Human DC-SIGN mediated recognition of mycobacteria in conventional and bacterial artificial chromosome transgenic mouse models [Elektronische Ressource] / Martin Schäfer

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 45
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
der Technischen Universität München
Human DC-SIGN mediated recognition of mycobacteria
in conventional and bacterial artificial chromosome
transgenic mouse models
Martin Schäfer


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Karl-Heinz Schleifer
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Alfons Gierl
2. Priv.-Doz. Dr. Carsten Kirschning




Die Dissertation wurde am 02.08.2006 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 06.11.2006 angenommen.

1 Abbreviations ..................................................................................................4
2 Introduction .....................................................................................................8
2.1 Mycobacterium tuberculosis..............................................................................8
2.2 The first encounters of M. tuberculosis-the macrophages.................................8
2.3 T cell response to mycobacterial infection.........................................................8
2.4 The role of dendritic cells in infection ................................................................9
2.5 Receptor mediated antigen recognition and uptake of mycobacteria................9
2.5.1 Role of TLRs in immune response to mycobacteria........................................10
2.5.2 DC-SIGN and its function in mycobacterial infection.......................................12
2.5.2.1 Expression and structure of DC-SIGN.............................................................12
2.5.2.2 Self and non-self recognition by DC-SIGN......................................................12
2.5.2.3 Crosstalk between TLRs and DC-SIGN in response to M. tuberculosis .........13
2.5.3 Aims of the project...........................................................................................15
3 Materials and Methods..................................................................................16
3.1 Materials..........................................................................................................16
3.1.1 Equipment .......................................................................................................16
3.1.2 Chemicals........................................................................................................17
3.1.3 Expandable items............................................................................................19
3.1.4 Primer..............................................................................................................19
3.1.5 Antibodies used for FACS ...............................................................................20
3.1.6 sed for Western Blot ....................................................................21
3.2 Methods...........................................................................................................21
3.2.1 Cell biology......................................................................................................21
3.2.1.1 Media used for the culture of eukaryotic cells .................................................21
3.2.1.2 Culture of HEK293 cells ..................................................................................22
3.2.1.3 Preparation and culture of Bone Marrow derived murine Macrophages .........22
3.2.1.4 mouse Dendritic Cells........22
3.2.2 Mice.................................................................................................................22
3.2.3 Culture of prokaryotic cells ..............................................................................23
3.2.3.1 Media and buffers for the culture of prokaryotic cells......................................23
3.2.3.2 Bacteria and Infection......................................................................................24
3.2.3.3 Uptake and binding assay ...............................................................................24
3.2.3.4 Colony enumeration assay24
3.2.4 Protein biochemistry........................................................................................25
3.2.4.1 Protein biochemistry buffers and solutions......................................................25
3.2.4.2 Cell lysis ..........................................................................................................26
3.2.4.3 Protein determination26
3.2.4.4 SDS-Polyacrylamid gel electrophoresis (PAGE) of proteins ...........................26
3.2.4.5 Western Blot analysis27
3.2.5 Molecular biology ............................................................................................28
3.2.5.1 Buffers and solutions for molecular biology.....................................................28
3.2.5.2 Basic tools for molecular genetic approaches.................................................28 3.2.5.3 Agarose gel electrophoresis............................................................................28
3.2.5.4 PCR.................................................................................................................28
3.2.5.5 EMSA - Electrophoretic Mobility Shift Assay...................................................29
®3.2.5.6 mRNA Isolation using TRIzol Reagent ..........................................................31
3.2.5.7 TaqMan primers and fluorogenic probes.........................................................31
3.2.5.8 TaqMan PCR procedure .................................................................................31
3.2.6 Immunology.....................................................................................................32
3.2.6.1 Enzyme linked immunosorbent assay (ELISA) ...............................................32
3.2.6.2 Flow cytometry ................................................................................................33
3.2.6.3 Analysis of cell surface antigens by flow cytometry.........................................34
3.2.6.4 Analysis of intracellular ry ........................................34
3.2.6.5 Isolation of mouse dendritic cells from spleen or lymphnodes on a discontinous
TMOptiPrep gradient.........................................................................................34
TM3.2.6.6 Isolation of lls from spleen on a discontinous NycoPrep
gradient ...........................................................................................................35
4 Results37
4.1 Generation of transgenic mice ........................................................................37
4.1.1 Generation of human DC-SIGN transgenic mouse models.............................37
4.2 Analysis of transgene expression....................................................................38
4.2.1 Transgene expression in BMDCs38
4.2.2 Human DC-SIGN expression on ex vivo purified splenic cells of naïve mice..39
4.2.3 xpreex vivo purifiedcells after IL-4
treatment .........................................................................................................41
4.2.4 Transgene expression in peritoneal lavage cells.............................................42
4.2.5 Splenic transgene expression after Flt3-ligand treatment ...............................43
4.2.6 Transgene expression in peripheral blood monocytes....................................44
4.2.7 Transgene transcript levels in CD11c-DC-SIGN mice.....................................45
4.3 Role of human DC-SIGN in vitro .....................................................................47
4.3.1 Cytokine response by human DC-SIGN transgenic BMDCs to infection with
M. bovis BCG ..................................................................................................47
4.3.2 GN transgenic BMDCs infected with
M. tuberculosis ................................................................................................49
4.3.3 Uptake of M. bovis BCG by BMDC..................................................................50
4.3.4 Activation of MAPK in M. bovis BCG infected BMDCs....................................51
4.3.5 IκBα activation in M. bovis BCG-infected BMDCs ..........................................53
4.3.6 NF-κB activation in DCs after mycobacterial stimulation.................................54
4.4 Role of transgenic human DC-SIGN in vivo ....................................................56
4.4.1 Bacterial burden in the spleen at d14 after infection with M. bovis BCG.........56
4.4.2 rden in the lung at d41 after infection with M. tuberculosis...........57
4.4.3 Survival after low dose M. tuberculosis infection.............................................58
4.4.4 r high dose infection ...........................................59
4.4.5 Survival of wild-type and human DC-SIGN transgenic mice after infection with
C. albicans.................................................................