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The role of the microbiome in rheumatoid arthritis Dargham Bayan Mohsen Hammad A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy December 2020 Keele University I Acknowledgement I would like to express my deepest gratitude to Dr Daniel Tonge, my lead supervisor, for his support and assistance in completing this study, starting from the thesis proposal up to the thesis manuscript as well as his encouragement. Without his guidance, endless advice and persistent help, this study would not have been possible. I am forever indebted to his kindness, moral support and assistance during the extremely difficult time that I faced throughout these four years. I am grateful to Dr Ray Amith (second supervisor), and I am extremely thankful to Dr Prof David Furness as an advisor of my study. I owe big thanks and gratefulness to Jayne Bromley, Nigel Bowers, Lisa Cartlidge and Chris Bain for providing the facilities throughout the academic study. II Abstract The human microbiome plays a vital role in both health and disease. The evolution of molecular techniques to characterise entire microbiome communities has renewed interest in the involvement of microorganisms in the pathogenesis of Rheumatoid arthritis (RA). In this thesis, 16S and ITS amplicon sequencing were used to characterise bacterial and fungal DNA present in a range of human and mouse samples. Firstly, characterisation of the microbiome present in blood samples obtained from human RA, ankylosing spondylitis, and psoriatic arthritis patients was carried out, relative to healthy controls. Results revealed that the bacterial population in the serum of RA patients was distinct from the healthy state. Through the analysis of paired RA patient blood taken before and three months after treatment, partial microbiome normalisation was identified and was particularly evident in seronegative arthritis patients. Next, the presence and identity of bacterial and fungal communities were investigated in samples of synovial fluid obtained from human RA patients and healthy controls. Our findings revealed that the synovial fluid microbiome of RA could be distinguished from control. Further, IL6, IL71A, IL22, IL23 were elevated in the blood and synovial fluid of RA subjects. The association of IL6 with bacteria and fungi microbiome was observed in the RA synovial fluid. Finally, a characterisation of the bacterial community members presents in the stool, urine, synovial fluid, blood, and serum from collagen-induced arthritis (CIA) and control mouse samples were undertaken. Here, we demonstrated that the bacterial community in CIA stool samples was distinct from the control. These data propose that the human blood and synovial fluid microbiome and gut microbiome of the mice is modulated by disease status (RA) and therefore have the III potential to serve as a novel biomarker in RA pathogenesis and treatment response. Further, studies are required to investigate these initial findings. IV Contents 1 Literature Review ........................................................................................................ 2 1.1 Introduction ...................................................................................................... 2 1.2 Rheumatoid Arthritis ........................................................................................ 3 1.2.1 Definition, symptoms, epidemiology, and aetiology .............................. 3 1.2.2 Pathophysiology and mechanisms underlying disease process .......... 6 1.2.3 Classification of Rheumatoid Arthritis ................................................... 10 1.2.4 Diagnosis of Rheumatoid Arthritis, therapy, and treatment ................ 13 1.2.5 Ankylosing spondylitis, and psoriatic arthritis .................................... 15 1.3 Immune cells .................................................................................................. 18 1.3.1 Innate immune cells ................................................................................. 18 1.3.2 Adaptive immunity ................................................................................... 22 1.3.2.1 T cells ................................................................................................ 22 1.3.2.2 B cells ............................................................................................... 29 1.3.2.3 Natural killer T cells ......................................................................... 33 1.3.2.4 Gamma delta T cells ........................................................................ 34 1.3.2.5 Mucosal-associated invariant T cells (MAIT) ................................. 35 1.3.2.6 Cytokines .......................................................................................... 36 1.3.2.6.1 IL-6 ................................................................................................. 36 1.3.2.6.2 IL-17 ............................................................................................... 37 1.3.2.6.3 IL-22 ............................................................................................... 39 1.3.2.6.4 IL-23 ............................................................................................... 40 1.4 Microbiome ..................................................................................................... 41 1.4.1 General introduction ................................................................................ 41 1.4.2 Microbiome changes in diseases ........................................................... 42 1.4.2.1 Ankylosing spondylitis, and psoriatic arthritis ............................. 43 V 1.4.2.2 Obesity .............................................................................................. 44 1.4.2.3 Diabetes mellitus ............................................................................. 45 1.4.2.4 Atherosclerosis ................................................................................ 47 1.4.2.5 Inflammatory bowel disease ........................................................... 47 1.4.2.6 Gout .................................................................................................. 48 1.4.2.7 Cancer ............................................................................................... 49 1.4.3 Microbial niches ...................................................................................... 50 1.4.4 Rheumatoid arthritis and dysbiosis ....................................................... 58 1.4.4.1 Gut microbiome and RA .................................................................. 58 1.4.4.2 Oral microbiome and RA ................................................................. 71 1.4.4.3 Blood microbiome and RA .............................................................. 74 1.4.4.4 Microbiome in other tissue sites .................................................... 82 1.4.5 Mouse studies in Autoimmune/inflammatory disorders ...................... 84 1.4.6 16S ribosomal RNA .................................................................................. 86 1.4.7 ITS2 ........................................................................................................... 87 1.4.8 Potential mechanisms of microbial populations in RA pathophysiology ..................................................................................................... 88 1.5 Aims and Objectives ...................................................................................... 92 2 Materials and methods .............................................................................................. 94 2.1 Overview ......................................................................................................... 94 2.2 Samples........................................................................................................... 94 2.2.1 Human blood samples ............................................................................. 95 2.2.2 Human synovial fluid samples................................................................ 98 2.2.3 Mice biological samples (stool, synovial fluid, urine, blood, and serum) 100 2.2.3.1 Biological samples from the mouse study .................................. 101 2.2.3.1.1 Blood Collection ......................................................................... 101 VI 2.2.3.1.2 Synovial fluid collection ............................................................ 102 2.2.3.1.3 Urine and faeces collection ....................................................... 102 2.3 Microbiome characterisation ....................................................................... 103 2.3.1 Microbiome characterisation of human blood and synovial fluid samples 103 2.3.1.1 16S rRNA and ITS2 PCR ................................................................ 104 2.3.1.2 DNA Gel electrophoresis ............................................................... 106 2.3.1.3 Clean-up of PCR product samples ..............................................