C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-κB activation

Abstract

Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6'-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.

Keywords: Innate Immunity; Lectin; NF-kappa B (NF-κB); NF-κB Transcription Factor; Signal Transduction.

FIGURE 1.

Dectin-3 is required for TDM-induced Mincle expression. A and B, BMDMs from WT and Dectin-3 KO mice were stimulated with 20 μg/ml plate-coated TDM for 3, 6, 9, and 12 h. A, cell lysates were analyzed by immunoblotting with the indicated antibodies. B, total mRNA was analyzed by RT-PCR for Mincle expression. C–E, BMDMs from WT mice were pretreated with control (Ctr) IgG or blocking antibodies to Dectin-3 (C) or Dectin-2 (D and E) for 45 min and then stimulated with plate-coated TDM for indicated time points. C, D, total RNA was prepared from these cells, and real-time PCR was performed to quantify Mincle mRNA. E, cell lysates were analyzed by immunoblotting with the indicated antibodies.

FIGURE 2.

Dectin-3 is required for TDM-induced NF-κB activation. A–C, BMDMs from WT and Dectin-3 KO mice were either untreated or stimulated with plate-coated TDM for indicated time points. A, total cell lysates were prepared from these cells and then subjected to immunoblotting analysis using indicated antibodies. B, nuclear extracts were prepared and analyzed by immunoblotting with p65, NFAT-c1, and proliferating cell nuclear antigen antibodies. C, immunoblotting with the indicated antibodies were done with the total cell lysates. D, BMDMs from WT mice were pretreated with Dectin-2 or Dectin-3 blocking antibodies for 45 min. Resulting cells were stimulated with plate-coated α-mannan (man) or TDM for 1 h. Nuclear extracts were prepared and analyzed by immunoblotting with p65 and proliferating cell nuclear antigen (PCNA) antibodies. Total cell lysates were immunoblotting with Dectin-2 or Dectin-3 antibodies. E, ELISA results for TNF-α, IL-6, and IL-1β in supernatants of BMDMs derived from WT or Dectin-3 KO mice, which were stimulated with plate-coated TDM for 8 h. Data shown are representative of three independent experiments. S.D. is indicated. **, p < 0.01 (t test). Ctr, control.

FIGURE 3.

NF-κB but not NFAT activation is essential for Mincle induction. A–C, BMDMs from WT mice were stimulated with plate-coated TDM for indicated time points. TPCA-1 (NF-κB inhibitor, 1 μm) or 11R-VIVIT (NFAT inhibitor, 10 μm) was added to cells when they were stimulated. A and B, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. C, total mRNA was analyzed by RT-PCR for Mincle expression. D, BMDMs were treated as described in A–C by plate-coated TDM for 8 h, and supernatants were subjected to ELISA analysis for TNF-α, IL-6, and IL-10. Data shown are representative of three independent experiments. S.D. is indicated. **, p < 0.01 (t test). Ctr, control.

FIGURE 4.

NF-κB p65 subunit binds with Mincle promoter to regulate Mincle expression. A, the structure for Mincle gene promoter region. −1589, −941, and −851 are the three predicted NF-κB-binding sites. Different luciferase reporter constructs are also shown. B, BMDMs were stimulated with plate-coated TDM for 2 h or left untreated (0h), followed by ChIP assay with the indicated antibodies and real-time PCR analysis for Mincle promoter. Results are presented as means plus S.D. after normalization to input. C, RAW264.7 cells were transfected with different luciferase reporter constructs together with Dectin-3 expression plasmid. The luciferase activities of the Mincle promoter were measured. Two putative NF-κB-binding sites were mutated and the mutant reporter was designated as −941m and −851m. Data shown are representative of two independent experiments. S.D. is indicated. **, p < 0.01 (t test). Ctr, control.

FIGURE 5.

Dectin-3 neither forms heterodimer nor synergistically induces signaling together with Mincle. A, RAW264. 7 cells stably expressing human Mincle (M), Dectin-3 (D3), or both (M + D3) were stimulated with plate-coated TDM for indicated time points and then lysed. Cell lysates were immunoprecipitated with anti-Dectin-3 or anti-Mincle antibodies (5 ug), and then the immunoprecipitated (IP) and lysate fractions were analyzed by immunoblotting with the indicated antibodies. B, RAW264.7 cells stably expressing human Dectin-3, Mincle, both (D3 + M), or control vector (Mock) were stimulated with plate-coated TDM for 60 min. Nuclear extracts (top and middle panel) and cytoplasmic fractions (bottom panel) of these cells were prepared and subjected to immunoblotting analysis. C, ELISA results for TNF-α and IL-6 in supernatants of RAW264.7 cells stimulated as described in B for 6 h, cells treated only with the solvent for TDM were used as unstimulated control (Unsti). Data shown are representative of three independent experiments. PCNA, proliferating cell nuclear antigen.

FIGURE 6.

Dectin-3 serves as the sensor for induction of Mincle expression. A and B, BMDMs were incubated with Dectin-3-blocking antibodies for 30 min at 37 °C and then stimulated with TDM (pre+α-D3) or stimulated with TDM for 3 h and then Dectin-3-blocking antibodies (3hr+α-D3). After another 6-h incubation, all cells were collected and stained with Dectin-3 antibody (A) or Mincle antibody (B), followed by FITC-labeled goat anti-mouse secondary antibodies. Samples were then examined by flow cytometry. C and D, the indicated cell supernatant were collected and measured by ELISA for TNFα and IL-6 production. Data shown are representative of three independent experiments. S.D. is indicated. **, p < 0.01 (t test). Ctr, control.

FIGURE 7.

CARD-9, Bcl10, and Malt1 are required for Mincle induction. A and B, BMDMs from WT and CARD9-deficient (KO) mice (A) or from WT, Bcl10-deficient (KO) and Malt1-deficient (KO) mice (B) were stimulated with plate-coated TDM for different time points. Cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. C and D, total mRNA from above stimulated cells was prepared and analyzed by RT-PCR for Mincle expression. E, BMDMs from different mice were stimulated with or without plate-coated TDM for 8 h. Supernatants from these cells were subjected to ELISA analysis for the level of TNF-α, IL-6, and IL-1β. Data shown are representative of three independent experiments. S.D. is indicated. **, p < 0.01 (t test). Ctr, control.

FIGURE 8.

Dectin-3- and CARD9-deficient mice produce much less cytokine and antibodies upon immunization using adjuvant containing TDM. CARD9- and Dectin-3-deficient mice and their controls were immunized with KLH emulsified in complete Freund's adjuvant at the base of the tail. A, forty-eight hours later, blood was collected from the tail and measured with ELISA for TNFα and IL-6 production. **, p < 0.01 (t test). B, seven days after immunization, sera from the indicated immunized mice were subjected to serial dilutions, and the concentrations of KLH-specific IgG and IgM antibodies were analyzed by ELISA and averaged for each group. Graphs show the means ± S.D. from three mice of each genotype.

FIGURE 9.

Proposed model for the mechanism by which Dectin-3 induces Mincle expression. Step 1, Dectin-3 functions as an initial sensor for detecting low level of TDM and triggers an intracellular signaling pathway leading to activation of NF-κB in a CARD9-Bcl10-Malt1-dependent manner. Step 2, The activated NF-κB induces the expression of Mincle gene, which results in significantly higher amount of Mincle expressed on the cell surface. Step 3, The high amount of Mincle further activates by TDM to induce the expression of proinflammatory cytokines.