Medicine (St Vincent's) - Theses
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ItemActivation of islet inflammation by cytokine signalling in pancreatic beta cells: understanding the role of protein tyrosine phosphatases( 2018)Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. Furthermore, PTPN1 inhibition reduced IFN-g induced MHC-I expression on the surface of NODPI beta-cells and reduced the ability of autoreactive NOD8.3 CD8+ T-cells to destroy isolated NOD/Lt islets. These studies showed that PTPN1 is a positive regulator of cytotoxic signalling in NIT-1 cells and NODPI islets and promotes immune cell mediated death, suggesting it may be a potential therapeutic target for type 1 diabetes. Chapters 5&6 study the role of PTPN6 in cytokine signalling regulation in NIT-1 cells. PTPN6 inhibition was found to enhance TNF-a induced NIT-1 cell death independent of IFN-g and IL-1b in vitro. TNF-a induced JNK signalling was enhanced with PTPN6 inhibition which resulted in reduced anti-apoptotic BCL-2 protein expression and enhanced caspase-3 cleavage. Pan-caspase inhibition prevented TNF-a induced cell death suggesting that cells were dying through apoptosis. TNF-a induced cell death was also prevented with RIPK1 inhibition which prevented enhanced caspase-8 cleavage in PTPN6 deficient cells. Collectively these studies showed that PTPN6 negatively regulates TNF-a induced intrinsic and extrinsic apoptosis of beta-cells in vitro. Overall, the data indicate that PTPs play a nonredundant role in the regulation of cytotoxic signalling in NIT-1 cells and NODPI islets. This finding is consistent with results in other disease pathologies. The results provide a mechanistic insight into how PTPN1 and PTPN6 have opposite roles in regulating cytokine signalling, highlighting how PTPN1 antagonism and PTPN6 agonism may prove beneficial in reducing beta-cell death in vitro. Whether these results are directly translatable into in vivo models of autoimmune diabetes remains undetermined. The use of PTPN1 inhibitors or PTPN6 agonists currently under development would allow direct translation of these results.