RNA recognition in immune cells [Elektronische Ressource] / presented by Tina von Thülen

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RNA recognition in immune cells Dissertation for the degree of Doctor of Natural Sciences (Dr. rer. nat.) submitted to the Faculties of Pharmacy of the Philipps-University Marburg presented by Tina von Thülen born in Wilhelmshaven Marburg/Lahn 2010 Accepted from the Faculties of Pharmacy of the Philipps-University Marburg: _________________________________ Referees: Prof. Dr. Stefan Bauer Prof. Dr. Roland Hartmann Oral examination: ___________________________ II Dedicated to my parents III Human subtlety will never devise an invention more beautiful, more simple, or more direct than does Nature - because in her inventions, nothing is lacking - and nothing is superfluous… Leonardo da Vinci IV Table of contents 1. Introduction ............................................................... 1 1.1. Immunity- an overview ..................................................................................... 1 1.1.1. Innate immunity................................................................................................... 1 1.1.2. Pattern recognition receptors (PRRs) ................................................................. 2 1.1.3. Nucleic acids recognition .................................................................................... 6 1.2.

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RNA recognition in immune cells




Dissertation
for
the degree of
Doctor of Natural Sciences
(Dr. rer. nat.)



submitted to the
Faculties of Pharmacy
of the Philipps-University Marburg
presented by


Tina von Thülen
born in Wilhelmshaven

Marburg/Lahn 2010














Accepted from the Faculties of Pharmacy
of the Philipps-University Marburg: _________________________________


Referees: Prof. Dr. Stefan Bauer
Prof. Dr. Roland Hartmann



Oral examination: ___________________________
II


Dedicated to my parents




















III
Human subtlety will never devise an
invention more beautiful, more simple, or
more direct than does Nature - because in
her inventions, nothing is lacking - and
nothing is superfluous…
Leonardo da Vinci












IV
Table of contents

1. Introduction ............................................................... 1
1.1. Immunity- an overview ..................................................................................... 1
1.1.1. Innate immunity................................................................................................... 1
1.1.2. Pattern recognition receptors (PRRs) ................................................................. 2
1.1.3. Nucleic acids recognition .................................................................................... 6
1.2. Antimicrobial peptides (AMPs) ...................................................................... 10
1.3. TLR ligands as adjuvants............................................................................... 12
1.4. Hydrolysis of RNA........................................................................................... 13
2. Goal of the project................................................... 17
3. Material..................................................................... 19
3.1. Machines and technical devices.................................................................... 19
3.2. Software...........................................................................................................20
3.3. Equipment........................................................................................................ 20
3.4. Chemicals 21
3.5. Biochemicals, kits and enzymes ................................................................... 23
3.6. Antibodies 25
3.7. Size markers.................................................................................................... 26
3.8. Buffers and media........................................................................................... 26
3.9. Mice ..................................................................................................................28
X
3.10. Embryonated chicken eggs ........................................................................... 29
3.11. Transfection reagents..................................................................................... 29
3.12. Peptides ........................................................................................................... 29
3.13. Plasmids .......................................................................................................... 30
3.14. Oligonucleotides............................................................................................. 30
3.15. Ligands for stimulation .................................................................................. 30
3.16. Primer...............................................................................................................31
4. Methods.................................................................... 32
4.1. Cell culture.......................................................................................................32
4.1.1. Cell culture material .......................................................................................... 32
4.1.2. Cell lines............................................................................................................33
4.1.3. Culture media.................................................................................................... 34
4.1.4. Passage of eukaryotic cells............................................................................... 35
4.1.5. A/PR/8 infection of MDCK cells......................................................................... 35
4.1.6. Viable cell counts .............................................................................................. 36
4.1.7. Freezing and thawing of cells............................................................................ 36
4.1.8. Mycoplasma test ............................................................................................... 36
4.1.9. Generation of primary cells ............................................................................... 37
4.2. General nucleic acids techniques ................................................................. 40
4.2.1. Nucleic acid gel electrophoresis........................................................................ 40
4.2.2. Detection of nucleic acids from gels.................................................................. 43
4.2.3. Photometric concentration determination of nucleic acids................................ 43
4.2.4. Alcohol precipitation.......................................................................................... 44
XI
4.2.5. Phenol/chloroform extraction ............................................................................ 44
4.2.6. Micro Bio-Spin® 30 Columns 45
TM4.2.7. XBRIDGE OST C columns.......................................................................... 45 18
4.3. RNA techniques .............................................................................................. 46
4.3.1. Trizol RNA preparation from eukaryotic cells.................................................... 46
4.3.2. Isolation of the 18S rRNA from eukaryotic total RNA........................................ 47
4.3.3. Virus isolation by inoculation in embryonated eggs .......................................... 47
4.3.4. In vitro transcription........................................................................................... 50
4.3.5. Design of the RIG-I ligand 5`-3P RNA .............................................................. 51
4.3.6. Hydrolysis of RNAs with different RNase types ................................................ 53
4.3.7. Removing the 2`,3`-cyclic phosphate at the 3`-end of RNA.............................. 53
2+ 2+4.3.8. Fragmentation of RNA with Zn or Pb ........................................................... 54
4.3.9. Ultrasonic treatment.......................................................................................... 55
4.3.10. RNase III treatment of RNA .............................................................................. 55
4.3.11. CIP treatment.................................................................................................... 56
4.4. DNA techniques .............................................................................................. 56
4.5. Transfection with DOTAP or Lipofectamine 2000 for “in vitro” stimulation
..........................................................................................................................57
4.6. Transfection with LL-37.................................................................................. 59
4.7. FACS ................................................................................................................59
4.8. Immunofluorescence staining of LL-37/RNA complexes............................ 60
4.9. Cytokine detection by ELISA ......................................................................... 61
4.10. HPLC (high performance liquid chromatography)....................................... 64
4.11. Immunization................................................................................................... 65
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4.12. Electroporation................................................................................................66
5. Results ..................................................................... 68
5.1. Influence of RNA modifications at the 2`-position of ribose on immune
stimulation....................................................................................................... 68
5.1.1. Purified “natural” 18S rRNA in contrast to in vitro transcribed 18S rRNA ......... 68
5.1.2. 2`-O-ribose methylated synthetic RNA sequences from 18S rRNA do not
stimulate TLR7, but still stimulate TLR8............................................................ 72
5.1.3. Summary........................................................................................................... 75
5.2. Analysis of the immunostimulatory capacity of RNA from virus-infected
cells complexed to cationic lipids or natural carriers ................................. 76
5.2.1. Stimulation with RNA from virus-infected cells.................................................. 77
5.2.2. The immunostimulatory ability of RNA complexed to cathelicidins in human
immune cells ..................................................................................................... 78
5.2.3. Monocytes are the main source of IFN- α upon recognition of RNA from A/PR/8-
infected cells. ....................................................................................................79
5.2.4. Analysis of important RNA features for recognizing the A/PR/8/MDCK-RNA... 81
5.2.5. Analysis of the receptor recognizing the A/PR/8/MDCK-RNA .......................... 86
5.2.6. Influence of the length of antimicrobial peptides for the immunostimulatory
ability................................................................................................................. 94
5.2.7. Characterization of synthetic LL-37/CRAMP as carrier for immunostimulatory
RNA...................................................................................................................95
5.2.8. Immunization of mice ........................................................................................ 96
5.2.9. Summary........................................................................................................... 99
5.3. Analysis of the immunostimulatory capacity of self-RNA ........................ 100
5.3.1. Comparing the immunostimulatory ability of RNA fragments generated by
different techniques......................................................................................... 100
XIII
5.3.2. Characterization of the immunostimulatory nature of self-RNA fragments ..... 114
5.3.3. Immunostimulatory RNA species.................................................................... 115
5.3.4. Identification of cell types recognizing RNase A-derived fragments ............... 119
5.3.5. Summary......................................................................................................... 126
6. Discussion ............................................................. 127
6.1. Recognition of 2`-O-ribose methylated RNA .............................................. 127
6.1.1. RNA modifications........................................................................................... 127
6.1.2. Analysis of the immunostimulatory potential of eukaryotic and in vitro
transcribed 18S rRNA ..................................................................................... 128
6.1.3. Effect of modifications at the 2`-position of ribose concerning the stimulatory
potential of ssRNA .......................................................................................... 128
6.2. Recognition of RNA from influenza-infected cells..................................... 130
6.2.1. Recognition of an influenza virus infection...................................................... 130
6.2.2. Role of cathelicidins in viral infections and autoimmune diseases.................. 131
6.2.3. Monocytes are responsible for recognition of RNA from A/PR/8-infected cells
........................................................................................................................133
6.2.4. Features of RNA from virus-infected cells which are important for induction of
IFN- α ............................................................................................................... 133
6.2.5. TLR-independent recognition of A/PR/8/MDCK-RNA ..................................... 136
6.2.6. IPS-dependent recognition of A/PR/8/MDCK-RNA......................................... 137
6.2.7. Escape mechanism of viruses ........................................................................ 138
6.2.8. Localization studies of RNA/LL-37 complexes................................................ 139
6.2.9. Immunization with natural carrier for RNA ...................................................... 140
6.2.10. Relevance of LL-37/RNA complexes and the influence of the microenvironment
........................................................................................................................141
XIV
6.3. Immunorecognition of self-RNA .................................................................. 142
6.3.1. Discrimination of self-versus non-self-nucleic acids........................................ 142
6.3.2. RNase treatment of self-RNAs........................................................................ 143
6.3.3. Fragments generated by different techniques................................................. 145
6.3.4. Double-stranded character is important for IFN- α signaling............................ 149
6.3.5. Identification of the immunostimulatory RNA species..................................... 149
6.3.6. Monocytes are responsible for recognition of RNase A-derived fragments .... 151
6.3.7. Receptor for recognition of fragments from self-RNA ..................................... 152
6.3.8. Cell types and transfection reagents............................................................... 154
6.3.9. Relevance of the recognition of self-RNA fragments ...................................... 154
7. Summary................................................................ 156
8. Zusammenfassung................................................ 158
9. Literature 160
9.1. Paper & Books............................................................................................... 160
9.2. Doctoral thesis .............................................................................................. 175
9.3. Diploma thesis and Master thesis ............................................................... 175
10. Abbreviations and Units....................................... 176

Acknowledgements
Publications arising from this work
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