Assessing the role of sCYLD and Bcl-3 in immune regulation [Elektronische Ressource] / vorgelegt von Joumana Masri

Joumana Masri

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

Assessing the role of sCYLD and
Bcl-3 in immune regulation





Dissertation
Zur Erlangung des Grades
“Doktor der Naturwissenschaften”
am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz




vorgelegt von
Joumana Masri
aus Köln

Mainz 2010









Dekan:
Berichterstatter 1:
Berichterstatter 2:
Tag der mündlichen Prüfung:
Table of Contents
1   Introduction ......................................................................................... 1  
1.1   The NF-κB signaling pathway.......................................1  
1.2   Regulation of the NF-κB pathway by ubiquitin signaling ...........................2  
1.3   CYLD................................................................................3  
1.4   The IκB -like molecule Bcl-3...........5  
1.5   The role of NF-κB signaling in cells of the immune system ......................6  
1.5.1   B lymphocytes..................................................................................................6  
1.5.2   T lymphocytes..6  
1.5.3   Dendritic cells (DCs).........................8  
1.5.4   Macrophages....9  
2   Specific aims of this thesis .............................................................. 11  
3   Materials and Methods...................................... 13
3.1   Chemicals and biological material.............................13  
3.2   Molecular biology.........................................................14  
3.2.1   Preparation of genomic DNA..........14  
3.2.2   Polymerase chain reaction (PCR)..................................................................14  
3.2.3   RNA isolation and Quantitative Real-Time PCR............15  
3.2.4   Amplification and isolation of plasmid DNA....................................................16  
3.2.5   Agarose gel electrophoresis and DNA gel extraction.....16  
3.2.6   Quantification of DNA and RNA .....................................................................16  
3.2.7   Cloning and sequencing.................16  
3.3   Biochemistry.................................17  
3.3.1   Preparation of protein extracts.......17  
3.3.2   Western Blot...................................................................18  
3.3.3   Electromobility shift assay..............18  
3.3.4   Luciferase assay ............................................................20  
3.4   Cell biology...20  
3.4.1   Preparation of single cell suspensions from lymphoid organs .......................20  
3.4.2   Cell counting...................................................................21  
3.4.3   Flow cytometry ...............................................................21  
3.4.4   Magnetic cell sorting.......................22  
3.4.5   CFSE labeling22  
3.4.6   Culture of ex vivo lymphocytes.......................................................................23  
3.4.7   T cell proliferation assay.................23  
3.4.8   Mouse bone marrow derived dendritic cells...................23  
3.4.9   Peritoneal and bone marrow derived macrophages (BMDMs) ......................24  
3.4.10   Cytokine release measurement with ELISA or CBA-Flex ..............................24  
3.4.11   Preparation of mouse embryonic fibroblasts (MEFs) .....................................24  
3.5   Mouse experiments......................................................................................25  
3.5.1   Mice................................................25  
3.5.2   Induction and assessment of EAE.25  
3.5.3   Colitis induction and/or scoring ......................................................................25  
3.6   Software........26  
4   Results ............................................................... 27  
4.1   Impact of sCYLD overexpression on dendritic cell and macrophage
function.........................................................................27  
ex7/8 ko4.1.1   Analysis of dendritic cell function in CYLD and CYLD mice...................27  
4.1.1.1 Over-expression of sCYLD enhances the expression of co-stimulatory molecules on
BMDCs ...................................................... 28
ex7/84.1.1.2 Increase of inflammatory cytokine secretion in CYLD BMDCs.............................29
ex7/84.1.1.3 BMDCs from CYLD mice induce an increased clonal T cell expansion................30
ex7/84.1.1.4 BMDCs from CYLD fail to induce peripheral tolerance.........................................32
ex7/84.1.1.5 Analyzing Bcl-3 expression and regulation in CYLD BMDCs ...............................33
I Table of Contents
ex7/84.1.1.6 Elevated Bcl-3 expression in CYLD BMDCs is accompanied by increased NF-κB
signaling......................................................................................................................36
floxed/floxed4.1.2   Analysis of the LysMcre Cyld strain..................38  
4.1.2.1 Overexpression of sCYLD in elicited peritoneal macrophages does not increase MHC
class II expression levels.........................................................................................................39
4.1.2.2 Analysis of bone marrow derived macrophages (BMDMs)........40
FL/FL4.1.2.3 MHC class II and CD86 surface expression on LysMcre Cyld BMDMs ...............41
FL/FL4.1.2.4 Measurement of IL-6 secretion in LysMcre Cyld macrophages ...........................42
FL/FL4.1.2.5 iNOS (inducible nitric oxide synthase) expression in LysMcre Cyld macrophages..
....................................................................................................................................43
FL/FL4.1.2.6 Increased EAE severity in LysMcre Cyld mice.....................43
4.2   Impact of cell-type specific Bcl-3 overexpression on dendritic cells .....47  
4.2.1   BMDCs from CD11c-Cre Bcl-3 mice overexpress Bcl-348  
4.2.2   Expression of activation markers on CD11c-Cre Bcl-3 BMDCs.....................49  
4.2.3   BMDCs from CD11c-Cre Bcl-3 mice do not show increased levels of
inflammatory cytokines...................................................................................50  
4.2.4   Analyzing the stimulatory capacity of CD11c-Cre Bcl-3 BMDCs....................51  
4.3   Investigating the impact of Bcl-3 overexpression on T cell development
and function..................................................................................................53  
4.3.1   CD4-Cre Bcl-3 mice develop spontaneous colitis..........54  
4.3.2   Ex vivo analysis of thymocytes and the peripheral T cell pool .......................54  
4.3.3   CD4-Cre Bcl-3 mice have less peripheral effector/memory T cells................56  
4.3.4   Ex vivo analysis of the Th1, Th17 and Treg subpopulations..........................57  
+4.3.5   In vitro differentiation of naïve CD4 T cells from CD4-Cre Bcl-3 mice ..........59  
+ -4.3.6   Naïve CD4 CD25 T cells from CD4-Cre Bcl-3 mice fail to induce colitis after
transfer to RAG mice......................................................................................61  
4.3.7   In vitro assay reveals proliferation defect in CD4-Cre Bcl-3 T cells ...............63  
5   Discussion and Outlook................................... 65  
5.1   Role of CYLD transcripts in dendritic cell and macrophage function ....65  
5.1.1   Impact of sCYLD overexpression on dendritic cell function ...........................65  
5.1.2   Analyzing the impact of sCYLD overexpression in macrophages..................68  
5.2   Bcl-3 overexpression in dendritic cells......................................................70  
5.3   T cell specific overexpression of Bcl-371  
6   Summary ............................................................................................ 74  
7   Zusammenfassung............................................................................ 75  
8   References......................... 77  
9   Acknowledgements........... 89  
10   Erklärung............................................................................................ 90  
11   Curriculum Vitae................ 91  
12   Publications....................... 92  

II Abbreviations
Abbreviations

Ab antibody
APC antigen presenting cell or allophycocyanin
approx. approximately
Bio biotinylated
β-ME β-mercaptoethanol
bp base pair
BSA bovine serum albumin
°C temperature in degrees Celsius
CD cluster of differentiation
CFA Complete Freund’s Adjuvant
cDNA complementary DNA
C