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Differential Gene Expression of Cd43 Wildtype and Knockout Macrophages in Response to Mycobacterial Stimulation

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Differential Gene Expression of Cd43 Wildtype and Knockout Macrophages in Response to Mycobacterial Stimulation DIFFERENTIAL GENE EXPRESSION OF CD43 WILDTYPE AND KNOCKOUT MACROPHAGES IN RESPONSE TO MYCOBACTERIAL STIMULATION by Audra Helena Vair B.Sc., The University of Ottawa, 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Pathology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2010 © Audra Helena Vair, 2010 Abstract Previous studies in our lab show CD43 deficient macrophages have reduced binding and uptake of M.tb bacilli but allow increased intracellular bacterial growth within the host macrophage due to decreased TNF‐α production. It was also found that there are two heat‐shock proteins found on the cell surface of M.tb that are capable of binding CD43: Cpn 60.2 and dnaK. The purpose of this study was to determine the intracellular signalling pathways activated by CD43 upon M.tb stimulation and to determine if Cpn 60.2 or dnaK could reproduce these responses. An RNA‐microarray was performed using mRNA isolated from wildtype (CD43 +/+) and gene‐deleted (CD43‐/‐) bone‐marrow derived murine macrophages that had been co‐incubated with one of M.tb, Cpn 60.2 or dnaK. There was a large quantity of differentially regulated genes for each treatment, ranging from 330 to 990. Only 113 of these genes were statistically significant and shared between treatment types, 21 genes down regulated in the knockout compared to the wild type and 92 genes were up regulated in CD43‐/‐ compared to CD43+/+. Pathway analysis of differential gene expression was performed using lists of statistically significant, differentially‐regulated genes, which indicated there were multiple pathways and cellular processes in which several genes were found to be affected by the absence of CD43. The pathways included p38/JNK/Erk MAP kinase signaling functioning, TNF‐α post‐transcriptional control, cytoskeletal remodeling, phagocytosis and apoptosis. Related genes from both the microarray data and interacting genes from pathway‐data associated with TNF‐α and apoptosis were selected to both validate the microarray and possibly determine if changes in TNF‐related regulation could be controlled by CD43. The microarray indicated several genes involved in TNF‐α regulation and apoptosis were differentially expressed in CD43 +/+ and CD43 ‐/‐ macrophages. Quantitative Reverse‐Transcriptase PCR was used on a selected number of these genes. Differential gene regulation was observed in the Q RT‐PCR results; however results conflicted with the microarray. In conclusion, CD43 influences the course of M.tb infection within the macrophage, possibly through the regulation of TNF‐α and apoptosis. There are implications for CD43‐related control of eicosanoid production and cytoskeletal regulation but these observations require further investigation. ii Table of contents Abstract……………………………………………………………………………………………………………………………………………...ii Table of contents……………………………………………………………………………………………………………………………….iii List of tables……………………………………………………………………………………………………………………………………….v List of figures…………………………………………………………………………………………………………………………………..…vi List of abbreviations………………………………………………………………………………………………………………………….vii Acknowledgements…………………..……………………………………………………………………………………………………….ix Dedication…………………………………………….………………………………………………………………………………………….…x 1 Introduction.......................................................................................................................................1 1.1 Mycobacterium tuberculosis .............................................................................................................. 1 1.1.1 Mycobacterium tuberculosis biology .......................................................................................... 2 1.1.2 Mycobacterium tuberculosis infection.........................................................................................2 1.1.3 Prevention ................................................................................................................................... 5 1.1.4 Treatment ................................................................................................................................... 9 1.2 Immune response to TB ................................................................................................................... 11 1.2.1 Macrophages ............................................................................................................................ 11 1.2.2 Cell‐mediated immunity ........................................................................................................... 13 1.3 Bacterial chaperonins and host cell response .................................................................................. 16 1.4 CD43 .................................................................................................................................................. 18 1.4.1 Structure ................................................................................................................................... 19 1.4.2 Expression .................................................................................................................................. 20 1.4.3 Function .................................................................................................................................... 20 1.4.4 Implications in disease .............................................................................................................. 22 1.5 Introduction of thesis ....................................................................................................................... 22 2 Materials and methods ........................................................................................................................... 25 2.1 Cell preparation and tissue culture ................................................................................................... 25 2.1.1 Mice ........................................................................................................................................... 25 2.1.2 Culture of bone‐marrow derived macrophages ........................................................................ 25 2.2 Bacterial culture ............................................................................................................................... 26 2.3 Recombinant molecular chaperones ................................................................................................ 26 2.4 In vitro assay for binding of particles to macrophages and M.tb infection ...................................... 27 2.5 RNA isolation of BMDMφ .................................................................................................................. 28 2.6 RNA amplification ............................................................................................................................. 29 iii 2.7 RNA microarray application ............................................................................................................. 30 2.7.1 Labelling and purification of nucleic acid ............................................................................. 30 2.7.2 Microarray chip hybridization ............................................................................................... 31 2.8 Microarray analysis – Genespring GX analysis ................................................................................ 32 2.8.1 Ingenuity pathway analysis ................................................................................................... 33 2.9 Quantitative reverse‐transcriptase PCR ........................................................................................... 33 2.9.1 Copy‐DNA (cDNA) synthesis .................................................................................................. 33 2.9.2 Quantitative PCR‐primer design and quality analysis ........................................................... 34 2.9.3 Quantiative RT‐PCR efficiency and protocol ........................................................................ 34 2.9.4 RNA isolation‐ for additional qPCR samples ......................................................................... 35 2.9.5 Quantitative reverse‐transcriptase PCR analysis .................................................................. 36 3 Identification of CD43‐mediated gene expression in response to Mycobacterium tuberculosis and Heat‐Shock proteins 65 and 70 using RNA microarray ............................................................................. 37 3.1 Introduction ...................................................................................................................................... 37 3.2 Rationale ........................................................................................................................................... 39 3.3 Results ............................................................................................................................................... 41 3.3.1 CD43 knockout and CD43 wildtype macrophages exhibit differential regulation to each other in response to mycobacterial stimuli ............................................................................................ 41 3.3.2 Mycobacterium tuberculosis, Cpn 60.2 and dnaK elicit differential responses in CD43‐/‐ and CD43+/+ BMDMΦ .................................................................................................................................. 41 3.3.3 CD43 deficiency affects multiple
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