Novel mouse models for use in IL-17A and Th17 research [Elektronische Ressource] / Andrew Lewis Croxford

Andrew Croxford

Sujets

• Biologie

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Novel Mouse Models for Use in IL-17A
and Th17 Research







Dissertation
Zur Erlangung des Grades
Doktor der Naturwissenschaft



Am Fachbereich Biologie
Der Johannes Gutenberg-Universität Mainz



Andrew Lewis Croxford
Geb. am 1. Januar 1981 in New York, USA



Mainz, 2009



ABBREVIATIONS.......................................................................................................................8
1 INTRODUCTION..............11
1.1 EAE, AN ANIMAL MODEL FOR MS.....................................................................................11
1.2 THE T HELPER CELL SUBSET PARADIGM............12
1.3 DIFFERENTIATION OF TH17 CELLS12
1.4 TRANSCRIPTIONAL CONTROL OF TH17 CELLS ...................................................................13
1.5 FOXP3 AND RORγT............................................14
1.6 IL-23 ..................................15
1.7 IL-17A...............................................................17
1.8 IL-17F................................................................17
1.9 IL-17A SIGNALING AND TISSUE INFLAMMATION.............................18
1.10 IL-17A AND AUTOIMMUNITY...........................19
1.11 G-PROTEIN SIGNALLING AND AUTOIMMUNITY...............................20
1.12 AIM OF THIS THESIS..........................................................................................................22
2 MATERIALS AND METHODS .......................................................................................23
2.1 CHEMICALS AND BIOLOGICAL MATERIAL.....................................................................23
2.2 MOLECULAR BIOLOGY.....................................25
2.2.1 COMPETENT CELLS AND ISOLATION OF PLASMID DNA.................25
2.2.2 ISOLATION OF GENOMIC DNA FROM ES CELLS AND MOUSE ORGANS .........................25
2.2.3 RT-PCR AND QUANTITATIVE REAL-TIME PCR.............................................................25
2.2.4 AGAROSE GEL ELECTROPHORESIS AND DNA GEL EXTRACTION ..................................26
2.2.5 DNA SEQUENCING..........................................26
2.2.6 QUANTIFICATION OF DNA..............................26
2.2.7 POLYMERASE CHAIN REACTION (PCR)..........................................27
2.2.8 SOUTHERN BLOT ANALYSIS ...........................................................28
2.2.9 PURIFICATION OF LINEARIZED BAC DNA.....................................29
2.3 CELL BIOLOGY .................................................30
2.3.1 EMBRYONIC STEM CELL CULTURE................................30
2.3.2 HTNC TREATMENT........31
2.3.3 PREPARATION OF MOUSE EMBRYONIC FIBROBLASTS (MEFS) ......................................31
2.3.4 PCELLS FROM LYMPHOID ORGANS......................................................31
2.3.5 CULTURE OF EX VIVO LYMPHOCYTES.............................................32
2.3.6 CELL COUNTING..............................................32
2.3.7 ADOPTIVE TRANSFER......................................32
2.3.8 FLOW CYTOMETRY.........33
2.3.9 MAGNETIC CELL SORTING AND FACS SORTING ...........................................................34
2.3.10 CULTURE OF EX VIVO SPLENOCYTES AND LYMPHOCYTES...........34
2.3.11 INDUCTION OF CRE RECOMBINASE ACTIVITY AND IL-17A EXPRESSION IN VIVO .........35
2.4 IN VIVO DEPLETION OF CD25+ CELLS.............................................................................35
2.5 HISTOLOGICAL ANALYSIS AND IMMUNOHISTOCHEMISTRY.........35
2.6 CYTOKINE DETERMINATIONS.........................36
2.6.1 FLOWCYTOMIX...............................................36
2
2.6.2 ELISA .............................................................................................................................36
2.7 MOUSE EXPERIMENTS.....................................36
2.7.1 INDUCTION AND ASSESSMENT OF EAE...........................................36
2.8 STATISTICS.........................37
3 RESULTS ............................................................................................................................38
IND3.1 IL-17A SCREENING AND TESTING.................38
3.1.1 IL-17A OVEREXPRESSION INDUCES GRANULOCYTOSIS AND ANAEMIA. ........................42
THE WHOLE BODY RESULTS IN GROWTH RETARDATION, GRANULOCYTOSIS, ANAEMIA AND
PREMATURE DEATH. ....................................................................................................................44
IND3.1.2 NEUTROPHILS ACCUMULATE IN THE LUNGS OF DELETER-IL-17A MICE. ...................45
3.1.3 SKIN-SPECIFIC IL-17A EXPRESSION RESULTS IN PSORIASIS-LIKE DERMATITIS.............46
3.1.4 IL-17A SIGNALING IN SKIN INDUCES SYNTHESIS OF INNATE CHEMOTACTIC PROTEINS.48
IND/+3.1.5 K14-IL-17A MICE HAVE SYSTEMIC GRANULOCYTOSIS ............................................50
ERT2 IND3.2 THE ROSA-CRE -IL-17A SYSTEM ................................52
ERT2 IND/+ 3.2.1 TAMOXIFEN ADMINISTRATION TO ROSA-CRE -IL-17A MICE INDUCES
GRANULOPOIESIS.........................................................................................53
IND3.3 CD4-CRE IL-17A CREATES AN IL-17A EVEREXPRESSING T CELL REPERTOIRE............54
IND/+ 3.3.1 CD4-IL17A T CELLS DO NOT OVERTLY AFFECT LYMPHOCYTE DEVELOPMENT,
HOMEOSTASIS OR PERIPHERAL TOLERANCE................................................................................56
IND/+ 3.3.2 CD4-CRE IL-17A MICE SHOW INCREASED GRANULOPOIESIS...57
3.3.3 IL-17A–OVEREXPRESSING T CELLS DO NOT ENHANCE THE PATHOGENESIS AND
CLINICAL DEVELOPMENT OF MYELIN OLIGODENDROCYTE GLYCOPROTEIN–INDUCED EAE......59
3.3.4 IL-17A OVEREXPRESSION DOES NOT EXACERBATE HAPTEN-INDUCED CHS-RESPONSES
60
EYFP4 IL-17F-CRE ....................................................................................................................63
4.1 GENERATION OF IL-17A-CRE AND IL-17F-CRE ALLELES.................64
EYFP4.1.1 TH17 CONDITIONS INDUCE IL-17F EXPRESSION IN BOTH CD4 AND CD8 IL-17F-CRE
T CELLS........................................................................................................................................67
+4.1.2 IL-17F EXPRESSION IS RESTRICTED TO CD4 T CELLS DURING MOG-INDUCED EAE...69
4.1.3 TH17 CELLS ARE RESISTANT TO EXPRESSION OF FOXP3 IN VIVO...71
4.1.4 TH17 CELLS DOWNREGULATE IL-17 CYTOKINES AND EXPRESS IFN-γ ..........................72
4.1.5 IFN-γ DOES NOT ACT ON MATURE TH17 CELLS TO INHIBIT TH17 DIFFERENTIATION. ...75
RFP4.2 THE IL-17F-CRE -DEREG DUAL REPORTER SYSTEM....................................................77
RFP4.2.1 TH17 CELLS ARE RESISTANT TO FOXP3 EXPRESSION IN THE IL-17F-CRE -DEREG
SYSTEM........................................................................................................79
FL/FL5 CD4-CRE IL-6R............................................81
5.1 IL-6 SIGNALING REGULATES THE TH17/TREG AXIS BY INHIBITING ITREG GENERATION IN
VIVO 81
FL/FL5.2 CD4-IL-6R MICE LACK EXPRESSION OF THE IL-6Rα PROTEIN AND IL-6 SIGNALING
CAPABILITY .................................................................................................................................82
FL/FL5.3 CD4-CRE-IL-6R MICE SHOW ENHANCED TREG RESPONSES AFTER MOG-CFA
IMMUNIZATION............................85
FL/FL 5.4 TREG DEPLETION RENDERS CD4-CRE IL-6R MICE SUSCEPTIBLE TO EAE...................87
.....................................................................................................................................................90
3
6 EBI2 RESULTS SECTION..................................................................................................91
6.1 CD4+ T CELLS PREFERENTIALLY EXPRESS EBI2..............................91
6.2 EBI2-DEFICIENT T CELLS PRODUCE CYTOKINES AND PROLIFERATE NORMALLY. ............92
6.3 EBI2-DEFICIENT MICE ARE RESISTANT TO EAE INDUCTION. ................................93
6.4 EBI2-DEFICIENT MICE ARE RESISTANT TO HAPTEN INDUCED EAR SWELLING...................95
6.5 EBI2-DEFICIENT T CELLS INEFFICIENTLY MIGRATE TO SECONDARY LYMPHOID ORGANS.
97
6.6 LOW EBI2 EXPRESSION CORRELATES WITH A REGULATORY PHENOTYPE. .......................99
-/-6.7 EBI2-DEFICIENT T CELLS DO NOT EFFICIENTLY INDUCE COLITIS IN RAG1 HOSTS. .....100
7 DISCUSSION....................................................................................................................102
IND/+7.1 IL-17A........................102
IND/+7.1.1 K14 IL-17A .............................................................................................................102
IND/+7.1.2 CD4-IL-17A...........104
IND/+7.1.3 DELETER IL-17A.....108
7.2 IL-17F-CRE.....................109
+ +7.2.1 IL-17A IFN-γ T CELLS ................................................................................................109
7.2.2 TH17 AND FOXP3 EXPRESSION110
FL/FL7.3 CD4-IL-6R .................................................................................................................114
7.4 EBI2.................................116
8 BIBLIOGRAPHY...............118
9 ACKNOWLEDGEMENTS.................................. ERROR! BOOKMARK NOT DEFINED.
VERSICHERUNG......................................................................................................