Chaperonin 10 (Cpn10) is a mitochondrial protein with protein folding function. There is substantial evidence that extracellular Cpn10 regulates the immune response. Prior research has shown that Cpn10 binds to T cells, inhibits LPS-induced RAW264.7 macrophage cell- and healthy donor peripheral blood mononuclear cell (PBMC)-activation, and downregulates lipopolysaccharide (LPS)-induced membrane distribution of the MHC II molecule on dendritic cells (DC). Recent Phase IIa rheumatoid arthritis (RA), psoriasis and multiple sclerosis (MS) clinical trials demonstrate improved disease amelioration with Cpn10. Despite compelling evidence of the anti-inflammatory properties of Cpn10, the precise mechanisms of action are unknown. The principal aim was to characterise the modulation of inflammation by Cpn10 and in the process create a bioassay that would allow for the reliable assessment of batch-to-batch variability of Cpn10 preparations. For this purpose, a Cpn10 bioassay was performed in the RAW264.7 cell line and expanded to DC and T cell lines. Furthermore, the analysis of gene expression in healthy donor PBMC was performed, as a mixed cell population experiment, to reflect possible involvement of cell-to-cell communication pathways. Initial data showed that Cpn10 reduced LPS-induced tumour necrosis factor ?? (TNF??) expression in RAW264.7 cells. However, the Cpn10 preparation was shown to contain trace lipid contaminants, which induced cellular tolerance, resulting in the observed reduction in TNF??. Experiments with a second batch of Cpn10 showed no reduction of LPS-induced TNF?? in the RAW264.7 cells, seen with the primary batch of Cpn10 and previously reported characterisation of Cpn10. The Cpn10 bioassay conducted in DC and T cell lines was shown to have the potential to decrease toll-like receptor 9 (TLR9) expression, suggesting that Cpn10 may attenuate immune responses by downregulating receptor recognition of bacterial components. The Cpn10 bioassay conducted in LPS-stimulated PBMCs revealed that Cpn10 downregulates gene expression of Th1 related genes including the polarising cytokines IL-7, IL-12B and IL-23A and Th2 related genes including the transcriptions factors GATA3, GFI1 and CEBPB. The downregulation of these genes may play an immuno-modulatory role, having improved efficacy of Cpn10 in T cell mediated autoimmune diseases, with possible therapeutic implications in Th2 mediated diseases such as asthma. The research carried out in this thesis provides insight into the success of Cpn10 in the RA, MS and psoriasis clinical trials. These results have also supported previously published data and provide additional insight into the mechanism of action of Cpn10. In addition, a Cpn10 bioassay has been established using healthy donor PBMCs stimulated with LPS and results show a reduced expression of Th1 and Th2 associated genes. The findings that in mixed cell populations, Cpn10 downregulates not only genes involved in Th1 polarisation mainly at the signal 3 level, but is also capable of downregulating Th2 polarising genes at the signal 1 level of TCR mediated transcription factors, are of particular interest. Ultimately, research from this project has confirmed the anti-inflammatory action of Cpn10 and given useful insight into how Cpn10 acts to modulate the inflammatory response.
| Identifer | oai:union.ndltd.org:ADTP/272945 |
| Date | January 2009 |
| Creators | Scott, Melissa Margaret Eve, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Scott Melissa Margaret Eve., http://unsworks.unsw.edu.au/copyright |
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