Mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analogue trehalose-dibehenate

Abstract

The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analogue trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in antigen presenting cells. Here, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein Fc receptor gamma chain (FcRγ) was essential for macrophage activation and Th17 adjuvanticity. The FcRγ-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 FcRγ were impaired in the inflammatory response to Mycobacterium bovis BCG. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.

Introduction

The development of recombinant subunit vaccines has been hampered by the lack of safe and effective adjuvants for human use. For many years research into adjuvants was neglected and only recently has been invigorated by the discovery of pattern recognition receptors (PRR) for microbial ligands (so-called PAMPs) on antigen presenting cells (APC). Modern, molecularly defined adjuvants may be used to induce appropriate types of immune responses through engagement of specific receptors and pathways.

TLRs are the best characterized family of PRR and recognize a wide spectrum of PAMPs from different pathogens, including mycobacteria (1). In the last few years, several C-type lectin receptors (CLEC) were shown to act as PRR that activate antigen presenting cells through a pathway comprising the kinase Syk and the adapter proteins Card9, Bcl10 and Malt1 (2, 3). Dectin-1, the first member of the CLEC PRR, binds β-glucans from yeast cell wall and directly recruits Syk (4-6). The CLEC Dectin-2 and Mincle both require the adapter protein Fc receptor gamma chain (FcRγ) for Syk activation (7-9), whereas others associate with the Dap12 adapter molecule (10). The binding specificity of CLEC is not restricted to fungal carbohydrate structures but encompasses various chemical structures and microbes, including dengue virus particles (Clec5a), the ectoparasite house dust mite (Dectin-2) (11) and the nuclear ribonucleoprotein SAP130 (Mincle) (9, 10). Of interest, Syk-Card9-dependent APC activation by triggering of Dectin-1 and Dectin-2 induces Th17 immunity to fungal infection (7, 12).

Complete Freund’s Adjuvant is a suspension of killed Mycobacterium tuberculosis (MTB) in mineral oil that has been used experimentally for many decades. The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM), also known as cord factor, and its synthetic analogue Trehalose-6,6-dibehenate (TDB), are effective adjuvants for induction of protective T cell immunity by recombinant proteins from MTB, Chlamydia and other pathogens (13-15). We have previously investigated how these glycolipid adjuvants elicit protective immunity. The development of a mixed Th1/Th17 response by TDB/TDM-containing adjuvants is linked to protection after challenge with MTB (4, 14). APC activation by TDB and TDM in vitro is independent of TLR/Myd88 signaling but instead requires the Syk-Card9-Bcl10-Malt1 signaling axis, which is also essential for adjuvanticity in vivo. We excluded Dectin-1 as essential receptor for TDB and TDM, but obtained evidence in vitro that the adapter protein FcRγ may couple a receptor protein to Syk-Card9 signaling (4).

Here, we show that the FcRγ-coupled C-type lectin receptor Mincle binds the glycolipids TDB and TDM, is essential for activation of macrophages and adjuvant activity, and plays a non-redundant role in the macrophage response to intact mycobacteria.

Materials and Methods

Reagents, mice and immunizations

TDB, TDM, CpG, DDA liposomes have been described (4). Myd88−/− and Fcerg1−/− (referred to as FcRγ−/− mice here) were used with permission from Drs. Akira and Takai, resp. (16, 17). Card9−/− and Mincle−/− mice have been described (2, 18). Animal experiments were approved by the Regierung von Oberbayern. Mice were immunized subcutaneously with Ag85B-ESAT-6 (H1) mixed with adjuvants as described (4).

Bacteria

Mycobacterium bovis BCG glycerol stocks were prepared at a density of 1.7 × 10⁹/ml. Escherichia coli (DH5α) and Staphylococcus aureus ATCC R5923 were cultured on blood agar, picked and adjusted to a density of 10⁸/ml before freezing. All bacteria were diluted in cDMEM before use for macrophage stimulation at the indicated MOI.

Generation of macrophages from bone marrow and stimulation with glycolipids

was done as described (4). The glycolipids TDB and TDM were coated directly to cell culture plates following the procedure described by (19).

Measurement of nitrites, cytokines and mRNA expression

was done as described (4).

Recombinant C-type lectin receptors and glycolipid-binding ELISA

TDB, TDM and Curdlan were coated onto 96-well cell culture plates at 40, 10, and 2.5 μg/ml. After washing and blocking with 3% BSA in HBSS, supernatant of Mincle-Fc transfected HEK 293 cells was added at a dilution of 1:4 in blocking buffer. Following incubation at 4°C overnight, plates were washed three times, bound protein detected by incubation with anti-human Fc antibody conjugated to peroxidase, washed again and developed with ELISA substrate.

Results and Discussion

FcRγ-dependent activation of macrophages by TDB and the cord factor

Previously, we demonstrated that the glycolipid adjuvants TDM and TDB activate the Syk-Card9-Bcl10-Malt1 pathway in APC (4). In addition, TDM/TDB-induced NO production is independent of Dectin-1, but requires the Fc receptor gamma chain (FcRγ) (4). To corroborate and extend these data, we tested whether the expression of G-CSF, IL-1β and Arginase-1 requires FcRγ (Supplemental Fig. 1). As expected, TLR9- as well as Dectin-1-triggered induction was not affected by the deletion of FcRγ, whereas TDB and TDM failed to induce G-CSF and IL-1β expression in FcRγ−/− BMM. FcRγ also associates with the activating Fcγ receptors (FcγRI (CD64), FcγRIII (CD16) and FcγRIV) that can also bind non-Ig ligands (20). However, while NO production was completely abrogated in the absence of FcRγ, none of the Fcγ receptor-deficient BMM genotypes showed a substantial impairment in NO release (Supplemental Figure 2). Thus, FcRγ is essential for transcriptional responses to TDM/TDB, but Fcγ receptors are not involved.

FcRγ-dependent Th17 adjuvant activity of TDB

The adjuvanticity of TDB/TDM is abrogated in Card9−/− mice, including the loco-regional inflammatory reaction and the induction of Th1/Th17 biased antigen specific T cells (4). As FcRγ-deletion closely mimicked the phenotype of Card9−/− in BMM, we next asked whether FcRγ is also essential for the induction of cellular immunity to subunit vaccination with the MTB fusion protein Ag85B-ESAT-6 (H1) by the adjuvant TDB in vivo. The moderate footpad swelling after injection of H1 in liposomes alone was not affected by the absence of FcRγ. In contrast, the large increase in footpad thickness in the TDB-containing adjuvant group was almost entirely abrogated in FcRγ−/− mice (Fig. 1A). The induction of IFNγ-producing T cells was reduced to the levels obained with liposomes alone in FcRγ−/− (Fig. 1B), and the strong induction of IL-17 producing H1-specific T cells by the adjuvant TDB was completely FcRγ-dependent (Fig. 1C). The similarity between the phenotype of FcRγ−/− and Card9−/− mice indicates that FcRγ is critical for the Syk-Card9-dependent activation of APC. The lack of residual adjuvant activity of TDB in FcRγ-deficient mice suggested that other adapter molecules or receptors that directly recruit Syk are most likely not involved.

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.

Mincle expression, regulation and binding to TDB and cord factor

In our search for a receptor for the TDM and TDB we focussed on the family of CLEC because we had excluded Fcγ receptors (Supplemental Fig. 2). We reasoned that the putative receptor should be expressed in macrophages and mined our microarray datasets from BMM (GSE10530 and GSE10532 in Gene Expression Omnibus, http://www.ncbi.nlm.nih.gov/sites/entrez?db=geo; see ref. 4) for expression of CLEC family members (Supplemental Fig. 3). Among the expressed CLEC, Dectin-1 had already been ruled out as a receptor (4); Clec5a associates with Dap12 (10), and the adapter protein coupling Clec4d/Clecsf8 to Syk is unknown. In contrast, Mincle and Dectin-2 stood out because they are FcRγ-associated receptors and were expressed and up-regulated in BMM by TDB. In a second microarray dataset, all TDB regulation of CLEC receptors was Card9-dependent (Supplemental Fig. 4). Validating expression of Mincle mRNA by qRT-PCR, we observed a complete dependence of the TDM/TDB-induced increase on FcRγ and Card9, but not on the TLR adapter protein Myd88 (Fig. 2A). Mincle can interact with ligands of diverse chemical nature (9, 18, 21). We therefore investigated whether it directly binds to TDM and TDB. A Mincle-Fc fusion protein specifically and dose-dependently bound to TDM and TDB, but not to curdlan (Fig. 2B). These data demonstrate that the mycobacteria-derived glycolipids TDM and TDB are newly recognized ligands of Mincle.

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.

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

Mincle−/− mice have been generated independently by two groups (18, 21). We first studied Mincle−/− macrophages in vitro and observed a complete abrogation of the TDM/TDB-induced expression of G-CSF and IL-6; in contrast, the response to Curdlan or CpG was unaltered (Fig. 3A; Supplemental Figure 5). Likewise, the production of NO by IFNγ-primed BMM specifically required Mincle when the cells were stimulated with TDM or TDB (Fig. 3B). The role of Mincle in generating the adjuvant effect of TDB in vivo was studied using again the H1 subunit vaccination protocol. The cellularity in the draining lymph node was significantly reduced in Mincle−/− mice when TDB was the adjuvant, but not when CpG was applied (Fig. 3C). The functional analysis of the T cell response to H1 revealed a significantly impaired Th1 response for TDB but not CpG or liposomes alone (Fig. 3D). Robust IL-17 production after immunization was only observed when TDB was included as adjuvant and strictly depended on Mincle (Fig. 3E). Thus, the deletion of any component of the Mincle-FcRγ-Card9 signaling pathway destroyed the capacity of TDB to drive Th1 and Th17 immune responses to protein antigen in vivo. These data identify Mincle as an innate pattern recognition receptor that is targeted by TDM and TDB and controls the generation of cellular immunity known to be linked to protection against mycobacterial infection (4).

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.

Mincle-FcRγ-dependent signaling in recognition of mycobacteria

The cord factor is a virulence factor of pathogenic mycobacteria that is sufficient to cause granuloma formation and tissue damage (22). Given the abundance of TDM in the mycobacterial cell wall, binding to Mincle may occur when macrophages encounter mycobacteria. Indeed, Mincle−/− BMM showed a significant reduction in the expression of G-CSF and IL-6 after stimulation with Mycobacterium bovis Bacille Calmette Guerin (BCG), that was not observed for E. coli or S. aureus (Fig. 4). Deletion of FcRγ caused a similar effect on the response to BCG, suggesting that Mincle-FcRγ-dependent recognition of TDM synergizes with Myd88-dependent activation by multiple mycobacterial TLR ligands (1) for full induction of gene expression in macrophages dealing with mycobacteria (Supplemental Fig. 6).

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.

Concluding remarks

The data presented here show that Mincle is a pivotal receptor for the mycobacterial cord factor, entirely in agreement with the findings of Ishikawa et al. published during the review process of this paper (23). It should be pointed out that additional receptors may bind TDM independently or in cooperation with Mincle. Candidates include other CLEC proteins such as Dectin-2 that also associates with FcRγ, is expressed in macrophages and binds to M. tuberculosis (24). The scavenger receptor MARCO interacts with TDM, yet lacks an intracellular domain for signal initiation (25). In contrast, Mincle can directly trigger Syk-Card9 signaling via its association with FcRγ. The impaired macrophage response to mycobacteria in the absence of Mincle raises the question about the function of Mincle and the subsequent Syk-Card9-dependent response for the control of mycobacteria in vivo: cord factor recognition by Mincle may be beneficial for the host by increasing cytokines and anti-microbial effector molecules, but a down-regulatory role, e.g. through G-CSF and IL-10 also seems possible (26).

Our data reveal the molecular mechanism underlying the Th1/Th17 adjuvanticity of the glycolipids TDM and TDB. The identification of Mincle as the TDB receptor provides the target structure for this promising snythetic glycolipid adjuvant that has entered clinical studies for TB subunit vaccination. These results also suggest new possibilities of modulating vaccination responses, e.g. by targeting Mincle with specific antibodies coupled to antigen as a dual function receptor for antigen delivery and APC activation.