E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor-mediated antifungal innate immunity

Abstract

Activation of various C-type lectin receptors (CLRs) initiates potent proinflammatory responses against various microbial infections. However, how activated CLRs are negatively regulated remains unknown. In this study, we report that activation of CLRs Dectin-2 and Dectin-3 by fungi infections triggers them for ubiquitination and degradation in a Syk-dependent manner. Furthermore, we found that E3 ubiquitin ligase Casitas B-lineage lymphoma protein b (Cbl-b) mediates the ubiquitination of these activated CLRs through associating with each other via adapter protein FcR-γ and tyrosine kinase Syk, and then the ubiquitinated CLRs are sorted into lysosomes for degradation by an endosomal sorting complex required for transport (ESCRT) system. Therefore, the deficiency of either Cbl-b or ESCRT subunits significantly decreases the degradation of activated CLRs, thereby resulting in the higher expression of proinflammatory cytokines and inflammation. Consistently, Cbl-b-deficient mice are more resistant to fungi infections compared with wild-type controls. Together, our study indicates that Cbl-b negatively regulates CLR-mediated antifungal innate immunity, which provides molecular insight for designing antifungal therapeutic agents.

Figure 1.

Activated Dectin-2 and Dectin-3 can trigger themselves for ubiquitination and degradation. (A and B) Hyphae-induced ubiquitination and degradation of Dectin-2 and Dectin-3 in mouse BMDMs. (C and D) α-Mannan­–induced ubiquitination of Dectin-2 and Dectin-3 in RAW264.7 cells overexpressing Dectin-2 or Dectin-3. (E) α-Mannan–induced K48 ubiquitination of Dectin-2 and Dectin-3 in RAW264.7 cells overexpressing Dectin-2 and Dectin-3. (F) Hyphae-induced K48 ubiquitination of Dectin-2 and Dectin-3 in RAW264.7 cells overexpressing mutant Dectin-2_K12A and Dectin-3_K9A. Immunoprecipitation was performed using anti–Dectin-2 or –Dectin-3 antibodies on solubilized protein extracts from BMDMs or RAW264.7 cells and subjected to immunoblotting with the indicated antibodies. (G and H) Hyphae-induced degradation levels in RAW264.7 cells expressing WT or mutant Dectin-2 (G) and Dectin-3 (H), which were pretreated with 10 µg/ml cycloheximide for 15 min and then stimulated with hyphae (multiplicity of infection [MOI] =1) for the indicated times. The cell lysates were subjected to immunoblotting using the indicated antibodies. The data shown are representative of three independent and reproducible experiments. IP, immunoprecipitation; Ly, lysis; Ub, ubiquitination.

Figure 2.

Cbl-b mediates ubiquitination and degradation of Dectin-2 and Dectin-3. (A and B) Association analyses of Cbl-b with Dectin-2 or Dectin-3 in mouse BMDMs, which were stimulated with C. albicans hyphae for the indicated times. (C and D) Hyphae-induced ubiquitination of Dectin-2 and Dectin-3 in WT and Cbl-b–deficient BMDMs. (E) Hyphae-induced degradation of Dectin-2 and Dectin-3 in WT and Cbl-b–deficient BMDMs. (F) Hyphae-induced degradation of Mincle, Syk, and SHP-2 in WT and Cbl-b–deficient BMDMs. Cell lysates were immunoprecipitated with anti–Dectin-2 or –Dectin-3 antibodies as indicated. The cell lysate and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. (G and H) Surface expression levels of Dectin-2 (G) or Dectin-3 (H) in WT and Cbl-b–deficient BMDMs, which were stimulated with C. albicans hyphae (MOI = 1) for the indicated times. BMDMs were incubated with anti–Dectin-2, anti–Dectin-3, or isotype control IgG for 30 min at 4°C and then stained with FITC-labeled goat anti–mouse secondary antibodies. Samples were then examined for FITC intensity by flow cytometry. The data shown are representative of three independent and reproducible experiments. IP, immunoprecipitation; Iso, isotype; Ly, lysis.

Figure 3.

Syk is required for Cbl-b–mediated ubiquitination and degradation of Dectin-2 and Dectin-3. (A) Hyphae-induced phosphorylation of Cbl-b in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. p-Cbl, phosphorylated Cbl. (B) Hyphae-induced association analyses of Cbl-b with Dectin-2 or Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (C and D) Hyphae-induced ubiquitination of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (E) Hyphae-induced degradation of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without Syk inhibitor (piceatannol) for 30 min. (C–E) Cell lysates were immunoprecipitated with the indicated antibodies. (F) HEK293 T cells were transfected with expression vectors encoding Flag–Dectin-2 (D2-Flag), Flag–Dectin-3 (D3-Flag), FcR-γ–HA, Myc-Syk, and Cbl-b in different combinations. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The cell lysate and immunoprecipitates were subjected to immunoblots using the indicated antibodies. (G and H) Hyphae-induced ubiquitination of Dectin-2 and Dectin-3 in WT and CARD9-deficient BMDMs. Cell lysates were immunoprecipitated with anti–Dectin-2 or –Dectin-3 antibodies as indicated. The cell lysate and immunoprecipitates were subjected to immunoblotting using the indicated antibodies. The data shown are representative of three independent and reproducible experiments. IP, immunoprecipitation; Ly, lysis; p-Syk, phosphorylated Syke; Ub, ubiquitination.

Figure 4.

Cbl-b negatively regulates in vitro CLR-mediated innate immune responses. (A) Nuclear p65 in WT and Cbl-b–deficient BMDMs that were stimulated with 40 µg/ml of precoated α-mannans for different times. The data shown are representative of three independent and reproducible experiments. (B) Quantification grayscale analysis of p65 versus internal control proliferating cell nuclear antigens (PCNA) in A. (C) ELISA results for TNF in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with precoated α-mannans at the indicated concentrations for 12 h. (D) ELISA results for IL-6, IL-1β, and IL-12p40 in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with 40 µg/ml of precoated α-mannans for 12 h. (E and F) ELISA results for TNF and IL-6 in supernatants of WT and Cbl-b–deficient BMDMs, which were stimulated with 40 µg/ml of precoated LAM (E) or TDM (F) at the indicated concentrations for 12 h. (G) ELISA results for TNF and IL-6 in supernatants of WT and Cbl-b–deficient BMDMs, which were pretreated with or without anti–Dectin-3 antibody (α–Dectin-3), α–Dectin-2, or an isotype-matched control IgG for 30 min before stimulation with C. albicans hyphae (MOI = 0.1) for 12 h. Usti., unstimulated. (H) ELISA results for TNF and IL-6 in supernatants of WT, Cbl-b KO, CARD9 KO, or Cbl-b/CARD9 dKO BMDMs, which were stimulated with C. albicans hyphae (MOI = 0.01 or 0.1) for 12 h. Data are means ± SD of triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test or ANOVA).

Figure 5.

Activated Dectin-2 and Dectin-3 are sorted into lysosomes by the ESCRT system for degradation. (A) Hyphae-induced degradation of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without proteasome inhibitor (MG132) or lysosome inhibitor (chloroquine). Cell lysates were probed with the indicated antibodies. (B) Hyphae-induced degradation of Dectin-2 and Dectin-3 in iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA using Trans–IT-TKO transfection reagent (Mirus). (C) Nuclear p65 in iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA before stimulation with C. albicans hyphae (MOI = 0.1) for the indicated times. (D) ELISA results for TNF, IL-6, and IL-12p40 in supernatants of iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA before stimulation with C. albicans hyphae (MOI = 0.1) for 12 h. (E) STAM protein amounts in WT and STAM-deficient iBMDMs. The ESCRT-0 subunit STAM gene was disrupted in iBMDM cells using the CRISPR/Cas9 system. (F–H) Nuclear p65 in WT and STAM-deficient iBMDMs upon stimulation with 40 µg/ml of precoated α-mannans (F), 40 µg/ml LAM (G), or 50 µg/ml TDM (H) for the indicated times. (I) ELISA results for TNF in supernatants of WT and STAM-, HRS-, and Vps28-deficient iBMDMs upon stimulation with 40 µg/ml of precoated α-mannans, 40 µg/ml LAM, or 50 µg/ml TDM for 12 h. (A–C and E–H) The data shown are representative of three independent and reproducible experiments. (D and I) Data are means ± SD of triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01 (Student'’s t test or ANOVA). Ctl, control; PCNA, proliferating cell nuclear antigen; Usti., unstimulated; Vec, vector.

Figure 6.

Cbl-b negatively regulates in vivo CLR-mediated antifungal immunity. (A) Survival of WT and Cbl-b KO mice (n = 10 per group) infected intravenously with 10⁵ CFU of C. albicans (SC5314). (B) Kidney CFU assay of WT and Cbl-b KO mice (n = 5 per group) infected intravenously with 10⁵ CFU of C. albicans (SC5314) for 24 h. (C) Amounts of TNF and IL-6 in kidneys of infected WT and Cbl-b KO mice after infection with 10⁵ CFU of C. albicans (SC5314) for 24 h. (D) Quantitative real-time PCR assay of chemokines including CXCL1, CXCL2, and CCL3 in kidneys of WT and Cbl-b KO mice after infection with 10⁵ CFU of C. albicans (SC5314) for 24 h. (E) Lung CFU assay of WT and Cbl-b KO mice (n = 3 per group) after trans-nasal infection with 10⁶ CFU of A. fumigatus conidia for 24 h. The data shown are representative of three independent and reproducible experiments. (F) Amounts of TNF and IL-6 in lungs of WT and Cbl-b KO mice after trans-nasal infection with 10⁶ CFU of A. fumigatus conidia for 24 h. (C, D, and F) Data are means ± SD of triplicate samples and are representative of three independent experiments. **, P < 0.01; ***, P < 0.001 (Student’s t test).

Figure 7.

CLR/CARD9 cascade is required for host Cbl-b–mediated antifungal immune regulation. (A) Survival of WT, Cbl-b KO, Dectin-3 KO, or Cbl-b/Dectin-3 dKO mice (n = 10 per group) infected intravenously with 10⁵ CFU of C. albicans (SC5314). (B) Kidney CFU assay of WT, Cbl-b KO, Dectin-3 KO, or Cbl-b/Dectin-3 dKO mice (n = 15 per group) infected intravenously with 10⁵ CFU of C. albicans (SC5314) for 24 h. The data shown are representative of three independent and reproducible experiments. (C) Amounts of TNF and IL-6 in kidneys of WT, Cbl-b KO, Dectin-3 KO, or Cbl-b/Dectin-3 dKO mice after infection with 10⁵ CFU of C. albicans (SC5314) for 24 h. Data are means ± SD of triplicate samples and are representative of three independent experiments. (D) Survival of WT, Cbl-b KO, CARD9 KO, or Cbl-b/CARD9 dKO mice (n = 10 per group) infected intravenously with 10⁵ CFU of C. albicans (SC5314). (E) Kidney CFU assay of WT, Cbl-b KO, CARD9 KO, or Cbl-b/CARD9 dKO mice (n = 5 per group) after intravenous infection with 10⁵ CFU of C. albicans (SC5314) for 24 h. The data shown are representative of three independent and reproducible experiments. (F) Amounts of TNF, IL-6, IL-1β, and IL-12p40 in kidneys of WT, Cbl-b KO, CARD9 KO, or Cbl-b/CARD9 dKO mice after infection with 10⁵ CFU of C. albicans (SC5314) for 24 h. Data are means ± SD of triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (ANOVA).