- and C. albicans–induced gene expression.

(a) BMDMs primed by IL-4 (10ng/ml) were stimulated by dectin-1 ligands Zymd (100μg/ml), ZymA (100μg/ml) or Curdlan (100μg/ml) for 24 h. Supernatants were collected for ELISA. (b) BMDCs were stimulated by Zymd (100μg/ml), MDP (10μg/ml), Pam3 (100ng/ml), CpGB (100nM), polyI:C (100μg/ml), or LPS (100ng/ml) for 24 h, and supernatants were collected for ELISA. (c) WT, FcRγ-KO and Mincle-KO BMDCs were stimulated with mannan (100μg/ml), TDB (50μg/well) or zymd (100μg/ml) for 24 h and secreted TNF was measured by ELISA. (d) BMDMs primed by IL-4 (10ng/ml) were stimulated by heat-killed yeast (MOI: 2) or hyphae of C. albicans (MOI: 1) for 24 h. Cytokines and chemokines were measured by ELISA. (e,f) BMDMs primed by GM-CSF (10ng/ml) were stimulated by dectin-2 ligand mannan (e) or Mincle ligand TDB (f) for 24 h. Supernatants were collected for ELISA. Data are presented as mean ± SEM from 3 samples for each group, and one representative experiment of three is presented. Note: * p<0.05, ** p<0.01, *** p<0.001.

Supplementary Figure 3 SHP-2 mediates Syk activation in a variety of signaling pathways.

(a,b) BMDMs primed by IL-4 (10ng/ml) were either untreated or treated by Zymd (100μg/ml) (a) or heat-killed C. albicans yeast (MOI, 2) (b) and cell lysates were probed by indicated antibodies. (c) B cells were isolated from Shp-2fl/fl or Vav-cre:Shp-2fl/fl spleens. Purified B cells were seeded into 12-well plate and stimulated with IgM (10μg/ml). (d) BMDMs from Shp-2fl/fl or MN-Shp-2-/- mice were incubated with anti-CD16/32 antibody (2.4G2, BD Biosciences,10μg/ml) at 4˚C for 30 min, followed by cross-linking with mouse anti-rat IgG (30μg/ml) for various times at 37˚C. (e) BMDMs from Shp-2fl/fl or MN-Shp-2-/- mice were stimulated by OVA-IgG immune complexes (50μg/ml) for 5 or 15 min. OVA-IgG immune complexes were prepared by incubating albumin (Cat.# 02191349.2, MP BIOMEDICALS) with rabbit anti-OVA IgG fraction (Cat.# 0855029, MP BIOMEDICALS) at 1:10 ratio at 37℃for 1 h. (f) BMDMs from Shp-2fl/fl or MN-Shp-2-/- mice were stimulated by heat-induced IgG aggregates (50μg/ml) for 5 or 15 min. IgG aggregates were produced by incubation of mouse IgG (Jackson labs, ImmunoResearch) in borate-buffered saline, pH8.0 at 63℃ for 30 min. Cell lysates were resolved by 10% SDS-PAGE and probed by indicated antibodies. All the experiments were repeated at least twice with similar results, and the representative data are shown.

Supplementary Figure 4 SHP-2 recruits Syk to dectin-1 and mediates Syk activation.

(a) HEK293T cells were transiently transfected by various combinations of plasmids expressing V5-Syk, dectin-1 or FLAG-SHP-2. 48 h after transfection, cells were left unstimulated or stimulated by heat-killed C. albicans yeast for 15 min. Cell lysates were probed by indicated antibodies. (b,c) HEK293T cells were transiently transfected by plasmids expressing Flag-dectin-1, V5-Syk, with or without Myc-SHP-2, Myc-SHP-2 phosphatase-inactive mutant (PD). 48 h after transfection, cells were left unstimulated or stimulated by heat-killed C. albicans yeast for 15 min. Cell lysates were immunoprecipitated by anti-Flag and probed by anti-V5, anti-Myc and anti-FLAG, respectively. (d-f) MN-Shp-2-/- BMDMs were transducted by lentiviral vector pCDH, or pCDH expressing wild type or phosphatase-inactive mutant of SHP-2, respectively. Stable pools of lentiviral-transducted cells were primed by IL-4 (10ng/ml) overnight and then stimulated by Zymd (100μg/ml) for 10 min. BMDMs were fixed by paraformaldehyde and stained by indicated antibodies and DAPI, and fluorescence images were collected by a confocal laser microscope (d, e) or fluorescence microscope (f). Representative image from at least 20 fields of three different samples for each transducted cell type are shown. The above experiments were repeated twice with similar results.

Supplementary Figure 5 SHP-2 recruits Syk to dectin-1 or FcRγ and mediates Syk activation in CLR signaling.

(a) Schematic presentation of a proposed model, in which SHP-2 operates as a scaffold protein recruiting Syk to dectin-1 through its N-SH2 and C-terminal ITAM motif. (b,c) HEK293T cells were transiently transfected by plasmids expressing HA-dectin-2, V5-dectin-3, Syk, FLAG-FcRγ along with mutants of Myc-SHP-2. 48 h after transfection, cells were left unstimulated or stimulated by mannan (M) or heat-killed C. albicans hyphae (H) for 15 min. Cell lysates were immunoprecipitated by anti-Flag and probed by indicated antibodies. These experiments were repeated twice with similar results. (d) HEK293T cells were transiently transfected by plasmids expressing HA-dectin-2, V5-dectin-3, Syk, Myc-SHP-2, FLAG-FcRγ or mutants. 48 h after transfection, cells were stimulated by heat-killed C. albicans hyphae for 15 min. Cell lysates were immunoprecipitated by anti-Myc and probed by anti-FLAG and anti-Myc sequentially. These experiments were repeated twice with similar results. (e) Schematic presentation of a proposed model, in which SHP-2 operates as a scaffold recruiting Syk to FcRγ in dectin-2/3 signaling through its N-terminal SH2 domains and C-terminal ITAM motif.

Supplementary Figure 6 DC-Shp-2−/− mice exhibited severer tissue damage and more fungal burden in infected kidneys.

(a) Live C. albicans SC-5314 (2×105 fungal cells in 0.1ml of PBS buffer) were i.v. injected into 6-8 weeks old littermates of distinct genotypes. 5 days after infection, kidneys were harvested and fixed by 10% formalin and paraffin-embedded sections were stained by hematoxylin and eosin or periodic acid-Schiff, respectively. (b) Shp-2fl/fl and DC-Shp-2-/- mice were infected by C. albicans SC-5314 (2×105 fungal cells per mouse) and kidneys were harvested in 3 days. Kidneys were embedded in OCT and frozen-sections were stained by anti-Gr-1 or anti-F4/80. UI: Uninfected. Representative images were shown and positive cells for each genotype were quantified over 15 fields from three independent samples.

Supplementary Figure 7 MN-Shp -2−/− macrophages exhibited impaired phagocytosis of zymosan particles.

(a-c) BMDMs from Shp-2fl/fl and MN-Shp-2-/- mice were incubated with Alexa Fluor 594 labled Zymosan for 2 h (a) or various times as indicated (b, c). Both zymosan positive cell percentage and zymosan particles per cell were quantified over 6 fields from each genotype. UI: Uninfected. Data are presented as mean ± SEM of two experiments. Note: * p<0.05, ** p<0.01, *** p<0.001.

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Deng, Z., Ma, S., Zhou, H. et al. Tyrosine phosphatase SHP-2 mediates C-type lectin receptor–induced activation of the kinase Syk and anti-fungal TH17 responses. Nat Immunol 16, 642–652 (2015). https://doi.org/10.1038/ni.3155

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