University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2014-07-11 Novel Insights Into The Mechanisms Of Post-Infectious Irritable Bowel Syndrome Using Experimental Giardiasis Halliez, Marie, C.M. Halliez, M. (2014). Novel Insights Into The Mechanisms Of Post-Infectious Irritable Bowel Syndrome Using Experimental Giardiasis (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27133 http://hdl.handle.net/11023/1627 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca THE UNIVERSITY OF CALGARY & THE UNIVERSITY OF ROUEN Novel Insights Into The Mechanisms Of Post-Infectious Irritable Bowel Syndrome Using Experimental Giardiasis by Marie C. M. Halliez A COTUTELLE THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES, UNIVERSITY OF CALGARY, CALGARY, ALBERTA, CANADA DEPARTMENT OF MEDICINE AND PHARMACY, UNIVERSITY OF ROUEN, ROUEN, FRANCE JULY, 2014 ©Marie C.M. Halliez 2014 ABSTRACT Irritable Bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder in humans characterized by abdominal pain and altered bowel habits. The major pathophysiological features of IBS include: visceral hypersensitivity, intestinal barrier dysfunction, low grade inflammation appear following acute gastroenteritis despite the clearance of the inciting pathogen. This post-infectious (PI)-IBS may occur in patients following infection with bacteria such as C. jejuni, E. coli, Salmonella spp. Recent studies have implicated protozoan parasites such as Giardia duodenalis in the appearance of PI-IBS. G. duodenalis, the most common enteropathogen worldwide, is responsible for giardiasis, a disease causing intestinal malabsorption and diarrhea in a wide variety of species including humans. Using in vivo and in vitro models, the present study, established a proof-of-concept between Giardia-infection and the development of PI-IBS. This study also characterized one of the contributing mechanisms leading to post-giardiasis IBS. In a new neonatal immunocompetent rat model, the human assemblages of Giardia duodenalis caused a significant visceral hypersensitivity 50 days post-infection in two parts of the gastrointestinal tract. Visceral hypersensitivity was associated with mucosal structure modifications: villus atrophy and crypt hyperplasia after clearance of the pathogen. It was also associated with activation of the mucosal immune system as shown by an increase in intraepithelial lymphocytes and mast cell counts during the post-infectious stage. And with activation of the nociceptive signaling pathway by induction of c-fos expression starting at day 7 post-infection and continuing until the post-infectious stage. This study showed a dysfunction of the intestinal barrier, in vivo and in vitro, characterized by the translocation of commensal bacteria. Giardia-induced bacterial translocation, further characterized in vitro, was shown to occur via the paracellular route in conjunction with the degradation of the tight junctional proteins occludin and claudin-4. In conclusion, this study presented a new animal model of giardiasis eliciting PI-IBS symptoms. This study also showed that Giardia was able to induce the translocation of commensal bacteria through the epithelial monolayer via the paracellular route by degrading tight junctional proteins. This model suggest that the host immune system reactivity toward its own microbiota due to impaired intestinal barrier function seems to be one of the mechanisms contributing to post- infectious irritable bowel syndrome following acute giardiasis. ii Acknowledgements First I would like to thank my supervisors Dr. André Buret, Dr. Gilles Gargala and Dr. Loic Favennec for giving me the opportunity to work in their lab in this co-tutelle program. Working in both France and Canada was for me an amazing opportunity to develop new skills, learn new techniques and a new language. But let's not forget that it was also for me an amazing time abroad, which has allowed me to discover this great country that is Canada and the opportunity to meet those wonderful people that are Canadians. I would like to thank all my colleagues both in France and in Canada, for their welcoming and their help through this PhD. I wasn't always the most enthusiastic person with my bad moods and complaints, but I'm French what can I do..... Let's start with the University of Rouen where I did the first two years of my PhD. Gilles and Loic, thank you for choosing me to be your student, for your help and your kindness. Laëtitia thank you for your help especially with all those crazy animal studies, for the smoke breaks and the pint of Guinness after work, I had a great time with you and I'm really happy I had the opportunity to know you. Marie-Laure, my little trainee, thank you for all your help with those PCR, without you I would never have made it through. Thanks to Françoise for her help with the sequencing. Moving on to the University of Calgary I would like to thank Andre first, thank you for your enthusiasm, your support, your help, your availability, you are amazing don't change a bit. It really was a privilege for me to be part of your team. Thank you to all my colleagues and especially Amol and James for all your help with the in vitro studies, Jennifer for your help with my writing and your support, Kristen thank you for your help with the bacterial culture and the PCR, Troy we had such great moments and laughs over my animal studies, thank you for being here and for your help over this past two years, and sorry again for the little PFA incident........Dr. T. for your kindness and your joy, and finally JP thank you for your help with my animal studies and the staining, you're a boss. I would like to thank Dr. Doug Morck, Dr. Simon Hirota, Dr. Lash Gedamu. Dr. Norm Neumann, Dr. Nathalie Kapel, Dr. Gilles Gargala, Dr. Loic Favennec and Dr. André Buret for accepting to serve on my PhD supervisory and examination committees (Candidacy and defense). Thanks also to Dr. Tamia Lapointe and Dr. Christophe Altier, for your collaboration iii and your help on the nociceptive part of this project, thank you both for your patience and understanding. Thanks to Dr. James Wasmuth, Dr. Jean-Paul Motta and Coralie Druelle for your help in editing parts of this document. Cause you can't do a PhD without a Master, Anna, our little princess fairy doctor, my mentor, you taught me so much, thank you for your help with our little Pneumocystis. Coming to work at 6 am, finish at 11pm, spend two hours watching the FACS in the freezing room, the 45 minutes centrifuges starting at 8pm....the substractive hybridization.... those are so good memories I wouldn't change it for the world. Finally, a huge thank you to Nicole and André for helping me in my move to Calgary, in finding a place to live and all the processes to adapt to this new life. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (individual operating and CREATE), the France-Canada Research Fund, and the "Ministère de l'enseignement supérieur et de la recherche" (French Ministry of secondary education and research). iv Personal acknowledgements Now I would like to thank all my friends and family for their support, I wouldn't be here today without you. Mom, Dad, and my "cat fish", thank you for always being here, helping me (especially with money, PhD student is really not a well paid job and life in Canada is quite expensive, I owe you big time), listening to me and keeping me on track. Even though our lives took different paths there is two persons that I would like to thank cause without their encouragement at some point in my life I wouldn't have made it to a PhD, Datchu, thank you for convincing me to enter a biology degree, you were right to convince me to follow my dream, I wouldn’t be there today if it wasn't for you. Hervé, even though you think you weren't of any help, trust me you gave me the courage and the envy to keep going and enter a master research, you were there to help me when I was struggling with my exams I will never forget all you did for me and all the wonderful moments that we spent together you always will be in my heart. To my "zozios" a huge thank you for being there, listening to my complaints, to sheer me up when I was feeling down, for the nights out, the drinks, the skypes, everything. Coralie my best friend my sister of heart thank you for all your help your support your kindness and your friendship. Georges, thank you for your craziness and love, you always make me laugh. Thank you for being there and listening to me so often. Thierry, just because you are you. Our holidays in Digne, your yogourt, your technique to get out of my car, the nights at the raz playing darts.... So many memories Mike, thank you for your kindness and your tenderness, you gave me strength when I needed it most, you'll always have a special place in my heart. "Lapin" thank you for those amazing years, we met in our first year of biology I will never forget our great time together, and that "le lapin est passé par là" in our lecture theatre, the "pouf le cul" in front of the SN1 (when there was still water in those kind of ponds.....).