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

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.

Figure 1

SHP-2 is tyrosine-phosphorylated and regulates pro-inflammatory gene expression upon dectin-1 activation. (a,b) Wild-type BMDCs differentiated by GM-CSF (20ng/ml) and IL-4 (10ng/ml) were either untreated or treated by Zymd (100µg/ml) for various times as indicated. Cell lysates were immunoblotted by respective antibodies. (c) Wild-type BMDMs primed with IL-4 (10ng/ml) overnight were either untreated or treated by Zymd (100µg/ml). Cell lysates were immunoprecipitated by control IgG, anti-SHP-2 or anti-p-Tyr, followed by immunoblotting with anti-p-Tyr or anti-SHP-2, respectively. (d) Wild-type BMDCs were pretreated by vesicle or inhibitors PP2 (3µM) and Piceatannol (15µM) for 1 h, followed by Zymd treatment for 15 min. Cell lysates were probed by indicated antibodies. (e) BMDCs derived from Shp-2^fl/fl and DC-Shp-2^−/− mice were stimulated by dectin-1 ligands Zymd (100µg/ml), ZymA (100µg/ml) or Curdlan (100µg/ml) for 24 h. Supernatants were collected and amounts of cytokines and chemokines were measured by ELISA. Data are presented as means ± SEM from three samples for each group in one representative experiment, and similar results were obtained from three independent experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 2

SHP-2 is required for CLR- and C. albicans-induced gene expression. (a) BMDCs derived from Shp-2^fl/fl and DC-Shp-2^−/− mice were stimulated by heat-killed yeast (MOI: 2) or hyphae of C. albicans (MOI: 1) for 24 h. (b) BMDCs derived from WT, FcRγ-deficient or Mincle-deficient mice were stimulated by mannan (100µg/ml) or TDB (50µg/well) for indicated times. Whole cell lysates were resolved by 10% SDS-PAGE and then probed by respective antibodies. (c,d) Wild-type and DC-Shp-2^−/− BMDCs were stimulated by mannan (100µg/ml) or TDB (50µg/well) for 24 h. Supernatants were collected and amounts of cytokines and chemokines were measured by ELISA. Data are presented as means ± SEM from three samples for each group in one representative experiment of three. * p<0.01, ** p<0.001.

Figure 3

SHP-2 mediates Syk activation in dectin-1 and C. albicans-induced signaling. (a,b) BMDCs were either untreated or treated by Zymd (100µg/ml) (a) or heat-killed C. albicans yeast (MOI: 2) (b), and cell lysates were immunoblotted by indicated antibodies. (c) BMDCs were either untreated or treated by Zymd (100µg/ml) for various times, and cell lysates were immunoprecipitated by control IgG or anti-SHP-2. (d) BMDCs were either untreated or treated by Zymd (100µg/ml) for 15 min before lysis. Whole cell lysates were incubated with nickel-beads or nickel-beads conjugated His-tagged Syk protein (1µg) for 4 h. Proteins pulled-down were probed by indicated antibodies. (e) BMDCs treated by Zymd for various times were subject to biochemical fractionation and cytosolic and cell membrane proteins were probed by anti-SHP-2 and anti-Syk, respectively. (f) Untreated or Zymd-treated BMDCs were fixed by paraformaldehyde and stained by indicated antibodies and DAPI. Representative data from three independent experiments are shown.

Figure 4

SHP-2 mediates Syk membrane translocation and colocalization with dectin-1 and FcRγ. (a) BMDCs derived from Shp-2^fl/fl and DC-Shp-2^−/− mice were stimulated by Zymd or Mannan for various times. Cytosolic and cell membrane fractions were resolved by SDS-PAGE and probed with anti-Syk. (b,c) WT and DC-Shp-2^−/− BMDCs were stimulated with Zymd (b) or mannan (c) for 10 min, followed by immune fluorescence staining with indicated antibodies. Representative data from two independent experiments are shown.

Figure 5

SHP-2 recruits Syk to dectin-1 upon C. albicans infection. (a,b) HEK293T were transiently transfected by plasmids expressing FLAG-tagged wild-type or various deletion mutants of SHP-2, along with plasmids expressing HA-dectin-1 or HA-dectin-1 Y15F. In 48 h, cells were left unstimulated or stimulated by heat-killed C. albicans yeast (MOI: 2) for 15 min. Cell lysates were immunoprecipitated by anti-FLAG and probed by indicated antibodies. (c,d,e) HEK 293T cells were transiently transfected by plasmids expressing V5-tagged wild-type and N-SH2 or C-SH2 deletion mutants of Syk along with plasmids expressing HA-tagged dectin-1 and Flag-tagged SHP-2 (c); or V5-tagged Syk and HA-tagged dectin-1, along with Flag-tagged wild-type and mutants of SHP-2 (d,e). 48 h after co-transfection, cells were stimulated with heat-killed C. albicans yeast (MOI: 2) for 15 min. Cell lysates were immunoprecipitated by anti-FLAG and probed by anti-HA or anti-p-Tyr. The representative results from three independent experiments were shown above. (f) BMDMs from M/N-Shp-2^−/− mice were transducted by lentiviral vector pCDH expressing wild-type or mutants of SHP-2. Stable pools of lentiviral-transducted cells were primed by IL-4 (10ng/ml) and stimulated by Zymd (100µg/ml) for 24 h, TNF induction was measured by ELISA. Data are presented as mean ± SEM of three samples for each group, and the representative data from two independent experiments are shown. * p<0.01, ** p<0.001.

Figure 6

SHP-2-mediated CLR signaling in DCs is indispensable for anti-fungal T_H17 responses. (a) 6–8 week-old littermates of Shp-2^fl/fl and DC-Shp-2^−/− mice were infected by C. albicans SC-5314 (2×10⁵ fungal cells/mouse) via i.v. injection. Weight loss and survival were monitored daily. Data was pooled from 14 pairs of sex- and age-matched littermates through three independent experiments. Statistical analysis was performed by log-rank test (p<0.0001). (b) Kidneys, livers and spleens from infected mice were homogenized, and colonies of C. albicans SC-5314 were counted by serial dilution. (c) Mice were infected by C. albicans SC-5314 (2×10⁵ or 1×10⁶ fungal cells/mouse), and sera were collected in 24 h for ELISA. (d) Kidneys from C. albicans SC-5314 (2×10⁵ fungal cells/mouse) infected mice for 5 days were homogenized, followed by RNA isolation. IL-17A, IL-17F and IFN-γ mRNAs were assessed by quantitative real-time PCR. (e) Total spleens were isolated from mice uninfected or infected by C. albicans SC-5314 (2×10⁵ fungal cells/mouse) for 5 days. Splenic cells were stimulated by heat-killed C. albicans (MOI: 1) for 2 days. IL-17A and IFN-γ from supernatants were measured by ELISA. UI: Uninfected. Data are presented as mean ± SEM from 5 mice per group, and representative data from three independent experiments are shown. Note: * p<0.05, ** p<0.01, *** p<0.001.

Figure 7

SHP-2-mediated CLR signaling in macrophages/neutrophils is critical for anti-fungal innate immune responses. (a,b) 6–8 week-old littermates of Shp-2^fl/fl and M/N-Shp-2^−/− mice were infected by C. albicans SC-5314 (2×10⁵ fungal cells/mouse) via i.v.. Weight loss and survival were monitored daily. Data was pooled from 16 pairs of sex- and age-matched littermates through three independent experiments. Statistical analysis was performed by log-rank test (day1–9, p<0.05; day10–21, p>0.05). (c) 24 h after C. albicans SC-5314 infection, sera were collected for ELISA. Data are presented as mean ± SEM from 7 mice per group, and representative data from two independent experiments are shown. (d) Spleens were isolated from mice uninfected or infected by C. albicans SC-5314 (2×10⁵ fungal cells/mouse) for 5 days. Splenic cells were stimulated by heat-killed C. albicans (MOI: 1) for 2 days. Supernatants were collected for ELISA. Data are presented as mean ± SEM from 5 mice per group, and representative data from two independent experiments are shown. (e) Kidneys from Shp-2^fl/fl and M/N-Shp-2^−/− mice 3 days after C. albicans SC-5314 infection were embedded in OCT, and frozen-sections were stained by anti-Gr-1, anti-F4/80 and DAPI. Representative images were shown and positive cells for each genotype were quantified over 20 fields from three independent samples. UI: Uninfected. Note: * p<0.05, ** p<0.001.