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An NLR Member with Impact on Adaptive and Innate Immune Mechanisms and Its Protective Role in the Progression of Colitis

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An NLR Member with Impact on Adaptive and Innate Immune Mechanisms and Its Protective Role in the Progression of Colitis NLRC5 An NLR member with impact on adaptive and innate immune mechanisms and its protective role in the progression of colitis Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel Vorgelegt von Manja Ehmke Kiel, 2015 CHRISTIAN-ALBRECHTS-UNIVERSITÄT ZU KIEL Diese Dissertation wurde angefertigt unter der Leitung von Prof. Dr. Philip Rosenstiel am Institut für Klinische Molekularbiologie (IKMB). PROF. DR. PHILIP ROSENSTIEL 1. GUTACHTER: PROF. DR. THOMAS ROEDER 2. GUTACHTER: 11. JANUAR 2015 DATUM DER DISPUTATION: 11. JANUAR 2015 ZUM DRUCK GENEHMIGT: DER DEKAN DER MATHEMATISCH- NATURWISSENSCHAFTLICHEN FAKULTÄT gez. Prof. Dr. Wolfgang J. Duschl i CHRISTIAN-ALBRECHTS-UNIVERSITÄT ZU KIEL EIDESSTATTLICHE ERKLÄRUNG Hiermit versichere ich, Manja Ehmke, geboren am 09. Oktober 1982 in Stralsund, dass ich die vorliegende Dissertation mit dem Titel „NLRC5 – An NLR member with impact on adaptive and innate immune mechanisms and its protective role in the progression of colitis“ selbst und selbständig gemäß den Regeln der guten wissenschaftlichen Praxis der Deutschen Forschungsgemeinschaft und ausschließlich unter Verwendung der hier angegebenen Hilfsmittel angefertigt habe. Darüber hinaus versichere ich, dass ich zeitgleich mit diesem Thema nicht an einer anderen Fakultät im In‐ oder Ausland die Zulassung zur Promotion beantragt habe oder beantragen werde. Dies ist mein erstes Promotionsvorhaben an einer Hochschule. Ich habe weder eine missglückte Promotion hinter mir, noch wurde mir je ein akademischer Grad entzogen. Manja Ehmke ii NLRC5 – An NLR member with impact on adaptive and innate immune mechanisms and its protective role in the progression of colitis TABLE OF CONTENTS 1 Introduction ....................................................................................................................................... 6 1.1 The Immune System.................................................................................................................6 1.2 Innate Immunity .......................................................................................................................6 1.2.1 Early Induced Responses: Inflammation & Cytokines ........................................................7 1.2.2 Pattern Recognition Receptors – Innate Immune Sensors ...................................................8 1.2.3 TLRs – TOLL-like Receptors ..............................................................................................9 1.2.4 NLRs – Nucleotide-Binding Domain And Leucine-Rich Repeats-Containing Receptors ...9 1.2.4.1 Discovery & Plant Homologues ...................................................................................9 1.2.4.2 Structure & Subfamilies ...............................................................................................9 1.2.4.3 Signaling.....................................................................................................................10 1.2.4.4 NLR Ligands & Expression .......................................................................................11 1.2.4.5 NLRs and Disease ......................................................................................................11 1.2.5 NLRC5 – An NLR Member With Ambiguous Functions..................................................12 1.2.5.1 Expression & Induction ..............................................................................................12 1.2.5.2 Structure .....................................................................................................................12 1.2.5.3 Proposed Functions ....................................................................................................13 1.3 Adaptive Immunity ................................................................................................................15 1.3.1 Generation & Maturation Of Lymphocytes .......................................................................15 1.3.2 Variant Lymphocyte Antigen Receptors ............................................................................16 1.3.3 B Lymphocytes & Humoral Adaptive Immunity ...............................................................16 1.3.4 T Lymphocytes & Cell-Mediated Adaptive Immunity ......................................................16 1.3.5 Antigen Presentation ..........................................................................................................17 1.3.5.1 MHC Class II Complex ..............................................................................................17 1.3.5.2 MHC Class I Complex ...............................................................................................18 1.4 Inflammatory Bowel Disease .................................................................................................19 1.4.1 Clinical Manifestation ........................................................................................................19 1.4.2 Pathogenesis .......................................................................................................................20 1.4.3 Therapy...............................................................................................................................20 1.4.4 DSS-Induced Colitis ...........................................................................................................21 1.5 Objective ................................................................................................................................22 2 Materials & Methods ...................................................................................................................... 23 2.1 Experimental Mice .................................................................................................................23 2.1.1 Generation of Nlrc5 Knockout Mice ..................................................................................23 2.1.2 Animal Husbandry .............................................................................................................24 2.1.3 Genotyping .........................................................................................................................24 Contents 2.1.4 Withdrawal of Murine Material for ex vivo Analyses ........................................................25 2.1.5 Experimental DSS Colitis ..................................................................................................25 2.2 Histology ................................................................................................................................25 2.2.1 Hematoxylin & Eosin Staining...........................................................................................25 2.2.2 Periodic Acid – Schiff Stain ...............................................................................................26 2.2.3 Microscopy .........................................................................................................................26 2.3 Cell Culture ............................................................................................................................26 2.3.1 Cell lines & Passage ...........................................................................................................26 2.3.2 Primary Cell Culture ..........................................................................................................27 2.3.3 Harvesting Cultured Cells ..................................................................................................28 2.3.4 Cell Stimulations ................................................................................................................28 2.4 Flow Cytometry .....................................................................................................................28 2.5 Enzyme-Linked Immunosorbent Assay .................................................................................29 2.6 Multiplex Assay .....................................................................................................................30 2.7 Isolation & Analyses of DNA ................................................................................................30 2.7.1 DNA Extraction ..................................................................................................................30 2.7.2 Polymerase Chain Reaction................................................................................................30 2.7.3 Agarose Gel Electrophoresis ..............................................................................................31 2.8 Isolation & Analyses of RNA ................................................................................................31 2.8.1 RNA Isolation & Determination of Concentration ............................................................31 2.8.2 Genomic DNA Contaminations & RNA Clean Up ............................................................32 2.8.3 Reverse Transcription PCR ................................................................................................32 2.8.4 Quantitative Real Time PCR ..............................................................................................33 2.9 Isolation & Analyses of Proteins ............................................................................................34 2.9.1 Protein Isolation & Determination of Concentration .........................................................35 2.9.2 SDS–PAGE
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