The role of mucus in the cross talk between gut bacteria and the host Olivia Kober, BSc (Hons) A thesis submitted in accordance with the requirements for the degree of Doctor of Philosophy (PhD) To The University of East Anglia Institute of Food Research Gut Health and Food Safety Norwich Research Park Colney NR4 7UA September 2013 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition any quotation or extract must include full attribution. Abstract The mammalian gastrointestinal tract is home to a complex microbial community engaged in a dynamic interaction with the immune system. Mucus is the first point of contact of the microbiota with the host, acting as a first line of defence. Furthermore γδ intraepithelial lymphocytes (IELs) respond to the invading bacteria that circumvent the mucus barrier. In this study two approaches were used to investigate the role of mucus in intestinal homeostasis; firstly the impact of γδ IELs on the mucus layer, and secondly the adhesion properties of the gut symbiont Lactobacillus reuteri to mucus. To study the impact of IELs on mucus properties, a γδ T cell-deficient (TCRδ-/-) mouse model was used. TCRδ-/- mice showed increased susceptibility to dextran sodium sulphate (DSS)-induced colitis, alterations in mucin expression, glycosylation and goblet cell numbers, but maintained an intact mucus layer in vivo. Moreover, TCRδ-/- mice showed reduced levels of interleukin-33 mRNA, a mediator of mucosal healing. An ex vivo SI organoid model using input cells from TCRδ-/- mice showed, upon addition of keratinocyte growth factor, increases in crypt length, and both goblet cell numbers and redistribution. These findings provide novel mechanisms by which γδ IELs may modulate mucus properties, explaining the increased susceptibility of TCRδ-/- mice to chemically-induced colitis. L. reuteri strains protect against DSS-induced colitis in mice. To investigate the importance of L. reuteri adhesion to the intestinal mucus layer, the mucus-producing HT29-MTX cell line as well as murine and human intestinal tissues were used in conjunction with chemical treatments. The mucus-binding protein MUB of L. reuteri ATCC 53608 was found to promote L. reuteri adhesion to mucins in a host and tissue- specific manner and display sialic acid-binding specificities. Together, these data provide insights into L. reuteri-mucus interactions; a key factor in influencing host response and exerting health effects. 2 Preface The work of this PhD project has resulted in the following publications: Scientific paper publications: Kober OI., Ahl D., Carding SC., Holm L., Juge N. γδ T cell-deficient mice show alterations in mucin expression, glycosylation and goblet cells but maintain an intact mucus layer. Under revision at Am J Physiol Gastrointest Liver Physiol. Fuell C., Kober OI., Hautefort I., Carding SC., Juge N. Mice deficient in intestinal γδ intraepithelial lymphocytes show a different mucin O-glycan profile compared to wild- type mice. Under revision at Glycobiology. Oral presentations: Kober O., Holm L., Carding SC. Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. Mucins in health and disease, 12th International Workshop on Carcinoma-associated Mucins, Robinson College, Cambridge, UK, 27th-31st July 2013. Kober O., Holm L., Carding SC. Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. Early careers British Society for Immunology Inflammation and Immunology Symposium, University of East Anglia, Norwich, UK, 26th March 2013. (Awarded prize for best oral presentation). Poster presentations: Kober O., Holm L., Carding SC. Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. British Society for Research on Ageing meeting, University of East Anglia, Norwich, UK, 3rd September 2013. Kober O., Hautefort I., Holm L., Carding SC., Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. Digestive Disease Week 2013, Orange County Convention Centre, Orlando, FL, 18th-21st May 2013. 3 Kober O., Hautefort I., Holm L., Carding SC., Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. Inaugural Annual Symposium, Gut Health and Food Safety ISP, John Innes Conference Centre, Norwich, UK, 5th March 2013. Kober O., Hautefort I., Holm L., Carding SC., Juge N. Impact of gamma delta T cells on murine intestinal mucus properties. Gamma delta T cell conference, Freiburg, Germany, 31st-2nd June 2012. Kober O., Wakenshaw L., Hautefort I., Carding SC., Holm L., Juge N. Modulation of the mucus component of intestinal barrier defence in mouse models. Mucins in Health and Disease, 11th International Workshop on Carcinoma-associated Mucins, Robinson College, Cambridge, UK, 9th-13th July 2011. Kober O., Wakenshaw L., Hautefort I., Holm L., Carding SC., Juge N. Modulation of the mucus component of intestinal barrier defence in mouse models. John Innes Centre, BBSRC conference, John Innes Conference Centre, Norwich, UK, 4th-6th July 2011. Kober O., Wakenshaw L., Hautefort I., Holm L., Carding SC., Juge N. Modulation of the mucus component of intestinal barrier defence in mouse models. IFR Student Science Showcase, Institute of Food Research, Norwich, UK, 1st July 2011. Kober O., Schreiber O., Jeffers F., Wakenshaw L., Roos S., Holm L., Juge N. Functional modulation of the rodent mucus layer by gut bacteria. The molecular biology of inflammatory bowel disease, Biochemical Society Conference, University of Durham, Durham, UK, 20th-22nd March 2011. Kober O., Jeffers F., Wakenshaw L., Hemmings A., MacKenzie D., Juge N. Functional modulation of the murine mucus layer by gut bacteria. British Society for Immunology Annual Congress, Arena and Conference Centre, Liverpool, UK, 6th-10th December 2010. Kober O., Wakenshaw L., Holm L., Carding SC., Juge N. Functional modulation of the murine mucus layer in response to gut bacteria. IFR Student Science Showcase, Institute of Food Research, Norwich, UK, 6th May 2010. 4 Acknowledgements This PhD was supported by a four-year Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Grant award to the Institute of Food Research. The first big thank you goes to my supervisors Dr Nathalie Juge and Prof Simon Carding, for supporting me throughout my PhD. Particular thanks go to Nathalie for her inexhaustible time and effort put in during the writing phase of my thesis. A second thank you goes to everyone who has helped and been involved in my PhD work throughout the four years, particularly Dr Louise Wakenshaw who was a big support during my first two years. This thank you to all extends beyond the work of this PhD, as I have thoroughly enjoyed my life in Norwich during these years. A huge thank you goes to Alex, who has provided lots of laughter and support during my PhD. You have my eternal gratitude for: being you. Being there. Being amazing. Finally I would like to thank my examiners Prof Dirk Haller and Dr Claudio Nicoletti for kindly agreeing to read and discuss my work with me. I would like to dedicate this thesis to my amazing parents. It is through their continuous love and support that I arrived at the stage where I had the option of doing a PhD. Importantly; my parents introduced me to the feel-good movie “The Best Exotic Marigold Hotel”, which taught me a saying that got me through my PhD: “Everything will be all right in the end…if it’s not all right, then it’s not yet the end.” 5 Table of contents ABSTRACT ............................................................................................................................................. 2 PREFACE ................................................................................................................................................ 3 ACKNOWLEDGEMENTS ......................................................................................................................... 5 TABLE OF CONTENTS ............................................................................................................................. 6 LIST OF FIGURES ...................................................................................................................................12 LIST OF TABLES .....................................................................................................................................15 ABBREVIATIONS ...................................................................................................................................16 CHAPTER 1 INTRODUCTION .................................................................................................................21 1.1 STRUCTURE AND FUNCTION OF THE VERTEBRATE GASTROINTESTINAL (GI) TRACT ............................................... 21 1.2 THE MAMMALIAN GI MUCUS LAYER .......................................................................................................... 24 1.2.1 Structure and organisation of the mammalian GI mucus layer ................................................ 24 1.2.2 Composition of the mammalian GI mucus ............................................................................... 27 1.2.2.1 Mucins ................................................................................................................................................29 1.2.2.2 Mucin glycosylation ............................................................................................................................30