Seroprevalence Rates of Antibodies to Phenolic Glycolipid-I Among ...
C)&-/'*0$ !# %(.,0+ · - unique mycobacterial glycolipid PIM Phosphatidyl inositol mannoside LAM...
Transcript of C)&-/'*0$ !# %(.,0+ · - unique mycobacterial glycolipid PIM Phosphatidyl inositol mannoside LAM...
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ITAM�
TCR�BCR�FcR�
Ig�� Ig��
ITAM�ITAM�
Acquired immunity�Innate immunity�
Multiple immune responses�
Immune receptors�
ITAM: Immunoreceptor Tyrosine-based Activation Motif�
Y�Y�
CLR�C-type lectin�
TLR�
NLR�
RLR�
TCR�BCR�FcR�
Y�Y�?�
Ig�� Ig��
TLR� CLR�
NLR�
RLR�
ITAM�
Acquired immunity�Innate immunity�
Immune receptors�
?�
ITAM�ITAM�ITAM�
Multiple immune responses�
C-type lectin�
ITAM: Immunoreceptor Tyrosine-based Activation Motif�
Mincle (Macrophage inducible C-type lectin)�
LSWTIAGASILFLSGCFITRCVV�LSWTMAGASILFLSVCFITRCVV�FLWTVAGIPILFLSACFITRCVV�Human�
Trans-membrane region�
+�
R�N� C�C-type lectin domain�
Y�Y�
Y�Y�
–�–�
?�Mincle�
+�
N�
C�
Matsumoto et al. J. Immunol. 1999�
adaptors�
Mouse�
Rat�
+�
FcR��DAP10�DAP12�
anti-Mincle�
0.01� 0.1� 1� 10�
LPS (ng/ml)�
0�
ITAM�
M� �
Mincle-FlagFcR� + +
WT R42I
anti-FlagmAb
IP: anti-FcR�
anti-FcR�
anti-Mincle�
anti-Flag�
anti-Mincle�
IP: anti-Flag�
Total lysates�
Mincle�
Mincle�Flag-DAP12�
Flag-FcR��Flag-DAP10�
+�+�–�–�
+�–�+�–�
+�–�–�+� FcR��
R�D� I� D�
FcR��
Mincle� MincleR42I�
IP : anti-FcR��
–�+� –�
Mincle is associated with FcR� by charge�
Mincle induces inflammatory cytokines�
FcR��
Mincle�
Inflammatory�cytokines�
Peritoneal M� �TLR�
MyD88�
Mincle induces inflammatory cytokines�
FcR��
Mincle�
Inflammatory�cytokines�
?�Peritoneal M� �TLR�
MyD88�
Peritoneal M� �
FcR��
Mincle�
NFAT-GFP�
–�
T cell�hybridoma�
TLR�
?�
Establishment of indicator cell lines�
non-myeloid�
Establishment of indicator cell lines�
Peritoneal M� �
FcR��
Mincle�
NFAT-GFP�
–�Y�
anti-Mincle�
Y�anti-�
Mincle�
Plate-coated�
T cell�hybridoma�
TLR�
–�
non-myeloid�
Mincle recognizes�
Dead cells�
PI �
Yamasaki, et al. Nat. Immunol. 2008�
Mincle recognizes�
DAMPs�Damage-associated molecular patterns�
PI �
Yamasaki, et al. Nat. Immunol. 2008�
Mincle recognizes�
Malassezia�
5 μm�
DAMPs�Damage-associated molecular patterns�
PAMPs�Pathogen-associated molecular patterns�
Yamasaki, et al. Proc. Natl. Acad. Sci. USA. 2009�Yamasaki, et al. Nat. Immunol. 2008�
PI �
Damaged-self� Non-self�Danger�
Mincle may be a dual sensor for the “danger” �
Ligand�Yamasaki, et al.� Nat. Immunol. 2008�
Yamasaki, et al.� PNAS. 2009�
Inflammation�
Mincle�
Fungus�SAP130�
Repair?�
Autoimmune?�
Host �Physiological�advantage�
Potential risk�
Defense?�
Pathogens�
Skin/Lung disease?�Granulomatous disease?�
Damaged-self� Non-self�Danger�
Mincle may be a dual sensor for the “danger” �
Ligand�Yamasaki, et al.� Nat. Immunol. 2008�
Yamasaki, et al.� PNAS. 2009�
Inflammation�
Mincle�
Fungus�SAP130�
Repair?�
Autoimmune?�
Host �Physiological�advantage�
Potential risk�
Defense?�
Pathogens�
Bacteria�
Skin/Lung disease?�Granulomatous disease?�
?�
Mycobacterium tuberculosis�
�
Research Institute of Tuberculosis�
Mincle recognizes mycobacteria�
Lab strain�
Mincle�
?�NFAT-GFP�
Yasu Morita�Taroh Kinoshita�
Mincle recognizes mycobacteria�
BCG strain� Virulent strain�
H37Rv�
Yasu Morita�Taroh Kinoshita�
Hisakata Yamada�Yasunobu Yoshikai�
Lab strain�
Mincle�
NFAT-GFP�
Y�Mycobacteria�
anti-Mincle�
Mycobacterium smegmatis�
NFAT-GFP
anti-Mincle�
rat IgG�
Mincle recognizes mycobacteria�
PIM �Phosphatidyl�inositol �mannoside�
LAM �Lipoarabino�mannan�
GMM�Glucose�monomycolate�
Myc
ola
te�
Peptideglycan�
Transmembrane�
Mycobacterial cell wall�
TDM�Trehalose�dimycolate�
MDP �Muramyl �dipeptide�
Arabinogalactan�
What is the ligand for Mincle?�
Soluble extract�Insoluble�
Ligand?�
- Chloroform:MeOH�- Hexane�- Acetone�- BuOH�
Solvents�
Ligand?�
PIM �Phosphatidyl�inositol �mannoside�
LAM �Lipoarabino�mannan�
GMM�Glucose�monomycolate�
Myc
ola
te�
Peptideglycan�
Transmembrane�
TDM�Trehalose�dimycolate�
MDP �Muramyl �dipeptide�
Arabinogalactan�
What is the ligand for Mincle?�
Whole�mycobacteria�
Mycobacterium smegmatis�
What is the ligand for Mincle?�
Insoluble fractions�
Mincle�
NFAT-GFP�?�
Mycobacterium smegmatis�
Insoluble�
What is the ligand for Mincle?�
C:M ; Chloroform:MeOH = 9:1�
Mycobacterium smegmatis�
Soluble fractions�
evaporate� reporter assay�
coated�
Insoluble�
What is the ligand for Mincle?�
C:M ; Chloroform:MeOH = 9:1�
Ligand was extracted into “lipophilic” fraction �
Mycobacterium smegmatis�
Soluble �
evaporate� reporter assay�
coated�
Insoluble�
C:M ; Chloroform:MeOH = 9:1�
Soluble fractions�
Mycobacterium smegmatis�
Soluble �
C:M ; Chloroform:MeOH�
Insoluble�
Lipid?�
Ligand was extracted into “lipophilic” fraction �
TLC (Thin Layer Chromatography)�
C:M = 9:1�
Solvent front�
Sample spot�
Yasu Morita�Tetsuaki Ishikawa�
C:M = 9:1�
TLC (Thin Layer Chromatography)�
Fra
ctio
n#�
Yasu Morita�Tetsuaki Ishikawa�
Fra
ctio
n#�
C:M = 9:1�
Fra
ctio
n#�
Orcinol stain�
purple�
Active fraction contains sugar and lipid moiety�
Rf�
Active fraction contains sugar and lipid moiety�
C:M = 9:1�
Glycolipid�Orcinol stain�
Fra
ctio
n#�
Fra
ctio
n#�
Active fraction contains sugar and lipid moiety�Glycolipid�
Rf�
C:M = 9:1�
TDM�Trehalose dimycolate�
Orcinol stain�
TDM (trehalose 6,6� dimycolate)�
TDM (trehalose 6,6� dimycolate)�
- unique mycobacterial glycolipid�
PIM �Phosphatidyl�inositol �mannoside�
LAM �Lipoarabino�mannan�
GMM�Glucose�monomycolate�
Mycolate�
Peptideglycan�
Transmembrane�
Mycobacteria�
TDM�Trehalose�dimycolate�
MDP �Muramyl �dipeptide�
Arabinogalactan�
TDM (trehalose 6,6� dimycolate)�
- unique mycobacterial glycolipid�
- immunostimulant called “cord factor”�
Kekkaku 9:521-4., 1955�
Cord factor�
Lung cavity�
(= TDM)�
TDM (trehalose 6,6� dimycolate)�
- unique mycobacterial glycolipid�
- major adjuvant component of CFA�
- immunostimulant called “cord factor”�
TDM (trehalose 6,6� dimycolate)�
- specific receptor??�
- major adjuvant component of CFA�
- unique mycobacterial glycolipid�
- immunostimulant called “cord factor”�
TDM (trehalose 6,6� dimycolate)�
- specific receptor??�
FcR��
Mincle�
- immunostimulant called “cord factor”�
- unique mycobacterial glycolipid�
- major adjuvant component of CFA�
Direct receptor?�
OD
450
nm�
Y�TDM�
Mincle-Ig�
anti-hIgG�
Mincle is a direct receptor for TDM�
TD
M b
indi
ng (O
D 4
50 n
m)�
Y�TDM�
Mincle-Ig�
anti-hIgG�
OD
450
nm�
TD
M b
indi
ng (O
D 4
50 n
m)�
Trehalose�
Mincle is a direct receptor for TDM�
Mincle-deficient mice?�
Mincle is essential for TDM-induced M�� activation�
BMM��
NO
�
Shizuo Akira �
In vivo response?�
TDM i.v.�Serum (day 1)�
TDM-induced systemic inflammation�
TDM i.v.�Lung (day 0, 1, 3, 7)�
TDM-induced lung inflammation�
Day 0� Day 1� Day 3� Day 7�
H&
E s
tain
ing�
1 mm�
TDM i.v.�Lung (day 7)�
WT�
Min
cle–
/–�
H&E staining / lung�
Granuloma�
Day 0� Day 7�Day 7�
0.1 mm�
TDM-induced lung inflammation�
�
TDM i.v.�Lung (day 7)�
WT�
Mincle–/–�
Day 0� Day 7�Day 7�
0.1 mm�
TDM-induced lung inflammation�
TDM i.v.�Lung (day 7)�
Mincle mediates granuloma-forming signal in vivo�
FcR��
Mincle�
Granuloma�
�
TDM�TDB�
Synthetic adjuvant, TDB�
Trehalose�dibehenate�
Trehalose�dimycolate�
TDB acts through Mincle�
TDB�Trehalose�
dibehenate�
BMM��
Mycobacteria�
Host immunity�
Evolutional struggle between host and mycobacteria?�
Mycobacteria�
Host immunity�
Matsunaga, et al. �J. Biol. Chem. 2008�
Moody, et al. �Nature 2000�
3. Convert�
Evolutional struggle between host and mycobacteria?�
Mycobacteria�
Host immunity�
3. Convert�
Matsunaga, et al. �J. Biol. Chem. 2008�
Evolutional struggle between host and mycobacteria?�
Moody, et al. �Nature 2000�
APC�
Mycobacteria�
Host immunity�
Matsunaga, et al. �J. Biol. Chem. 2008�
3. Convert�
Evolutional struggle between host and mycobacteria?�
Moody, et al. �Nature 2000�
APC�
3. Escape?�
Trehalase�
TDM� GMM�
In vitro mimic�
OH�
HO�
Myc
olat
e�
Myc
olat
e�
In vitro mimic of bacterial conversion�
Glucose�Glucose�
Trehalose dimycolate� Glucose monomycolate�
+ Glucose�
Mycobacteria�
Mycolyl-�transferease�
Glucose�Glucose�
Myc
olat
e�
Conversion�
HO�
OH�
Trehalase�In vitro mimic�
Myc
olat
e�
Myc
olat
e�
Glucose�Glucose�
GMM conversion may be an escape strategy �
OH�
HO�
Mincle�
NFAT-GFP�
TDM� GMM�Trehalose dimycolate� Glucose monomycolate�
Conversion�
Evolutional struggle between host and mycobacteria?�
Mycobacteria�
Host immunity�
Matsunaga, et al. �J. Biol. Chem. 2008�
Moody, et al. �Nature 2000�
3. Escape�
APC�
Mycobacteria�
Host immunity�
Matsunaga, et al. �J. Biol. Chem. 2008�
3. Convert�
Evolutional struggle between host and mycobacteria?�
APC�
Adjuvant�
3. Escape�
Prime?�
Moody, et al. �Nature 2000�
Acknowledgments�
Machie Sakuma�Takashi Saito�
Hisakata Yamada�Akiko Oyamada�Yasunobu Yoshikai�
(RCAI, RIKEN)�
(Kyushu Univ.)�
Makoto Matsumoto�Osamu Takeuchi�Kiyoshi Takeda�Shizuo Akira�
(Osaka Univ.)�
Eri Ishikawa�Tetsuaki Ishikawa�Kenji Toyonaga�Yasunobu Miyake�Daiki Mori�Masahiro Nagaat�
(Kyushu Univ.)�
Yasu Morita�Taroh Kinoshita�
(Osaka Univ.)�