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The differential gene expression of CD43 wildtype and knockout macrophages in response to mycobacterial stimulation Vair, Audra Helena


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 o served 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.

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