C)&-/'*0$ !# %(.,0+ - NIBIOHN · FcR BCR TCR? Y Y Ig Ig TLR CLR NLR RLR ITAM Innate immunity Acquired immunity Immune receptors? ITAM ITAM ITAM Multiple immune responses C-type lectin

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C �

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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.)�