Detection of bacterial infection [Elektronische Ressource] : analysis of specificity and regulation as well as therapeutic blockade of toll-like receptor 2 and toll-like receptor 4 / Stephan Spiller

Stephan Strogoff

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Technische Universität München
Lehrstuhl für Technische Mikrobiologie



Detection of bacterial infection: Analysis of specificity and
regulation as well as therapeutic blockade of Toll-like receptor 2
and Toll-like receptor 4


Stephan Spiller


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. K. – H. Engel

Prüfer der Dissertation: 1. Univ.-Prof. Dr. R. F. Vogel
2. Priv.-Doz. Dr. C. Kirschning
3. Priv.-Doz. Dr. F. Ebel
(Ludwig-Maximilians-Universität München)


Die Dissertation wurde am 19.06.2008 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt am 05.08.2008 angenommen.
TABLE OF CONTENTS 2
TABLE OF CONTENTS
TABLE OF CONTENTS..............................................................................................2
INDEX OF FIGURES ..................................................................................................6
INDEX OF TABLES ....................................................................................................8
ABBREVIATIONS.......................................................................................................9
1 INTRODUCTION...................................................................................................13
1.1 Innate and adaptive immunity..................................................................................13
1.2 Innate immunity and the pattern recognition receptors (PRRs)...........................15
1.2.1 Toll-like receptors (TLRs)..................................................................................... 16
1.2.1.1 TLR signalling ............................................................................................. 18
1.2.1.2 Bacterial recognition through TLR4 and TLR2............................................ 22
1.2.1.3 Regulation of TLR signalling ....................................................................... 26
1.2.2 Non-Toll-like pattern recognition receptors .......................................................... 27
1.2.2.1 The NOD-like receptors (NLRs) .................................................................. 27
1.2.2.2 The RIG-like helicases (RLHs).................................................................... 28
1.3 Sepsis.........................................................................................................................29
1.3.1 Pathogenesis of sepsis ........................................................................................ 30
1.3.2 The role of TLRs in sepsis pathology................................................................... 31
1.4 Aims of the study......................................................................................................32
2 MATERIAL AND METHODS................................................................................33
2.1 Material.......................................................................................................................33
2.1.1 Equipment............................................................................................................ 33
2.1.2 Kits and enzymes................................................................................................. 34
2.1.3 Reagents.............................................................................................................. 34
2.1.4 Primers and probes.............................................................................................. 36
2.1.5 Vectors................................................................................................................. 38
2.1.6 Antibodies used for Western blot ......................................................................... 40
2.1.7 sed for flow cytometry ...................................................................... 40 TABLE OF CONTENTS 3
2.1.8 Antagonistic antibodies used for in vitro and in vivo experiments........................ 41
2.2 Methods .....................................................................................................................42
2.2.1 Cell biology........................................................................................................... 42
2.2.1.1 Culture of murine RAW 264.7 and human THP-1 macrophages ................ 42
2.2.1.2 Culture of HEK 293 cells ............................................................................. 42
2.2.1.3 Cryopreservation of cells 43
2.2.1.4 Generation/cultivation of human peripheral mononuclear cells (PBMCs)... 43
2.2.1.5 Transient and stable transfection of HEK293 cells...................................... 44
2.2.1.6 Generation of murine bone marrow-derived macrophages (BMDMos)....... 44
2.2.1.7 Generation of hybridoma, producing TLR4 specific antibodies................... 45
2.2.2 Molecular biology ................................................................................................. 47
2.2.2.1 Basic tools................................................................................................... 47
2.2.2.2 Agarose gel electrophoresis........................................................................ 48
2.2.2.3 Polymerase chain reaction (PCR)............................................................... 48
2.2.2.4 Restriction digestion, dephosphorylation and ligation of DNA fragments.... 49
2.2.2.5 Site-Directed Mutagenesis .......................................................................... 50
2.2.2.6 Heat-shock transformation of E. coli ........................................................... 51
2.2.2.7 DNA sequencing ......................................................................................... 51
2.2.3 Protein biochemistry............................................................................................. 51
2.2.3.1 Cell lysis ...................................................................................................... 51
2.2.3.2 Immunoprecipitation.................................................................................... 52
2.2.3.3 SDS-polyacrylamid gel electrophoresis (PAGE) of proteins ....................... 53
2.2.3.4 Western blot analysis .................................................................................. 54
2.2.3.5 Luciferase reporter gene assay................................................................... 55
2.2.4 Immunology.......................................................................................................... 56
2.2.4.1 Enzyme linked immunosorbent assay (ELISA) ........................................... 56
2.2.4.2 NO-Assay.................................................................................................... 58
2.2.4.3 Flow cytometry ............................................................................................ 58
2.2.5 Bacteria................................................................................................................ 59
2.2.6 Mice...................................................................................................................... 60
2.2.7 Animal experiments.............................................................................................. 60
2.2.7.1 Organ withdrawal ........................................................................................ 60
2.2.7.2 Blood withdrawal ......................................................................................... 60
2.2.7.3 OVA-Immunization using Myr CSK as an adjuvant.................................... 60 3 4
2.2.7.4 Sterile, PAMP-induced shock...................................................................... 61
2.2.7.5 Model for bacteria induced septic shock ..................................................... 61 TABLE OF CONTENTS 4
3 RESULTS .............................................................................................................62
3.1 Overlap in ligand specificity of TLR2 and TLR4.....................................................62
3.1.1 Myr CSK and LPS activate both overexpressed TLR2 and TLR4 ...................... 62 3 4
3.1.2 Analysis of endogenous TLR2 and TLR4 in terms of LPS and Myr CSK3 4
recognition............................................................................................................ 64
3.1.3 Blockade of murine and human TLR2 does not inhibit responsiveness to
Myr CSK 66 3 4
3.1.4 Systemic responsiveness towards Myr CSK and LPS ....................................... 66 3 4
3.1.5 TLR2 and TLR4 activation is not due respective contamination.......................... 68
3.1.6 Tri-myristoylation is required for TLR4 stimulation............................................... 69
3.1.7 Synthetic pneumolysin analogues are TLR2-specific stimuli ............................... 69
3.1.8 Exchange of relevant LRRs to analyze structural requirements determining ligand
specificity................................................................