The Role of Probiotic Lactobacillus in Immune Regulation and Modulation of the Vaginal Microbiota During Pregnancy by Siwen Yang A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Physiology University of Toronto © Copyright by Siwen Yang 2015 The Role of Probiotic Lactobacillus in Immune Regulation and Modulation of the Vaginal Microbiota During Pregnancy Siwen Yang Doctor of Philosophy Department of Physiology University of Toronto 2015 Abstract Preterm birth (PTB) occurs in 10% of all pregnancies globally. Premature babies have a mortality rate 40 times higher than term infants. Approximately 25-30% of PTB can be attributed to intrauterine infection/inflammation. A disturbance of the vaginal microbiota as observed in bacterial vaginosis (BV) is associated with an increased risk of PTB. Treatment of preterm labor with antibiotics is largely ineffective, and probiotic lactobacilli have been proposed as a potential preventive therapy for BV and PTB. The objectives of this thesis were to assess 1) the effect of Lactobacillus rhamnosus GR-1 (GR-1) and its supernatant (GR-1 SN) on the prevention of lipopolysaccharide (LPS)-induced PTB and systemic and intra-uterine cytokine and chemokine profiles in pregnant CD-1 mice, 2) the effect of GR-1 on the mouse vaginal microbiota, and 3) the effect of GR-1 and L. reuteri RC-14 on the cervico-vaginal cytokine profile and vaginal microbiota in pregnant women with an abnormal Nugent score. Pregnant mice were pre-treated with intra-peritoneal injections of ii GR-1 SN or oral GR-1 live bacteria prior to intrauterine injection of LPS in two separate studies. The expression of cytokines and chemokines in the maternal plasma, amniotic fluid and intrauterine tissues were then measured. The vaginal microbiota was also determined in animals treated with oral GR-1 live bacteria. Pre-treatment with GR-1 SN, but not with GR-1 live bacteria, reduced LPS-induced PTB and inflammation in pregnant mice. The vaginal microbiota of pregnant mice was altered with oral GR-1 live bacteria. A randomized, double blind placebo-controlled trial was conducted, in which pregnant women with an abnormal Nugent score in their first trimester of pregnancy received orally either placebo or GR-1 and RC-14 for 12 weeks. Their cervico-vaginal cytokine profile and vaginal microbiota was then determined. Oral GR-1 and RC-14, at the dose and duration used, did not change the cytokine profile and vaginal microbiota of pregnant women with an abnormal Nugent score. We conclude that L. rhamnosus GR-1 supernatant, but not the live bacteria, may have the potential to serve as a prophylactic therapy for inflammation-associated conditions during pregnancy, including PTB. iii Dedication To my beloved parents, for their continuous support and unconditional love. iv Acknowledgements With sincere respect, I would like to express my gratitude to my supervisor, Dr. Alan Bocking, for his continuous guidance and unfaltering belief in me for the past years. Your professional work ethics served as a role model to me. I appreciate your patience, understanding and support through the tough times. I am forever thankful for your encouragement and valuable ideas that make my PhD experience meaningful and productive. My deepest thanks to my co-supervisor, Dr. John Challis, for sharing his wealth of knowledge in physiology and providing his continuous support throughout the years. I am grateful for your constructive recommendations while challenging me to think beyond my intellectual comfort zone. Many thanks to members of my advisory committee for keeping me on the right track to the completion of my projects. I would like to thank Dr. Stephen Lye, for offering his advice on the physiological aspect of my project. I would also like to thank Dr. Sung Kim, for sharing his knowledgeable insights in immunology. I am thankful to Dr. Gregory Gloor for his advice on interpreting sequencing data. His excellent teaching skills made learning R less nerve wrecking. I would like to extend my gratitude to Dr. Gregor Reid for sharing his knowledge on probiotics and his willingness to devote time to engage in my work. I would like to acknowledge members of his laboratory, Shannon, Jordan, Grace, Leslie, Amy, Camilla and Yige for making me feel at home during my stay in London. Special thanks to Ms Shannon v Seney, Mr Rod McPhee and Ms Amy McMillian for providing Nugent scores for my project. I would like to acknowledge members of Dr. Gloor’s laboratory, Jean and Julia, for helping me learn R. Special thanks to David Carter at the London Research Institute for his help with Illumina Sequencing. Sincere thanks to examiners of my qualifying exam, Drs Michelle Letarte and Theodore Brown, and examiners of my CIHR grant proposal course, Drs Lee Adamson, Denise Belsham and Clifford Librach, for your critical evaluations of my project and for your valuable suggestions at the examinations. I would like to thank members of the VOGUE team who have generously contributed their ideas and time discussing my project. I would also like to thank Dr. Laurent Briollais, for offering his help with the statistical analysis of my project. I would like to thank the research nurses, Ms Mary-Jean Martin and Ms Tara Maria Rocco, of Mount Sinai Hospital for the recruitment of participants and collection of vaginal swabs, as well as the volunteer participants for their generous contribution of samples for the project. I am lucky to have the great companion from members of the Bocking lab and the Lye lab. Thank you all for providing such a supportive and enjoyable working environment. My special thanks to Dr. Wei Li for mentoring me during times of technical difficulties, and my deepest gratitude to Dr. Oksana Shynolva, for both your scientific insights and for offering me emotional support. Many thanks to executive assistants, Ms Elaine Dwek and Ms Beverly vi Bessey, for being miracle creators. No matter how busy their bosses’ schedules were, you can always accommodate my continuous requests to schedule meetings. I would like to thank the Department of Physiology for being a haven of intellectual freedom, and the wonderful staffs of the department, especially Ms Colleen Shea and Ms Rosalie Pang for your continuous support with administrative issues. I would also like to acknowledge the support of the funding agencies, the Genesis Research Foundation, the University of Toronto and Mount Sinai Hospital, for supporting my education and recognizing the importance of my project. I am grateful for the company and steadfast support of my best friends, Sally Shi and Lydia Zhou. Thank you girls for the wonderful moments we had together and for always believing in me. Special thanks to Han Li, for taking care of me like a big sister. Finally, I would like to dedicate my work to my beloved parents, who are my source of strength. Thank you for your unconditional love and for your support in realizing my dreams. Thank you for your guidance in life and for teaching me the most important aspects of life are to have Peace, Happiness and Health. vii Table of Contents 3. Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... iv Acknowledgements ............................................................................................................. v Table of Contents ............................................................................................................. viii List of Abbreviations ........................................................................................................ xii List of Figures .................................................................................................................. xiv List of Tables ................................................................................................................... xvii 1 General Introduction ........................................................................................................ 2 1.1 Human Pregnancy and Parturition ......................................................................... 2 1.1.1 Anatomy of the Intra-uterine Environment ........................................................... 3 1.2 Mechanisms of Human Parturition ............................................................................ 8 1.2.1 Prostaglandins (PGs) ............................................................................................. 12 1.2.2 Matrix Metalloproteinase (MMPs) ....................................................................... 12 1.2.3 Cytokines and Chemokines ................................................................................... 15 1.3 Preterm Birth .............................................................................................................. 24 1.3.1 Epidemiology ......................................................................................................... 24 1.3.2 Etiology .................................................................................................................. 25 1.3.3 Infection Routes ..................................................................................................... 25 1.3.4 Infection and/or Inflammation- induced PTB ........................................................ 26 1.3.5 Current