Plasmacytoid dendritic cells and neutrophils – underestimated cell populations during the onset of atherosclerosis [Elektronische Ressource] / Yvonne Döring

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

PLASMACYTOID DENDRITIC CELLS AND NEUTROPHILS –
UNDERESTIMATED CELL POPULATIONS DURING THE ONSET OF
ATHEROSCLEROSIS


VON DER FAKULTÄT FÜR MATHEMATIK, INFORMATIK UND NATURWISSENSCHAFTEN DER
RWTH AACHEN UNIVERSITY ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINER
DOKTORIN DER NATURWISSENSCHAFTEN GENEHMIGTE DISSERTATION

VORGELEGT VON


MASTER OF SCIENE
YVONNE DÖRING
AUS LAUTERBACH IN HESSEN, DEUTSCHLAND



BERICHTER:
UNIVERSITÄTSPROFESSOR DR. MED. CHRISTIAN WEBER
UNIVERSITÄTSPROFESSOR DR. RER. NAT. MARTIN ZENKE


TAG DER MÜNDLICHEN PRÜFUNG: 07.07.2011

DIESE DISSERTATION IST AUF DER INTERNETSEITE DER HOCHSCHULBIBLIOTHEK ONLINE
VERFÜGBAR.

THE RESULTS OF THIS WORK WILL BE PARTLY PUBLISHED IN:


Döring, Y.*, Soehnlein, O.*, Drechsler, M., Meiler, S., Shagdarsuren, E., Hartwig, H.,
Hieronymus, T., Hristov, M., Koenen, R.R., Zenke, M., Weber, C., and Zernecke, A.,
Chronic myelogenous leukemia-like disease due to hematopoietic IRF8-deficiency fuels
atherosclerosis in mice. Circulation Research, 2011 (in revision).

Döring, Y., Manthey, H., Drechsler, M., Lievens, D., Manca, M., Hartwig, H., Busch, M.,
Koenen, R.R., Soehnlein, O., Zenke, M., Daemen, M.J., Weber, C., Lutgens, E., and
Zernecke, A., Autoantigenic protein-DNA complexes stimulate plasmacytoid dendritic
cells to promote atherosclerosis. Circulation, 2011 (in revision).
















TABLE OF CONTENTS

TABLE OF CONTENTS
TABLE OF CONTENTS ....................................................................................................... III
ABBREVIATIONS .............. VI
1 INTRODUCTION ............... 1
1.1 THE IMMUNE SYSTEM ............................................................................................... 1
1.2 CELLS OF THE IMMUNE SYSTEM ................................................................................ 2
1.2.1 NEUTROPHILS ... 2
1.2.2 PLASMACYTOID DENDRITIC CELLS....... 7
1.2.3 OTHER CELLS ................................................................................................. 13
1.3 ATHEROSCLEROSIS ............................... 15
1.3.1 NEUTROPHILS IN ATHEROSCLEROSIS 17
1.3.2 PLASMACYTOID DENDRITIC CELLS IN ATHEROSCLEROSIS ................................... 18
1.4 AIM OF THE STUDY ................................................................. 20
2 MATERIALS AND METHODS ........................... 21
2.1 GENERAL EQUIPMENT ............................ 21
2.2 MICE ..................................................................................................................... 22
2.3 CYTOKINES AND RECOMBINANT PROTEINS ............................... 22
2.4 MISCELLANEOUS REAGENTS ................... 23
2.5 TOLL LIKE RECEPTOR STIMULI ................................................................................. 23
2.4 ANTIBODIES........................................... 24
2.4.1 PRIMARY ANTIBODIES ...................... 24
2.4.2 DIRECTLY CONJUGATED ANTIBODIES ................................................................ 24
2.4.3 SECONDARY ANTIBODIES ................................................................................. 25
2.4.4 DEPLETION ANTIBODIES .................. 25
2.5 CELL CULTURE ...................................................................................................... 26
2.5.1 PREPARATION OF PRIMARY CELL SUSPENSIONS ................ 26
2.5.2 ISOLATION OF DCS .......................................................................................... 27
+2.5.3 ISOLATION OF CD4 T CELLS ............ 27
2.5.4 CELL SORTING ................................ 27
2.5.5 BLOOD SMEAR STAINING ................................................................................. 28
2.6 MOLECULAR METHODS .......................... 28
2.6.1 ISOLATION OF DNA ......................... 28
2.6.2 ISOLATION OF RNA ................................................................ 28
III TABLE OF CONTENTS

2.6.3 QUANTIFICATION OF DNA AND RNA ................................................................. 28
2.6.2 QUANTATIVE REAL-TIME POLYMERASE CHAIN REACTION (PCR) .......................... 28
2.7 PROTEIN ASSAYS .................................. 30
2.7.1 FLOW CYTOMETRY ................................ 30
2.7.2 BEAD ARRAY ................................... 31
2.7.3 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) ........... 31
2.7.4 ANALYSIS OF MPO AND MMP-9 ACTIVITY AND ROS FORMATION ........................ 32
2.7.5 HISTOCHEMISTRY ............................................................................................ 32
2.7.6 IMMUNOFLUORESCENCE .................. 33
2.8 FUNCTIONAL ASSAYS ............................. 33
2.8.1 APOPTOSIS AND CELL CYCLE ASSAYS ............................................................... 33
2.8.2 PHAGOCYTOSIS ASSAYS .................................................. 33
2.8.3 IN VITRO STIMULATION OF PDCS ....................................... 34
2.9 ANIMAL MODELS .................................................................... 34
2.9.1 MOUSE MODELS OF ATHEROSCLEROTIC DISEASE .............. 34
2.9.2 BONE MARROW TRANSPLANTATION .................................. 35
2.9.3 PERITONITIS ................................................................... 35
2.9.4 SUBCUTANEOUS AIR POUCH ............ 36
2.9.5 T CELL PROLIFERATION IN VIVO ........................................ 36
2.10 DATA ILLUSTRATION AND STATISTICAL ANALYSIS .................... 36
3. RESULTS ................................................................................................ 37
3.1 RESULT OUTLINE ................................................................................................... 37
3.2 ROLE OF HEMATOPOIETIC IRF8 IN ATHEROSCLEROTIC LESION FORMATION AND ITS
EFFECTS ON MONOCYTE/MACROPHAGE AND PMN FUNCTIONS RELATED TO
ATHEROSCLEROSIS ...................................................................................................... 39
-/- -/-3.2.1 APOE MICE TRANSPLANTED WITH IRF8 BM DISPLAY A CML-LIKE PHENOTYPE .. 39
-/- -/-3.2.2 INCREASED ATHEROSCLEROTIC LESION FORMATION IN IRF8 ►APOE MICE ....... 41
3.2.3 MACROPHAGE FUNCTIONS BUT NOT ACCUMULATION OR APOPTOSIS ARE IMPAIRED
-/- IN IRF8 MICE .......................................................................................................... 45
-/-3.2.4 THE INFLAMMATORY ACCUMULATION OF IRF8 PMN IS DUE TO ENHANCED
EXTRAVASATION ...... 49
-/-3.2.5 ROS FORMATION AND GRANULE DISCHARGE ARE NOT IMPAIRED IN IRF8 PMN .. 51
-/- -/-3.2.6 PMN-DEPLETION IN IRF8 ►LDLR MICE PREVENTS AGGRAVATED ATHEROS-
CLEROTIC LESION FORMATION ................................................................................... 53
IV TABLE OF CONTENTS

3.3 ROLE OF PLASMACYTOID DENDRITIC CELLS IN THE DEVELOPMENT OF
ATHEROSCLEROSIS ...................................................................................................... 56
-/-3.3.1 PDCS ARE PRESENT IN ATHEROSCLEROTIC LESIONS OF APOE MICE ................. 56
3.3.2 MANIPULATION OF PDC COUNTS AND ACTIVITY CORRELATES WITH
ATHEROSCLEROTIC LESION FORMATION ..................................................................... 58
3.3.3 ALTERATIONS OF PDC FUNCTIONS IN THE PRESENCE OF OXLDL TREATMENT ...... 62
3.3.4 ACTIVATION OF PDCS BY AUTOIMMUNE MECHANISMS ........ 65
4. DISCUSSION................................................................................................................ 69
4.1 IRF8-DEFIENCY EXACERBATES ATHEROSCLEROSIS .................. 69
-/-4.1.1 IRF8 MACROPHAGES DISPLAY IMPAIRED FUNCTIONALITY ................................... 69
4.1.2 MONOCYTE EXTRAVASATION IS NOT AFFECTED BY IRF8 DEFIENCY ..................... 70
4.1.3 PMN ACCUMULATION ACCOMPANIED BY GRANULE PROTEIN DISCHARGE DRIVES
-/- -/-ATHEROSCLEROSIS IN IRF8 ►APOE MICE ............................................................... 71
4.1.4 MYELOPROLIFERATIVE DISORDERS DRIVE ATHEROGENESIS 72
4.1.5 PERSPECTIVES ................................ 73
4.2 AUTOANTIGENIC PROTEIN-DNA COMPLEXES STIMULATE PLASMACYTOID DENDRITIC
CELLS TO PROMOTE ATHEROSCLEROSIS ....................................................................... 74
4.2.1 ACTIVATED PDCS INITIATE ATHEROSCLEROTIC LESION FORMATION .................... 74
4.2.2 OXLDL CRITICALLY INFLUENCES PDC FUNCTIONS ............. 76
4.2.3 AUTOIMMUNE MECHANISMS DRIVE PDC ACTIVATION IN ATHEROSCLEROSIS ......... 77
4.2.4 PERSPECTIVES ............................................................................................... 79
5. SUMMARY ................................................... 81
6. ZUSAMMENFASSUNG ................................... 83
7. REFERENCES .............................................. 85
8. ACKNOWLEDGEMENTS ............................................................... 106
9. CURRICULUM VITAE................................... 108





V ABBREVIATIONS

ABBREVIATIONS
Ab antibody
AD atopic dermatitis
Ag antigen
APCs antigen presenting cells
Apoe Apolipoprotein E
BM bone marrow