An Investigation of the Intestinal Epithelial Damage Response to Cytokines Produced During Inflammatory Bowel Disease and Sepsis
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An investigation of the intestinal epithelial damage response to cytokines produced during inflammatory bowel disease and sepsis Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in philosophy Felix Irewole Ikuomola February 2020 Declaration I hereby declare that this thesis is a presentation of my original work. Wherever contributions of others are involved, every effort has been made to indicate this clearly, with due reference to the literature. Work was performed under the guidance of project supervisors’ Dr Carrie Duckworth* and Professor Pritchard**. *Gastroenterology Research Unit, Department of Molecular Physiology and Cell Signalling, Institute of Systems, Molecular and Integrative Biology, University of Liverpool ** Gastroenterology Research Unit, Department of Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool. 2 Acknowledgements Firstly, I am truly grateful and thankful to Dr Carrie Duckworth who has supported me immensely throughout the course of my PhD studies, and without her in-depth knowledge, patience, perseverance and guidance this doctoral thesis would not have been pragmatically possible to complete. I would like to thank her for lecturing me comprehensively in scientific writing and for the opportunity given me to participate in the UK/Northern Ireland NF-kappaB meeting of which I won one of the best poster awards (2019). Secondly, I am also grateful to my second supervisor, Professor Mark Pritchard for his thought-provoking supervision, advice and immeasurable support throughout the scale and scope of my PhD studies and thesis preparation. I gratefully acknowledge Juan Hernandez-Fernand from the University of Warwick for his help with proteomics. Special thanks to all members of the Henry Wellcome Laboratories at the University of Liverpool and Department of Cellular and Molecular Physiology for their help. Last but not the least, I am very gratefully to the Almighty God for life, sustenance and provision. And I would like to thank Wendy, my beloved wife for her inspiration, encouragement and support and Alexa Bisi, my daughter for being a source of relief from stress. 3 Abstract An investigation of the intestinal epithelial damage response to cytokines produced during inflammatory bowel disease and sepsis. Felix Irewole Ikuomola Sepsis is a life-threatening host response to severe systemic infection that can result in multi-organ dysfunction syndrome (MODS) and organ failure. Globally, there are around 30 million cases of and 6 million deaths from sepsis annually. The intestine is one of the first organs to be damaged during the sepsis-induced proinflammatory cytokine production and hyperinflammatory responses. Similar processes occur in the intestinal mucosa during active inflammatory bowel disease (IBD). Under normal physiological conditions, the intestinal epithelium promotes gut homeostasis via mechanical protection and maintenance of structural integrity, and provides a physical barrier comprised of a continuous single cell layer of intestinal epithelial cells. Intestinal barrier dysfunction or compromised intestinal integrity potentially facilitates the passage of intestinal bacteria into the gut mucosa and blood circulation. Compromised intestinal barrier function occurs as a result of excessive epithelial cell shedding and apoptosis resulting from the direct effects of pro-inflammatory cytokines such as tumour necrosis factor (TNF) on intestinal epithelia. Understanding the mechanisms responsible for intestinal barrier function break down will potentially enable future prevention and treatment of sepsis, IBD and other conditions associated with intestinal barrier disruption. Recent advances in intestinal stem cell culture have enabled the generation of three-dimensional organoid cultures of the intestinal epithelium (termed enteroids) which can auto-propagate, auto-renew and contain similar cell populations to those found in the intestinal epithelium in vivo. This culture system represents a more physiological model of the intestinal epithelium than has previously been achievable using standard tissue culture techniques. It enabled investigation of tissue-level dynamics and the importance of NFκB signalling specifically in the intestinal epithelium in response to TNF (chapter 3). TNF treatment was associated with asynchronous expansions and contractions, increased motility, claudin-7 remodelling, reduced epithelial thickness and early increased surface area of enteroids. The alternative NFκB signalling pathway was demonstrated to regulate the intestinal epithelial response to injury and further exploration of other cytokines known to activate alternative pathway NFκB signalling was undertaken. We identified that other activators of NFκB signalling that are upregulated in the serum of patients with inflammatory conditions, such as TWEAK and LIGHT, were also capable of initiating the enteroid damage response and that Nfκb2-/- enteroids were resistant to these stimuli (chapter 4). Proteomic analyses were used to identify potential mechanisms responsible for altered intestinal epithelial dynamics in response to TNF including TNFaip3 and saa3. Proteomics elucidated the potential mechanistic drivers of the intestinal damage response during perturbed NFκB signalling (chapter 5). The enteroid inflammatory model was finally tested with corticosteroids, a non-steroidal anti-inflammatory drug and a natural anti-inflammatory compound found in the diet known to modulate NFκB signalling to determine whether this newly established in vitro model could be used to assess novel putative therapeutic interventions for sepsis and IBD (chapter 6). Further investigation is now warranted to determine the importance of TNF, other pro-inflammatory cytokines and alternative pathway NFκB signalling in regulating intestinal tissue level dynamics during health and disease and how these parameters may be modulated therapeutically to ameliorate the severity of IBD and sepsis. 4 Table of contents Declaration ………………………………...………………………………………………2 Acknowledgements ………………………………...……………………………………..3 Abstract......................................................................................................................4 List of figures …..………...........................................................................................11 List of tables …..………............................................................................................15 Abbreviations …...……….........................................................................................17 1 Introduction …...………...................................................................................... 23 1.1 Sepsis …..…...……….................................................................................... 23 1.1.1 Clinical course of sepsis …...…………………........................................ 24 1.1.2 Pathogenesis of sepsis …...………......................................................... 25 1.1.3 Intestinal manifestation of sepsis …...……….......................................... 28 1.2 Therapeutic approaches for sepsis ……………………………………………. 29 1.2.1 General measures ………………………………………………………….. 29 1.2.2 Specific measures ……………………………….……………….………… 30 1.2.2.1 Antimicrobial antibiotics ………..…………………….……………….. 30 1.2.2.2 Corticosteroids ………………….………………………….………….. 30 1.2.2.3 PRR Antagonist …………………………………….…………..……… 32 1.2.2.4 HMGB-1 inhibitors ………………………………….…………….……. 32 1.2.2.5 NFκB inhibitors ………………….…………….……….………….…… 32 1.2.2.6 Immunosuppressants ……….…………………………...……………. 33 1.2.2.7 Biomedical therapy ……………………………………….…………… 33 1.2.2.8 Natural products …………………………………….…………………. 34 1.2.2.9 Surgery………………………………………………………………….. 34 1.3 Inflammatory bowel diseases …........………................................................. 35 1.4 Similarities between sepsis and inflammatory bowel diseases ……….…….. 36 1.5 Anatomy and physiology of the small intestine……………………………….. 37 1.6 Intestinal mucosal epithelium …………………………………………………… 39 1.6.1 Intestinal tight junctions ………………… ……………………………….. 35 1.6.1.1 Claudins …...………........................................................................ 40 1.6.1.2 Occludin …...………........................................................................ 42 1.6.1.3 Junctional adhesion molecule A (JAM-A) ……………..………..…… 43 5 1.6.1.4 ZO-1 …...…….................................................................................. 44 1.7 Cell dynamics of the small intestinal epithelium ……..…………………..…… 45 1.7.1 Stem cells and epithelial cell turnover ……..…………..……………….…. 45 1.7.2 Enterocytes …........................................................................................ 47 1.7.3 Paneth cells …........................................................................................ 49 1.7.4 Goblet cells .…...……............................................................................. 50 1.7.5 Enteroendocrine cells …........................................................................ 51 1.7.6 Tuft cells …...…….….............................................................................. 52 1.7.7 M-cells ………….……………………………………………………………. 52 1.8 Organoids …...……….................................................................................... 53 1.8.1 Organoid definition …...………............................................................... 53 1.8.2 Small intestinal epithelial organoids ………………………………………. 53 1.8.3 Advantages of 3D enteroid