The role of CYLD in dendritic cell function [Elektronische Ressource] / Cathy Cecilia Srokowski

Cathy Palmer

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

The Role of CYLD in Dendritic Cell
Function

Dissertation
to achieve the degree
“Doktor der Naturwissenschaften”

in the Faculty of Biology
at the Johannes Gutenberg-Universität Mainz

Cathy Cecilia Srokowski



Mainz, November 2008
























This thesis is dedicated to my mother













2 Acknowledgements:

Thank you to all the members of the laboratory for their scientific advice and friendship.

3 TABLE OF CONTENTS
LIST OF FIGURES AND TABLE.................................................................... 7
ABBREVIATIONS USED............... 8
ABSTRACT ................................................................................................... 11
1. INTRODUCTION..................................................................................... 12
1.1. Dendritic Cells......................................13
1.1.1. The role of dendritic cells in the immune system: immunity and tolerance...14
1.1.2. Antigen Processing: the choice between two pathways..................................16
1.1.3. Toll like receptors on dendritic cells ...............................................................18
1.1.4. Dendritic cells and DALIS: ubiquitin aggregates with DRiPs........................20
1.2. Ubiquitination.......................................................................................................................................21
1.3. NF-κB...................22
1.3.1. NF-κB signaling pathways..............................................................................................................23
1.3.2. NF-KB, IκB and IKK protein families..........................24
1.3.3. Bcl-3 ................................................27
1.4. DUB.......................................................................................................................29
1.4.1. CYLD..............30
1.4.2. CYLD mouse models......................31
1.4.3. A20 ..................................................32
1.5. Goals of this Thesis..............................................................................................................................33
2. MATERIALS & METHODS...... 36
2.1. Cell lines, Antibodies and Reagents ..................................................................................................36
2.2. Mice........................................................................................36
ex7/8 2.2.1. Genotyping CYLD mice...........................................................................36
2.3. Mouse bone marrow derived dendritic cells....................37
2.3.1. BMDC cytokine release measurement with ELISA or CBA-Flex .................................................38
2.3.2 BMDC immunizations.....................................................................................38
2.4. Endogenous DC purification from mouse organs..........................................38
2.5. RNA isolation and cDNA preparation..............................................................................................39
2.6. Realtime PCR .......................................................................40
2.6.1. Real-time Primer List ......................................................................................40
2.7. FACS staining.......................................................................41
2.8. LCMV infection....................................................................41
2.9. DEC-205:OVA mediated tolerance....................................................................41
4 2.10. T cell isolation.....................................................................................................................................41
2.10.1. Treg culture...42
2.11. Western Blot........42
2.11.1. Whole cell lysates (WCL) ..............................................................................................................42
2.11.2. Isolation of Cytoplasmic and Nuclear Cellular Fractions.............................43
2.12. Immunofluorescence .........................................................................................................................43
2.13. Antigen Processing/ Antigen Phagocytosis...................43
2.14. CFSE labeling .....................................................................................................................................44
2.15. Intracellular Cytokine Staining.........44
2.16. BMDC transfection and NF-κB luciferase assay..........................................................................44
2.17. S5a-GST as a bait to purify poly-ubiquitinated proteins...............................45
2.18. Software Programs Used ...................................................................................................................45
2.19. Statistical analysis...............................45
3. RESULTS................................................................................................... 46
ex7/8 3.1. CYLD BMDCs exhibit a hyper-reactive phenotype in terms of cell surface expression
kocompared to CYLD and WT counterparts............................46
ex7/83.2. Cytokine secretion in CYLD CD11c+ve BMDCs reflects the high expression values of B7,
TNF and MHC class II cell surface receptors........................................................................................49
3.3. sCYLD confers a stimulatory phenotype in BMDCs which leads to increased T cell expansion
and an enhanced T cell cytotoxicity activity...........................51
ex7/8 3.4. Investigation of DC function in CYLD BMDCs .......................................................................53
ex7/8 3.4.1. Processing and phagocytic activity in CYLD BMDCs.............................53
3.4.2. NH Cl and chloroquine markedly inhibited the processing of DQ-OVA in BMDCs ..................56 4
3.4.3. Investigating MHC class II presentation efficiency confirms the hyper-reactive phenotye in
ex7/8 CYLD BMDCs when co-cultured with TCR Ag specific OT-II T cells ...........................................58
ex7/8 3.5. PDL-2 expression in CYLD BMDCs..........................................................................................61
ex7/8 3.6. Treg interaction with CYLD BMDCs does not result in suppression ...................................63
3.7. Ubiquitination and CYLD targets .....................................................................66
3.8. A20 and CYLD the determinants of disparate phenotype in CYLD mutations?........................69
ex7/8 3.9. Endogenous CD11c+populations in CYLD mice .....................................................................71
ex7/8 3.10. CYLD mice respond normal to LCMV infection....73
ex7/8 3.11. DEC-205:OVA mediated peripheral tolerance in CYLD mice ..............................................75
3.12. The effect of sCYLD on NF-κB family members.........................................78
3.13. sCYLD expression correlates with stimulation in WT BMDCs ..................................................80
5 3.14. Searching for ubiquitin targets of FL-CYLD and sCYLD............................................................82
4. DISCUSSION............................................................................................. 85
4.1. Incorporation of results in thesis to a model hypothesis:...............................................................94
5. FUTURE DIRECTIONS ........................................... 96
6. REFERENCES .......................................................................................... 98

6 List of Figures and Table
Figure 1. Outcomes from DC and T cell interactions………………………………………… 15
Figure 2. MHC class I and II antigen processing pathways………………………………….... 17
Figure 3. Ligand specificities of TLR 1-9……………………………………………………... 19
Figure 4. MyD88 dependent TRL4 signaling pathway………………………………………... 20
Figure 5. Protein ubiquitination and deubiquitination……………………………………….... 21
Figure 7. NF-κB activation pathways………………………………………………………… 24
Figure 8. NF-κB/Rel family members………………………………………………………... 25
Figure 9. IκB family………………………………………………………………………….. 26
Figure 10. IKK complex……………………………………………………………………... 27
Figure 11. Bcl-3 in NF-κB activation…………………………………………………………. 29
Figure 12. The protein structure of FL-CYLD and sCYLD…………………………………... 31
Figure 13. CYLD targets considered………………………………………………………….. 34
Figure 14. The effect of sCYLD over-expression in BMDCs confers a
hyper-reactive phenotype……………………………………………………………………... 48
ex7/8 Figure 15. CYLD BMDCs secrete significantly higher pro-inflammatory cytokines and lower
anti-inflammatory cytokines………………………………………………………………….... 50
ex7/8 Figure 16. CYLD BMDCs accentuate T cell expansion and T cell cytotoxic
capacity in vivo…………………………………………………………………………………. 52
ex7/8 Figure 17. The capacity to process antigen in CYLD BMDCs is altered but not the capacity
to phagocytose……………………………………………………………………………… 55
Figure 18. Addition of NH4Cl + chloroquine, antigen processing inhibitors, attenuates DQ-
ex7/8 OVA processing in WT BMDCs to a re