Factors regulating cell shedding in mouse and rat small intestine Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in philosophy Ahmed Hamed Elramli August 2018 0 Acknowledgements I am truly grateful and thankful to Professor D Mark Pritchard who has supported me throughout my PhD studies, and without his patience, guidance and immense knowledge, this doctoral thesis would not have been completed. I would like to thank him for teaching me scientific writing and for the opportunity to participate in the British Society of Gastroenterology meeting (2016). I am also grateful to my second supervisors, Professor Barry J Campbell and Dr Carrie Duckworth for their supervision, advice and valuable support throughout all my PhD studies and thesis preparation. I gratefully acknowledge Dr Jonathan Williams for his help at the start of my PhD studies. I am also grateful to Dr Stamatia Papoutsopoulou, Dr Felix Ikuomola and Dr Esam Shahout from the Department of Cellular and Molecular Physiology and Juan Hernandez-Fernaud from the University of Warwick for their help with proteomics and Mrs Alison Beckett from EM unit, Department of Cellular and molecular physiology for the help in tissue preparation for EM. Thanks also to all members of the Henry Wellcome Laboratories at the University of Liverpool for their help. I would like to acknowledge the University of Benghazi via the Ministry of Education in Libya for my PhD funding. Last but not the least, I would like to thank my father, mother, wife and kids for their sincere encouragement and inspiration throughout this study. 1 Factors regulating apoptosis and shedding in mouse and rat small intestine Ahmed Hamed Elramli The gut barrier is composed of a single layer of intestinal epithelial cells which are connected together by junctional complexes and are covered by a layer of glycocalyx. Its function is to prevent the passage of harmful and toxic substances from the gut lumen into the blood circulation. Structural homeostasis in the small intestine is maintained by the shedding of intestinal epithelial cells at the villus tip and generation of new cells within the crypt compartment. Increased gut permeability can result when the rate of intestinal epithelial cell shedding is greater than the rate of generation of new crypt cells, and this phenomenon is associated with several gastrointestinal diseases including inflammatory bowel disease (IBD). Lipopolysaccharide (LPS) is an integral cell wall component of Gram negative bacteria and following intraperitoneal administration induces the pathological shedding of murine small intestinal epithelial cells (SIEC). This thesis characterises the pathological apoptosis and shedding of SIECs induced by systemic LPS administration in wild-type mouse strains of different genetic background including C57BL/6J, Balb/C, CD1, FVB/N and DBA/2. We have also determined whether murine age and LPS derived from different bacterial species are factors that modulate the severity of SIEC apoptosis and shedding in C57BL/6J mice. Comparative studies were also conducted in Ludwig Olac Wistar rats following to determine whether this phenomenon occurred to the same extent within another species. SIEC apoptosis and shedding was additionally induced by the administration of other systemic stimuli to C57BL/6J mice including anti-CD3 antibody (to activate T cells), polyinosinic:polycytidylic acid (to mimic viral infection) and the putative downstream effector tumour necrosis factor (TNF). The roles of NFκB transcription factor subunits in regulating SIEC apoptosis and shedding in villi and crypts induced by all stimuli at 1.5 and 24 hours were assessed and compared with the responses to LPS. SIEC apoptosis and shedding was assessed morphologically and quantified by immunohistochemistry for active caspase-3. Ultrastructural analysis of SIEC apoptosis and shedding following LPS administration was conducted using transmission electron microscopy to determine the action of LPS on the intracellular components and how they may contribute to the breakdown of barrier function associated with this stimulus by modulating NFκB signalling. A broad spectrum proteomic analysis was also conducted to determine how LPS alters the small intestinal mucosal proteome to modulate susceptibility to SIEC apoptosis and shedding. Conclusion: In conclusion, this study has described the SIEC apoptosis and shedding that are induced by different stimuli including LPS, anti-CD3, poly I:C and TNF. Particularly it has indicated that LPS from different bacterial sources induces SIEC apoptosis and shedding in different mouse strains, at different ages and in both mouse and rats, but with different degrees of effectiveness. It has also indicated the role of the NFҡB signalling pathway in regulating this process. 2 Table of contents Acknowledgement..………………………........................................................................……1 Abstract ……...................................................................................................................2 List of figures...………..………............................................................................................7 List of tables…….…..…………...........................................................................................11 Abbreviation….….........................................................................................................14 1. Introduction............................................................................................................17 1.1 Anatomy and histology of the small intestine….....................................................17 1.2 Disease of the intestinal tract...............................................................................19 1.2.1 Inflammatory bowel disease (IBD)……….………………………………………….………………19 1.2.2 Cancer of the intestine……….……………………………………………………………………………20 1.2.3 Sepsis……….…………………………………………………………………………………………………….22 1.3 Intestinal mucosal epithelium……..........................................................................23 1.4 Cell types in the intestinal epithelium....................................................................24 1.4.1 Intestinal epithelial stem cells……........................................................................25 1.4.2 Absorptive cells (mature enterocytes)……...........................................................26 1.4.3 Goblet cells………................................................................................................27 1.4.4 Paneth cells……….……….......................................................................................27 1.4.5 Enteroendocrine cells……….………………………………….............................................28 1.4.6 Tuft cells……….………………………………………………………………………………………………..28 1.5 Proliferation and differentiation of the small intestinal epithelium….....................29 1.5.1 Wnt signalling pathway….……………………………………………………………………............31 1.5.2 Notch signalling pathway……….……………………………………………………………………….32 1.6 Cell death…….…………………………………………………………………………………………………….34 1.6.1 Necrosis……….…………………………………………………………………………………………………34 1.6.2 Apoptosis………..…………………………………………………………..…………………………………35 1.6.3 Necroptosis……….……………………………………………………………………………………………36 1.6.4 Pyroptosis………..…………………………………………………………………..………………………..37 1.6.5 Autophagy……….………………………………………………………………………..…………………..37 1.7 Cell death in the small intestine…….…………………………………………………………………..38 1.7.1 Mechanism of the IEC shedding during homeostasis……….……………………………..39 1.8 Genetic regulation of apoptosis in the gastrointestinal tract……………………………….44 1.8.1 Bcl2 and related genes……….………………………………………………………………………..…45 1.8.2 p53……….…………………………………………………………………………………………………..……46 1.8.3 Tumour necrosis factor family………..…………………………………………………………….47 3 1.8.4 C-myc proto-oncogene………..………………………………………………………………………….48 1.9 Stimuli that cause pathological intestinal epithelial cell apoptosis and shedding………………………………………………………………………………………………………………...48 1.9.1 Bacterial lipopolysaccharide (LPS)……………………..…………………………………….49 1.9.2 Anti-mouse CD3e antibody……….…………………………………………………………………….51 1.9.3 Polyinosinic:polycytidylic acid (poly I:C)……….…………………………………………………52 1.9.4 Tumour necrosis factor (TNF)……….…………………………………………………………………53 1.10 Nuclear factor kappa B (NFκB) signalling…….……………………………………………………55 1.10.1 Canonical NFκB signalling pathway………………………………………………………………56 1.10.2 Non-canonical NFκB signalling pathway……….……………………………………………….56 1.11 proteomics approaches in the intestine…….…………………………………………………….58 1.12 Aims of the study……............................................................................................61 2. Materials and methods……......................................................................................63 2.1 Animals…..............................................................................................................63 2.1.1 Mice…...............................................................................................................63 2.1.1.1 C57BL/6J mice…………......................................................................................63 2.1.1.2 Balb/C mice………………………………………………………………………………………………….65 2.1.1.3 CD1 mice……………..………………………………………………………………………………………65 2.1.1.4 FVB/N mice……………..…………………………………………………………………………………..65 2.1.1.5 DBA/2 mice……………….……………………………………………………………………………….66 2.1.2 Ludwig Olac Wistar Rats………............................................................................66 2.2 Animal procedures……..........................................................................................66