The Contribution of Mast Cells to Antiviral Immune Responses: Characterization and Functional Definition of Receptor Transporter Protein 4 (RTP4)
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Aus dem Institut für Geflügelkrankheiten des Fachbereichs Veterinärmedizin der Freien Universität Berlin in Kooperation mit dem Forschungszentrum Borstel Leibniz-Zentrum für Medizin und Biowissenschaften Laborgruppe Immunbiologie The contribution of mast cells to antiviral immune responses: Characterization and functional definition of receptor transporter protein 4 (RTP4) Inaugural-Dissertation zur Erlangung des Grades eines Doktors der Veterinärmedizin an der Freien Universität Berlin vorgelegt von Diego Andrés Goyeneche Patiño Tierarzt aus Bogotá, Kolumbien. Berlin, 2012 Journal-Nr.: 3608 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Hafez Mohamed Hafez Zweiter Gutachter: PD. Dr. Holger Heine Dritter Gutachter: Univ.-Prof. Dr. Susanne Hartmann Deskriptoren (nach CAB-Thesaurus): Mast cells, immune response, viral infections, interferon, G proteins Tag der Promotion: 19.03.2013 A mis madres y a mi Toto (R.I.P) V Table of contents Page. Table of contents ................................................................................................................. V List of figures ..................................................................................................................... IX List of Tables ...................................................................................................................... XI List of abbreviations ........................................................................................................ XII 1. Introduction .................................................................................................................. 1 2. Literature review .......................................................................................................... 2 2.1 Immunity ................................................................................................................. 2 2.1.1 Antiviral innate immune responses ..................................................................... 2 2.1.1.1 Molecular mechanisms for viral recognition ............................................... 2 2.1.1.2 Viral recognition through toll like receptors ................................................ 3 2.1.1.3 Viral recognition through RIG-I like receptors ........................................... 6 2.1.1.4 Viral recognition by other receptors ............................................................ 9 2.1.1.5 Interferons .................................................................................................. 10 2.1.1.6 Induction of type I interferons ................................................................... 11 2.1.1.7 Modulatory functions of type I interferons ................................................ 12 2.1.2 Antiviral adaptive immune responses ............................................................... 14 2.2 Mast cells ............................................................................................................... 15 2.2.1 Mast cell development and classification.......................................................... 15 2.2.2 Mast cells activation through the IgE high affinity receptor ............................. 16 2.2.3 Mast cells and their role in immunity................................................................ 17 2.2.4 Mast cells and their role in viral infections ....................................................... 18 2.3 G protein coupled receptors .................................................................................. 20 2.3.1 Receptor transporter protein 4 ........................................................................... 21 3. Materials and methods............................................................................................... 23 VI Table of contents 3.1 Materials ................................................................................................................ 23 3.1.1 Mice ................................................................................................................... 23 3.1.2 RTP4 Deficient Mice Generation ...................................................................... 23 3.1.3 Viruses ............................................................................................................... 24 3.1.3.1 Newcastle disease virus ............................................................................. 24 3.1.3.2 Influenza A virus ....................................................................................... 25 3.1.4 Chemicals and reagents ...................................................................................... 25 3.1.5 Lab Supplies ....................................................................................................... 27 3.1.6 Antibodies .......................................................................................................... 28 3.1.7 Primers ............................................................................................................... 29 3.1.8 Solutions ............................................................................................................ 29 3.1.8.1 Biochemistry and Molecular Biology Buffers........................................... 29 3.1.8.2 Cell Culture Medium ................................................................................. 31 3.1.8.3 Flow Cytometry (FCM) and Microscopy Buffers ..................................... 32 3.1.9 Laboratory Equipment ....................................................................................... 33 3.1.10 Software ............................................................................................................. 35 3.2 Methods ................................................................................................................. 35 3.2.1 Cell counting ...................................................................................................... 35 3.2.2 Generation and culture of bone marrow derived mast cells (BMMC) .............. 35 3.2.3 Generation and culture of bone marrow derived dendritic cells (BMDC) ........ 36 3.2.4 Generation and culture of bone marrow derived macrophages (BMM) ............ 36 3.2.5 Culture of cell lines ............................................................................................ 37 3.2.6 Isolation of cells from primary and secondary immune organs ......................... 37 3.2.7 Detachment of adherent cells from cell culture flasks ....................................... 37 3.2.8 Purification of cell populations by magnetic associated cell sorting ................. 38 3.2.8.1 B cells separation ....................................................................................... 38 3.2.9 Stimulation of mast cells for experimental procedures ...................................... 39 3.2.10 Flow cytometry .................................................................................................. 40 3.2.10.1 Identification of cell subsets .................................................................. 41 3.2.10.2 Use of flow cytometry for assessment of mast cells degranulation ....... 42 3.2.11 Confocal microscopy ......................................................................................... 42 3.2.12 Cytokine quantification in culture supernatants by ELISA ............................... 43 3.2.13 RNA isolation .................................................................................................... 44 Table of contents VII 3.2.14 Complementary DNA (cDNA) synthesis .......................................................... 44 3.2.15 Analysis of gene expression by microarrays ..................................................... 45 3.2.16 Quantitative real-time polymerase chain reaction (qRT-PCR) ......................... 45 3.2.17 Protein extraction and sample preparation ........................................................ 46 3.2.18 SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) ................................... 46 3.2.19 Western Blotting ............................................................................................... 47 3.2.20 Immunodetection of proteins ............................................................................ 47 3.2.21 Fractionation...................................................................................................... 48 3.2.22 Molecular cloning of RTP4 ............................................................................... 48 3.2.23 Cell transfection for expression of RTP4 .......................................................... 49 3.2.24 Immunoprecipitation ......................................................................................... 50 3.2.25 Induction and isolation of lipid bodies .............................................................. 51 3.2.26 Statistical analysis and data management ......................................................... 52 3.2.26.1 General data processing ......................................................................... 52 3.2.26.2 Quantification of the gene expression for qRT-PCR ............................. 52 3.2.26.3 Calculation of relative density for Western blot bands .......................... 52 3.2.26.4 Analysis of microarray data ................................................................... 53 4. Results ........................................................................................................................