Lipoteichoic acid anchor triggers Mincle to drive protective immunity against invasive group A Streptococcus infection

Article abstract of DOI:10.1073/pnas.1809100115

Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen that causes a range of diseases, including fatal invasive infections. However, the mechanisms by which the innate immune system recognizes GAS are not well understood. We herein report that the C-type lectin receptor macrophage inducible C-type lectin (Mincle) recognizes GAS and initiates antibacterial immunity. Gene expression analysis of myeloid cells upon GAS stimulation revealed the contribution of the caspase recruitment domain-containing protein 9 (CARD9) pathway to the antibacterial responses. Among receptors signaling through CARD9, Mincle induced the production of inflammatory cytokines, inducible nitric oxide synthase, and reactive oxygen species upon recognition of the anchor of lipoteichoic acid, monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS infection, Mincle-deficient mice exhibited impaired production of proinflammatory cytokines, severe bacteremia, and rapid lethality. GAS also possesses another Mincle ligand, diglucosyldiacylglycerol; however, this glycolipid interfered with MGDG-induced activation. These results indicate that Mincle plays a central role in protective immunity against acute GAS infection.

Full text of DOI:10.1073/pnas.1809100115

Lipoteichoic acid anchor triggers Mincle to drive
protective immunity against invasive group A
Streptococcus infection
Takashi Imaia,b,c, Takayuki Matsumurad,1, Sabine Mayer-Lambertze,1, Christine A. Wellsf, Eri Ishikawab,g
,
Suzanne K. Butcherf, Timothy C. Barnetth,i, Mark J. Walkerh,i, Akihiro Imamuraj, Hideharu Ishidak,l, Tadayoshi Ikebem,
Tomofumi Miyamoton, Manabu Atod, Shouichi Ohgac, Bernd Lepeniese, Nina M. van Sorgeo, and Sho Yamasakia,b,g,p,2
aDivision of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, 812-8582 Fukuoka, Japan; bDepartment of Molecular
Immunology, Research Institute for Microbial Diseases, Osaka University, 565-0871 Osaka, Japan; cDepartment of Pediatrics, Graduate School of Medical
Sciences, Kyushu University, 812-8582 Fukuoka, Japan; dDepartment of Immunology, National Institute of Infectious Diseases, 162-8640 Tokyo, Japan;
eImmunology Unit, Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, 30559 Hannover, Germany; fThe Centre for
Stem Cell Systems, Anatomy and Neuroscience, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Melbourne, VIC 3010, Australia;
gDepartment of Molecular Immunology, Immunology Frontier Research Center, Osaka University, 565-0871 Osaka, Japan; hSchool of Chemistry and
i
Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia; Australian Infectious Diseases Research Centre, The University of
j
Queensland, Brisbane, QLD 4072, Australia; Department of Applied Bio-organic Chemistry, Gifu University, 501-1193 Gifu, Japan; kFaculty of Applied
Biological Sciences, Gifu University, 501-1193 Gifu, Japan; lCenter for Highly Advanced Integration of Nano and Life Sciences, Gifu University, 501-1193 Gifu,
Japan; mDepartment of Bacteriology I, National Institute of Infectious Diseases, 162-8640 Tokyo, Japan; nDepartment of Natural Products Chemistry,
Graduate School of Pharmaceutical Sciences, Kyushu University, 812-8582 Fukuoka, Japan; oMedical Microbiology, University Medical Center Utrecht,
Utrecht University, 3584 CX Utrecht, The Netherlands; and pDivision of Molecular Immunology, Medical Mycology Research Center, Chiba University,
260-8673 Chiba, Japan
Edited by Lewis L. Lanier, University of California, San Francisco, CA, and approved September 26, 2018 (received for review June 1, 2018)
GAS challenge (12). The upstream pattern recognition receptor
(PRR) that triggers MyD88-dependent responses has not been
clearly elucidated despite the fact that individual TLRs recognize
and respond to isolated GAS components in vitro. For example,
TLR2 recognizes GAS lipoteichoic acid (LTA), an essential
glycerol phosphate polymer with a glycosyldiacylglycerol mem-
brane anchor, to induce myeloid cell activation (13). In addition,
mouse TLR13 interacts with bacterial rRNA (14) and is involved
in GAS recognition in vitro (15). However, the in vivo role of
TLRs is not fully understood, as mice lacking individual TLRs
did not show consistent results regarding GAS susceptibility (7,
16, 17). In addition, analysis of GAS-susceptible patients sug-
gests the involvement of MyD88-independent receptor(s) (18).
Group A Streptococcus (GAS) is a Gram-positive bacterial pathogen
that causes a range of diseases, including fatal invasive infections.
However, the mechanisms by which the innate immune system rec-
ognizes GAS are not well understood. We herein report that the
C-type lectin receptor macrophage inducible C-type lectin (Mincle)
recognizes GAS and initiates antibacterial immunity. Gene expres-
sion analysis of myeloid cells upon GAS stimulation revealed the
contribution of the caspase recruitment domain-containing protein
9 (CARD9) pathway to the antibacterial responses. Among receptors
signaling through CARD9, Mincle induced the production of inflam-
matory cytokines, inducible nitric oxide synthase, and reactive oxy-
gen species upon recognition of the anchor of lipoteichoic acid,
monoglucosyldiacylglycerol (MGDG), produced by GAS. Upon GAS
infection, Mincle-deficient mice exhibited impaired production of
proinflammatory cytokines, severe bacteremia, and rapid lethality.
GAS also possesses another Mincle ligand, diglucosyldiacylglycerol;
however, this glycolipid interfered with MGDG-induced activation.
These results indicate that Mincle plays a central role in protective
immunity against acute GAS infection.
Significance
Group A Streptococcus (GAS) causes invasive streptococcal in-
fections in humans, resulting in high mortality. Thus, GAS is
also known as “killer bacteria” or “flesh-eating bacteria.” The
mechanism by which the immune system recognizes this po-
tent pathogen remains elusive. In this study, we showed that
the innate immune receptor Mincle (macrophage inducible
C-type lectin) plays pivotal roles against invasive GAS infec-
tion through the recognition of monoglucosyldiacylglycerol
(MGDG), a component of the lipoteichoic acid anchor. MGDG
induced proinflammatory cytokines, ROS, and NO production
in a Mincle-dependent manner. In an invasive GAS infection
model, Mincle-deficient mice exhibited severe bacteremia and
rapid lethality. These results indicate that Mincle plays a central
role in protective immunity against acute GAS infection.
innate immunity bacterial infection C-type lectin receptors glycolipids
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adjuvant
roup A Streptococcus (GAS, Streptococcus pyogenes) is a
G
Gram-positive bacterial pathogen that causes various self-
limiting diseases, including pharyngitis, impetigo, and scarlet
fever. Importantly, GAS also causes severe invasive infections such
as streptococcal toxic shock syndrome, necrotizing fasciitis, and
bacteremia (1). Recurrent GAS infection increases the risk for
GAS-associated autoimmune diseases, such as rheumatic fever and
rheumatic heart disease (2). Invasive infections and autoimmune
complications are associated with high mortality despite the sen-
sitivity of GAS to antibiotics, including penicillins (3). To control
such devastating GAS-mediated disease outcomes, it is crucial to
improve our understanding of the mechanism(s) by which the im-
mune system senses GAS and triggers protective responses.
A number of studies have demonstrated that innate immune
responses play a predominant role in host protection against
GAS (4–6). Neutrophils and myeloid cells are important classes
of innate immune cells that fulfill nonredundant roles in pre-
venting GAS dissemination (7, 8). The common TLR adaptor
MyD88 substantially contributes to GAS-induced cytokine pro-
duction (9–11), and MyD88-deficient mice are susceptible to
Author contributions: T. Imai, T. Matsumura, M.A., B.L., N.M.v.S., and S.Y. designed re-
search; T. Imai, T. Matsumura, S.M.-L., E.I., S.K.B., T.C.B., T. Miyamoto, and N.M.v.S. per-
formed research; C.A.W., M.J.W., A.I., H.I., T. Ikebe, and S.O. contributed new reagents/
analytic tools; T. Imai, T. Matsumura, C.A.W., M.J.W., T. Miyamoto, M.A., B.L., and N.M.v.S.
analyzed data; and T. Imai, T. Miyamoto, N.M.v.S., and S.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1T. Matsumura and S.M.-L. contributed equally to this work.
2To whom correspondence should be addressed. Email: yamasaki@biken.osaka-u.ac.jp.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1809100115/-/DCSupplemental.
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Indeed, several PRRs other than TLRs are involved in immune
responses to GAS, including the Nod-like receptor NLRP3 (11,
19). However, NLRP3-deficient mice are still capable of elimi-
nating GAS (11).
mock
GAS
C. albicans
S. typhimurium
LPS
A
2000
1 x 109
5 x 108
0
Recently, an additional family of PRRs, termed C-type lectin
receptors (CLRs), was defined as receptors that recognize gly-
cosylated pathogen-associated molecular patterns (PAMPs) (20,
21). Myeloid cells express many CLRs that activate inflammatory
responses through an immunoreceptor tyrosine-based activation
motif (ITAM) located in their own cytoplasmic tails, or by
coupling with ITAM-bearing signaling subunits (22). In either
case, the ITAM undergoes phosphorylation upon ligand en-
gagement and recruits tyrosine kinase Syk to activate the pivotal
adaptor complex CARD9-Bcl10-Malt1. This signaling cascade
triggers multiple innate responses, including the production of
inflammatory mediators and up-regulation of costimulatory
molecules (23, 24). Among the ITAM-coupled CLRs, we pre-
viously reported that macrophage inducible C-type lectin (Min-
cle), MCL, Dectin-2, and DCAR are FcRγ-coupled receptors
that preferentially recognize glycolipids from pathogens (25–29).
In particular, Mincle recognizes diverse glycolipids such as my-
cobacterial trehalose dimycolate (TDM) (25), pneumococcal
glucosyl diacylglycerol (30), and other glucose- or mannose-
containing glycolipids (31). Based on the importance of mye-
loid cells in GAS immune defense, and the fact that no previous
studies have probed the role of CLRs in GAS recognition in host
defense, we hypothesized that a specific CLR may recognize
GAS through glycolipid ligand(s) to initiate immune responses.
In the present study, we show that the C-type lectin Mincle
recognizes GAS through the LTA anchor glycolipid mono-
glucosyldiacylglycerol (MGDG) of GAS to initiate cytokine and
reactive oxygen species (ROS) production. We further demon-
strate, using Mincle-deficient mice, that this innate immune
pathway plays a pivotal role in protective immunity against in-
vasive GAS infection. Finally, GAS also produces a glycolipid
that blocks MGDG-induced Mincle activation.
0
-2000
1 2 3 4
10
0
2000
6000
-6000
-2000
Contributing component
PC1
WT
CARD9
WT
CARD9
10
B
C
8
6
4
2
0
10
8
30
20
10
0
8
6
6
4
2
0
4
2
0
0
1
10
LPS
LPS
0
1
10
Live GAS (MOI)
Live GAS (MOI)
D
WT
CARD9
8
6
4
2
0
16
12
8
4
0
0
0.03 0.3
C:M ( g/well)
3
0
10 100 1000
M:W ( g/ml)
LPS
Fig. 1. GAS infection of human monocytes activates CARD9 signaling. (A)
Principal component (PC) analysis on data from CAGE experiments (Left). Pri-
mary monocytes from three donors were stimulated with the following for
2 h: GAS, C. albicans, S. typhimurium, LPS, mock-treated. Data were repre-
sented as 2D plot (PC1 vs. PC2), as scree plot indicates major contribution of the
first two components to variance in the CAGE data (Right). Symbols shown in
the same color (green or blue) indicate the same cluster. (B and C) BMDCs from
WT or CARD9−/− mice were infected with GAS (MOI = 1 and 10) or left un-
infected. LPS was used as a positive control. At 16 h after infection, the
supernatants were collected, and the concentrations of (B) TNF and (C) MIP-
2 were determined by ELISA. (D) BMDCs from WT or CARD9−/− mice were
stimulated with indicated concentrations of plate-coated lipophilic fraction (C:M)
or hydrophilic fraction (M:W) of GAS for 48 h. Supernatants were collected, and
the concentration of TNF was determined by ELISA. C:M, chloroform:methanol;
M:W, methanol:water. In B−D, data are presented as mean SD and are rep-
resentative of three independent experiments with similar results.
Results
Monocytes Display a CARD9 Signature upon Exposure to GAS. To
explore the signaling pathway(s) involved in immune responses
against GAS, we carried out capped analysis of gene expression
(CAGE) (32, 33). Primary human monocytes were stimulated
with human bacterial or fungal pathogens including GAS, Can-
dida albicans, Salmonella typhimurium, and lipopolysaccharide
(LPS). We identified a diverse set of proximal promoters that
were induced in response to these stimuli (SI Appendix, Fig. S1A
and Dataset S1). Application of principal component analysis to
the dataset highlighted that the transcriptional profile induced by
GAS largely overlapped with the profile of C. albicans (Fig. 1A),
for which the involvement of CLR–CARD9 signaling is well
established (34–36).
Mincle Is the CLR That Recognizes the Lipophilic Component from
GAS. We next sought to identify the innate immune receptor(s)
upstream of CARD9 that recognizes a component in the lipo-
philic fraction from GAS. We utilized cells expressing NFAT-
GFP reporters and a range of innate immune receptors (37).
Among the assessed PRRs, cells bearing the CLR Mincle
showed high GFP reporter expression in response to the hy-
drophobic extract from GAS (Fig. 2A). This ability was highly
conserved across species, as Mincle orthologs derived from
mouse, rat, guinea pig, and human all retained activity in this
assay (Fig. 2A). In addition, the reporter cells expressing mouse
Mincle reacted to this fraction in a dose-dependent manner (Fig.
2B), allowing the use of Mincle-deficient BMDCs to further
examine the contribution of endogenously expressed Mincle to
immune signaling. Induction of TNF production by the C:M
fraction was decreased in Mincle−/− cells to an extent similar to
that observed in CARD9−/− cells (Fig. 2C). Consistent with this
observation, a Mincle-Ig fusion protein directly bound to GAS
(Fig. 2D). Furthermore, upon in vitro infection of BMDCs with
GAS, TNF production was significantly lower in Mincle−/−
BMDCs compared with WT cells (Fig. 2E). The contribution of
Mincle to BMDC cytokine responses was observed with several
GAS isolates (SI Appendix, Fig. S2). Correspondingly, evaluation
CARD9 Is Required for the Production of Proinflammatory Cytokines
in Response to GAS. To validate the observation that CARD9
signaling is involved in the induction of inflammatory re-
sponses to GAS infection, we performed in vitro experiments
with bone marrow-derived dendritic cells (BMDCs) from WT
and CARD9−/− mice. Infection of CARD9−/− BMDCs with GAS
demonstrated significantly lower production of TNF and MIP-2
compared with WT (Fig. 1 B and C).
To identify the molecular triggers for these inflammatory
cytokines and chemokines, we extracted hydrophilic and hy-
drophobic components from GAS using a CHCl3:MeOH:H2O
(C:M:W) solvent and evaluated each extract for its ability to
stimulate BMDCs. Only the hydrophobic C:M extract induced
the production of TNF in WT BMDCs, which was decreased
in CARD9−/− BMDCs (Fig. 1D). These results suggest that a
component in the lipophilic fraction from GAS is recognized by
an innate immune receptor(s) that initiates CARD9-dependent
signaling upon GAS infection.
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Imai et al.

50
40
30
20
10
0
A
Unstim.
C:M extract
Syk
FcR
DAP12
+
FcR
+
FcR
+ FcR
hLox1/CD3
mLox1/CD3
hMICL/CD3
mMICL/CD3
hDCIR/CD3
mClec2/CD3
gMCL hMCL
mMCL
FcR
hSiglec5/hCSDig3lec7/hCSDig3lec9/CD3
mSiglecF/CD3
mClec1a/CD3
Syk
rMincle
+ FcR
+
FcR
gMincle +hMFcinRcle
+
FcR
mDCAR1/CD3
hClec12bm/CSDig3lecE/CD3
mClec12b/CD3
hDectin-1
+ Syk
hDNGR-1
mDNGR-1 Syk
hDectin-1B Syk
mDectin-1B + Syk
+
mMincle
hBDCA2
+
FcR
hDectin2
mDectin2 FcR
+
FcR
mDectin-1
+
Syk
+
mDCAR1
m
+
DFC
c
A
R
R2
+
+
+
mLMIR5
+
DAP12
mMDL-1
+
DAP12
Fig. 2. Mincle recognizes the lipophilic fraction of
GAS. (A) NFAT-GFP reporter cells expressing vari-
ous PRRs were left unstimulated or stimulated by
plate-coated lipophilic fraction from GAS for 16 h.
Induction of GFP was analyzed by flow cytometry,
and percent positive cells are plotted. (B) The li-
pophilic fraction (C:M) was plate-coated to stimu-
late NFAT-GFP reporter cells expressing FcRγ only or
mouse Mincle + FcRγ. (C) BMDCs from WT, CARD9−/−, or
Mincle−/− mice were stimulated with the indicated
concentrations of plate-coated lipophilic fraction for
16 h. The concentration of TNF was determined by
ELISA. (D) Live GAS were incubated with human IgG1-
Fc (Ig) or Mincle-Ig fusion protein (Mincle-Ig) (1 μg/mL)
for 1 h at 4 °C. Bound proteins were detected with
anti−hIgG-Alexa 488 by flow cytometry. (E) BMDCs
from WT or Mincle−/− mice were infected with live GAS
(MOI = 1 and 10) or exposed to LPS (10 ng/mL) for 16 h.
The concentrations of TNF produced were determined
by ELISA. *P < 0.05. Data are presented as mean SD
and are representative of (A) two or (B−E) three in-
dependent experiments with similar results.
mDCAR2m/DDCCIARR12m+/DDFCcCRIARR22m+/DDFCcCRIARR32+/DFCcRIR4 + FcR
FcR
FcR
Mincle
B
D
C 8
WT
CARD9
Mincle
16
12
8
30
20
10
0
+
6
4
2
0
4
0
0
0.03 0.3
C:M ( g/well)
3
*
0
0.03 0.1 0.3
1
LPS
C:M ( g/well)
200
E
16
12
8
WT
Mincle
160
120
80
Mincle-Ig
Ig
*
4
40
0
0
0
1
10
LPS
hIgG-Alexa 488
Live GAS (MOI)
of the initial CAGE experiment similarly highlights the in-
volvement of Mincle pathway in response to GAS (SI Appendix,
Fig. S1 B and C).
MS (GC-MS) analysis following methanolysis (SI Appendix, Fig.
S3B). The GC-MS chromatogram of fatty acid methyl esters
(FAMEs) showed the major fatty acids of Spot 1 are C16:0,
C16:1, C18:1, and C18:0 (Fig. 4B and SI Appendix, Fig. S3B).
RuCl3/NaIO4 oxidation followed by GC-MS analysis revealed the
positions of C16:1 and C18:1 double bonds as C11 and C12 (SI
Appendix, Fig. S3C). The combined NMR, ESI-TOF-MS, and
GC-MS analyses identify the major component of Spot 1 as
MGDG (16:0/18:1) (Fig. 4C).
Spot 1, hereafter referred to as MGDG, bound to the Mincle-
Ig protein as did the known ligand, TDM (Fig. 4D) (25), and
the structure of MGDG fulfills the minimum criteria of Mincle
ligand: a polar head consisting of glucose or mannose and a
hydrophobic chain (38, 39). MGDG was also recognized by hu-
man Mincle in reporter cells and myeloid cells (SI Appendix, Fig.
S3 D and E). Thus, we conclude that the Mincle ligand in GAS is
MGDG. MGDG constitutes an anchor moiety of the TLR2 ligand
membrane-anchored glycopolymer LTA; however, MGDG did
not activate NF-κB reporter cells via TLR2 (Fig. 4E), and, con-
versely, Mincle was not activated by intact LTA (Fig. 4F).
These results suggest that Mincle is a major receptor for GAS
acting upstream of the CARD9 signaling pathway.
Identification of MGDG as a Mincle Ligand. To identify the GAS-
derived Mincle ligand, we separated the C:M fraction of GAS
into 16 subfractions by high-performance TLC (HPTLC) fol-
lowed by copper acetate staining. Each subfraction was assessed
for stimulation of the Mincle reporter cells. Spot 1 (subfraction
#12) possessed potent ligand activity in Mincle-expressing re-
porter cells, whereas the most abundant Spot 2 (subfractions
#8 and #9) did not have substantial activity (Fig. 3A). Purified
Spot 1 could be detected as a single spot upon staining with ei-
ther copper acetate or orcinol, which visualizes organic mole-
cules and sugar moieties, respectively. However, it was not visible
after molybdenum blue staining, suggesting that Spot 1 repre-
sents a glycolipid lacking phosphate moieties (Fig. 3B). Spot 1
activated Mincle-expressing reporter cells in a dose-dependent
manner, and its specific activity per milligram of lipid was in-
creased (Fig. 3C) compared with that of crude C:M extract (Fig.
2B). Furthermore, Mincle-Ig fusion protein directly bound to
purified Spot 1 (Fig. 3D), further demonstrating that Spot 1
derived from GAS contains a Mincle ligand.
MGDG Activates Dendritic Cells Through Mincle. We next examined
the role of Mincle in MGDG-induced innate immune responses.
MGDG stimulated BMDCs to produce proinflammatory cyto-
kines and chemokines, such as TNF, MIP-2, and IL-6, which
were completely suppressed in Mincle-deficient cells (Fig. 5 A–C).
Nitric oxide (NO) and ROS are additional potent effectors that
are involved in the bactericidal action of myeloid cells (40–42).
MGDG also induced the expression of NO synthase 2 (NOS2/
iNOS), an NO-producing enzyme, and ROS production in a
Mincle-dependent manner (Fig. 5 D and E), consistent with the
finding that the CARD9 pathway induces ROS (24). Importantly,
To determine the chemical structure of the compound present
in Spot 1, we used NMR and mass spectrometry (MS) analysis.
1
The H-NMR and 13C-NMR spectra of Spot 1 showed proton
signals characteristic of α-glucopyranose, glycerol, and unsatu-
rated fatty acid (Fig. 4A and SI Appendix, Fig. S3A). High-
resolution electrospray ionization time-of-flight MS (ESI-TOF-MS)
analysis of Spot 1 showed molecular-related ion peaks at m/z
749.5191, 751.5336, 777.5495, and 779.5656 (Fig. 4B). We also the induction of iNOS and ROS upon infection with live GAS was
determined the fatty acid composition by gas chromatography
also impaired in the absence of Mincle (SI Appendix, Fig. S4).
Imai et al.
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16
15
14
13
12
11
10
9
8
7
6
5
affected by Spot 2 (Fig. 6C), suggesting that this spot inhibits
Mincle-mediated cell activation. Spot 2 also down-regulated the
expression of costimulatory molecules in BMDCs induced by
MGDG (Fig. 6D) or TDM (Fig. 6E), and suppressed T-cell ac-
tivation triggered by MGDG-stimulated BMDCs (Fig. 6F).
Mincle-Ig protein directly bound Spot 2, although the binding
appeared weaker compared with that of MGDG and TDM (Figs.
4D and 6G and SI Appendix, Fig. S6).
Using NMR and MS, we determined the chemical structure of
Spot 2 to be diglucosyldiacylglycerol (DGDG) (mainly 16:1/18:1;
16:0/18:1) (Fig. 7 A–F). Similar to MGDG, DGDG is produced
as an LTA anchor molecule of GAS (43, 44). Indeed, synthetic
DGDG suppressed the activation of reporter cells and BMDCs
stimulated with MGDG (Fig. 7 G and H). These results suggest
that, although DGDG binds Mincle, it lacks agonistic activity.
MGDG and DGDG were detected in the invasive M1 serotype
strains 5448 and NIH44, and the M3 serotype strain NIH34, in
addition to the noninvasive M1 serotype strains SF370 and
Se235, and the M3 serotype strain K33, suggesting that these are
shared immunomodulators across different GAS serotypes. In-
terestingly, DGDG/MGDG ratio varied among the tested strains,
which may partly contribute to the different pathogenicity of GAS
strains (SI Appendix, Figs. S7 A–C and S8).
Copper
acetate
Molybdenum
blue
A
B
Orcinol
Spot 1
Spot 2
FcR
FcR
Mincle
+
4
3
2
1
0
0
25
25
50
50
NFAT-GFP (%)
Ig
FcR
FcR + Mincle
C
D
Mincle-Ig
100
1.2
0.8
0.4
0
80
60
40
20
0
0
0.03 0.3
3
0
0.03 0.3
3
Mincle Is Critical for Controlling GAS Infection. Finally, we examined
the effect of Mincle on GAS infection in vivo. To gain insight
into early responses, WT and Mincle−/− mice were infected in-
traperitoneally with the invasive GAS strain NIH34. Upon in-
fection, early induction of serum inflammatory cytokines and
chemokines, such as IP-10, IL-6, and CXCL1, was significantly
suppressed in Mincle−/− mice compared with WT mice (Fig. 8A).
Regarding the susceptibility to GAS, Mincle−/− mice demon-
strated a dramatic increase in mortality compared with WT mice
(Fig. 8B). Consistent with these results, Mincle−/− mice exhibited
severe GAS bacteremia (Fig. 8C) and abscess formation in the
kidneys, which was not detected in WT mice (Fig. 8 D and E).
Within the abscesses, GAS bacteria were identified as chains of
cocci, by staining with hematoxylin (Fig. 8F). Mincle−/− mice also
suffered exacerbated liver damage, as the degree of hepatocel-
lular vacuolation was higher in Mincle−/− mice than WT mice
(Fig. 8 G and H). Furthermore, Mincle−/− mice developed
hypercytokinemia when bacteremia was observed (SI Appendix,
Fig. S7D), suggesting that the rapid increase of bacterial number
in mice lacking Mincle-mediated early immune responses even-
tually causes septic shock and multiple organ failure. Importantly,
the role of Mincle in initial cytokine responses and reduced
mortality to GAS infection was confirmed by using other GAS
strains (SI Appendix, Figs. S7E and S8). Taken together, these
results suggest that Mincle is crucial for protection against early
GAS infection.
Spot 1 ( g/well)
Spot 1 ( g/well)
Fig. 3. A GAS glycolipid activates Mincle. (A) The lipophilic fraction of GAS
was analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol), stained with
copper acetate reagent, and divided into 16 subfractions (Left). Each sub-
fraction was coated onto a plate to stimulate reporter cells. Induction of
NFAT-GFP was analyzed by flow cytometry (Right). (B) The C:M extract was
analyzed by HPTLC using C:M:W (65:25:4; vol/vol/vol). The HPTLC plates were
stained with copper acetate (Left), orcinol (Middle), or molybdenum blue
(Right) reagent. (C) Reporter cells expressing FcRγ only or Mincle + FcRγ were
stimulated with the indicated concentrations of plate-coated purified Spot
1 for 16 h. Induction of NFAT-GFP was analyzed by flow cytometry. (D) Ig or
Mincle-Ig was incubated with the indicated concentrations of plate-coated
purified Spot 1. Bound proteins were detected with anti−hIgG-HRP. Data are
presented as mean SD and are representative of three independent ex-
periments with similar results.
Since MGDG had such a pronounced Mincle-dependent effect
on BMDC cytokine production, we additionally investigated the
downstream consequences for T-cell activation. First, we observed
that stimulation of WT BMDCs with MGDG up-regulated the
expression of costimulatory molecules, such as CD40, CD80, and
CD86 (Fig. 5F, Upper), which was abolished in the absence of
Mincle (Fig. 5F, Lower). Second, BMDCs were pulsed with the
ovalbumin (OVA) antigen peptide and cocultured with T cells
from OVA-specific OT-II TCR transgenic mice in the presence or
absence of MGDG. Antigen-specific secretion of IL-17 by CD4+
OT-II T cells was significantly augmented when the cells were
cocultured with MGDG-treated WT BMDCs. However, this ef-
fect was eliminated if the MGDG-treated BMDCs were Mincle−/−
(Fig. 5G). The effect of MGDG treatment on antigen-dependent
IFNγ production was less potent, compared with TDM treatment
(Fig. 5H). Collectively, these in vitro results suggest that MGDG
enhances antigen-presenting cell functions to promote potent
Th17 response in a Mincle-dependent manner.
Discussion
In this manuscript, we have demonstrated that Mincle plays a
central role in protective immunity against lethal GAS infection.
However, GAS can counteract this protective response by pro-
duction of DGDG.
Previous studies have reported several innate immune recep-
tors that are involved in GAS sensing and signaling, most notably
those receptors that signal through the MyD88-dependent path-
way (10–12, 16). However, the roles of individual TLRs in GAS
recognition were mainly characterized in vitro, whereas the in vivo
susceptibility to GAS infection of mice lacking individual recep-
tors is not clear (7, 16, 17). Strikingly, we show that Mincle de-
ficiency resulted in impaired immune responses and increased
susceptibility to GAS infection both in vitro and in vivo. Given
that Mincle expression is robustly up-regulated by various
stimuli in a MyD88-dependent manner (45, 46), MyD88 path-
ways may potentially contribute to protective immunity through
the induction of Mincle expression. In line with this idea, future
experiments may investigate whether introduction of a Mincle
transgene into MyD88-deficient mice restores GAS resistance.
Diglucosyldiacylglycerol Inhibits MGDG-Induced Activation. Given
the significant role of Mincle in immunity against GAS, it is
possible that GAS may target Mincle for immune evasion. The
increase in specific activity during the course of MGDG purifi-
cation suggested the presence of an inhibiting molecule in the
crude lipid fraction of GAS. Indeed, although Spot 2, the most
abundant spot, did not activate Mincle reporter cells (Fig. 3A
and Fig. 6A, Left) or BMDCs (Fig. 6B, Left), it prevented
Mincle-mediated activation induced by MGDG (Fig. 6 A and B,
Right) and TDM (SI Appendix, Fig. S5). In contrast, TLR4-
mediated or Dectin-2−mediated BMDC activation were not
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A
B
-(CH2)n-
solvent
-CH3
gly-3’
G6
-CO-CH2-
=CH-CH2-
G3
G2
G5
gly-3
-CH=CH-
gly-2
G1
gly-1
=CH-CH2-CH2-
G4
gly-1’
ppm
0.5
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Intens.
x 104
1.0
779.5656 (16:0&18:1)
777.5495 (16:1&18:1)
749.5191 (16:1&16:1)
0.8
0.6
0.4
0.2
0.0
751.5336 (16:0&16:1)
Fig. 4. Identification of MGDG as a Mincle ligand.
The (A) 1H-NMR spectrum and (B) ESI-TOF-MS spec-
trum of purified Spot 1. ESI-TOF-MS data of MGDG:
m/z = 749.5191 [M+Na]+ (calculated for C41H74NaO10
Δmmu = −1.7), 751.5336 [M+Na]+ (calculated for
761.1845
770.5272
765.5235
742.3877
745 750
m/z
755
760 765 770 775
780 785
D
1.6
1.2
0.8
0.4
C
41H76NaO10 Δmmu =
−0.5), 777.5495 [M+Na]+
Ig
1.6
1.2
0.8
0.4
0
Mincle-Ig
(calculated for C43H78NaO10 Δmmu = −0.8), and
779.5656 [M+Na]+ (calculated for C43H80NaO10
Δmmu = +1.2). (C) Chemical structure of MGDG (Spot
1) from GAS. The Z geometry of the double bond in the
fatty acid (FA) side chain was determined by allylic car-
bon chemical shift at δC 28.2, which was assigned by
HMBC experiments. (D) Ig or Mincle-Ig were incubated
with the indicated concentrations of plate-coated TDM
or purified MGDG. Bound proteins were detected with
anti−hIgG-HRP. (E) HEK293 cells expressing FcRγ only,
Mincle + FcRγ, TLR2, or TLR4 were stimulated with the
indicated concentrations of plate-coated purified MGDG
(Spot 1) for 16 h. Pam3, LPS, and TDM were used as
positive controls. Activation of NF-κB was measured by
SEAP activity in the supernatants. (F) HEK293 cells
expressing FcRγ only, Mincle + FcRγ or TLR2 were stim-
ulated with the indicated concentrations of LTA from
GAS for 16 h. Pam3 and TDM were used as positive
controls. Activation of NF-κB was measured by SEAP
activity in the supernatants. Data are presented as
mean SD and are representative of (F) two or (D and
E) three independent experiments with similar results.
C
E
Exact Mass: 756.57515
0
100 102 104 106
MGDG (fmol/well)
100 102 104 106
TDM (fmol/well)
F
TLR2
1.6
1.6
1.2
0.8
0.4
0
0.6
1.6
1.2
0.8
0.4
0
TLR2
TLR4
1.2
0.8
0.4
0
0.4
0.2
0
0
1
10 100
Pam3 TDM
Pam3 LPS TDM
Recent reports have highlighted a critical role of the NLRP3 unidentified (57). As Mincle is an inducible protein that signals
inflammasome and IL-1β in protective immunity against GAS through ITAMs and as ITAM-coupled receptors trigger IFNγ
(47–49). Downstream signaling of CLR, including Dectin-1 and production in lymphocytes, it is plausible that Mincle might also
Mincle, can directly activate NLRP3 (50, 51). Therefore, GAS-
induced inflammasome activation may be partly mediated by
the MGDG−Mincle axis in conjunction with the previously
reported effector proteins, such as M protein or streptolysin O
(11, 19).
contribute to the robust IFNγ production in this myeloid subset
upon sensing GAS-derived MGDG.
We identified two Mincle ligands in the GAS lipophilic frac-
tion: MGDG, which has agonistic activity, and DGDG, which
interacts with Mincle but does not initiate signaling. In-
terestingly, both molecules are membrane anchors for LTA, a
common structural feature of the Gram-positive cell envelope
(43, 44). As such, glycosyldiacylglycerol is also present in other
pathogens (58), and Mincle may play a role in response to such
targets. Determination of these immunomodulatory glycolipids
together with the expression analysis of the synthase candidates in
various microorganisms may help to predict their immunological
properties.
GAS infection induces the production of various proin-
flammatory cytokines, including TNF from myeloid cells (42),
which are critical factors in controlling the dissemination of GAS
(7). The increased susceptibility of Mincle-deficient mice to early
GAS infection is likely due to their reduced ability to produce
these key cytokines. NO and ROS are also direct effectors that
enhance the elimination of GAS early in infection (41, 42). The
Mincle-dependent activation of these pathways may thus syner-
gistically contribute to suppressing the GAS infection process.
Mincle is expressed in a wide range of hematopoietic cells (52–
55), and multiple immune cells contribute to immune protection
against GAS (5, 8, 56). It is therefore critical to clarify which
Mincle-expressing cells are the main contributors to protection
against GAS. A recent report showed that, in the early stage of
GAS infection, an IFNγ-producing Gr1+CD11b+ myeloid subset
rapidly appears in the periphery and plays a protective role,
whereas the receptor that triggers IFNγ production remains
The precise role of DGDG in streptococci has been elusive.
Disruption of the glucosyltransferase in group B Streptococcus
(GBS), ΔiagA (invasion-associated gene A), impaired the invasive
capacity in mice, which pointed to a requirement for DGDG in
promoting invasion of GBS in host endothelial cells (59). Although
no studies have examined DGDG function in GAS, our study
herein proposes an action of DGDG as a suppressor of Mincle-
mediated responses. The molecular mechanism(s) of this sup-
pression is unknown; however, as DGDG does not suppress the
Imai et al.
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A
D
B
C
Fig. 5. MGDG induces innate immune responses in a
Mincle-dependent manner. (A–C) BMDCs from WT or
Mincle−/− mice were stimulated with the indicated
concentrations of plate-coated purified MGDG or LPS
for 48 h. The concentrations of (A) TNF, (B) MIP-2,
and (C) IL-6 were determined by ELISA. (D) BMDCs
from WT or Mincle−/− mice were stimulated with
plate-coated purified MGDG (10 μg per well) or LPS
(10 ng/mL) for 24 h. Intracellular iNOS staining was
measured by flow cytometry. (E) BMDCs from WT or
Mincle−/− mice were left unstimulated (dotted line)
or stimulated with plate-coated purified MGDG (10
μg per well; solid line) for 24 h. ROS production was
monitored using H2DCFDA staining followed by
FACS analysis. (F) BMDCs from WT (Upper) or Min-
cle−/− (Lower) mice were left unstimulated (dotted
line) or stimulated with plate-coated purified MGDG
(3 μg per well; solid line) for 48 h. The surface ex-
pressions of CD40, CD80, and CD86 were analyzed by
flow cytometry. (G and H) BMDCs from WT (open
circle) or Mincle−/− (closed circle) mice were pulsed
with OVA323–339 peptide and cocultured with CD4+
OT-II T cells for 4 d in the presence of plate-coated
purified MGDG (3 μg per well) or TDM (30 ng per
well). The concentrations of IL-17 and IFNγ secreted
into the supernatant were determined by ELISA.
Data are presented as mean SD and are represen-
tative of three independent experiments with similar
results.
E
G
H
F
signaling of other PRRs (Fig. 6C), it is unlikely that DGDG de- quired immune responses during recurrent infection. GAS in-
livers intrinsic inhibitory signals. Rather, DGDG seems to in- fection typically induces a Th17 response, which leads to the
establishment of protective acquired immunity against GAS in
the later phase (63–65). As Mincle ligands potently promote
Th17 responses (Fig. 5G) (66, 67), which are inhibited by
DGDG, the use of a component GAS vaccine retaining MGDG
and less DGDG may induce protective immunity against both
invasive and recurrent GAS infections. Indeed, in a mouse
model, vaccination with liposomes bearing Mincle ligands suc-
cessfully controlled infections after GAS challenge (68). Hence,
GAS-derived MGDG and its derivatives may represent attractive
adjuvants for preventing invasive infection and diseases associ-
ated with recurrent infection (2, 69, 70).
terfere with the interaction of agonistic ligands to Mincle. For
example, some low-affinity/-valency CLR ligands act antagonisti-
cally, as exemplified by Dectin-1 ligands (37, 60, 61). Alternatively,
as DGDG possesses an α-1,2-linked glucose instead of an α-1,1-
linked glucose as found in TDM, it is possible to speculate that the
second glucose in DGDG interacts with Mincle in a different way,
which may lead to the inefficient receptor clustering. It would be
intriguing to speculate that bacteria may regulate the composition
of their LTA anchor to escape immune triggering of Mincle. In
this respect, it is important that the relative content of MGDG and
DGDG in clinical GAS isolates in various stages of infection and
diseases be investigated, although virulence is determined by
multiple factors. The blockade of DGDG-synthesizing enzyme
might provide a therapeutic option by increasing Mincle sensi-
tivity to invasive GAS.
Materials and Methods
Mice. Mincle-deficient mice (53) were backcrossed for at least nine gen-
erations with C57BL/6 mice. CARD9-deficient mice were provided by
H. Hara, Kagoshima University, Kagoshima, Japan (23). OVA-specific TCR
OT-II transgenic mice were used on a C57BL/6 background. C57BL/6 mice
were obtained from Japan Clea or Kyudo. All mice were maintained in a
filtered-air laminar-flow enclosure and given standard laboratory food
and water ad libitum. Animal protocols were approved by the committee
of Ethics on Animal Experiment, Faculty of Medical Sciences, Kyushu Uni-
versity and Research Institute for Microbial Diseases, Osaka University. All
work in GAS infection performed using mice was carried out in accordance
with the guidelines for animal care approved by the National Institute of
Infectious Diseases.
An important unsolved question is how and where Mincle
interacts with MGDG/DGDG expressed by GAS. Given that
diacylglycerols act as membrane anchors for LTA, it is thought
that these glycolipids are localized in the plasma membrane,
which lies beneath the thick peptidoglycan layer (43). However,
our finding that Mincle-Ig can interact with intact GAS raises the
possibility that MGDG/DGDG may be exposed on the cell
surface. Additionally, Mincle may recognize degraded moieties
of LTA that are shed from or exposed on streptococci (59, 62).
Indeed, we could detect MGDG and LTA activity in the GAS
culture supernatant (SI Appendix, Fig. S9).
Bacterial Strains. GAS strains used in this study were SF370 (ATCC 700294;
American Type Culture Collection), 5448, NIH44, and Se235 of the
M1 serotype, and NIH34 and K33 of the M3 serotype. The NIH44, Se235,
In addition to the protective innate immune responses via
Mincle in acute GAS infection, Mincle may also promote ac-
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log phase (OD600 = 0.7 to 0.9) at 37 °C in 5% CO2, pelleted by centrifugation,
washed twice with sterile PBS, and suspended in sterile PBS.
A
B
C
Reagents. TDM (T3034), LTA (L3140), and LPS (L4516) were purchased from
Merck. Lipoarabinomannan (LAM; 02449-61) was from Nacalai Tesque.
Pam3CSK4 (Pam3; tlrl-pms) was from InvivoGen. OVA323–339 peptide was
obtained from ABGENT. ELISA kits for TNF (558534), IL-6 (555240), and IFNγ
(551866) were from BD Bioscience; CXCL10/IP-10/CRG-2 (DY466), CXCL1/KC
(DY453-05), MIP-2 (DY452), and IL-17 (DY421) were from R&D Systems; and
human IL-8 (88-8086-22) was from eBioscience. Th1/Th2 10 plex Flow-
Cytomix (BMS820FF), MCP-1 (BMS86005FF), IP-10 (BMS86018FF), and CXCL1/
KC (BMS86019FF) were from eBioscience. Anti-CD80 (16-10A1) and
CD86 (GL-1) antibodies (Abs) were from BioLegend. Anti-CD40 (3/23) antibody
(Ab) was from BD Bioscience. Anti-human IgG (H+L) Ab was from Jackson
ImmunoResearch. Anti-NOS2 (CXNFT) Ab was from eBioscience. H2DCFDA was
from Molecular Probes. Anti-mouse Mincle Ab (1B6) was described previously (72).
Cells. The 2B4-NFAT-GFP reporter cells expressing various receptors were prepared
as previously described (37). BMDCs and human monocyte-derived dendritic cells
were also generated as previously described (27, 73). Human embryonic kidney
(HEK293) cells expressing secreted alkaline phosphatase (SEAP) under the control
of an NF-κB response element were purchased from InvivoGen. The collection
and use of human peripheral blood mononuclear cells under written informed
consent obtained from all donors were approved by the institutional review
boards of Research Institute for Microbial Diseases, Osaka University.
D
E
F
Transcriptome Analysis. Buffy coats were obtained from the Australian Red
Cross Blood Service (University of Queensland ethics number HREC2011000232),
and CD14+ monocytes, isolated from three donors as previously described (74),
were immediately stimulated with GAS strain 5448; or C. albicans strain 3630, a
clinical isolate obtained from the Australian Medical Mycology Reference Lab-
oratory or S. typhimurium. LPS (Invivogen) was used at 10 ng/mL. All pathogens
were heat-killed and used at multiplicity of infection (MOI) 10, for 2 h at 37 °C
in serum-free RPMI 1640 medium. Cell lysates were collected for RNA (miRNeasy
kit; Qiagen). CAGE sequencing was generated as part of the Functional An-
notation of Mammalian Genomes 5 (FANTOM5) consortium (32), and primary
data are available from the FANTOM5 database (33). For the data described
here, Relative log expression normalized expression data (75) were filtered to
include only those tags that mapped to an Entrez or HUGO Gene Nomenclature
Committee (HGNC) identifier. Multimapped CAGE tags were excluded.
To be considered as input for differential expression analysis, a CAGE tag
was required to be expressed at greater than 1 tags per million (TPM) in all
donors of a treatment condition, and have a mean expression level greater
than in the control condition (i.e., up-regulated), or be expressed at greater
than 1 TPM in all donors of the control condition and have a mean expression
level greater than in the treatment condition (i.e., down-regulated). Dif-
ferential expression was assessed by the Rank Product method (76), using the
“RankProd” package in R. One thousand permutations were conducted per
comparison. Statistically significant differential expression was defined as
CAGE tags having a percentage of false prediction value of less than
0.05 and a raw fold change of greater than 1.5-fold. Hypergeometric Opti-
mization of Motif EnRichment (77) was used to analyze enrichment of
transcription factor binding sites in the region 500 nucleotides upstream and
downstream of the center of a CAGE peak. Enrichment was assessed against
a background of all CAGE tags ( 500 nucleotides) expressed above 1 TPM in
all three Mock donors. Where genomic regions overlapped or book-ended
on the same strand, they were merged into a single region using bedtools.
Principal component analysis was conducted in R (version 3.2.1) on all
expressed TSSs using the “plotPCA” function within the “affycoretools”
package. Functional enrichment was conducted using the Protein Analysis
Through Evolutionary Relationships (PANTHER) tool (78) interface with
Reactome and Gene Ontology (GO).
G
Fig. 6. Identification of
a
GAS glycolipid that interferes with Mincle-
mediated signaling. (A) Reporter cells expressing FcRγ only or Mincle +
FcRγ were stimulated with the indicated concentrations (0.3, 1, and 3 μg per
well) of plate-coated purified Spot 2 in the absence (Left) or in the presence
(Right) of purified MGDG (0.3 μg per well) for 16 h. Induction of NFAT-GFP
was analyzed by flow cytometry. (B) BMDCs from WT mice were stimulated
with indicated concentrations (0.03, 0.1, 0.3, and 1 μg per well) of plate-
coated purified Spot 2 for 48 h. The concentration of TNF was determined
by ELISA (Left). BMDCs from WT mice were stimulated with indicated con-
centrations (0.03, 0.1, 0.3, and 1 μg per well) of plate-coated purified Spot 2
in the presence of purified MGDG (0.3 μg per well) for 24 h. The concen-
tration of TNF was determined by ELISA (Right). (C) Effect of Spot 2 on LPS
(Upper) or LAM (Lower)-induced TNF production in BMDCs. (D and E) BMDCs
from WT mice were stimulated with plate-coated (D) purified MGDG (30 μg
per well) or (E) TDM (100 ng per well) in the presence of Spot 2 (30 μg per
well) for 48 h. The surface expressions of CD40, CD80, and CD86 were ana-
lyzed by flow cytometry. (F) BMDCs from WT or Mincle−/− mice were pulsed
with OVA323–339 peptide and cocultured with CD4+ OT-II T cells for 4 d in the
presence of plate-coated purified MGDG (3 μg per well) or plate-coated
purified MGDG (3 μg per well) and Spot 2 (3 μg per well). The concentra-
tions of IL-17 secreted into the supernatant were determined by ELISA. (G) Ig
or Mincle-Ig were incubated with the indicated concentrations of plate-
coated purified Spot 2. Bound proteins were detected with anti−hIgG-
HRP. Data are presented as mean SD and are representative of three in-
dependent experiments with similar results.
Lipid Extraction and Purification. GAS was washed with distilled deionized H2O
and then partitioned by C:M:W (8:4:3; vol/vol/vol) into lower organic phase
(C:M) and upper aqueous phase (M:W). Lower organic phase was filtered and
further fractionated by HPTLC. Each fraction including MGDG and DGDG was
developed using the developing solvent C:M:W (85:15:1; vol/vol/vol) and
C:M:W (65:25:4; vol/vol/vol), respectively, and collected from the HPTLC
plate and dissolved in C:M (2:1; vol/vol).
NIH34, and K33 clinical isolates were collected by the Working Group for
Beta-hemolytic Streptococci in Japan (71). Stock cultures were maintained
in 10% glycerol/PBS or 10% skim milk at −80 °C, and SF370 was routinely
cultured at 37 °C in Brain Heart Infusion (BHI) broth for 24 h. The number
of viable bacteria was determined by counting colony-forming units
(CFUs) after diluting and plating onto BHI agar plates. NIH34, K33, NIH44,
and Se235 were cultured in Todd Hewitt broth supplemented with 0.5%
yeast extract (THY medium). The growth of NIH34 was turbidimetrically mon-
itored at 600 nm, using a MiniPhoto 518R (Taitec). Cultures were grown to late-
Synthesis of DGDG. Synthetic DGDG was chemically prepared via 17 reaction
steps starting from D-glucose. First, D-glucose was converted into the glucosyl
trichloroacetimidate as the glycosyl donor, which possessed two benzyl
groups (at C4 and C6 positions) and the 1,1,3,3-tetraisopropyldisiloxanylidene
Imai et al.
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A
B
D
Fig. 7. Chemical identification of Spot 2 as DGDG.
The (A) 1H-NMR spectrum, (B) 13C-NMR spectrum,
and (C) ESI-TOF-MS spectrum of purified lipids Spot
2. ESI-TOF-MS data of Spot 2: m/z = 911.5792 [M+
Na]+ (calculated for C47H84NaO15 Δmmu = −9.0),
913.5907 [M+Na]+ (calculated. for C47H86NaO15
Δmmu = −4.8), 939.6103 [M+Na]+ (calculated for
C49H88NaO15 Δmmu = −8.8), and 941.6216 [M+Na]+
(calculated for C49H90NaO15 Δmmu = −4.4). (D) GC-
MS chromatogram of FAMEs prepared by the meth-
anolysis of Spot 2: methyl hexadecenoate (16:1), tR
[min] = 14.2(7.2%), m/z = 268 (M+); methyl hex-
adecenoate (16:1), tR [min] = 14.3(17.9%), m/z = 268
(M+); methyl hexadecenoate (16:1), tR [min] = 14.4
(2.7%), m/z = 268 (M+); methyl hexadecanoate (16:0),
C
E
tR [min]
= 14.5(19.0%), m/z =
270 (M+); methyl
F
octadecenoate (18:1), tR [min] = 16.3(7.0%), m/z =
296 (M+); methyl octadecenoate (18:1), tR [min] =
16.4(41.5%), m/z = 296 (M+); methyl octadecenoate
(18:1), tR [min] = 16.5(2.8%), m/z = 296 (M+); and
methyl octadecenoate (18:0), tR [min] = 16.6(1.9%),
m/z = 298 (M+). (E) FAMEs of Spot 2 after RuCl3/
NaIO4 oxidation: dimethyl undecanedioate (11:0), tR
[min] = 12.6, m/z = 213 (M+−31). (F) Chemical struc-
ture of DGDG (Spot 2) from GAS. (G and H) Effect of
synthetic DGDG (SynDGDG) on MGDG-induced acti-
vation of (G) Mincle reporter cells and (H) BMDCs.
SynDGDG (G: 0.3, 1, 3 μg per well), (H: 0.03, 0.1, 0.3,
1 μg per well) was coated together with MGDG
(0.3 μg per well).
G
H
group (bridged between C2 and C3) as protecting groups. The glucosyl donor
was next coupled with (S)-(+)-1,2-isopropylideneglycerol in the presence of
trimethylsilyl triflate in CH2Cl2 at –80 °C, affording the glucosyl glycerol de-
rivative in 61% yield (α/β ratio = 1:3). Subsequent hydrogenolysis of the iso-
lated β-glucosyl glycerol provided the corresponding glucosyl glycerol
acceptor, which was then subjected to the second glycosylation with the
A
D
E
7
6
5
4
3
2
1
0
WT
Mincle
*
*
40
30
20
10
0
*
4
3
2
1
0
12
8
4
0
WT Mincle WT Mincle
Uninf.
GAS
Uninf.
GAS
Uninf.
GAS
Uninf.
GAS
B
C
F
G
H
*
WT
Mincle
500
400
300
200
100
0
Mincle
WT
*
100
50
0
6
4
2
0
CV
CV
p < 0.01
WT Mincle WT Mincle
Uninf.
GAS
0
2
4
6
8
WT Mincle
Days post infection
Fig. 8. Critical role of Mincle in GAS infection. (A) The concentrations of IP-10, IL-6, and CXCL1 in the serum from uninfected or infected mice 3 h after i.p. infection
with 1.5 × 108 CFUs of NIH34. The concentrations of cytokines and chemokines were determined by FlowCytomix and ELISA. *P < 0.05. (B) Survival curves of WT (solid
line) and Mincle−/− (dotted line) mice after i.p. infection with 2.3 × 107 CFU of NIH34. Groups of 5 Mincle−/− or WT mice were infected, and survival was monitored over
8 d. Comparison of survival curves was performed using the log-rank test. Data are representative of two independent experiments with similar results. (C) Kinetics of
GAS bacterial growth in the blood of WT (n = 5) or Mincle−/− (n = 5) mice at 48 h after i.p. infection with 1.4 × 107 CFU of NIH34. The number of bacteria was compared
by the Mann–Whitney U test. *P < 0.05. (D) Histopathological changes in the kidney of WT (Left) or Mincle−/− (Right) mice infected with 1.4 × 107 CFU of NIH34. Tissues
were collected at 48 h after the i.p. injection of GAS and stained with hematoxylin and eosin. The black arrowheads indicate abscesses infiltrated with inflammatory
cells. (Scale bar: 500 μm.) (E) Quantification of abscesses in the kidney sections of WT and Mincle−/− mice. (F) The clusters of bacteria in the kidney. (Scale bar: 20 μm in
Upper and 2 μm in Lower.) (G) Histopathological changes in the liver of WT (Left) or Mincle−/− (Right) mice infected with 1.4 × 107 CFU of NIH34. Tissues were collected
at 48 h after i.p. injection of GAS and stained with hematoxylin and eosin. The black arrows indicate hepatocellular vacuolation. (Scale bar: 20 μm.) CV, centrilobular
vein. (H) Quantification of hepatocellular vacuolation in the liver sections of WT and Mincle−/− mice. *P < 0.05.
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Imai et al.

above-mentioned glucosyl donor. The glycosylation gave the gentiobiosyl
glycerol derivative in 67% yield along with good β-stereoselectivity (α/β ratio =
1:19). Subsequent hydrolysis with 85% aqueous acetic acid removed the iso-
propylidene group on the glycerol moiety. Then, both regioselective acylation
of the primary alcohol with heptadecanoic acid and subsequent acylation of
the secondary alcohol on the glycerol moiety with pentadecanoic acid were
achieved by the action of N,N′-dicyclohexylcarbodiimide and 4-(dimethyla-
mino) pyridine in CH2Cl2 at −5 °C, affording the desired DGDG framework.
Finally, global deprotection furnished the targeted synthetic DGDG.
subjected to GC-MS [Shimadzu QP-2010SE with INERTCAP 5MS/SIL (0.25 mm
i.d., ×30 m); GL Science Inc.; column temperature 100 °C to 280 °C; rate
of temperature increase: 10 °C/min]. Part of the FAMEs sample was dissolved in
CCl4/CH3CN/H2O (1/1/1, each 50 μL), with added RuCl3/NaIO4 (1/1, each 50 μg),
and vigorously stirred at room temperature. After 1 h, the reaction mixture was
diluted with H2O (100 μL) and extracted with Et2O (100 μL). The Et2O was dried
with N2 gas and incubated in 20% MeOH/benzene (50 μL) and trimethylsilyl-
CH2CN (50 μL) at 50 °C for 30 min. The reaction mixture was dried with N2 gas,
and was diluted with n-hexane and subjected to GC-MS. The position of the
double bond in major unsaturated FAME (16:1) was determined by the identi-
fication of dimethyl undecanedioate [MeOCO-(CH2)9-COOMe] using RuCl3/NaIO4
oxidation followed by GC-MS analysis.
In Vitro Infection. BMDCs were plated in a 12-well plate and infected with the
GAS strains and incubated for 2 h in antibiotic-free medium at 37 °C in 5%
CO2. BMDCs were washed with PBS to remove unbound bacteria and further
incubated with complete medium in the presence of streptomycin (100 μg/mL)
and penicillin (57.6 μg/mL) at 37 °C in 5% CO2 for 16 h.
CD4+ T-Cell Responses. BMDCs from WT or Mincle-deficient mice were left
untreated or stimulated with indicated amounts of plate-coated purified
MGDG and TDM in the presence of OVA323–339 peptide. CD4+ T cells from
OT-II Tg mice were purified with anti−CD4-conjugated magnetic beads
(Miltenyi Biotec) and cocultured with OVA-pulsed BMDCs in 96-well plates.
On day 4, the supernatants were collected, and the concentrations of IFNγ and
IL-17 were determined by ELISA.
In Vitro Stimulation. GAS lipid extracts and TDM were dissolved in C:M (2:1;
vol/vol) or methanol, diluted with isopropanol, and added on wells, followed
by evaporation of the solvent as previously described (25). GAS hydrophilic
extracts were dissolved in distilled deionized H2O and added to the culture
medium. The 2B4-NFAT-GFP reporter cells were stimulated for 16 h, and the
activation of NFAT-GFP was monitored by FACSCalibur flow cytometry (BD
Biosciences). BMDCs (1 × 105 cells per well) were stimulated for 2 d, and the
culture supernatants were collected to determine the concentrations of each
cytokine by ELISA. Activation of BMDCs was evaluated using surface staining
of the costimulatory molecules such as CD40, CD80, and CD86 by flow
cytometry. For iNOS measurement, BMDCs were stimulated for 2 h and further
incubated for 16 h with 2 μM Brefeldin A. Intracellular iNOS was detected with
anti-NOS2 Ab by flow cytometry. For ROS measurement, BMDCs were stimu-
lated for 24 h and further incubated with 10 μM H2DCFDA at 37 °C for 30 min
followed by flow cytometry analysis.
GAS Infection. GAS strains were grown to late-log phase (OD600 = 0.7 to 0.9),
resuspended in PBS and injected intraperitoneally, as previously described
(57), into 5- to 6-wk-old Mincle-deficient or C57BL/6 male mice. Survival
curves were compared using a log-rank test in GraphPad Prism 6. For mea-
surement of bacterial load, at 48 h postinfection, 20 μL of peripheral blood
was removed from the tail vein by phlebotomy. The blood was diluted at
1:10 to 1:1,000 with PBS and spread on a Columbia agar plate containing
5% sheep blood (BD). To determine the number of CFUs in peripheral blood,
the plates were incubated for 20 h at 37 °C in 5% CO2, and the colonies
were counted. The number of CFUs was compared statistically using the
Mann–Whitney U test. The concentrations of cytokines and chemokines in
plasma were determined by FlowCytomix (eBioscience) according to the
manufacturer’s instructions.
In Vitro Mincle Binding Assay. Mincle-Ig fusion proteins were prepared as
described previously (72). In brief, the C terminus of the extracellular domain
of mouse Mincle (a.a. 46 to 214) was fused to the N terminus of hIgG1 Fc
region. Then 10 μg/mL of hIgG1-Fc (Ig) or Mincle-Ig in binding buffer (20 mM
Tris·HCl, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, pH 7.0) was incubated with
plate-coated lipids. Bound protein was detected with anti−hIgG-HRP fol-
lowed by the addition of colorimetric substrate. Peroxidase activity was
measured spectrophotometerically. For Mincle-Ig binding to intact GAS, live
GAS was incubated with Mincle-Ig (1 μg/mL) for 1 h at 4 °C. Bound proteins
were detected with anti−hIgG-Alexa 488 by flow cytometry.
Histology Analysis. The tissues of kidney, liver, and lung from GAS-infected
mice were fixed in 10% formalin/PBS. The paraffin-embedded sections
were stained with hematoxylin and eosin. The semiquantitative analysis
included measurement of abscesses in the kidney, and hepatocellular vac-
uoles in the liver using BZ-Χ analyzer (Keyence).
Statistics. An unpaired two-tailed Student’s t test was used for all statistical
analyses unless otherwise specified.
ESI-TOF-MS and NMR. ESI-TOF-MS was measured with a Bruker microTOF mass
spectrometer in the positive ESI mode (Bruker Daltonics). For NMR experi-
ments, 1H- and 13C-NMR spectra were recorded on an Agilent INOVA
600 spectrometer. The operating conditions were as follows: 1H: 600 MHz,
298 K, CDCl3/CD3OD/D2O (60:35:5; vol/vol/vol); 13C: 125 MHz, 298 K, CDCl3/
CD3OD/D2O (60:35:5; vol/vol/vol). The 1H- and 13C chemical shifts were
assigned by 2D-NMR (COSY, TOCSY, HSQC, HMBC) experiments. The 1H
chemical shift was referenced to trimethylsilane (δH 0), and the 13C chemical
shift was referenced to CD3OD (δC 49.0).
ACKNOWLEDGMENTS. We thank T. Hara for supervision; M. Kurata, S. Iwai,
X. Lu, and K. Motomura for technical support; M. Tanaka, Y. Baba, K. Kaseda, and
M. Ikawa for embryonic engineering; and the Cooperative Research Project
Program of the Medical Institute of Bioregulation, Kyushu University. This re-
search was supported by Japan Society for the Promotion of Science (JSPS)
KAKENHI Grants JP26293099 (to S.Y.), JP26110009 (to S.Y.), and JP16K09952
(to T. Matsumura); Japan Agency for Medical Research and Development
(AMED) under Grants JP17gm0910010 (to S.Y.), JP17ak0101070 (to S.Y.),
JP18fk0108075 (to S.Y.), and JP18fk0108044 (to T. Ikebe and T. Matsumura);
Vidi Grant 91713303 from the Netherlands Organization for Scientific Re-
search (to N.M.v.S.); National Health and Medical Research Council (NHMRC)
Grants APP1041294 and APP1057846 (to M.J.W. and C.A.W.); and the Austra-
lian Research Council Grant SRI110001002 (to C.A.W.). S.Y. acknowledges
funding from Takeda Science Foundation. B.L. acknowledges funding from
the Niedersachsen-Research Network on Neuroinfectiology.
GC-MS Analysis of MGDG and DGDG. For methanolysis, MGDG or DGDG (each
ca. 100 μg) was heated with 10% HCl/MeOH (100 μL) in a sealed tube at 90 °C
for 3 h. The reaction mixture was diluted with MeOH (0.4 mL) and extracted
with n-hexane (200 μL × 2), and the n-hexane extract was concentrated in
vacuo to give a mixture of FAMEs. The FAMEs were dissolved in acetone and
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