Medicine (St Vincent's) - Theses
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ItemStudies investigating the clinical impact and immunological host response to viral eradication in hepatitis C infection( 2019)Australia now has improved access to HCV treatment for individuals with all stages of liver disease. There is limited data however, documenting any long-term clinical benefit of viral eradication in those with early stage liver disease. We report on the long-term outcomes of a well-characterised cohort of CHC subjects with predominantly early stage liver disease, whom paired liver fibrosis assessments were performed more than 10 years apart. We show in a real-world setting, that both early and curative HCV treatment halts fibrosis progression. Our data supports the early treatment of all people with CHC regardless of liver fibrosis stage, to prevent long-term liver sequelae. In subjects with acute / subacute HCV infection, we identify predictors of innate immunological response in peripheral blood mononuclear cells, to differentiate spontaneous clearers to those who progress to chronic infection. Our data strongly implicates TLR4 signaling in the persistence of HCV infection. Those who developed chronic infection had higher TLR4 expression on peripheral monocytes and NK cells and increased IFN-gamma response to TLR4 stimulation. We also observed increased TLR7 responsiveness in this group. We confirm the previously noted associations of IFNL4 genotype and plasma IP-10. Our data presents TLR4 as a potential biomarker for predicting clearance. This would clinically translate to individuals presenting with acute / subacute HCV infection, being able to defer drug therapy and its associated morbidity and cost. In subjects with chronic HCV infection, we demonstrate a clear effect of direct antiviral agent (DAA) mediated viral suppression therapy on patterns of TLR signaling in subjects with chronic HCV-1. We show that peripheral monocytic TLR2, TLR4 and TLR7 signaling is down-regulated early on in treatment, with a strong trend to higher baseline TLR signaling being associated with viral clearance with the DAAs. We are the first study to demonstrate a relationship between TLR signaling activity and IFN-free therapy for HCV. We hypothesise that the HCV virus directly stimulates the TLR pathways to induce an antiviral effect, with higher TLR signaling evident among those who respond to DAA therapy. This was accompanied by a reduction in PBMC ISG expression, NK activation markers and plasma levels of inflammatory cytokines / chemokines, with restoration of the innate immune response. Despite excellent treatment options, the mechanisms responsible for viral eradication in HCV have remained poorly defined. Previous data suggest a link between innate immunity and HCV pathogenesis, as well as spontaneous viral clearance. The data suggested that antiviral therapy alone was not sufficient to clear virus and supported a key role for innate immunity contributing to viral clearance. Our results strongly implicate TLR signaling / expression in HCV viral clearance. We believe TLR agonists must be considered as a potential HCV vaccine candidate and further research in this area is required.
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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.