MICRORNAS AND IMMUNOMODULATION BY VITAMIN D By DANYANG LI A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Metabolism and Systems Research College of Medical and Dental Sciences University of Birmingham January 2019 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 The active form of vitamin D, 1,25(OH)2D3, plays well-established roles in calcium regulation and bone formation. 1,25(OH)2D3 is also thought to exert immunoregulatory effects upon cells of the innate (dendritic cell) and adaptive (T cell) immune systems, that may impact health and disease. In recent years, the role of 1,25(OH)2D3 has been implicated in autoimmune diseases such as rheumatoid arthritis (RA). 1,25(OH)2D3 brings about genetic and epigenetic changes within immune cells, the latter which may include effects of microRNAs (miRNAs); small non-coding RNAs with an important regulatory role. To study the role of 1,25(OH)2D3 on miRNAs in RA, we utilised n=20 (RA) and n=7 (reactive arthritis, ReA) matched patient serum and synovial fluid (SF) samples to derive measurements of vitamin D metabolite concentrations by LC-MS/MS, vitamin D binding protein abundance by ELISA, and circulating miRNA expression by qPCR. To study the role of 1,25(OH)2D3 on miRNAs in healthy immune cells, we also generated in vitro models of dendritic cells (DC) and CD4+ T cells, treated with 1,25(OH)2D3 or vehicle at different stages of development. An unbiased array approach was then used to screen 372 miRNAs closely related to inflammation in the DC and T cell models. Bioinformatic analyses were used to identify predicted gene targets of significantly regulated miRNAs in both DC and T cells. Results showed that 1,25(OH)2D3 in SF was low or undetectable in 13/20 RA and 4/7 ReA samples. MiR-146a and miR-155 was up-regulated in RA SF compared to serum, but did not significantly correlate with RA disease markers. In DC, miR-155 but not miR-146a was up-regulated by LPS-induced cell maturation in the presence or absence of 1,25(OH)2D3. Global down-regulation of miRNAs was observed after either short or long-term treatment of DC with 1,25(OH)2D3. This was due, in part, to suppression of expression for miRNA processing genes. In contrast to DC, global miRNA down-regulation was not observed in T cells treated with 1,25(OH)2D3. Notably, MiR-155 was up-regulated by cell activation but not 1,25(OH)2D3, and miR- 212-3p was up-regulated by activation and 1,25(OH)2D3. Together, these results suggest that any 1,25(OH)2D3 generated within the synovial microenvironment may be restricted to the cells involved in immunoregulation within this tissue. The role of miR-146a and miR-155 in immune cells is still unclear; it is unlikely that these miRNAs are actively mediating gene silencing to cause inflammation within the local environment, but rather they are maintained as a reserve of miRNAs not associated with their target gene. Global down-regulation of miRNAs following 1,25(OH)2D3 in DC, but not T cells, suggests a role for 1,25(OH)2D3-mediated miRNA down-regulation as opposed to decreased miRNA synthesis. Coupled with bioinformatic tools and gene ontology analysis, there is potential to identify novel roles for 1,25(OH)2D3-responsive miRNAs in the prediction and pathogenesis of inflammatory disease. Acknowledgements Thank you to the Medical Research Council for funding this PhD Studentship research project. Firstly, I would like to express my sincerest gratitude to my PhD supervisors Professor Martin Hewison and Professor Karim Raza, for their continued advice and support throughout my PhD. Professor Hewison has been an outstanding supervisor and his experience, patience and approachability has been paramount to the success of this project. Furthermore his enthusiasm, motivation and immense knowledge has inspired me to continue pursuing a career in academia. Professor Raza has been a source of inspiration by providing fresh insights on my project and encouraging me to think critically from a wider perspective. The clinical chapter of this thesis would not have been possible without his input and sample sourcing. I would also like to thank a number of academics at the University of Birmingham for their assistance and contributions to this project, in particular Dr Louisa Jeffery and Dr Carl Jenkinson from the Institute of Metabolism and Systems Research, and Dr Peter Nightingale from the Institute of Translational Medicine. Additionally thank you to Dr Dean Larner and Amadeo Muñoz García for their input at various stages of data collection and analysis. I feel extremely fortunate to have a group of wonderful colleagues within the department who have been a joy to work with. A massive thank you to my friends with whom I have laughed through the highs and been supported through the lows of my PhD – you guys are amazing. Finally, this acknowledgement would not be complete without a heartfelt thank you to my family for their unwavering love, advice and support. From the bottom of my heart, I am thankful for everything. I hope I have made you all proud. CHAPTER 1. TABLE OF CONTENTS, LIST OF FIGURES AND LIST OF TABLES 1.1 Table of contents CHAPTER 1. TABLE OF CONTENTS, LIST OF FIGURES AND LIST OF TABLES…………………………………………………………………………………….1 1.1 Table of contents ........................................................................................ 1 1.2 List of figures and tables ........................................................................... 1 Abbreviations………………………………………………………...………….9 CHAPTER 2. GENERAL INTRODUCTION .......................................................... 11 2.1 Vitamin D metabolism, signalling and endocrine regulation .................... 12 2.2 Vitamin D transport and bioavailability…………………………………......19 2.3 Vitamin D-sufficiency and -deficiency ..................................................... 22 2.4 Vitamin D status and human health………………………………………...23 2.5 Quantification of vitamin D metabolites…………………………………....27 2.6 Vitamin D and the immune system ........................................................ 29 2.7 Vitamin D and microRNAs ..................................................................... 38 2.8 A role for miRNAs in the immunomodulatory actions of vitamin D ......... 42 2.9 Project aims and objectives ................................................................... 47 CHAPTER 3. MATERIALS AND METHODS ........................................................ 48 3. Introduction ............................................................................................ 49 3.1 Primary cell culture ................................................................................ 53 3.1.1 Ethics ..................................................................................................... 53 3.1.2 Isolation of PBMC from whole blood ...................................................... 53 3.1.3 Isolation of CD14+ monocytes ............................................................... 54 3.1.4 Induction of monocyte-derived DC phenotypes ..................................... 55 3.1.5 Isolation of naïve CD4+ CD25- T cells ................................................... 56 3.1.6 Naïve CD4+ T cell activation .................................................................. 57 3.1.7 T cell 1,25(OH)₂D3 time-course study ................................................... 58 3.2 Flow cytometry ....................................................................................... 60 3.2.1 DC and T cell surface antigen staining................................................... 62 3.2.2 Cell fixation with Paraformaldehyde (PFA) ............................................. 62 3.2.3 CD4+ T cell intracellular staining of CTLA-4 expression (using the Saponin method) .................................................................................... 63 3.2.4 CD4+ T cell intracellular staining of FoxP3 expression (using the eBioscience FoxP3 method) .................................................................. 63 3.3 DC and T cell gene expression analysis ................................................ 64 3.3.1 RNA isolation ......................................................................................... 64 3.3.2 Reverse transcription ............................................................................. 66 3.3.3 qRT-PCR analysis of mRNA expression ................................................ 67 3.4 DC and T cell candidate miRNA expression analysis ............................ 69 3.5 MiScript HC qPCR array analysis of miRNA expression ........................ 70 3.5.1 Reverse transcription for miRNA PCR array .......................................... 71 3.5.2 Pre-amplification of cDNA for miRNA array ........................................... 72 3.5.3 qPCR of miScript HC miRNA qPCR arrays ............................................ 73 3.6 Profiling serum and SF in rheumatoid arthritis......................................