Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle

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DOI: 10.1084/jem.20091750 · Source: PubMed
Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6'-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.
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Many pathogens are directly recognized by
pattern recognition receptors such as Toll-like
receptors (TLRs), RIG-I–like helicases, or
NOD-like receptors of the host cells (Akira
et al., 2006), most of which sense the charac-
teristic signatures of pathogens. Recently, some
members of the C-type lectin family have also
been identied as pattern recognition receptors
for bacteria or fungi; however, the ligands of
most C-type lectin receptors remain unidenti-
ed (Robinson et al., 2006).
Among these C-type lectin receptors is
Mincle (macrophage inducible C-type lectin,
also called Clec4e or Clecsf9), which is ex-
pressed in macrophages subjected to several
types of stress (Matsumoto et al., 1999). Mincle
possesses carbohydrate recognition domain
(CRD) within the extracellular region. We re-
cently reported that Mincle is associated with
an immunoreceptor tyrosine-based activation
motif –containing Fc receptor chain (FcR)
and functions as an activating receptor for
damaged self- and non–self-pathogenic fungi
(Yamasaki et al., 2008, 2009).
Tuberculosis is caused by Mycobacterium
tuberculosis that infects one third of the world’s
population (Hunter et al., 2006). M. tuberculosis
contains various unique components that affect
the host immune system through both iden-
tied and unidentied receptors (Jo, 2008).
Among these, the cord factor is the rst immuno-
Sho Yamasaki:
Abbreviations used: C:M,
chloroform:methanol; CRD,
carbohydrate recognition
domain; HPTLC, high-perfor-
mance thin-layer chromatog-
raphy; iNOS, inducible NO
synthase; LAM, lipoarabinoman-
nan; LWI, lung weight index;
Mincle, macrophage inducible
C-type lectin; NO, nitric oxide;
PIM, phosphatidylinositol
mannoside; TDB, trehalose
dibehenate; TDM, trehalose-
6,6’-dimycolate; TLR, Toll-like
receptor; TMM, trehalose
E. Ishikawa and T. Ishikawa contributed equally to this work.
Direct recognition of the mycobacterial
glycolipid, trehalose dimycolate, by C-type
lectin Mincle
Eri Ishikawa,
Tetsuaki Ishikawa,
Yasu S. Morita,
Kenji Toyonaga,
Hisakata Yamada,
Osamu Takeuchi,
Taroh Kinoshita,
Shizuo Akira,
Yasunobu Yoshikai,
and Sho Yamasaki
Division of Molecular Immunology,
Division of Host Defense, Medical Institute of Bioregulation, Kyushu University,
Fukuoka 812-8582, Japan
Cell Signaling, Chiba University School of Medicine, Chiba 260-8670, Japan
Department of Immunoregulation,
Department of Host Defense, Research Institute for Microbial Diseases,
of Immunoglycobiology,
Laboratory of Host Defense, WPI Immunology Frontier Research Center, Osaka University,
Suita 565-0871, Japan
Tuberculosis remains a fatal disease caused by
Mycobacterium tuberculosis
, which contains
various unique components that affect the host immune system. Trehalose-6,6’-dimycolate
(TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most stud-
ied immunostimulatory component of
M. tuberculosis
. Despite ve decades of research on
TDM, its host receptor has not been clearly identied. Here, we demonstrate that macro-
phage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed myco-
bacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of
the bacteria; analysis of the lipid extracts identied TDM as a Mincle ligand. TDM activated
macrophages to produce inammatory cytokines and nitric oxide, which are completely
suppressed in Mincle-decient macrophages. In vivo TDM administration induced a robust
elevation of inammatory cytokines in sera and characteristic lung inammation, such as
granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-
decient mice. Whole mycobacteria were able to activate macrophages even in MyD88-
decient background, but the activation was signicantly diminished in Mincle/MyD88
double-decient macrophages. These results demonstrate that Mincle is an essential recep-
tor for the mycobacterial glycolipid, TDM.
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The Journal of Experimental Medicine
2880 Mincle is a receptor for mycobacterial glycolipid | Ishikawa et al.
through an unknown ligand (Yadav and Schorey, 2006;
Rothfuchs et al., 2007). Other C-type lectins could poten-
tially recognize mycobacteria, but the activating lectin re-
ceptors for mycobacteria in macrophages have not been
clearly identied.
In this study, we show that Mincle is a direct receptor
for mycobacterial TDM. We further demonstrate, through
Mincle-decient mice, that Mincle is an essential receptor for
TDM-dependent inammatory responses.
Mincle can recognize mycobacteria
We rst investigated whether Mincle recognizes mycobac-
teria through the use of NFAT-driven GFP reporter cells
that express Mincle and its signaling subunit, the FcR
chain (Yamasaki et al., 2008). The heat-killed mycobacte-
rial species Mycobacterium smegmatis and Mycobacterium bovis
Bacille de Calmette et Guerin clearly activated NFAT-GFP
in reporter cells expressing FcR with Mincle (Fig. 1,
A and B). Importantly, the virulent strain M. tuberculosis
H37Rv also showed substantial ligand activity against
Mincle (Fig. 1 C).
The recognition of mycobacteria by Mincle was shown
to require the Glu-Pro-Asn (EPN) sequence, a putative
mannose-binding motif within CRD, as the activity was
eliminated by introducing a mutation of EPN into QPD
stimulatory component to be identied that might elicit pul-
monary inammation, a characteristic feature of mycobacterial
infection (Bloch, 1950; Yamaguchi et al., 1955). In 1956, the
chemical structure of the cord factor was established as treha-
lose-6,6’-dimycolate (TDM; Noll et al., 1956), which is the
most abundant glycolipid in the mycobacterial cell wall
(Hunter et al., 2006). The long-chain lipids of TDM repre-
sent a structural component of the hydrophobic cell wall that
is critical for the survival of mycobacteria within the phago-
some of host cells (Indrigo et al., 2002). TDM has been
shown to possess unique immunostimulatory activity, such as
granulomagenesis, adjuvant activity for cell-mediated im-
mune responses, humoral responses, and tumor regression
(Hunter et al., 2006). Recently, Marco and TLR are pro-
posed as TDM receptor (Bowdish et al., 2009), whereas other
group suggested that FcR-coupled receptor(s) are candi-
dates (Werninghaus et al., 2009). Thus, the receptor for
TDM is still controversial.
Some C-type lectins are involved in the recognition of
M. tuberculosis (Jo, 2008). Mannose receptor expressed on
macrophages is reported to mediate phagocytosis of myco-
bacteria (Kang et al., 2005). Another C-type lectin receptor,
DC-SIGN, is known to recognize mycobacteria (Tailleux
et al., 2003), but its binding results in the down-regulation of
dendritic cell–mediated immune responses (Geijtenbeek
et al., 2003). Dectin-1 is reported to recognize mycobacteria
Figure 1. Mincle recognizes mycobacterial species. (A–C) NFAT-GFP reporter cells expressing FcR only (FcR), Mincle + FcR, and Mincle
FcR were co-cultured for 18 h with heat-killed M. smegmatis (A), M. bovis (B), or M. tuberculosis H37 Rv (C). Induction of NFAT-GFP was analyzed by
ow cytometry. (D) Reporter cells expressing Dectin-2 + FcR were stimulated with M. smegmatis, M. bovis, and Candida albicans (C. a.). (E) Reporter cells
expressing Mincle + FcR were stimulated with 10
M. smegmatis together with anti-Mincle mAb and rat IgG1 as a control. (F) Cells were stimulated with
wild-type M. smegmatis (WT), pimE-decient M. smegmatis (pimE), and pimE-decient M. smegmatis reconstituted with pimE (pimE + pimE). All data
are means ± SD for triplicate assays, and representative results from three independent experiments with similar results are shown.
JEM VOL. 206, December 21, 2009
Br ief Definitive Repor t
; Fig. 1, B and C; Drickamer, 1992). However,
the similar immunoreceptor tyrosine-based activation motif–
coupled C-type lectin Dectin-2 (Sato et al., 2006) was not
capable of recognizing mycobacteria (Fig. 1 D), suggesting
selective recognition for Mincle. Indeed, activation of NFAT
by mycobacteria was completely blocked in the presence of
anti-Mincle mAb (Fig. 1 E).
As a putative mannose-binding motif within Mincle was
essential for the recognition process (Fig. 1, B and C), we
initially assumed that Mincle could recognize terminal 1,2
mannose residues of mycobacterial molecules, such as phos-
phatidylinositol mannoside (PIM), lipomannan, or lipoara-
binomannan (LAM). To examine the contribution of PIM,
we used mutant strains of mycobacteria lacking the terminal
1,2 mannose residues. M. smegmatis pimE, a deletion
mutant of mannosyl-transferase PimE, lacks mature PIMs
(Fig. S1; Morita et al., 2006). However, pimE still re-
tained stimulatory activity at a comparable level to PimE-
sucient strains such as wild-type M. smegmatis and
pimE-reconstituted pimE strain (Fig. 1 F). In addition, a
mutant M. smegmatis strain decient in 1,2 mannose of
lipomannan and LAM could be recognized normally by
Mincle, and puried LAM alone did not activate Mincle-
expressing cells (unpublished data).
These results show that mycobacteria can be recognized
by Mincle, but their 1,2 mannose-containing glycolipids
appears not to be necessary for recognition to occur.
Identication of mycobacterial glycolipids
recognized by Mincle
Mycobacteria have a wealth of unique lipids on their cell
walls, some of which presumably protect the bacteria from
the host’s defense system. To examine the contribution of
other mycobacterial lipids as a potential candidate for a Min-
cle ligand, we extracted the lipids from M. smegmatis using
various organic solvents (Fig. 2 A). M. smegmatis treated with
chloroform:methanol (C:M) selectively lost their Mincle-
stimulating activity (Fig. 2 B). Simultaneously, we analyzed
the activity of extracted lipid fractions in plate-coated form
and found that only the C:M phase after C:M extraction
showed strong stimulatory activity (Fig. 2 C). We further an-
alyzed this active fraction by means of high-performance
thin-layer chromatography (HPTLC) and separated it into 22
subfractions to identify the active lipid components. Puried
extracts from these subfractions showed strong ligand activity
peaked at subfractions #7-9 (Fig. 2 D). Orcinol staining re-
vealed a purple-red band at the position corresponding to
subfractions #7-9 (Fig. 2 D, left lane), indicating that the
ligand contains a sugar moiety. Furthermore, the purple-red
band migrated to a position similar to that of puried TDM
derived from M. tuberculosis (Fig. 2 D, right lane). From these
characteristics, we hypothesized that TDM could be a candi-
date for the Mincle ligand. Subfraction #2 also showed weak
activity, implying that trehalose monomycolate (TMM), a
precursor of TDM biosynthesis, could also act as weak li-
gand, as previously suggested (Sueoka et al., 1995).
Figure 2. Mincle recognizes mycobacterial trehalose-6,6’-dimyco-
late. (A) Schematic diagram of delipidation of M. smegmatis. Delipidated
bacteria (gray boxes) and lipid extracts (open boxes) were applied for li-
gand assays in B and C, respectively. (B and C) Heat-killed M. smegmatis
treated with C:M, hexane, acetone, or 1-butanol (BuOH; B), and plate-
coated lipid extract (C) were co-cultured with reporter cells expressing
Mincle + FcR. (D) C:M phase of C:M extract was analyzed by HPTLC and
divided into 22 subfractions. Each subfraction was coated onto a plate to
stimulate reporter cells. Puried TDM was used as a reference (right lane).
Arrowheads show the origin and solvent front. Data are means ± SD for
triplicate assays (B and C) or means for duplicate assays (D). Representa-
tive results from three independent experiments with similar results
are shown.
2882 Mincle is a receptor for mycobacterial glycolipid | Ishikawa et al.
generated TMM by partial alkaline deacylation of TDM.
As shown in Fig. 3 E, TMM could potentially activate
Mincle-expressing cells, albeit less potent than TDM. Sol-
uble trehalose had no stimulatory activity, and a large
excess of trehalose did not block TDM-mediated NFAT
activation (unpublished data). Thus, a combination of both
the sugar and lipid moieties appears to be critical for the
ligand activity of TDM.
Next, to verify the direct interaction between Mincle and
TDM, we prepared soluble Mincle protein (Mincle-Ig). As
TDM as a Mincle ligand
Indeed, we found that puried TDM, the structure of which
is shown in Fig. 3 A, dramatically activated Mincle-expressing
cells in plate-coated form (Fig. 3 B). The TDM analogue
trehalose dibehenate (TDB), which is also used as a synthetic
adjuvant, was a strong ligand for Mincle, as well (Fig. 3 C).
TDM consists of a trehalose moiety and two mycolate
chains (Fig. 3 A), but puried mycolate did not itself
activate Mincle-expressing cells (Fig. 3 D). To examine
whether TMM also possesses ligand activity or not, we
Figure 3. Puried TDM is recognized by Mincle. (A) Chemical structure of TDM. -Mycolate (shown), methoxy-mycolate, and keto-mycolate are the
major subclasses of mycolate found in M. tuberculosis TDM. (B and C) Reporter cells were stimulated with the indicated amount of plate-coated TDM
(B) or TDB (C). (D) Reporter cells were stimulated with the indicated amount of TDM, methyl -mycolate (-mycolate), or methyl keto-mycolate (keto-
mycolate). (E) Reporter cells were stimulated with the indicated amount of TDM and TMM. (F) ELISA-based detection of TDM by Mincle-Ig. hIgG1-Fc (Ig),
Mincle-Ig, and Dectin2-Ig were incubated with 0.1 nmol/0.32 cm
of plate-coated TDM. Bound protein was detected with anti–hIgG-HRP followed by the
addition of colorimetric substrate. (G) Effect of trehalose (100 µg/ml), EDTA (10 mM), rat IgG (10 µg/ml), and anti-Mincle mAb (10 µg/ml) on TDM recogni-
tion by Mincle-Ig. ELISA-based detection was performed as in E. (H) Reporter cells were stimulated with TDM, which was treated with trehalase as de-
scribed in Materials and methods. Cells were also stimulated with plate-coated anti-Mincle mAb treated with trehalase as a negative control. All data are
means ± SD for triplicate assays and representative results from three independent experiments with similar results are shown.
JEM VOL. 206, December 21, 2009
Br ief Definitive Repor t
ably to escape from Mincle-mediated host immunity (Fig. S2;
Matsunaga et al., 2008). Intriguingly, it has also been reported
that as a possible counterdefense, host cells present this GMM
on group1 CD1 molecules in human to provoke TCR-
mediated acquired immune responses against mycobacteria
(Moody et al., 1997; Matsunaga et al., 2008). Importantly,
we found that TDM is recognized by human Mincle as well
as murine Mincle (unpublished data).
Collectively, these results suggest that, among various
mycobacterial components, Mincle is specic for the ester
linkage of a fatty acid to trehalose.
Mincle is essential for TDM-dependent
macrophage activation
TDM also activated macrophages to produce nitric oxide
(NO), which is critical for direct killing of mycobacteria.
However, NO production was almost completely suppressed
in Mincle
macrophages (Fig. 4 A). In contrast, LPS in-
duced a similar response in Mincle
cells to that in WT
cells. Inducible NO synthase (iNOS) is the enzyme responsi-
ble for production of NO. The transcriptional induction of
detected by anti-hIgG, Mincle-Ig, but not other control pro-
teins such as Ig or Dectin2-Ig, selectively binds to plate-coated
TDM (Fig. 3 F). This biochemical data provides further proof
that TDM is a direct Mincle ligand. This binding was blocked
by EDTA and anti-Mincle mAb but not by excess trehalose,
suggesting that Mincle recognizes specic glycolipid structures
in a cation-dependent manner (Fig. 3 G).
It was recently proposed that TDM on mycobacterial cell
wall could be converted into glucose monomycolate (GMM)
in the host cell environment (Matsunaga et al., 2008). To test
the hypothesis that this conversion may be of advantage to
mycobacteria in escaping from Mincle-mediated recognition,
we attempted to mimic the conversion in vitro by using tre-
halase, which hydrolyzed trehalose into two glucose units
(Asano et al., 1996). Intriguingly, the trehalase treatment im-
paired the ligand activity of TDM (Fig. 3 H). Note that this
enzymatic treatment did not grossly disrupt Mincle-mediated
responses because stimulation by plate-coated anti-Mincle
mAb was not aected by the trehalase treatment (Fig. 3 H).
This nding is consistent with the idea that mycobacteria
convert TDM into GMM upon infection into host, presum-
Figure 4. Lack of TDM-mediated activation in Mincle-decient macrophages. (A) BMM from Mincle
and Mincle
were primed with IFN-
(10 ng/ml) and stimulated with plate-coated TDM or LPS (10 ng/ml) as a control. Culture supernatants were collected at 48 h and concentration of NO
was measured. (B) IFN-–primed BMM were stimulated with plate-coated TDM for 36 h as in A, and mRNA expression of iNOS was analyzed by real-
time PCR. (C and D) IFN-–primed BMM were stimulated with plate-coated TDM (C) or TDB (D) for 24 h. Culture supernatants were collected and con-
centrations of TNF and MIP-2 were determined by ELISA. All data are means ± SD for triplicate assays and representative results from three independent
experiments with similar results are shown.
2884 Mincle is a receptor for mycobacterial glycolipid | Ishikawa et al.
ammatory cells. However, these cytokines were not pro-
duced upon TDM stimulation in the absence of Mincle
(Fig. 4 C). Similarly, TDB-induced cytokine production was
also eliminated in Mincle
macrophages (Fig. 4 D).
We have previously reported that Mincle transduces sig-
nal through the FcR–CARD9 signaling axis (Yamasaki
et al., 2008). In line with these observations, it was recently
reported that TDM and TDB activates macrophage in an
iNOS by TDM stimulation was also completely suppressed
in Mincle
mice (Fig. 4 B).
The crucial role of TNF has been reiterated by the recent
observation of patients suering from the reactivation of la-
tent tuberculosis infection upon anti-TNF therapy for auto-
immune diseases (Winthrop, 2006). TDM stimulation
induced the production of TNF and MIP-2 (also called
CXCL2), which are potent chemoattractant factors for in-
Figure 5. Mincle is essential for TDM-induced inammation in vivo
(A) Mincle
, Mincle
, FcR
and Myd88
mice were injected intravenously
with an oil-in-water emulsion containing TDM (150 µg). Emulsion without TDM was injected as a vehicle control. At day 1 after injection, IL-6 and TNF concentra-
tions in sera were determined by ELISA. Each symbol represents an individual mouse. (B) Lungs of TDM-injected mice were removed at day 7 and inammatory
intensity was evaluated by calculation of LWI. (C) Proinammatory mediator mRNA levels in lungs at day 7 after TDM administration were evaluated by real-time
PCR. Relative expression levels are shown as 2
. Each symbol represents an individual mouse. Ct, cycle threshold. *, P < 0.05; **, P < 0.001. NS, not signicant.
(D) Histology of the lungs from untreated (control) and TDM-injected (TDM) mice was examined by hematoxylin-eosin staining at day 7. Bar, 0.1 mm. (E) Number
of lung granulomas. Granuloma in lungs from mice injected with TDM was counted as described in Materials and methods. Data are means ± SD for the mean
number for at least three independent mice. Representative results from two independent experiments with similar results are shown.
JEM VOL. 206, December 21, 2009
Br ief Definitive Repor t
FcR- and CARD9-dependent manner (Werninghaus et al.,
2009). Thus, Mincle is an essential receptor for macrophage
activation elicited by TDM and TDB.
TDM-induced lung granuloma formation requires Mincle
In vivo administration of TDM is capable of inducing in-
ammatory symptoms characteristic of tuberculosis (Yama-
guchi et al., 1955; Hunter et al., 2006). Single injection of
TDM into mice induced the robust production of inamma-
tory cytokines, such as IL-6 and TNF, in sera. However,
these were completely eliminated in Mincle
mice (Fig. 5 A).
FcR is an essential signaling subunit for Mincle (Yamasaki
et al., 2008). Indeed, FcR
mice did not detectably
respond to TDM in vivo (Fig. 5 A). In contrast, TDM was
able to induce cytokine production in MyD88
mice, sug-
gesting that TLRs are not essential for TDM recognition
(Fig. 5 A; Werninghaus et al., 2009), although potential roles
of TLR2 and TLR4 have been proposed (Bowdish et al.,
2009). TDM also induced inammatory lung swelling as as-
sessed by lung weight index (LWI), but this was totally de-
pendent on the Mincle–FcR axis (Fig. 5 B and Fig. S3). In
concert with the acute lung inammation, the up-regulation
of mRNA for inammatory cytokines/chemokines upon
TDM treatment was severely impaired in Mincle
(Fig. 5 C). In addition, TDM induced thymic atrophy in
mice, and was also Mincle dependent (Fig. S4).
Granulomas are complex aggregates of immune cells, and
are widely believed to constrain mycobacterial infection by
physically surrounding the infecting bacteria (Adams, 1976).
TDM alone dramatically induced granuloma formation,
which is a characteristic of mycobacterial infection at day 7
after administration (Fig. 5 D). Strikingly, no granuloma for-
mation was observed in the lungs of TDM-treated Mincle
mice (Fig. 5 D). Quantitative analysis on multiple sections
revealed that granuloma was completely eliminated in
and FcR
mice, but was induced normally in
mice (Fig. 5 E). Thus, Mincle is a critical recep-
tor for TDM-induced granuloma formation, most likely
through the production of inammatory cytokines/chemo-
kines to recruit inammatory cells (Welsh et al., 2008).
These results show that Mincle is an essential receptor for
TDM-mediated inammatory responses in vivo.
Role of Mincle in response to whole mycobacteria
Finally we investigated the role of Mincle in response to
whole mycobacteria using virulent strain, M. tuberculosis
H37Rv. Heat-killed mycobacteria induced vigorous produc-
tion of TNF and MIP-2 in macrophages, whereas Mincle
Figure 6. Mincle is responsible for the TLR-independent pathway of anti-mycobacterium responses. (A and B) BMM from WT, Mincle
, and MyD88
(B) mice were stimulated with indicated number of heat-killed M. tuberculosis H37Rv. Cells were also stimulated
with zymosan as a positive control. At day 2 after stimulation, production of TNF and MIP-2 in supernatants was determined. Data are means ± SD for
triplicate assays and representative results from three independent experiments with similar results are shown. *, P < 0.05; **, P < 0.01.
2886 Mincle is a receptor for mycobacterial glycolipid | Ishikawa et al.
bacteria is still controversial (Hunter et al., 2006). Given its
potent granuloma-inducing capacity, Mincle might also be
involved in diseases characterized by granulomas.
Recent studies have suggested that T cells can respond
to TDM, despite little evidence of CD1-mediated TDM
presentation (Guidry et al., 2004; Otsuka et al., 2008). Min-
cle is also expressed in T cells upon activation (unpublished
data), and some T cell populations use FcR (Ohno et al.,
1994). It is tempting to speculate that such T cells may di-
rectly recognize TDM to produce T cell–specic cytokines,
such as IFN- or IL-17, in a “TCR-independent but
“Mincle-dependent” manner.
We recently discovered that Mincle also recognizes the
pathogenic fungus Malassezia (Yamasaki et al., 2009). Our
current ndings could suggest that Mincle uniquely recog-
nizes glycolipid through the motifs that have generally been
considered as mannose-binding motifs. We therefore specu-
late that the Mincle–Malassezia interaction might be mediated
by unique fungal glycolipids similar to TDM. Interestingly,
Malassezia species among other fungi uniquely require lipid
for their growth (Schmidt, 1997).
TDM and its synthetic analogue, TDB, have been exten-
sively studied because they are eective adjuvants (Azuma
and Seya, 2001). It is believed that TDM accounts for part of
the eect of CFA (Billiau and Matthys, 2001). Identication
of the host receptor for TDM/TDB will provide valuable in-
formation related to the design of vaccine adjuvants, because
rational screening of synthetic Mincle ligands is now feasible
and could potentially lead to the development of an ideal
synthetic adjuvant. Such a synthetic Mincle ligand could al-
low ecient vaccination against tuberculosis, other infectious
diseases, and cancers.
Mice. Mincle-decient mice were used as C57BL/6 and 129 mixed genetic
background (Yamasaki et al., 2009). MyD88-decient mice were purchased
from Oriental Yeast. FcR-decient mice on a C57BL/6 background were
provided by T. Saito (RIKEN, Yokohama, Japan; Park et al., 1998).
C57BL/6 mice were obtained from Japan Clea or Kyudo. All mice were
maintained in a ltered-air laminar-ow enclosure and given standard labo-
ratory food and water ad libitum. Animal protocols were approved by the
committee of Ethics on Animal Experiment, Faculty of Medical Sciences,
Kyushu University.
Bacteria. M. smegmatis strain mc
155 were cultured in Middlebrook 7H9
broth as previously described (Morita et al., 2005). M. bovis Bacille de
Calmette et Guerin were cultured in Middlebrook 7H9 supplemented with
Middlebrook ADC enrichment and 0.05% Tween-80. M. smegmatis pimE,
which is a mutant decient in mannosyltransferase PimE, lacks 1,2-mannose
moiety of PIM. M. smegmatis and M. bovis were heat-killed by pasteurization
at 63°C for 40 min. Virulent strain M. tuberculosis H37Rv was autoclaved
before use. Candida albicans (IFM No. 54349) was provided by T. Gonoi
(Chiba University, Chiba, Japan).
Delipidation. M. smegmatis was delipidated with C:M (2:1), hexane, ace-
tone, or 1-butanol (BuOH). Insoluble fractions were collected as delipidated
bacteria. Soluble fractions were further partitioned by C:M:W (8:4:3; vol/
vol) into lower organic phase (C:M) and upper aqueous phase (M:W). Up-
per aqueous phase (M:W) was further partitioned by 1-butanol:water (1:1;
vol/vol) into upper butanol phase (BuOH) and lower aqueous phase (water).
deciency did not detectably impair the production of TNF
and only partially impaired MIP-2 production (Fig. 6 A).
This is probably because whole mycobacteria possess many
immunostimulatory components other than TDM, including
TLR ligands (Jo, 2008). Indeed, cytokine production was
markedly decreased, but was substantially induced in
macrophages (Fig. 6 B; Fremond et al., 2004).
However, the level of these cytokines was signicantly re-
duced in MyD88
double-decient macro-
phages when compared with MyD88
macrophages (Fig.
6 B). In contrast, the response to zymosan, which is known
to be recognized by Dectin-1 and TLR2, in MyD88
macrophages was the same as that in MyD88
cells (Fig. 6). These results suggest that Mincle plays a major
role in TLR-independent recognition of mycobacteria. The
remaining production of cytokines by mycobacteria observed
in MyD88
macrophages (Fig. 6 B) may reect
a possible contribution of the NOD-like receptor family
NOD2, which was reported to recognize mycobacteria
through muramyl dipeptide (Coulombe et al., 2009).
Concluding remarks
TDM (also called cord factor) is an essential component that
permits the survival of mycobacteria within the host cells
(Indrigo et al., 2002), whereas it is barely present in verte-
brates. It would therefore be a reasonable strategy for the host
to recognize TDM as a signal of mycobacterial infection. In
this study, we identied C-type lectin Mincle as an essential
receptor for TDM.
It was demonstrated that cyclopropane within mycolate
chain of TDM is critical for its immunostimulatory activity
(Rao et al., 2005). However, TDB, which lacks the cyclo-
propane in the carbon chain, is still capable of activating mac-
rophages and Mincle-expressing cells (Fig. 3 C and Fig. 4 D;
Werninghaus et al., 2009). On the other hand, it has been
suggested that specic conguration of TDM is necessary to
exert its activity (Retzinger et al., 1981). Therefore the
“kink” in the carbon chain of TDM may contribute to the
optimal presentation of polar head to Mincle, rather than as a
direct binding site for Mincle. It could be hypothesized that
two Mincle receptors recognize one disaccharide head of
TDM, presumably together with a proximal region of myco-
late, although further structural analysis is needed to clarify
this issue.
TDM has been known to induce lung granuloma in vivo,
and we demonstrated that it is totally dependent on Mincle.
We have recently found that Mincle also senses dead cells and
recruits inammatory cells (Yamasaki et al., 2008). Because
the presence of necrotic cells in the center of a granuloma is a
characteristic feature of tuberculosis (Adams, 1976), both
TDM and dead cells may contribute cooperatively to the for-
mation of granuloma through Mincle-mediated secretion of
inammatory cytokines/chemokines. The physiological role
of Mincle during virulent infection is a critical issue that needs
to be claried and is now under investigation using Mincle
mice, as the contribution of TDM in the virulence of myco-
JEM VOL. 206, December 21, 2009
Br ief Definitive Repor t
assistance; and Y. Nishi and H. Yamaguchi for secretarial assistance. We also thank
N. Kinoshita (Laboratory for Technical Support, Medical Institute of Bioregulation,
Kyushu University) for histochemical staining.
This work was supported by Grant-in-Aid for Young Scientists (S) and Takeda
Science Foundation.
The authors have no conicting nancial interests.
Submitted: 12 August 2009
Accepted: 3 November 2009
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Each fraction was dried, resuspended in DMSO relative to the original cell
pellet weight, and tested as lipid extracts (Morita et al., 2005).
Reagent. TDM, Methyl -mycolate, Methyl keto-mycolate, and LAM
were purchased from Nakalai tesque and TDB was obtained from Avanti
Polar Lipids, Inc. D-Trehalose dihydrate (T3663), Trehalase (T8778), LPS
(L4516), and zymosan (Z4250) were purchased from Sigma-Aldrich. For
stimulation of reporter cells and bone marrow–derived macrophages
(BMM), TDM and TDB dissolved in chloroform at 1 mg/ml were diluted
in isopropanol and added on 96-well plates at 20 µl/well, followed by evap-
oration of the solvent as previously described (Ozeki et al., 2006). For gen-
eration of TMM, TDM was partially acylated with 0.4 M NaOH for 10
min, and the amount of TDM and TMM was determined by orcinol stain-
ing on HPTLC. Corresponding fraction to TDM and TMM was collected
and used for assay.
Cells. 2B4-NFAT-GFP reporter cells expressing WT Mincle or Mincle
(E169Q/N171D) mutant and BMM were prepared as previously described
(Yamasaki et al., 2008). BMM pretreated with 10 ng/ml IFN- for 4 h and
reporter cells were stimulated with various bacteria and bacterial cell wall
components. Activation of NFAT-GFP was monitored by ow cytometry.
The levels of cytokines were determined by ELISA. NO production was
measured by Griess assay.
Antibodies. Anti-Mincle mAbs were established as described previously
(Yamasaki et al., 2008) and clone 1B6 (IgG1, k) was used in this study.
HRP-conjugated anti–human IgG (109–035-088) was from Jackson Immuno-
Research Laboratories.
Ig fusion protein. The extracellular domain of Mincle (a.a. 46–214) and
Dectin2 (a.a. 43–209) was fused to hIgG1 Fc region and prepared as de-
scribed previously (Yamasaki et al., 2009).
Trehalase treatment. 0.8 µg TDM was coated on a 96-well plate as de-
scribed in the Reagents section, followed by incubation with 1 to 10 mU/ml
of Trehalase in 10 mM Tris-HCl buer (pH 5.9) at 37°C for 6 h.
Quantitative RT-PCR. Total RNA was isolated from stimulated BMM
or lungs of TDM-administrated mice subjected to real-time PCR (Applied
Biosystems). Sequence of gene-specic primers is available upon request.
Administration of TDM. TDM was prepared as oil-in-water emulsion
consisting of mineral oil (9%), Tween-80 (1%), and saline (90%) as previ-
ously described (Numata et al., 1985). 100 µl of emulsion containing 150 µg
of TDM was injected intravenously into 6–11-wk-old mice. Emulsion with-
out TDM was injected as a vehicle control. At day 7, thymocyte number
was calculated and lungs were weighed and xed in 10% formaldehyde for
hematoxylin-eosin staining. A part of lungs was frozen for quantitative RT-
PCR. LWI was calculated as described (Guidry et al., 2004). The number of
granulomas was determined by counting focal mononuclear cell inltrations
in randomized 10 microscopic elds (0.8 mm
) per mouse.
Statistics. An unpaired two-tailed Student’s t test was used for all the statis-
tical analyses.
Online supplemental material. Fig. S1 shows schematic structure of
PIM in M. smegmatis mutant. Fig. S2 shows schematic representation of
hypothetical evolutional struggle between mycobacteria and host immu-
nity. Fig. S3 shows lack of TDM-induced pulmonary inammation in
mice. Fig. S4 shows impaired TDM-induced thymic atrophy in
mice. Online supplemental material is available at http://www
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    • "Mincle appears to be selectively associated with the Fc gamma receptor (FcγR) and activates macrophages to produce inflammatory cytokines and chemokines [6]. Mincle is a key receptor for the mycobacterial cord factor trehalose dimycolate (TDM) thus being an important modulator of the antimicrobial immunity [7,8]. Mincle has a crucial role in antifungal immunity as it recognizes some pathogenic fungi such as Candida albicans, Malassezia species and Fonsecaea pedrosoi, the causative agent of chromoblastomycosis91011. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Mincle, macrophage-inducible C-type lectin, is a member of C-type lectin receptors. It plays an important role in anti-mycobacterial and anti-fungal immunity. Furthermore it senses dead cells through its primary ligand SAP130. Materials and findings: We examined ten urothelial tumors of the urinary bladder of cattle. Eight of them expressed E5 cDNA of bovine papillomaviruses type 2 (BPV-2) and type 13 (BPV-13) that belong to Deltapapillomavirus genus. Two of them were not examined for detection of E5 cDNA. Mincle expression appeared to occur in urothelial neoplastic cells only. No mincle expression was detected in urothelial cells from healthy cattle. Mincle expression was characterized by a membranous pattern in papillary urothelial cancers; isolated and/or clustered urothelial cells showing a strong cytoplasmic immunoreactivity were primarily seen in invasive urothelial cancers. Conclusion: This is the first study about the expression of mincle in veterinary oncology and the first report which describes the expression of functional mincle receptor in neoplastic cells in medical literature. As it has been shown that urothelial cancer cells have the ability to function as antigen-presenting cells (APCs), it is conceivable that mincle expression is involved in the presentation of cancer cell antigens to cells of the immune system. Furthermore, since expression of mincle contributes to the control of Mycobacterium bovis BCG infection, this study has exciting clinical implications in comparative medicine keeping in mind that Bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment of non-muscle invasive bladder cancer in man. Mincle expression in urothelial tumor cells warrants further study to better understand the role, if any, of this receptor in bladder cancer. Future studies will provide insights in the role of mincle receptor of urothelial cancer cells in antitumor immunotherapy.
    Full-text · Article · Oct 2015
    • "Cao and colleagues demonstrate an important role for the C terminus in CARD9 signaling and show that ubiquitination of CARD9 by TRIM62 regulates CARD9-mediated anti-fungal immunity. INTRODUCTION CARD9 is a central adaptor protein in innate immune signaling via C-type lectin receptors (CLRs), such as Dectin-1, Dectin-2, and Mincle (Goodridge et al., 2009; Gross et al., 2006; Ishikawa et al., 2009; Roth and Ruland, 2013; Saijo et al., 2010; Schoenen et al., 2010; Werninghaus et al., 2009), and has more recently been reported to regulate cytokine production induced by cytosolic nucleic acid sensors RIG-I and Rad50 in mouse models (Abdullah et al., 2012; Poeck et al., 2010; Roth et al., 2014). CLRs sense components of fungal and bacterial cell walls, linking signaling from these immune receptors to nuclear factor-kB (NF-kB) activation through a series of sequential phosphorylation events. "
    [Show abstract] [Hide abstract] ABSTRACT: CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.
    Full-text · Article · Oct 2015
    • "After many years as an orphan receptor, the macrophage inducible C-type lectin, known as mincle or CLEC-4E, has recently been recognized as a receptor for glycans on pathogens licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( It is of particular interest because it can bind to trehalose dimycolate, an unusual glycolipid in the outer wall of mycobacteria. "
    [Show abstract] [Hide abstract] ABSTRACT: Mincle, the macrophage-inducible C-type lectin also known as CLEC-4E, binds to the mycobacterial glycolipid trehalose dimycolate and initiates a signaling cascade by serving as a receptor for Mycobacterium tuberculosis and other pathogenic mycobacterial species. Studies of the biological functions of human mincle often rely on mouse models, based on the assumption that the biological properties of the mouse receptor mimic those of the human protein. Experimental support for this assumption has been obtained by expression of the carbohydrate-recognition domain of mouse mincle and characterization of its interaction with small molecule analogs of trehalose dimycolate. The results confirm that the ligand-binding properties of mouse mincle closely parallel those of the human receptor. These findings are consistent with the conservation of key amino acid residues that have been shown to form the ligand-binding site in human and cow mincle. Sequence alignment reveals that these residues are conserved in a wide range of mammalian species, suggesting that mincle has a conserved function in binding ligands that may include endogenous mammalian glycans or pathogen glycans in addition to trehalose dimycolate.
    Full-text · Article · Apr 2015
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