Activation of innate immunity by ribonucleic acids [Elektronische Ressource] / presented by Florian Eberle

Florian Eberle

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

ACTIVATION OF INNATE IMMUNITY BY
RIBONUCLEIC ACIDS



DISSERTATION
submitted to the Combined Faculties for the
Natural Sciences and for Mathematics
of the Ruprecht-Karls University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















presented by
Dipl.-Biochem. Florian Eberle
born in Tübingen



DISSERTATION





submitted to the Combined Faculties for the
Natural Sciences and for Mathematics
of the Ruprecht-Karls University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences











presented by
Dipl. Biochem. Florian Eberle
born in Tübingen

Oral examination: ………………….
ACTIVATION OF INNATE IMMUNITY BY
RIBONUCLEIC ACIDS



















Referees: Prof. Dr. Ralf Bartenschlager
Prof. Dr. med. Alexander Dalpke




MEINEN ELTERN
Table of contents
TABLE OF CONTENTS........................................................................................................................... I
LIST OF ABBREVIATIONS...................................................................................................................IV
1 SUMMARY.................................................................................................................................... 1
1 ZUSAMMENFASSUNG................................................................................................................ 2
2 INTRODUCTION........................................................................................................................... 3
2.1 Innate immunity 3
2.2 Recognition principles within the innate immune system...................................................... 3
2.2.1 Pattern recognition receptors......................................................................................... 4
2.2.2 Cooperative recognition in innate immunity................................................................... 6
2.3 Ligands of pattern recognition receptors................................................................................. 6
2.3.1 Nucleic acids: pathogen associated molecular patterns................................................ 6
2.3.2 Recognition of RNA ....................................................................................................... 7
2.4 RNA interference....................................................................................................................... 12
2.4.1 Small interfering RNA as molecular tool for gene suppression ................................... 13
2.5 Objectives of this work............................................................................................................. 14
3 MATERIALS AND METHODS ................................................................................................... 15
3.1 Materials..................................................................................................................................... 15
3.1.1 Chemicals and enzymes.............................................................................................. 15
3.1.2 Kits 16
3.1.3 Buffers and solutions 16
3.1.4 Primer for quantitative real-time RT-PCR.................................................................... 18
3.1.5 Primer for in vitro transcription..................................................................................... 19
3.1.6 Plasmids ...................................................................................................................... 20
3.1.7 Markers ........................................................................................................................ 20
3.1.8 Oligonucleotides .......................................................................................................... 20
3.1.9 Antibodies .................................................................................................................... 22
3.1.10 Bacteria and cell culture .............................................................................................. 22
3.1.11 Mouse strains............................................................................................................... 24
3.2 Methods...................................................................................................................................... 24
3.2.1 Cell biology .................................................................................................................. 24
3.2.2 Molecular biology......................................................................................................... 28
3.2.3 Biochemistry ................................................................................................................ 32
3.2.4 Statistical analysis, data transformations and structure predictions............................ 34
I Table of contents
4 RESULTS.................................................................................................................................... 35
4.1 Characterization of siRNA-mediated immunostimulation .................................................... 35
4.1.1 RNA oligonucleotide mediated immunostimulation ..................................................... 35
4.1.2 Immunostimulation of fuctional siRNAs ....................................................................... 36
4.1.3 Analysis of immunostimulatory sequence motifs......................................................... 38
4.1.4 Analysis of nucleotide modifications and their effects on immunostimulation............. 38
4.1.5 Gene-silencing activity of modification-containing siRNAs.......................................... 41
4.1.6 Effect of thymidine residues in both strands of the siRNA duplex............................... 43
4.1.7 Antagonistic effects on RNA-induced interferon- α secretion....................................... 43
4.1.8 Secondary structure formation of RNA oligonucleotides............................................. 44
4.1.9 Positional influence of thymidine modifications on immunostimulation....................... 46
4.1.10 Summary...................................................................................................................... 47
4.2 Immunorecognition of prokaryotic RNA................................................................................. 47
4.2.1 Purification and validation of prokaryotic RNA ............................................................ 47
4.2.2 Bacterial RNA induces a type-I interferon response in human PBMCs ...................... 48
4.2.3 Bacterial RNA induces IL-12p40 in murine dendritic cells........................................... 51
4.2.4 Bacterial RNA induces an immune response in non-professional immune cells ........ 56
4.2.5 Summary 59
4.3 Ligand specificity of RIG-I........................................................................................................ 60
4.3.1 Characterization of RIG-I expressing Huh7.5 cells...................................................... 60
4.3.2 Recognition of different RNAs by RIG-I....................................................................... 61
4.3.3 RIG-I-ligand interactions .............................................................................................. 66
4.3.4 Summary...................................................................................................................... 68
5 DISCUSSION.............................................................................................................................. 69
5.1 siRNA-mediated immunostimulation ...................................................................................... 69
5.1.1 Sequence-dependent recognition of siRNAs............................................................... 70
5.1.2 Nucleotide modifications within the minor groove of RNA duplexes reduce
immunostimulation....................................................................................................... 70
5.1.3 2’-O-methyl-modifications inhibit TLR7 signalling........................................................ 72
5.1.4 Nucleotide modifications influence RNAi differentially................................................. 72
5.1.5 TLR7 might detect double-stranded structures ........................................................... 73
5.2 Immunorecognition of prokaryotic RNA................................................................................. 74
5.2.1 TLR7-mediated recognition of prokaryotic RNA.......................................................... 75
5.2.2 Prokaryotic RNA triggers (a) receptor(s) apart from TLR7.......................................... 76
5.2.3 Viral RNA receptors and NALP3 are dispensable for prokaryotic RNA–induced
NF κB activation............................................................................................................ 76
5.2.4 Bacterial RNA induces a general danger signal 77
II Table of contents
5.3 Ligand specificity of RIG-I.................