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Theroles of C5a Receptor 1 and Chemokine Receptor 5 In From the Institute of Systemic Inflammation Research of the University of Lübeck Director: Prof. Dr. med. Jörg Köhl The roles of C5a receptor 1 and chemokine receptor 5 in Toxoplasma gondii infection Dissertation for Fulfillment of the Requirements for the Doctoral Degree of the University of Lübeck From the Department of Natural Sciences Submitted by Daria Briukhovetska from Dnipropetrovsk Lübeck 2017 First referee: Prof. Dr. med. Jörg Köhl Second referee: Prof. Dr. Rainer Duden Date of oral examination: 29.08.2017 Approved for printing: 29.08.2017 List of contents Summary ............................................................................................................................VI Zusammenfassung ...........................................................................................................VIII 1 INTRODUCTION ............................................................................................................ 1 1.1. Innate and adaptive immunity as two important parts of the immune system ........ 1 1.2. The complement system is a major player of humoral immunity ............................ 2 1.2.1. Canonical activation of the complement cascade ............................................. 2 1.2.2. Non-canonical activation of the complement components ............................... 4 1.2.3. Regulation of the complement system .............................................................. 5 1.2.4. The anaphylatoxin C5a and its cognate receptors............................................ 6 1.2.4.1. The anaphylatoxin C5a .................................................................................. 7 1.2.4.2. Receptors for C5a .......................................................................................... 7 1.2.4.3. Expression of C5aR1 ..................................................................................... 8 1.2.5. Crosstalk between the C5a receptors and Toll-like receptors .......................... 9 1.2.6. Dimerization of C5aR1 with other G-protein coupled receptors ....................... 9 1.3. Dendritic cells as a key component of the pathogen recognition system.............. 10 1.3.1. Dendritic cell subpopulations in humans and mice ......................................... 10 1.3.2. Chemokine receptors of dendritic cells ........................................................... 13 1.3.3. Interleukin-12 family cytokines ........................................................................ 13 1.4. Biology and immune recognition of Toxoplasma gondii ........................................ 15 1.4.1. Recognition of the T. gondii parasite by the immune system ......................... 16 1.4.2. Innate immune responses to Toxoplasma gondii infection ............................. 18 1.4.3. Adaptive immune responses to Toxoplasma gondii infection ......................... 21 1.4.3.1. B cell response ............................................................................................. 21 1.4.3.2. T cell responses ........................................................................................... 22 1.4.1. Latent T. gondii infection in the brain .............................................................. 22 I List of contents 1.4.2. The intracellular niche of T. gondii that allows parasite survival and spreading in the host.......................................................................................................... 23 1.4.3. Resolution of inflammation in response to T. gondii infection......................... 24 1.4.4. Activation of the complement system during T. gondii infection ..................... 25 1.5. Hypothesis and specific aims of the project ........................................................... 26 2 MATERIAL .................................................................................................................... 27 2.1. Material ................................................................................................................... 27 2.1.1. Mouse strains ................................................................................................... 27 2.1.2. Parasites and cell lines .................................................................................... 27 2.1.3. Chemicals and reagents .................................................................................. 27 2.1.4. Buffers, solutions and media ........................................................................... 29 2.1.5. Antibodies for flow cytometry ........................................................................... 32 2.1.6. Plastic ware and disposable items .................................................................. 34 2.1.7. Commercially available kits ............................................................................. 35 2.2. Equipment and software ......................................................................................... 35 2.2.1. Laboratory equipment ...................................................................................... 35 2.2.2. Computer software........................................................................................... 37 3 METHODS .................................................................................................................... 38 3.1. Protein expression in bacterial culture ................................................................... 38 3.1.1. Heat-shock transformation of chemically competent bacteria ........................ 38 3.1.2. Plasmid preparation ......................................................................................... 39 3.1.3. Purification of GST-tagged protein from bacterial biomass ............................ 39 3.1.4. Endotoxin removal from the recombinant protein preparation ........................ 40 3.1.5. Determination of endotoxin content ................................................................. 40 3.1.6. Analysis of protein homogeneity by SDS-PAGE ............................................. 40 3.1.7. Staining protein gels with Coomassie blue ..................................................... 40 3.1.8. Staining protein gels with silver staining.......................................................... 40 3.1.9. Peptidyl-prolyl isomerase activity assay .......................................................... 41 3.2. Immune cell isolation from mouse tissues ............................................................. 41 II List of contents 3.2.1. Isolation of cells from mouse peritoneal cavity................................................ 42 3.2.2. Isolation of cells from spleen ........................................................................... 42 3.2.3. Isolation of cells from mesenteric lymph node ................................................ 42 3.2.4. Isolation of immune cells from mouse brain .................................................... 42 3.2.5. Determination of cell numbers ......................................................................... 43 3.2.6. Magnetic-activated cell sorting ........................................................................ 43 3.3. Receptor labelling and confocal microscopy.......................................................... 44 3.3.1. Generation of dendritic cells from bone marrow culture (BMDCs) ................. 44 3.3.2. Transfection of BMDC culture ......................................................................... 44 3.3.3. Laser scanning confocal microscopy .............................................................. 45 3.4. Maintenance of T. gondii tachyzoites in vitro ......................................................... 45 3.4.1. Vero cell culture ............................................................................................... 45 3.4.2. Preparation of soluble toxoplasma antigen (STAg) ........................................ 46 3.5. T. gondii mouse infection model............................................................................. 46 3.5.1. Determination of brain cyst numbers in T. gondii-infected mice ..................... 46 3.5.2. Intraperitoneal T. gondii infection .................................................................... 47 3.6. Determination of serum cytokines in T. gondii-infected mice ................................ 48 3.6.1. Determination of cytokine production by enzyme-linked immunosorbent assay (ELISA) .............................................................................................................. 48 3.6.2. Determination of serum cytokine concentrations using the Bio-Plex ProTM (BioRad) assay.................................................................................................. 49 3.6.3. Determination of serum cytokine concentrations using the Multiplex (MSD) assay ................................................................................................................. 49 3.6.4. Determination of serum Alanine amino transferase (ALT) activity ................. 50 3.7. Flow cytometry........................................................................................................ 50 3.7.1. Intracellular cytokine staining .......................................................................... 51 3.7.2. Tetramer staining ............................................................................................. 51 3.8. Statistical analysis .................................................................................................. 52 4 RESULTS.....................................................................................................................
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