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Pranvera Sadiku Thesis Final Submission.Pdf The regulation of myeloid cell survival: Emerging roles for the pro-survival protein HAX1 Pranvera Sadiku Academic Unit of Respiratory Medicine Department of Infection and Immunity University of Sheffield Thesis submitted for the degree of Doctor of Philosophy September 2014 Abstract The regulation of myeloid cell apoptosis is critical in both the control of inflammation and homeostasis. HAX1, a ubiquitously expressed multifunctional protein is thought to play a key role in cell survival. HAX1 mutations in the human result in premature apoptosis of bone marrow myeloid progenitor cells and a profound PMN (peripheral blood neutrophils) deficiency, suggesting HAX1 plays a critical role in PMN homeostasis. Human PMN are genetically intractable and Hax1 homozygous mice have been shown to die at 14 weeks of age. The molecular pathways in which HAX1 is involved are therefore yet to be elucidated. We hypothesised that HAX1 would play a critical role in constitutive cell death through effects on mitochondrial membrane stability and that hax1 deficient zebrafish embryos would have defective PMN development and survival. HAX1 expression was studied in primary PMN (prepared from venous blood of healthy volunteers), myeloid leukaemia cell line PLB-985 and wild type zebrafish embryos by western blotting, RT-PCR and in situ hybridisation. HAX1 was knocked down in the human myeloid leukaemia cell line PLB-985 and hax1 deficient zebrafish mutants were created in an MPO driven GFP transgenic line using antisense morpholino oligonucleotides (MO) or TALEN technology. PLB-985 and primary PMN express multiple HAX1 isoforms at the mRNA level, including the full-length isoform. I present data revealing the modulation of HAX1 in response to inflammatory stimuli in PLB-985 and show that HAX1 expression decreases during constitutive ageing of PMN. Zebrafish express multiple isoforms of hax1, providing novel evidence for alternative splicing of the gene in the model organism. hax1 MO injected embryos exhibit a reduction in PMN numbers, which may be due either to a loss of Hax1 or to the concomitant developmental delay observed. The hax1 targeting TALEN generated efficiently modified the hax1 gene and was successfully transmitted through the germline. Preliminary data suggest that the HAX1 knockdown in PLB-985 cells and the hax1 homozygous mutation generated in the zebrafish are not important in PMN lifespan. 1 Acknowledgements Firstly, I would like to thank my primary supervisor Lynne Prince and co- supervisor Stephen Renshaw for their continued guidance and support throughout the course of my studies. Thanks to Lynne Prince for my training, for the constant motivation in our meetings and through emails and most of all for her patience. Thanks to Steve Renshaw for his input and help. I am really happy and grateful that I have had the chance to be supervised by both of you. I also wish to give special thanks to the Medical Research Council for funding my project. I would like to thank all of the members of the Infection and Immunity department and of the Centre for Developmental and Biomedical Genetics in Sheffield for making it a pleasant environment to work in. Thank you to Julie Bennett for her help with molecular biology, Kathryn Higgins for help with PMN preps and Vanessa Singleton for always ensuring that reagents arrived on time and the lab ran smoothly. Thank you to the blood donors for making our research possible and everyone who carried out PMN preps and spared cells for my work. I would also like to thank Nikolay Ogryzko, Catherine Mottram, Kate Hammond and Anne Robertson for their help and training in zebrafish work and data analysis using Volocity® software. Thanks to James Robertson for the help with and input into the polymerosome experiments. Very big thanks to Stone Elworthy for his guidance and help with the ZFN and TALEN work. Thank you to Marzieh Fanaei, Selina Parmar, Jamil Jubrail, Kirsty Wilson for their moral support and Martin Bewley for the funny (mostly science) jokes that always made me laugh. I would also like to thank my family for their unconditional love and support. Thanks to my brothers, sister (Ilirida) and cousins/best friends (Hana, Kreni and Lulja) for listening to me, being interested in what I had to say about my research (even when they didn’t quite understand it) and for always putting a smile on my face. A final and very special thanks goes to my parents for their endless encouragement, patience and making me believe that I could get this far. 2 Table of Contents Abstract ......................................................................................................................... 1 Acknowledgements ................................................................................................... 2 List of Figures ........................................................................................................... 12 Figure 4.12 Analysis of i114 embryos co-injected with hax1 and p53 MO 158 14 List of Abbreviations .............................................................................................. 17 1 General introduction ...................................................................................... 20 1.1 Immunity .................................................................................................................. 20 1.2 Inflammation .......................................................................................................... 20 1.2.1 Leukocyte recruitment ............................................................................................ 20 1.2.2 Pattern recognition receptors .............................................................................. 21 1.3 PMN ............................................................................................................................ 22 1.3.1 PMN terminal differentiation ............................................................................... 23 1.3.2 PMN migration to the site of infection/injury ............................................... 24 1.3.3 The role of PMN in infection ................................................................................. 26 1.3.3.1 PMN granule content and action against pathogens ......................................... 27 1.3.3.2 PMN apoptosis ......................................................................................................................... 27 1.4 Inflammation resolution..................................................................................... 29 1.5 PMN in disease ........................................................................................................ 31 1.5.1 Inflammatory disease .............................................................................................. 31 1.5.2 Accelerated PMN apoptosis ................................................................................... 32 1.6 Molecular mechanisms of apoptosis .............................................................. 33 1.6.1 The extrinsic Apoptotic pathway ........................................................................ 33 1.6.2 Intrinsic (mitochondrial) apoptotic pathway ................................................ 34 1.6.3 The regulation of the mitochondrial apoptotic pathway by the Bcl-2 protein family ............................................................................................................................. 35 1.6.3.1 Pro-apoptotic proteins .................................................................................................... 35 1.6.3.2 Anti-apoptotic proteins................................................................................................... 36 1.7 HCLS-1 associated protein X1 (HAX1) ........................................................... 38 1.7.1 HAX1 genetic organization, transcription and conservation ................... 38 1.7.2 Molecular and physiological roles of HAX1 protein .................................... 41 1.7.2.1 Domain structure, function and cellular localisation ........................................ 41 1.7.2.2 HAX1 putative binding sites for RNA....................................................................... 44 3 1.7.2.3 Regulation of cell migration by HAX1 ...................................................................... 44 1.7.2.4 Interactions with viral proteins .................................................................................. 45 1.7.2.5 HAX1 involvement in cell fate ...................................................................................... 47 1.7.2.6 Interactions with factors involved in the apoptotic pathway ........................ 47 1.7.3 Roles of HAX1 in PMN .............................................................................................. 51 1.7.3.1 Involvement of HAX1 in Ca2+ signalling ................................................................. 51 1.7.3.2 The importance of HAX1 in PMN apoptosis .......................................................... 51 1.8 Zebrafish as a model of PMN inflammation ................................................. 53 1.9 Hypothesis, aims and Objectives: .................................................................... 57 2 Materials and Methods ............................................................................................ 59 2.1 In-vitro techniques ............................................................................................... 59 2.1.1 Reagents .......................................................................................................................
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