Cutting edge: Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate

Abstract

The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analog trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in APCs. In this study, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein FcRgamma was essential for macrophage activation and Th17 adjuvanticity. The FcRgamma-associated C-type lectin receptor Mincle was expressed in macrophages and upregulated by TDM and TDB. Recombinant Mincle-Fc fusion protein specifically bound to the glycolipids. Genetic ablation of Mincle abolished TDM/TDB-induced macrophage activation and induction of T cell immune responses to a tuberculosis subunit vaccine. Macrophages lacking Mincle or FcRgamma were impaired in the inflammatory response to Mycobacterium bovis bacillus Calmette-Guérin. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.

Figure 1. FcRγ-dependent adjuvanticity of TDB

Mice were immunized subcutaneously with 2 μg H1 protein adsorbed to DDA liposomes (250 μg) containing TDB (50 μg) or not at d0 and d21. (A) Footpad swelling was measured on d6 with a caliper. Baseline values were subtracted individually for both feet of each mouse. (B) IFNγ and (C) IL-17 production by lymph node cells after restimulation with H1 (10, 2, 0.4 μg/ml) for 96h in vitro. (A-C) Mean and SEM of five mice per group from a representative experiment of two to three with similar results. * p<0.05, ** p<0.01.

Figure 2. Mincle expression, regulation and binding to TDM and TDB

(A)Increased mRNA expression of Mincle in BMM after stimulation with TDB or TDM (4 μg/ml, resp.) for 48 h requires FcRγ and Card9, but not Myd88. Shown are mean and SD of quadruplicate qRT-PCR data. (B) Binding of recombinant Mincle-Fc fusion protein to glycolipids and Curdlan. TDB, TDM and Curdlan were coated at 40, 10 and 2.5 μg/ml in 96-well plates. Binding of Mincle-Fc was detected with peroxidase-conjugated anti-human Fc antibody. Mean and SD of triplicate wells from a representative experiment of two.

Figure 3. Mincle is required for macrophage activation and Th1/Th17 induction by TDM and TDB

(A)Mincle−/− and wt BMM were stimulated with plate-coated TDB (5 μg/ml), TDM (5 μg/ml) or Curdlan (500 μg/ml), or with CpG (1 μM) in solution for 24 hours. Changes in the expression of G-CSF and IL-6 mRNA were analysed by qRT-PCR. Mean and SD of quadruplicate samples from a representative experiment of three performed. (B) As in (A), except that glycolipids were titrated (0.015 – 4 μg/ml), IFNγ (10 ng/ml) was added and the supernatants were analysed for the accumulation of nitrite using the Griess assay. (C-E) Mice were immunized once subcutaneously at the base of the tail with 2 μg H1 protein adsorbed to DDA liposomes containing TDB or not, or with H1 mixed with 5 nmol CpG ODN, at d0 and sacrificed at d7. (C) Cell numbers in the draining inguinal lymph nodes. (D, E) Production of IFNγ (D) and IL-17 (E) by 3×10⁵ lymph node cells/well restimulated with H1 (10, 2, 0.4 μg/ml) for 96 h in 96-well plates. Mean and SEM of data obtained in three independent experiments (n=8 (Mincle−/−) and 10 (wt) for DDA, n=15 for DDA+TDB, n=7 for CpG). * p<0.05, ** p<0.01 in Student’s t-test.

Figure 4. Mincle−/− macrophages have a defect in the response to BCG

BMM were stimulated with live bacteria for 24 h at MOI of 30, 10 and 3, followed by preparation of RNA from the cell lysate. Expression of G-CSF and IL-6 was analysed by qRT-PCR. Shown are mean and SD of quadruplicate samples from a representative of two experiments.