Activation of Islet Inflammation by Cytokine Signalling in Pancreatic b-cells Understanding the Role of Protein Tyrosine Phosphatases William James Stanley ORCID: 0000-0001-8001-2359 Student Number: 515467 Submitted in Total Fulfilment of The Degree of Doctor of Philosophy March 2020 St. Vincent’s Institute of Medical Research Department of Medicine (St. Vincent’s Hospital) The University of Melbourne Abstract: Type 1 diabetes is characterised by the autoimmune destruction of insulin producing β- 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 b-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 β-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 β-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. i 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 β- 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 β-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 ii 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 β-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. iii Declaration: I. The thesis comprises only their original work towards the Doctor of Philosophy except where indicated in the preface. II. Due acknowledgement has been made in the text to all other material used. III. The thesis is fewer than the maximum word limit in length, exclusive of tables, maps, bibliographies and appendices or that the thesis is 80,000 words as approved by the Research Higher Degrees Committee. iv Preface: I assess my contribution to the results described in each chapter to be: Chapter 3: 95% Chapter 4: 90% Chapter 5: 95% Chapter 6: 100% The following list outlines the experiments performed by others or with assistance that have been included in the results section of this thesis: Chapter 3: § Oxidised PTP immunoblot was performed by Dr. Esteban Gurzov. Chapter 4: § PTPN1 deficient NIT-1 cells were generated in collaboration with Dr. Andrew Sutherland. § 51Chromium release assay was performed with assistance by Dr. Prerak Trivedi. Chapter 5: § PTPN6 deficient NIT-1 cells were generated in collaboration with Dr. Andrew Sutherland. v The following are a list of publications completed during the course of my PhD degree: 1. Stanley WJ, Trivedi PM, Sutherland AP, Thomas HE, Gurzov EN (2017). Differential regulation of pro- inflammatory cytokine signalling by protein tyrosine phosphatases in pancreatic β-cells. Journal of molecular endocrinology 59(4):325-337. 2. Stanley WJ, Litwak SA, Quah HS, Tan SM, Kay TW, Tiganis T, de Hann JB, Thomas He, Gurzov EN (2015). Inactivation of protein tyrosine phosphatases enhances interferon signalling in pancreatic islets. Diabetes 64(7):2489-96. 3. Pilkington EH, Lai M, Ge X, Stanley WJ, Wang B, Wang M, Kakinen A, Sani MA, Whittaker MR, Gurzov EN, Ding F, Quinn JF, Davis TP, Ke PC (2017). Star polymers reduce IAPP toxicity via accelerated amyloid aggregation. Biomacromolecules 18(12):4249-4260. 4. Litwak SA, Pang L, Galic S, Stanley WJ, Turatsinze JV, Loh K, Gough DJ, Eizirik D, Gurzov EN (2017). JNK activation of BIM promotes hepatic oxidative stress, steatosis and insulin resistance in obesity. Journal of Cell Biology 66(12):2973-2986. 5. Litwak SA, Loh K, Stanley WJ, Pappas EG, Wali J, Selck C, Strasser A, Thomas HE, Gurzov EN (2016). P53- upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity. Scientific reports 6:23802. 6. Gurzov EN, Stanley WJ, Pappas EG, Thomas HE, Gough DJ (2016). The JAK/STAT pathway in obesity and diabetes. FEBS J 283:3002-15. 7. Pilkington EH, Gurzov EN, Kakinen A, Litwak SA, Stanley WJ, Davis TP, Ke PC (2016). Pancreatic β-cell membrane fluidity and toxicity induced by human islet amyloid polypeptide species. Scientific reports 6:21274. 8. Gurzov EN, Wang B, Pilkington EH, Chen P, Kakinen A, Stanley WJ, Litwak SA, Hanssen EG, Davis TP, Ding F, Ke PC (2016). Inhibition of hIAPP amyloid aggregation and pancreatic β-cell toxicity by OH- terminated PAMAM dendrimer. Small 12(12):1615-26. 9. Nedumpully-Govindan P, Gurzov EN, Chen P, Pilkington EH, Stanley WJ, Litwak SA, Davis TP, Ke PC, Ding F (2016). Graphene oxide inhibits hiAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells. Physical Chemistry Chemical Physics 18(1):94- 100. 10. Litwak SA, Wali JA, Pappas E, Saadi H, Stanley WJ, Kay TW, Thomas HE, Gurzov EN (2015). Lipotoxic stress induces pancreatic beta cell death through modulation of Bcl-2 proteins by the ubiquitin/proteasome system. Journal of Diabetes Research 2015:280615. 11. Gurzov EN, Stanley WJ, Brodnicki TC, Thomas HE (2015). Protein tyrosine phosphatases: molecular switches in metabolism and diabetes. Trends in Endocrinology & Metabolism 26:30-9. vi Acknowledgements: I am very thankful to my supervisors Dr. Esteban Gurzov and Prof. Helen Thomas for allowing me to undertake my PhD studies in the Immunology and Diabetes Unit at St. Vincent’s Institute of Medical Research. I didn’t realise how difficult completing a PhD can be but their guidance and support pushed me to finish when I thought it would never end. I would also like to thank Prof. Natalie Sims, A/Prof Jörg Heierhorst and Dr. Amanda Edgley for their wisdom and assistance when I needed it as well. The Immunology and Diabetes Unit has been a pleasure to work in and I’m incredibly grateful to all the researchers for creating a wonderful work environment. In particular I’d like to thank Dr. Sara Litwak, Dr. Kate Graham and Stacey Fynch for technical assistance in the lab when I needed it. I also want to thank Dr. Andrew Sutherland and Dr. Prerak Trivedi for helping me with experiments I couldn’t have done alone. Working at St. Vincent’s Institute of Medical Research has been fantastic and the staff are a pleasure to be around. I’m grateful for Dr. Rachel Mudge, Dr. Anne Johnston and the foundation for helping me apply for scholarships and rewarding me with a top up scholarship. I also thank the Harold Mitchell Foundation for awarding me a travel grant to travel to Belgium and present my work at the European Cell Death Organisation. I’d also like to thank the students for keeping my optimism up and general comradery. I’d also like to thank my parents, family and friends for making this whole process slightly less tedious and more enjoyable. vii Table of Contents: Literature Review..................................................................................... 1 1.1 Literature review methodology: ....................................................................... 2 1.2 Type 1 diabetes overview: ............................................................................... 3 1.2.1 Human islet immunopathology in type 1 diabetes: ................................... 4 1.3 Mouse models of type 1 diabetes: ...................................................................