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

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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........................................................................................................................... ILIST OF ABBREVIATIONS...................................................................................................................IV 1 SUMMARY.................................................................................................................................... 1 1 ZUSAMMENFASSUNG................................................................................................................ 2 2 INTRODUCTION.....

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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........................................................................................................ 78
5.3.1 Double-stranded RNA is recognized by RIG-I............................................................. 78
5.3.2 Sensitivity of RIG-I to dsRNA is length-dependent...................................................... 79
5.3.3 Contribution of the terminal 5’-triphosphate to RIG-I signalling................................... 80
5.3.4 Single-stranded RNA is a ligand for RIG-I................................................................... 81
5.3.5 Outlook.........................................................................................................................83
6 BIBLIOGRAPH ........................................................................................................................... 85
7 PUBLICATIONS AND PRESENTATIONS................................................................................. 98
7.1 Publications............................................................................................................................... 98
7.2 Presentations............................................................................................................................. 98
8 ACKNOWLEDGMENT.............................................................................................................. 100


III List of abbreviations
List of abbreviations

APC antigen presenting cell
APS ammonium persulfate
Aqua dest aqua destillata

β-gal/BG β-galactosidase
BMDC bone marrow-derived dendritic cell
bp basepair(s)

Cardif Caspase activation recruitment domain inducing IFN- β
CD cluster of differentiation
cDNA complementary DNA
CLR C-type lectin receptor
c threshold cycle t

DAI DNA-dependent activator of IFN-regulatory factors
DEPC diethylpyrocarbonate
DC dendritic cell
DMSO dimethylsulfoxide
DNA deoxyribonucleicacid
DNase Deoxyribonuclease
dNTP deoxynucleoside triphosphate
DOTAP N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propan methyl-sulfate
ds double-stranded
DTT dithiothreitol

E.coli Escherichia coli
ECD extracellular domain
EDTA ethylene diamine tetraacetic acid
EGTA ethylene glycol tetraacetic acid
EGFP Enhanced green fluorescent protein
EIF2A eukaryotic translation initiation factor 2A
ELISA Enzyme-linked immunosorbent assay

FACS Fluorescence activated cell sorting
FCS Fetal calf serum
FITC Fluoresceinisothiocyanat
FLT3L FMS-like tyrosine kinase 3 ligand
fw forward
GAPDH Glycerinaldehyd-3-phosphate-dehydrogenase
GM-CSF Granulocyte macrophage-colony stimulating factor

IV List of abbreviations
h hour(s)
HCV hepatitis C virus
HEK293 human embryonal kidney cells
Huh human hepatocyte cell line

IFN interferon
IL interleukin
IPS-1 Interferon- β promoter stimulator 1
IRF Interferon regulatory factor
ISG interferon stimulated gene
ISRE mulated response element

K concentration required for half-maximal signalling 50
kDa kilo-dalton

LB Luria Bertani
LF Lipofectamine
LGP2 Laboratory of genetics and physiology 2
LPS Lipopolysaccharide
LRR Leucine rich repeat
LTA Lipoteichoic acid

MAPK Mitogen-activated kinase
MAVS Mitochondrial antiviral signalling
MDA5 Melanoma differentiation-associated gene 5
mDC myeloid dendritic cell
min Minute
mRNA messenger RNA
MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide
MyD88 Myeloid differentiation primary response protein

NALP NACHT-, LRR- and PYRIN-domain containing protein
n.d. not detected
NF κB Nuclear factor κB
NK cell Natural killer cell
NLR NOD (nucleotide-binding and oligomerization domain)-like receptor
n.t. not tested
nt nucleotide(s)

ODN Oligodeoxynucleotide

PAGE Polyacrylamide gel electrophoresis
PAMP Pathogen-associated molecular pattern
PBMC Peripheral blood mononuclear cell
PBS Phosphate buffered saline
V