The role of dendritic cells and their chemokine TARC in the pathogenesis of atherosclerosis [Elektronische Ressource] / Svenja Meiler

Svenja Hofert

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2010
• Nombre de lectures: 49
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

The role of dendritic cells and their chemokine TARC in
the pathogenesis of atherosclerosis




Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von




Diplom-Biologin
Svenja Meiler

aus Esslingen am Neckar, Deutschland


Berichter: Universitätsprofessor Christian Weber
Universitätsprofessor: Rainer Fischer
Tag der mündlichen Prüfung: 18.01.2010


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.The results of this work were in part published in:

Svenja Meiler, Tobias Junt, Remco T.A. Megens, Marc A. M. J. van Zandvoort, Mihail
Hristov, Christoph J. Binder, Yvonne Döring, Maik Drechsler, Ivett Jelinek, Steffen Jung,
Irmgard Förster, Martin Zenke, Thomas Hieronymus, Christian Weber, Alma Zernecke
(2009). CCL17-expressing dendritic cells drive atherosclerosis Nat Med in revision.
Table of contents
Table of Contents
Tabel of contents…………………………………………………………...…………….i
Abbreviations…………………………………………………………………….............v

I Introduction ......................................................................................................... 1
I.1 Dendritic cells ........................................................................................................ 1
I.2 T helper (Th) cells ................................................................................................. 4
I.3 Chemokines ........................................................................................................... 7
I.3.1 The DC-derived chemokine Ccl17 (thymus-and activation-regulated
chemokine, TARC) ....................................................................................................... 9
I.4 Atherosclerosis .................................................................................................... 11
I.4.1 Immune mechanisms in atherosclerosis ................................................... 11
I.5 Aim of this study ................................................................................................. 15
II Materials and Methods ..................................................................................... 17
II.1 General equipment ............................................................................................... 17
II.2 Mice ..................................................................................................................... 18
II.3 Chemokines and recombinant proteins................................................................ 19
II.4 Antibodies ............................................................................................................ 20
II.4.1 Primary Antibodies ................................................................................... 20
II.4.2 Directly conjugated antibodies ................................................................. 20
II.4.3 Secondary antibodies ................................................................................ 22
II.4.4 Blocking antibodies .................................................................................. 22
II.4.5 Depletion antibodies ................................................................................. 22
i Table of contents
II.5 Cell culture .......................................................................................................... 22
II.5.1 Culturing of bone marrow-derived myeloid DCs (BMDCs) .................... 23
II.5.2 Cell isolation after tissue digestion ........................................................... 23
+II.5.3 Isolation of CD4 T cells .......................................................................... 24
II.5.4 Cell sorting ............................................................................................... 24
II.6 Biomolecular methods ......................................................................................... 25
II.6.1 Quantification of RNA ............................................................................. 25
II.6.2 Quantitative real-time polymerase chain reaction (PCR) ......................... 25
II.6.3 Oligo microarray....................................................................................... 28
II.7 Protein assays ...................................................................................................... 29
II.7.1 Flow cytometry ......................................................................................... 29
II.7.2 Bead array ................................................................................................. 30
II.7.3 Enzyme-linked immunosorbant assay (ELISA) ....................................... 31
II.7.4 Histochemistry .......................................................................................... 31
II.7.5 Immunofluorescence ................................................................................ 32
II.7.6 Multiphoton microscopy .......................................................................... 32
II.8 Functional assays ................................................................................................. 34
II.8.1 Phagocytosis ............................................................................................. 34
II.8.2 Chemotaxis ............................................................................................... 34
II.8.3 Transmigration ......................................................................................... 34
II.8.4 Proliferation .............................................................................................. 35
II.9 Animal experiments ............................................................................................. 36
II.9.1 Mouse model of atherosclerotic disease ................................................... 36
II.9.2 Bone marrow transplantation ................................................................... 36
+II.9.3 Adoptive CD4 T cell transfer .................................................................. 36
II.9.4 Recruitment assays ................................................................................... 37
II.9.5 Proliferation in vivo .................................................................................. 37
II.10 Data illustration and statistical analysis............................................................... 38
III Results ................................................................................................................. 39
ii Table of contents
III.1 CCL17-producing myeloid DCs and the role of this chemokine in DC
function ......................................................................................................................... 39
+III.1.1 CCL17 DCs belong to the most mature myeloid DC population ........... 39
III.1.2 CCL17 expression does not influence other DC-specific genes .............. 41
III.1.3 CCL17 does not influence phagocytic ability or DC migration ............... 43
+III.1.4 CCL17 attracts CD4 T cells .................................................................... 45
+III.1.5 CCL17 is required for efficient CD4 T cell priming and polarization .... 46
III.2 Involvement of DC-derived CCL17 in atherosclerosis ....................................... 51
III.2.1 CCL17-expressing DCs are not located within the vessel wall of healthy
mice .................................................................................................................. 51
III.2.2 CCL17-expressing DCs accumulate in atherosclerotic lesions ................ 53
-/-III.2.3 Deficiency of CCL17 reduces atherosclerosis in Apoe mice ................. 55
-/-III.2.4 CCL17 contributes to lesion progression in Apoe mice ......................... 58
III.2.5 Vascular DCs home to lymphoid organs .................................................. 60
+III.2.6 T cells primed by CCL17 DCs mediate atherogenesis ........................... 61
IV Discussion ........................................................................................................... 64
+ +IV.1 CCL17 DCs are mature and recruit CD4 T cells .............................................. 64
IV.2 CCL17 is required for efficient T cell activation ................................................ 65
IV.3 CCL17 is required for efficient T cell priming and polarization ......................... 67
IV.4 Involvement of DC-derived CCL17 in atherosclerosis ....................................... 68
+IV.5 T cells primed by CCL17 DCs mediate atherogenesis ...................................... 71
IV.6 Perspectives ......................................................................................................... 73
V Summary ............................................................................................................ 75
V.1 Summary (English) .............................................................................................. 75
V.2 Zusammenfassung ............................................................................................... 76
VI Acknowledgement ................................