FUNCTIONAL CHARACTERIZATION OF ANNEXIN A1 IN TOLL LIKE RECEPTOR 7 SIGNALLING & INFLUENZA VIRUS INFECTION Suruchi Arora B. Tech , JIIT A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information, which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _________________________________________ Suruchi Arora 22nd Januray, 2014 2 Publications Pradeep Bist, Shinla Shu, Huiyin Lee, Suruchi Arora, Sunitha Nair, Jyue Yuen Lim, Jivanaah Dayalan, Stephan Gasser ,Subhra K. Biswas, Anna-Marie Fairhurst and Lina H. K. Lim . Annexin-A1 regulates TLR-mediated IFN-beta production through an interaction with TANK-binding kinase 1. J Immunol. 2013, 191: 4375-4382. 3 Contents Acknowledgements ....................................................................................................... 8 Summary ..................................................................................................................... 12 List of Figures .............................................................................................................. 13 List of Tables ............................................................................................................... 16 List of Abbreviations ................................................................................................... 17 Chapter 1 Introduction ................................................................................................. 20 1.1. Annexin 1 .................................................................................................... 20 1.1.1. Structure of ANXA1 ............................................................................ 20 1.1.2. Functions of ANXA1 ........................................................................... 21 1.1.2.1. Interaction with Phopholipase -2 (PLA2) ........................................ 21 1.1.2.2. Mediation of Glucocorticoid (GC) Actions ..................................... 22 1.1.2.3. Proliferation and Apoptosis ............................................................. 23 1.1.2.4. Annexin 1 Null mouse ..................................................................... 24 1.1.2.5. Pro-inflammatory actions ................................................................ 25 1.1.2.6. Phagocytosis “EAT me” signals ...................................................... 25 1.1.3. The ANXA1 receptor .......................................................................... 28 1.1.4. ANXA1 and APCs............................................................................... 28 1.2. Toll like Receptors (TLRs) .......................................................................... 29 1.2.1. MyD88 dependent pathway ................................................................. 31 1.2.2. TRIF-dependent pathway .................................................................... 31 1.2.3. Intracellular TLRs................................................................................ 32 1.2.4. ANXA1 and TLRs ............................................................................... 34 1.3. Influenza Virus ............................................................................................ 34 1.3.1. Health threat and Global Impact of Influenza Viruses ........................ 35 1.3.2. Signs and Symptoms ........................................................................... 37 1.3.3. Structure of Influenza virus ................................................................. 37 1.3.4. Viral Life Cycle ................................................................................... 43 1.3.5. Influenza Tropism................................................................................ 45 1.3.6. Immune responses against Influenza virus .......................................... 46 1.3.7. Humoral Response ............................................................................... 51 1.3.8. T cell Response .................................................................................... 51 1.3.9. Annexins and viruses ........................................................................... 52 4 1.4. Apoptosis ..................................................................................................... 53 1.4.1. The Receptor mediated/ Extrinsic Pathway ......................................... 54 1.4.2. The Intrinsic pathway .......................................................................... 55 1.4.3. The Execution Pathway ....................................................................... 60 1.4.4. Influenza and Apoptosis ...................................................................... 61 1.5. Aims of the study ......................................................................................... 63 1.6 Hypothesis ......................................................................................................... 63 Chapter 2 Materials and Methods ................................................................................ 64 2.1 Media and buffers .............................................................................................. 64 2.1.1. PBS ............................................................................................................ 64 2.1.2. FACS buffer .............................................................................................. 64 2.1.3. Red Blood Cell (RBC) Lysis Buffer .......................................................... 64 2.1.4. Wash buffer for Western Blotting (TBST) ................................................ 65 2.1.5. Buffer for ELISA ....................................................................................... 65 2.1.6. Complete DMEM for cell culture .............................................................. 65 2.1.7. Complete F-12K media for cell culture ..................................................... 65 2.1.8 Plain DMEM at 2X concentration .............................................................. 66 2.2. Cell Culture ...................................................................................................... 66 2.2.1. Cell Lines ................................................................................................... 66 2.2.2. Preparation of L929 Conditioned Media ................................................... 66 2.2.3. Generation of Bone Marrow Derived Macrophages (BMMOs) ................ 67 2.2.4. Generation of Bone Marrow Dendritic Cells (BMDCs) ............................ 67 2.2.5. Isolation of splenic cells ............................................................................ 68 2.2.6. Lung cell culture ........................................................................................ 68 2.2.7. Cell staining with CFSE and proliferation analysis ................................... 68 2.2.8. Thymidine Assay ....................................................................................... 69 2.2.9. Transfection ............................................................................................... 69 2.3. Mice .................................................................................................................. 69 2.3.1. Infection of mice with Influenza virus....................................................... 70 2.3.2. Cellular Infiltration .................................................................................... 70 2.3.3. Lung Histology .......................................................................................... 71 2.4 Influenza Virus Culture and Infection ............................................................... 72 2.4.1. Virus culture in chicken Embryos ............................................................. 72 2.4.2 Concentration of Influenza virus ................................................................ 73 5 2.4.3 Plaque forming assay .................................................................................. 73 2.4.4 Infection of cells with Influenza virus ........................................................ 74 2.5 Measurement of Cytokines ................................................................................ 74 2.6 Western Blot ...................................................................................................... 75 2.6.1. Protein lysis ............................................................................................... 75 2.6.2. Protein Concentration Quantification ........................................................ 76 2.6.3 Blotting ....................................................................................................... 76 2.7. Real Time PCR ................................................................................................ 78 2.7.1. RNA Isolation from cells ........................................................................... 78 2.7.2. RNA isolation from lungs ......................................................................... 78 2.7.3. Viral RNA (vRNA) isolation from cell supernatant .................................. 79 2.7.4. cDNA Synthesis ....................................................................................... 79 2.7.5. Real time PCR .........................................................................................