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Vitamin D in Pregnancy: Understanding Immune

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Vitamin D in Pregnancy: Understanding Immune Vitamin D in pregnancy: understanding immune effects in the decidua by Jennifer Ann Tamblyn A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Metabolism and Systems Research (IMSR) College of Medical and Dental Sciences University of Birmingham, 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Epidemiology has linked preeclampsia (PET) to vitamin D deficiency. To date, studies have focused upon serum 25-hydroxyvitamin D3 (25(OH)D3) alone as the marker of vitamin D status. We provide strong evidence comprehensive analysis of vitamin D metabolites in pregnancy is highly informative, particularly within the context of PET. Uniquely, analysis of maternal urinary metabolites provides a novel insight into vitamin D and the kidney, with lower 25(OH)D3 and 24,25(OH)2D3 excretion early indicators of a predisposition towards PET. Since vitamin D is a potent regulator of immune function, and the decidua appears a key extra-renal site for vitamin D metabolism, we investigated effects of 1,25(OH)2D3 upon decidual uterine natural killer cells and macrophages. We show both express a functional vitamin-D system and demonstrate differential sensitivity to 1,25(OH)2D3 compared to their peripheral counterparts. To understand the functional impact of vitamin D, whole transcriptomic analysis of 1,25(OH)2D3- mediated effects upon uNK and macrophages was performed. We show the actions of vitamin D extend far beyond simple immuno-regulation, targeting major cellular functions including migration, adhesion and apoptosis. In particular, our data support effects highly relevant to decidualisation. We anticipate these findings to be highly relevant within the context of vitamin D deficiency, malplacentation and PET. 2 Acknowledgements I would like to dedicate this thesis to my husband, Lee James, and my parents Nick and Sue Tamblyn for their patience, support and belief throughout both my academic and clinical career so far. Thank you also to my sister and brother-in-law, Claire and Kevin Young, and my close friends, Laura Brown, Katie Ruck, Fran Vince and Francesca Chappell, who have all together ensured I kept smiling and believed I would actually reach this point. I would particularly like to thank my co-supervisors, Professor Mark Kilby and Professor Martin Hewison, for their continued support throughout my fellowship. Professor Kilby has been an unfailing source of inspiration and guidance since the early clinical and academic stages of my career in obstetrics and gynaecology. Professor Hewison has been an exemplary supervisor and with his experience, enthusiasm and accessibility has greatly encouraged my development as a reproductive basic scientist. I am particularly grateful to a number of current and past College of Medical and Dental Sciences members at University of Birmingham for their assistance and contributions to this work, in particular Louisa Jeffery, Radhika Susarla, Carl Jenkinson, and Dean Larner. Thank you also to David Lissauer, Richard Powell, Omi Ohizua and the CHHIP research team at Birmingham Women’s & Children’s Foundation Hospital Trust. Without their continued support with CHHIP participant recruitment and sample collection this work simply would not have been possible. I would also like to thank my collaborators Judith Bulmer, Gendie Lash and Barbara Innes at the University of Newcastle, Konstantin Knoblich and Anne Fletcher at the University of Birmingham, and Susan Coort, from Maastricht University. The knowledge and technical expertise they have together provided has greatly facilitated the work achieved during this fellowship, and I am extremely grateful for each of their individual contributions to this work. Thank you finally to my principal funders Wellbeing of Women from whom I was awarded my prestigious 3-year clinical research training fellowship. Thank you also to Mason Medical Research, who specifically funded the RNA-sequence analysis component of this project. Vitabiotics Pregnacare 3 also generously contributed to this work through Wellbeing of Women, which independently selected the research through its AMRC accredited peer review process led by its Research Advisory Committee. 4 1 Contents 1 Contents .......................................................................................................................................... 5 1.1 Figures and Table Summary ................................................................................................. 12 Figures summary ........................................................................................................................... 12 Tables summary ............................................................................................................................ 16 1.2 Abbreviations ........................................................................................................................ 20 2 General Introduction ..................................................................................................................... 25 2.1 Introduction ........................................................................................................................... 26 2.1.1 Vitamin D metabolism and signalling........................................................................... 26 2.1.2 DBP and vitamin D transport ........................................................................................ 29 2.1.3 Vitamin D epimerisation, an alternative pathway ......................................................... 30 2.1.4 Extra-renal vitamin D metabolism ................................................................................ 31 2.1.5 Non-classical immune effects of vitamin D .................................................................. 33 2.1.6 Vitamin D status and assessment .................................................................................. 34 2.1.7 Overall study aims and objectives ................................................................................ 35 3 General Methods ........................................................................................................................... 37 3.1.1 Ethics ............................................................................................................................. 38 3.1.2 Participant recruitment .................................................................................................. 38 3.1.3 Sample preparation ....................................................................................................... 40 3.1.4 Analysis of serum and urine vitamin D metabolites by LC MS-MS ............................ 43 3.1.5 Analysis of serum DBP ................................................................................................. 48 3.1.6 Analysis of serum albumin ........................................................................................... 48 5 3.1.7 Analysis of free serum 25(OH)D3 ................................................................................ 49 3.1.8 Creatinine Jaffe reaction ............................................................................................... 49 3.1.9 Positive selection immuno-magnetic isolation of immune cell subsets ........................ 50 3.1.10 Cell culture .................................................................................................................... 51 3.1.11 LC MS-MS conversion assay ....................................................................................... 52 3.1.12 Flow cytometry ............................................................................................................. 52 3.1.13 Cytotoxicity assay ......................................................................................................... 55 3.1.14 RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR) ...................................................................................................................... 56 3.1.15 Fluorescence-activated cell sorting (FACS) ................................................................. 58 3.1.16 RNA extraction ............................................................................................................. 60 3.1.17 cDNA library preparation ............................................................................................. 62 3.1.18 RNA sequence analysis ................................................................................................. 67 3.1.19 Pathway analysis ........................................................................................................... 76 3.1.20 Statistics ........................................................................................................................ 77 4 Analysis of Vitamin D Metabolism in Pregnancy and Malplacentation ....................................... 78 4.1 Introduction
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