Natural Antimicrobials in Pregnancy Sarah JE Stock BSc (Hons) MBChB Jennifer Brown Research Fellow Edinburgh University Department of Obstetrics and Gynaecology Centre for Reproductive Biology Queen’s Medical Research Institute 47 Little France Crescent Edinburgh EH16 4TJ Thesis submitted to the University of Edinburgh for the Degree of Doctor of Philosophy March 2008 i Abstract Natural antimicrobials are peptides that are essential components of the innate immune system, providing broad-spectrum protection against bacteria, yeasts and some viruses. In addition to their innate immune activity, they exhibit properties suggesting they interact with the adaptive immune system. These functions imply they may be of particular importance in pregnancy. Intrauterine infection is responsible for approximately one third of cases of preterm labour, and normal labour is considered an inflammatory process, associated with leukocyte invasion of the uterine tissues and increased cytokine production. Little is known, however, about natural antimicrobial expression in pregnant reproductive tract. The aim of this thesis was thus to characterize natural antimicrobial production in pregnancy. The study focused on two main areas – the lower genital tract, comprised of the vagina and cervix; and the innermost fetal membrane, the amnion. In the lower genital tract, levels of natural antimicrobials were determined in samples of cervicovaginal secretions collected from pregnant women, using enzyme linked immunosorbance assay (ELISA). In addition Taqman quantitative PCR and ELISAs were used to investigate natural antimicrobial production by cell lines derived from endocervical, ectocervical and vaginal epithelium. It was found that elafin and secretory leucocyte protease inhibitor (SLPI) were found at high concentrations in cervicovaginal secretions, but levels were diminished in women with the common vaginal infection bacterial vaginosis (p<0.05). Cells derived from the vaginal epithelium expressed greater amounts of elafin than cervically derived cells. However, elafin and SLPI production could be stimulated in endocervical cells by the bacterial product lipopolysaccharide, a response that was not seen in the vaginal cell line. Natural antimicrobial production in the amnion was examined in tissue explants and primary cultured amnion cells, using a combination of Taqman PCR and ELISAs. In addition, cDNA microarray was carried out to investigate factors controlling amniotic antimicrobial production, and the involvement of signalling pathways was ii studied using specific pathway inhibitors. It was shown that the amnion expressed five antimicrobials: human beta defensins (HBD) 1, 2 and 3, SLPI and elafin. Expression of HBD2 was significantly upregulated following normal labour (p<0.05), with production in primary amnion epithelial cells dramatically increased by IL-1ß. The pattern of HBD2 expression in response to IL-1ß was biphasic, which suggested involvement of a secondary gene product. Several putative influential factors were identified by cDNA micorarray, including the NF-kappaB cofactor NF- kappaBinhibitorζ. Its relationship to HBD2 was explored. The involvement of both NF-kappaB and mitogen activated protein (MAP) kinase p38 signalling appeared crucial in the response. This work has shown that natural antimicrobials are expressed by both the lower genital tract, where infections that are associated with preterm labour originate, and in the amnion, which is the fetus’ last line of defence to infection. They may have an important role in the prevention of infection associated preterm labour. Further characterization of these responses may increase understanding of the physiology, and pathophysiology of labour, and lead to strategies for the prevention of premature delivery. iii Declaration Except where due acknowledgment is made by reference the studies undertaken in this thesis were the unaided work of the author. The work described in this thesis has not been previously accepted for, or is currently being submitted in candidature for another degree. Chapter 2 I acknowledge the assistance of Dr Elena Facenda who designed the majority of the primer/probe sets for Taqman quantitative PCR. I acknowledge the assistance of Professor Hilary Critchley, who provided the three endometrial RNA samples for use as positive controls. Chapter 3 I acknowledge the assistance of Rosie Branford, a medical student, who helped perform initial feasibility trials for the collection of cervicovaginal secretions. I also acknowledge the assistance of Leanne Duthie, another medical student, who collected most of the cervicovaginal secretion samples, helped with sample processing and performed some of the total protein, HBD2 and SLPI assays. Chapter 5 I acknowledge the assistance of the staff at the Scottish Centre for Genomics Technology and Informatics, particularly Marie Craigon, Thorsten Foster and Professor Peter Ghazal, who performed the cDNA microarray and helped with data analysis. My gratitude is extended to each of them. Sarah Stock March 2008 iv Acknowledgements I am indebted to Professor Rodney Kelly and Dr Simon Riley for their supervision of this project. I must also extend thanks to Professors Andrew Calder and Hilary Critchley for their guidance over the past three years. I would not have been able to perform any of the experiments described without help and instruction from the staff in the Queen’s Medical Research Institute. I am grateful to have had the opportunity to work in such a stimulating, productive and supportive environment. I am especially grateful to Dr Elena Facenda, Vivien Grant, Dr Phil Driver and Dr Nicole Kane (the “RWK Lab Group” as was) for their knowledge and patience. Nicole, in particular, was a brilliant lab partner, who was greatly missed when she left. Rose Leask not only provided a sympathetic ear when things didn’t go according to plan, but invariably came up with some practical tips to solve the problem. Dr Adam Pawson’s help was invaluable and he provided new approaches to seemingly unanswerable questions. Many thanks also to the staff at Genomics, Technology and Informatics, and Mick Rae, Anne King and Mike Miller, for their frequent advice. This project was generously supported by the Jennifer Brown Research Fund. It has been a privilege to have been involved in this new iniative, and to work with Anne Armstrong, Cath Dhaliwal and Hannah Shore – the other members of the Jennifer Brown Group. Amy Robb and Scott Fegan have also been fantastic office companions. I could not have asked for better colleagues or better friends, and I am sure they will remain as such throughout my career. I would like to say a big thankyou to my parents for their encouragement, and in particular to my mum for her diligent proof-reading of this manuscript. Finally, thankyou to Jim, for his unfaltering support, which I could not have done without. v Publications, Presentations and Posters Relating to this Thesis PUBLICATIONS Stock, S., Kelly, R.W., Riley, S.C., Calder, A.A. (2007) “Natural Antimicrobial Production by the Amnion”Am J Obst Gynecol 196 (3): 255e1-6 (Appendix 5) ORAL PRESENTATIONS Microarray analysis of the effects of interleukin-1 beta on the amnion. British Maternal and Fetal Medicine Society Annual Conference: Belfast April 2007 Elafin is Produced by Cells Derived from the Vagina and Cervix, and Levels are Diminished in Bacterial Vaginosis. Society of Gynaecological Investigation (SGI) Annual Conference: Reno March 2007 • SGI President’s Presenter Awards for Abstract Natural antimicrobials and bacterial vaginosis in pregnancy. Blair Bell Research Society Meeting: Leicester November 2006 • Best Oral Presentation Natural antimicrobials produced by the amnion: a possible role in defence against ascending infection. British Maternal and Fetal Medicine Society Annual Conference: Cardiff April 2006 • Best Oral Presentation (Labour and Delivery) Natural antimicrobials in the amnion. Blair Bell Research Competition: Royal College of Obstetricians and Gynaecologists, London November 2005 • Highly Commended vi POSTERS Natural Antimicrobials in the Amnion. Biochemical Society Meeting: “Antimicrobial peptides: mediators of innate immunity in the development of anti-infective, therapeutic and vaccination strategies”, Edinburgh, November 2005 (Abstract in Biochemical Society Transactions Vol 34 Part) MANUSCRIPTS IN PREPARATION Levels of Elafin are Diminished in Cervicovaginal Secretions in Association with Bacterial Vaginosis The effect of IL-1β on the amniotic epithelium vii Contents Abstract ii Declaration iv Acknowledgments v Publications, presentations and prizes relating to this thesis vi Contents viii List of figures xv Abbreviations xviii Chapter 1: LITERATURE REVIEW 1.1. Introduction 2 1.2. The Reproductive Tract in Pregnancy 3 1.2.1. The Uterus 3 1.2.2. The Cervix 4 1.2.3. The Fetal Membranes 5 1.2.3.1. Structure of the Fetal Membranes 5 1.2.3.2. Functions of the Fetal Membranes 6 1.2.4. The Vagina 8 1.3. The Innate Immune Response and Inflammation 9 1.3.1. Mediators of Inflammation 11 1.3.1.1. Cytokines 11 1.3.1.2. Chemokines 17 1.3.1.3. Prostaglandins 18 1.3.1.4. Matrix metalloproteinases 19 1.3.1.5. Toll-like receptors 20 1.3.2. Inflammatory Signalling 21 1.3.2.1. Receptor activation and adaptor recruitment 24 1.3.2.2. Activation of protein kinase cascades 24 1.3.2.3. Transcription factors 26 1.3.2.4. mRNA stabilization and post-transcriptional modifications 28 viii 1.4. Inflammation, Infection and Preterm Labour 28 1.4.1.Labour as an inflammatory process 28 1.4.2.The onset of normal labour 29 1.4.3.The onset of preterm labour 30 1.4.4.Infection as a cause of preterm labour 31 1.4.5.Bacterial Vaginosis and preterm labour 36 1.4.6.Clinical aspects of preterm labour and infection 39 1.4.6.1. Outcomes 39 1.4.6.2. Antibiotics for the prevention of preterm labour 40 1.4.6.3. Antibiotics for the treatment of preterm labour 41 1.5. Natural Antimicrobials 42 1.5.1.Human beta-defensins (HBDs) 43 1.5.2.The antileukoproteinases- SLPI and elafin 46 1.5.2.1. Structure and distribution 46 1.5.2.2. Antiprotease activity 47 1.5.2.3.