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Mechanism of the adjuvant activity of the synthetic mycobacterial cord factor analog trehalose-6,6-dibehenate (TDB) [Elektronische Ressource] / Kerstin Althaus

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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Biofunktionalität der Lebensmittel Mechanism of the adjuvant activity of the synthetic mycobacterial cord factor analog Trehalose-6,6-dibehenate (TDB) Kerstin Althaus Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. M. Schemann Prüfer der Dissertation: 1. Univ.-Prof. Dr. D. Haller 2. Univ.-Prof. Dr. R. H. Lang (Universität Erlangen-Nürnberg) Die Dissertation wurde am 31.08.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 18.12.09 angenommen. Per aspera ad astra TABLE OF CONTENTS Table of contents 3 Index of figures 5 Index of tables 8 Abbreviations 9 1 INTRODUCTION 13 1.1 Vaccines and Vaccination 13 1.2 Adjuvants 16 1.3 Combination of a synthetic cord factor analog with DDA liposomes - a powerful new adjuvant system (CAF01) 20 1.3.

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Published 01 January 2009
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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Biofunktionalität der Lebensmittel






Mechanism of the adjuvant activity of the synthetic
mycobacterial cord factor analog Trehalose-6,6-dibehenate (TDB)



Kerstin Althaus





Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines



Doktors der Naturwissenschaften


genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. M. Schemann


Prüfer der Dissertation: 1. Univ.-Prof. Dr. D. Haller
2. Univ.-Prof. Dr. R. H. Lang
(Universität Erlangen-Nürnberg)




Die Dissertation wurde am 31.08.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 18.12.2009 angenommen.
















































Per aspera ad astra
TABLE OF CONTENTS

Table of contents 3

Index of figures 5

Index of tables 8

Abbreviations 9

1 INTRODUCTION 13

1.1 Vaccines and Vaccination 13

1.2 Adjuvants 16

1.3 Combination of a synthetic cord factor analog with DDA
liposomes - a powerful new adjuvant system (CAF01) 20
1.3.1 Biological effects of the mycobacterial cord factor
Trehalose-6,6-dimycolate (TDM) 20
1.3.2 Immunological analysis of the adjuvant system CAF01 22

1.4 Activation of antigen-presenting cells 24
1.4.1 Toll-like receptors and Toll-like receptor signaling 24
1.4.1.1 Myd88-dependent pathway 26
1.4.1.2 Myd88-independent pathway 28
1.4.2 C-type lectin receptors and their signaling 28

2 AIMS OF THE STUDY 35

3 MATERIAL AND METHODS 36
3.1 Material 36
3.1.1 Equipment 36
3.1.2 Consumable items, kits and enzymes 37
3.1.3 Reagents 38
3.1.4 Primers and probes 40
3.1.5 Antibodies 40
3.2 Methods 41
3.2.1 Cell biology 41
3.2.1.1 Media used for eukaryotic cell culture 41
3.2.1.2 Generation of murine bone-marrow-derived macrophages 41
3.2.1.3 Coating of cell culture dishes with TDB and TDM 42
3.2.1.4 Coating of polystyrene beads with TDB and TDM 42
3.2.2 Mice 42
3.2.3 Animal experiments 43
3.2.3.1 Preperation of adjuvant formulas 43
3.2.3.2 Immunization of mice with the fusion protein Ag85B-ESAT6 43
3.2.3.3 Removal of lymph nodes and heart blood 44
3.2.3.4 Measurement of antigen-specific IFN-γ and IL-17 production 44
3.2.3.5 Determination of antigen-specific antibody response 44
3.2.4 Molecular biology 45
3.2.4.1 Buffers and solutions 45
3.2.4.2 Agarosegel electrophoresis 45
3.2.4.3 Standard PCR 45
3.2.4.4 Total RNA extraction 46
3.2.4.5 First strand cDNA synthesis 46

3 TABLE OF CONTENTS

3.2.4.6 Quantitative real-time PCR 46
3.2.4.7 Microarray analysis of gene expression 47
3.2.5 Immunology 48
3.2.5.1 Enzyme linked immunosorbent assay (ELISA) 48
3.2.5.2 MTT assay 49
3.2.5.3 NO assay 50
4 RESULTS 51
4.1 Differential induction of Th-1 and Th-17 responses by CpG ODN
and TDB adjuvants 51

4.2 TDB and TDM are potent activators of APCs 52

4.3 In vitro, TDB/TDM induced APC activation is independent of TLR signaling 57

4.4 TDB and TDM activate APCs via a Syk-Card9-Malt1-dependent
signaling pathway 61

4.5 The TDB/TDM effect is independent of the C-type lectin Dectin-1 74

4.6 Recognition of TDB/TDM requires FcRγ 76

4.7 Adjuvanticity of TDB in vivo requires Myd88, but is largely
independent of TLR signaling 78

4.8 Adjuvanticity of TDB in vivo requires inflammasome formation,
but is independent of IL1R signaling 89

4.9 The Myd88-dependency of TDB/TDM in vivo is restricted to myeloid cells 95

4.10 Induction of protective immunity to challenge with TB requires Card9 97

5 DISCUSSION 101
5.1 TDB/TDM potently activate macrophages and DCs 101

5.2 Macrophages and DCs recognize TDB/TDM via
a FcRγ-Syk-Card9 pathway 103

5.3 TDB/TDM induce a Th-1 and Th-17 response 105

5.4 Myd88 is essentially required for TDB adjuvanticity in vivo,
but dispensable for APC activation in vitro 112

5.5 Outlook: Possible receptors for TDB and TDM 115

6 SUMMARY 120
7 REFERENCES 122
8 ACKNOWLEDGEMENTS 139
9 CURRICULUM VITAE 140


4 INDEX OF FIGURES

Index of Figures

Introduction

Figure1 Where do adjuvants act? 17
Figure 2 Properties of adjuvants 19
Figure 3 Chemical structure of Trehalose-6,6-dimycolate (TDM) 21
Figure 4 Chemical structure of Trehalose-6,6-dibehenate (TDB) 22
Figure 5 Toll-like receptors 25
Figure 6 Distinct myeloid C-type lectin receptors (CLRs) use
distinct proximal signaling mechanisms 30
Figure 7 The C-type lectin receptor Mincle triggers inflammatory responses
to necrotic cells 32
Figure 8 Intracellular consequences of dectin-1 stimulation 34

Results
Figure 1 Immunization scheme 51
Figure 2 TDB induces a Th-1/Th-17 response 52
Figure 3 TDB and TDM are potent activators of macrophages 53
Figure 4 TDB and TDM are potent inducers of NO production by macrophages 54
Figure 5 TDB and TDM are potent inducers of IL-6 and IL-1β
production by macrophages 56
Figure 6 TDB/TDM induced NO production by macrophages is independent
of Myd88 57
Figure 7 TDB/TDM induced NO production by macrophages is independent
of TLR2, 3, 4, 7 and 9 59
Figure 8 TDB/TDM induced IL-6 and IL-1β expression by macrophages is
independent of Myd88 60
Figure 9 TDB/TDM induced IL-6 expression by macrophages is independent
of TLR2, 3, 4, 7 and 9 61
Figure 10 TDB/TDM induced macrophage activation requires Syk activity 63
Figure 11 TDB/TDM induced NO production by macrophages depends on Card9 64
Figure 12 TDB/TDM induced IL-6 and IL-1β expression by macrophages depends
on Card9 65
Figure 13 TDB induced IL-6 and IL-1β expression by macrophages
depends on Malt1 66
Figure 14 TDB induced expression profile in macrophages depends on Card9 67
Figure 15 TDB induced expression of Mincle and Dectin-2 depends on
Syk-Card9-Malt1 activation 73
5 INDEX OF FIGURES

Figure 16 TDB induced activation of macrophages is independent of Dectin-1 75
Figure 17 TDB induced expression of IL-6 and IL-1β in macrophages is
independent of Dectin-1 76
Figure 18 TDB-induced expression of IL-6 and iNOS is independent of DAP12
but requires the adaptor protein FcRγ 77
Figure 19 TDB/TDM-induced activation of macrophages requires the adaptor
protein FcRγ 78
Figure 20 Deficiency of Myd88, but not of TLR2, 3, 4, 7 and 9 results in a
reduced footpad swelling after TDB-immunization 80
Figure 21 Deficiency of Myd88 and TLR9 results in reduced cell numbers
in draining LNs after TDB-immunization 82
Figure 22 Development of a Th-1 response depends on Myd88 and partial
on TLR9, but is independent of TLR2, 3, 4 and 7 84
Figure 23 Development of a Th-17 response depends on Myd88, but is
independent of TLR2, 3, 4, 7 and 9 86
Figure 24 Production of IgG2a antibodies is strictly dependent on Myd88,
but largely independent of TLR2, 3, 4, 7 and 9 88
Figure 25 Deficiency of ASC, but not of IL-1R results in a reduced footpad
swelling after TDB-immunization 90
Figure 26 Deficiency of ASC, but not of IL-1R results in reduced cell numbers
in draining LNs after TDB-immunization 91
Figure 27 Induction of a Th-1 response by TDB is independent of IL-1R
signaling and inflammasome formation 92
Figure 28 Induction of a Th-17 response by TDB is independent of IL-1R
signaling but requires inflammasome formation 93
Figure 29 TDB induces production of Th-1 specific IgG2a antibodies
independent of IL-1R signaling and inflammasome formation 94
Figure 30 The Myd88-dependence of the TDB adjuvant effect
is independent of lymphoid cells 96
Figure 31 Deficiency of Card9 results in reduced footpad swelling after
TDB immunization 97
Figure 32 Deficiency of Card9 results in reduced cell numbers in LNs after TDB,
but not after CpG immunization 98
Figure 33 Deficiency of Card9 results in a reduced Th-1/Th-17 response after
TDB/TDM immunization 99
Figure 34 Deficiency of Card9 results in reduced Th-1 specific antibody titers
after TDB immunization 100
6 INDEX OF FIGURES

Discussion
Figure 1 Schematic overview of the TDB/TDM induced effects on
APC activation and T cell priming 119
















































7 INDEX OF TABLES

Index of Tables

Material and Methods
Table 1 Oligonucleotides for real-time PCR 40
Table 2 Universal library probes for real-time PCR 40
Table 3 Detection antibodies for ELISA 40

Results
Table 1 Expression levels of TDB induced genes in bone-marrow-derived
macrophages of Card9-deficient and wild-type mice 69/70
Table 2 Expression levels of TDB and CpG induced genes in bone-marrow-
derived macrophages of wild-type mice 71

Discussion
Table 1 Receptor candidates for TDB/TDM recognition 118































8 ABBREVIATIONS

Abbreviations
°C Centigrade
µg Microgram
µl Microlitre
µM Micromolar
Ag85B Antigen 85B
AP-1 Activator protein 1
APC Antigen-presenting cell
ASC Apoptosis-associated speck-like protein
containing a Card domain
ATF-2 Activating transcription factor 2
BCG Bacillus Calmette-Guérin
BMDC Bone-marrow-derived dendritic cell
BMDM Bone-marrow-derived macrophage
BSA Bovine serum albumine
CCl CC motif chemokine ligand
CD Cluster of development
CFA Complete Freund’s adjuvant
CLD C-type lectin domain
Clec C-type lectin like receptor
CLR C-type lectin receptor
CpG Cytosine-guanosine oligonucleotide
CRD Carbohydrate recognition domain
CTL Cytotoxic T lymphocyte
CXCl CXC motif chemokine ligand
DC Dendritic cell
DDA Dimethyldioctadecylammonium bromide
ddH O Double distilled water 2
DMEM Dulbecco’s modified Eagle medium
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide tri-phosphate
ELISA Enzyme-linked immunosorbent assay
ERK Extracellular signal-regulated protein kinase
ESAT6 Early secretory antigenic target 6
9 ABBREVIATIONS

f.p. Footpad
FcγR Fc gamma receptor
h Hour
hep A Hepatitis A
HIV Human immunodeficiency virus
HPLC High-performance liquid chromatography
HPRT Hypoxanthine-guanine phosphoribosyltransferase
HPV Human papilloma virus
Hsp65 Heat shock protein 65
ICAM Intercellular adhesion molecule
IFA Incomplete Freund’s adjuvant
IFN Interferon
IKK Inhibitor of κB kinase
IL Interleukin
IL-1R Interleukin 1 receptor
iNKT cell Invariant natural killer T cells
iNOS Inducible nitric oxide synthase
IRAK IL-1 receptor-associated kinase
IRF Interferon regulatory factor
ISCOM Immunostimulating complexes
ITAM Immunoreceptor tyrosine-based activation motif
ITIM Immunoreceptor tyrosine-based inhibitory motif
JNK C-JUN N-terminal kinase
kDa Kilodalton
KO Knock out
LPS Lipopolysaccharide
LRR Leucin rich repeats
LysM Lysozm M
MAL Myd88-adaptor-like
Man-LAM Mannosylated lipoarabinomannan
MAPK Mitogen-activated protein kinase
MAPKK Mitogen-activated protein kinase kinase
MBL Mannose binding lectin
MCSF Macrophage colony-stimulating factor
10