Immune receptors Y Y. Multiple immune responses CLR TLR NLR ITAM RLR. C-type lectin. ITAM: Immunoreceptor Tyrosine-based Activation Motif
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1 C
2 Immune receptors Innate immunity Acquired immunity TLR CLR FcR BCR TCR C-type lectin Y Y Ig Ig NLR ITAM ITAM ITAM RLR Multiple immune responses ITAM: Immunoreceptor Tyrosine-based Activation Motif
3 Immune receptors Innate immunity Acquired immunity TLR CLR FcR BCR TCR C-type lectin? Y Y Ig Ig NLR ITAM ITAM ITAM ITAM RLR? Multiple immune responses ITAM: Immunoreceptor Tyrosine-based Activation Motif
4 Mincle (Macrophage inducible C-type lectin) Matsumoto et al. J. Immunol M 0 LPS (ng/ml) anti-mincle Mincle C? N + R C-type lectin domain C + ITAM adaptors Mouse Rat Human LSWTIAGASILFLSGCFITRCVV LSWTMAGASILFLSVCFITRCVV FLWTVAGIPILFLSACFITRCVV + Trans-membrane region N Y Y Y Y FcR DAP10 DAP12
5 Mincle is associated with FcR by charge IP: anti-flag Mincle Mincle R42I Mincle Flag-DAP12 Flag-DAP10 Flag-FcR R D I D FcR FcR anti-mincle Mincle IP: : anti-fcr anti-fcr anti-flag WT FcR + + Mincle-Flag R42I Total lysates anti-flag mab anti-mincle anti-fcr
6 Mincle induces inflammatory cytokines Peritoneal M Mincle TLR FcR MyD88 Inflammatory cytokines
7 Mincle induces inflammatory cytokines Peritoneal M Mincle? TLR FcR MyD88 Inflammatory cytokines
8 Establishment of indicator cell lines Peritoneal M? Mincle TLR non-myeloid T cell hybridoma FcR NFAT-GFP
9 Establishment of indicator cell lines Peritoneal M Plate-coated anti-mincle Y Y Mincle TLR non-myeloid T cell hybridoma FcR NFAT-GFP anti- Mincle
10 Mincle recognizes Dead cells PI Yamasaki, et al. Nat. Immunol. 2008
11 Mincle recognizes DAMPs Damage-associated molecular patterns PI Yamasaki, et al. Nat. Immunol. 2008
12 Mincle recognizes DAMPs Damage-associated molecular patterns PAMPs Pathogen-associated molecular patterns Malassezia PI 5 μm Yamasaki, et al. Nat. Immunol Yamasaki, et al. Proc. Natl. Acad. Sci. USA. 2009
13 Mincle may be a dual sensor for the danger Danger Damaged-self Mincle Non-self Pathogens Ligand SAP130 Fungus Yamasaki, et al. Nat. Immunol Yamasaki, et al. PNAS Inflammation Physiological advantage Repair? Host Defense? Potential risk Autoimmune? Skin/Lung disease? Granulomatous disease?
14 Mincle may be a dual sensor for the danger Danger Ligand Damaged-self SAP130 Mincle Non-self Fungus Pathogens Bacteria? Yamasaki, et al. Nat. Immunol Yamasaki, et al. PNAS Inflammation Physiological advantage Repair? Host Defense? Potential risk Autoimmune? Skin/Lung disease? Granulomatous disease?
15 Mycobacterium tuberculosis Research Institute of Tuberculosis
16 Mincle recognizes mycobacteria Lab strain Mincle NFAT-GFP? Yasu Morita Taroh Kinoshita
17 Mincle recognizes mycobacteria Lab strain BCG strain Virulent strain H37Rv Yasu Morita Taroh Kinoshita Hisakata Yamada Yasunobu Yoshikai
18 Mincle recognizes mycobacteria Mycobacteria rat IgG anti-mincle Y Mincle anti-mincle NFAT-GFP Mycobacterium smegmatis
19 What is the ligand for Mincle? TDM Trehalose dimycolate GMM Glucose monomycolate LAM Lipoarabino mannan Mycolate MDP Muramyl dipeptide Arabinogalactan Peptideglycan PIM Phosphatidyl inositol mannoside Transmembrane Mycobacterial cell wall
20 What is the ligand for Mincle? TDM Trehalose dimycolate Mycolate GMM Glucose monomycolate LAM Lipoarabino mannan MDP Muramyl dipeptide Solvents - Chloroform:MeOH - Hexane - Acetone - BuOH Whole mycobacteria Arabinogalactan Peptideglycan PIM Phosphatidyl inositol mannoside Transmembrane Insoluble Soluble extract Ligand? Ligand?
21 What is the ligand for Mincle? Mycobacterium smegmatis Insoluble fractions Mincle NFAT-GFP?
22 What is the ligand for Mincle? Mycobacterium smegmatis Insoluble C:M ; Chloroform:MeOH = 9:1
23 What is the ligand for Mincle? Mycobacterium smegmatis Insoluble C:M ; Chloroform:MeOH = 9:1 Soluble fractions evaporate reporter assay coated
24 Ligand was extracted into lipophilic fraction Mycobacterium smegmatis Insoluble Soluble fractions Soluble evaporate reporter assay coated C:M ; Chloroform:MeOH = 9:1
25 Ligand was extracted into lipophilic fraction Mycobacterium smegmatis Insoluble C:M ; Chloroform:MeOH Soluble Lipid?
26 TLC (Thin Layer Chromatography) Solvent front Sample spot C:M = 9:1
27 TLC (Thin Layer Chromatography) Fraction# C:M = 9:1 Yasu Morita Tetsuaki Ishikawa
28 Active fraction contains sugar and lipid moiety Orcinol stain purple Fraction# C:M = 9:1 Yasu Morita Tetsuaki Ishikawa
29 Active fraction contains sugar and lipid moiety Orcinol stain Glycolipid Fraction# R f C:M = 9:1
30 Active fraction contains sugar and lipid moiety Orcinol stain Glycolipid TDM Trehalose dimycolate Fraction# R f C:M = 9:1
31 TDM (trehalose 6,6 dimycolate)
32 TDM (trehalose 6,6 dimycolate) - unique mycobacterial glycolipid TDM Trehalose dimycolate GMM Glucose monomycolate LAM Lipoarabino mannan Mycolate MDP Muramyl dipeptide Arabinogalactan Peptideglycan PIM Phosphatidyl inositol mannoside Transmembrane Mycobacteria
33 TDM (trehalose 6,6 dimycolate) - unique mycobacterial glycolipid - immunostimulant called cord factor
34 Kekkaku 9:521-4., 1955 Lung cavity Cord factor (= TDM)
35 TDM (trehalose 6,6 dimycolate) - unique mycobacterial glycolipid - immunostimulant called cord factor - major adjuvant component of CFA
36 TDM (trehalose 6,6 dimycolate) - unique mycobacterial glycolipid - immunostimulant called cord factor - major adjuvant component of CFA - specific receptor??
37 TDM (trehalose 6,6 dimycolate) - unique mycobacterial glycolipid - immunostimulant called cord factor - major adjuvant component of CFA - specific receptor?? Mincle FcR
38 Direct receptor?
39 Mincle is a direct receptor for TDM TDM OD450 binding nm (OD 450 nm) Y anti-higg Mincle-Ig TDM
40 Mincle is a direct receptor for TDM anti-higg Mincle-Ig TDM Y Trehalose TDM OD450 binding nm (OD 450 nm)
41 Mincle-deficient mice?
42 Mincle is essential for TDM-induced M activation BMM NO Shizuo Akira
43 In vivo response?
44 TDM-induced systemic inflammation TDM i.v. Serum (day 1)
45 TDM-induced lung inflammation TDM i.v. Lung (day 0, 1, 3, 7) Day 0 Day 1 Day 3 Day 7 H&E staining 1 mm
46 TDM-induced lung inflammation TDM i.v. Lung (day 7) Day 0 Day 7 Day 7 WT Mincle / 0.1 mm H&E staining / lung Granuloma
47 TDM-induced lung inflammation TDM i.v. Lung (day 7) Day 0 Day 7 Day 7 WT 0.1 mm Mincle /
48 Mincle mediates granuloma-forming signal in vivo TDM i.v. Lung (day 7) Mincle FcR Granuloma
49 Synthetic adjuvant, TDB TDB Trehalose dibehenate TDM Trehalose dimycolate
50 TDB acts through Mincle BMM TDB Trehalose dibehenate
51 Evolutional struggle between host and mycobacteria? Mycobacteria Host immunity
52 Evolutional struggle between host and mycobacteria? Matsunaga, et al. J. Biol. Chem Mycobacteria 3. Convert Moody, et al. Nature 2000 Host immunity
53 Evolutional struggle between host and mycobacteria? Matsunaga, et al. J. Biol. Chem Mycobacteria 3. Convert APC Host immunity Moody, et al. Nature 2000
54 Evolutional struggle between host and mycobacteria? Matsunaga, et al. J. Biol. Chem Mycobacteria 3. Escape? 3. Convert APC Host immunity Moody, et al. Nature 2000
55 In vitro mimic of bacterial conversion TDM Trehalose dimycolate Conversion GMM Glucose monomycolate Mycobacteria Mycolyl- transferease + Glucose Glucose Mycolate OH HO Glucose Glucose OH HO Glucose In vitro mimic Mycolate Trehalase Mycolate
56 GMM conversion may be an escape strategy TDM Trehalose dimycolate Conversion GMM Glucose monomycolate Glucose HO Glucose OH In vitro mimic Trehalase Mycolate Mycolate Mincle NFAT-GFP
57 Evolutional struggle between host and mycobacteria? Matsunaga, et al. J. Biol. Chem Mycobacteria 3. Escape APC Host immunity Moody, et al. Nature 2000
58 Evolutional struggle between host and mycobacteria? Matsunaga, et al. J. Biol. Chem Mycobacteria 3. Escape 3. Convert Adjuvant APC Host immunity Moody, et al. Nature 2000 Prime?
59 Acknowledgments Eri Ishikawa Tetsuaki Ishikawa Kenji Toyonaga Yasunobu Miyake Daiki Mori Masahiro Nagaat (Kyushu Univ.) Yasu Morita Taroh Kinoshita (Osaka Univ.) Machie Sakuma Takashi Saito (RCAI, RIKEN) Makoto Matsumoto Osamu Takeuchi Kiyoshi Takeda Shizuo Akira (Osaka Univ.) Hisakata Yamada Akiko Oyamada Yasunobu Yoshikai (Kyushu Univ.)
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Subject Index Adaptive immunity dependence on innate immunity 9, 10 evolution 10 Aging anti-inflammatory agents in counteraction 202 beneficial polymorphisms 199 201 definition 18, 189 innate immunity
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