Medicine (St Vincent's) - Theses
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ItemDissecting the interferon response required for triggering autoimmune diabetes( 2016)Type 1 diabetes (T1D) is characterized by the autoimmune destruction of β cells in the islets of Langerhans by immune cells. The interferon (IFN) family of cytokines has been implicated in diabetes pathogenesis, possibly via the activation of innate immune pathways. However, how IFNs contribute to the pathogenesis of autoimmune diabetes remains unclear. The overall aim of this thesis is to dissect the role of IFNs in the triggering of autoimmune diabetes using the non-obese diabetic (NOD) mouse model. Chapter 2 describes the contribution of type I IFN to the development of autoimmune diabetes in NOD mice. The islets of NOD mice displayed a unique mRNA expression pattern of IFN-induced genes that peaked at 3-4 weeks of age. Genetic ablation of type I IFN receptor in NOD mice (NOD.Ifnar1-/-) significantly reduced the expression of these genes. However, lack of IFNAR1 did not affect the development of diabetes. The overlapping function of type III IFN could be a reason for the lack of effect of IFNAR1-deficiency. Another reason could be that the natural level of type I IFN produced in wild-type NOD mice is insufficient to have an effect on diabetes development. In Chapter 3, the possible role of type III IFN in autoimmune diabetes was examined. β cells isolated from humans and mice expressed the type III IFN receptor and β cells were able to respond to type III IFN stimulation. Type III IFN was detected in the islets in NOD mice. These results indicate that type III IFN may contribute to diabetes development in NOD mice. Future experiments using NOD mice deficient in the type III IFN receptor will determine whether type III IFN contributes to the development of autoimmune diabetes. Engagement of pattern recognition receptors (PRRs) with exogenous and endogenous danger signals can trigger type I IFN production. Endogenous danger signals, such as aberrant cytosolic DNA, are normally eliminated to prevent unnecessary induction of PRRs. Nucleases, such as the 3’ exonuclease TREX1, are important in degrading aberrant DNA, and have been implicated in protection from autoimmunity. TREX1 is normally found in the SET complex, and one protease that can activate this complex is granzyme A. In Chapter 4, I show that granzyme A deficiency resulted in increased diabetes in NOD mice. Single-stranded DNA accumulated in the cytoplasm of dendritic cells and NK cells in the islets and spleens, and this was observed more frequently in NOD.Gzma-/- mice. Consistent with poor clearance of DNA and increased PRR activation, expression of IFN-induced genes was higher in the islets of NOD.Gzma-/- mice than NOD mice at 4 weeks of age. When NOD.Gzma-/- mice were crossed with NOD.Ifnar1-/- mice, diabetes returned to the rate observed in NOD mice. Overall, the data indicate that the natural level of type I IFN produced in wild-type NOD mice is not sufficient to cause diabetes, but excessive type I IFN can increase diabetes development in NOD.Gzma-/- mice. The results provide mechanistic insight into the triggering of autoimmune diabetes, suggesting that aberrant accumulation of cytoplasmic DNA and type I IFN production are important. In this study, these were caused by loss of granzyme A, but it is also possible that virus infection could similarly activate type I IFN and possibly also type III IFN to trigger T1D.
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ItemThe effect of inhibiting pro-apoptotic BH3-only proteins on beta cell loss and glucose homeostasis in type 2 diabetes( 2014)Type 2 diabetes is caused by a combination of insulin resistance, as well as dysfunction and loss of beta cell mass. Around 30-70% of beta cell mass is lost in type 2 diabetes and this is due to apoptosis, induced mainly by chronic hyperglycaemia. High concentrations of glucose activate the intrinsic apoptosis pathway in beta cells and this requires the pro-apoptotic Bcl-2 family proteins Bim and Puma. Glucose-induced production of reactive oxygen species may also activate the NLRP3-inflammasome pathway, resulting in caspase-1 cleavage, and production of IL-1β. This thesis aimed to identify the stress pathways that mediate glucose toxicity of beta cells. In addition, possible benefits of apoptosis inhibition on glucose homeostasis and beta cell function were examined using mouse models. Chapter 2 describes the role of endoplasmic reticulum (ER) stress and oxidative stress in mediating glucose toxicity of islets. Exposure of islets to high concentrations of glucose induced oxidative and ER stress and increased expression of the pro-apoptotic ER factor CHOP. This caused downstream activation of Bim and Puma-mediated apoptosis and was inhibited by chemical chaperones or antioxidant treatment. Further, increased expression of Bim and Puma mRNA was observed in human islets in type 2 diabetes. Inhibition of ER and oxidative stress could be an effective strategy to reduce beta cell apoptosis in type 2 diabetes. In Chapter 3, the controversial subject of activation of the NLRP3-inflammasome in islets by glucose toxicity-induced ER and oxidative stress was studied. Apoptosis induced by ER or oxidative stress inducing agents was similar in islets isolated from wild-type, NLRP3 or caspase-1 deficient mice. Loss of NLRP3 did not protect islets from glucose, ribose or gluco-lipotoxicity. The presence of a hyperactive mutant of NLRP3 in beta cells had no effect on ribose toxicity or ribose-induced IL-1β secretion. These results suggest that activation of the inflammsome does not mediate glucose toxicity in islets. In Chapter 4, the metabolic effects of apoptosis inhibition were studied in Bimdeficient mice. Bim deficiency resulted in reduced adiposity, improved insulin sensitivity and glucose tolerance in mice. Islet size and function was normal suggesting that phenotype was not beta cell-dependent. Study of mice in metabolic cages revealed that loss of Bim resulted in increased lipid oxidation and this may explain the metabolic changes. The effects of apoptosis inhibiton in beta cells were studied in Chapter 5 by analysing Bim-deficient Leprdb/db mice. Loss of Bim resulted in increased body weight but normalized fasting blood glucose and improved glucose tolerance in Leprdb/db mice. These changes were likely due to a two-fold increase in islet area and volume, and a similar increase in serum insulin concentration. Therefore, loss of Bim alters the balance between apoptosis and replication, thereby making it a possible therapeutic target to prevent beta cell loss in type 2 diabetes. Overall, work presented in this thesis shows that inhibition of apoptosis or its upstream activators ER and oxidative stress may prevent beta cell loss in type 2 diabetes. Inhibiting pro-apoptotic proteins could also have additional benefits such as increased lipid oxidation and improved insulin sensitivity.