The immunomodulatory impact of multipotential stromal cells on monocytes in healthy people and patients with rheumatoid arthritis Priyanka Dutta Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Faculty of Medicine and Health School of Medicine September 2020 Primary Supervisor: Professor Michael McDermott Secondary Supervisor: Dr Elena Jones Secondary Supervisor: Professor Graham Cook The candidate confirms that the work submitted is her own work and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and no quotation from the thesis may be published without proper acknowledgement. The right of Priyanka Dutta to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. i Acknowledgements I would like to express my deep gratitude to my supervisors Professor Michael McDermott, Dr Elena Jones and Professor Graham Cook for providing me with this opportunity. Thank you for all your understanding, guidance and immense support inside and outside of the lab. Thanks for sharing your knowledge and ideas to support this PhD work, most of all thank you for your patience. Also, thanks to Chi Wong, Rekha Parmar, Adam Davidson and Elizabeth Straszynski for their great support in the lab, as well as the staff and patients at Chapel Allerton Hospital for providing patient samples. Thank you to Professor Paul Genever for providing the Y201 and Y202 MSC immortalised cell lines and to Dr Andreia Riberio and Professor Rhodri for their expert advice on optimisation of the whole blood assay. As well as my postgraduate tutor's Dr Ai Lyn Tan and Dr Frederique Ponchel, thank you. To my friends and family, I would like to thank them for not giving up on me and providing me with great inspiration and strength to persevere through. Particularly, to my grandparents: Balraj Krishan Sharma, Dharm Vir Dutta and Pushpa Rani Dutta, they have always set a high standard on what it means to work hard and to never give up. I could not have done this without their inspirational teachings and advice. Thank you to my dear friends, Viktoriya Damyanova, Sheetal Maisuria, Shelly Pathak, Manoharini Radha and Sandra Gate your support and love has been invaluable – thank you for always believing in me. A special thanks to my trainer and mentor Kyle Green, I would not have been able to do this PhD without your positive input in helping me to keep a healthy mind and body during this time. Finally, I would like to dedicate this PhD in loving memory of my dear grandmother Pushpa Rani Dutta. For the little indian girl from a small village in India, who was deprived of a basic education simply for being a female. This is for you. ii Abstract Multipotent stromal cells (MSCs) have the capacity for multilineage differentiation and are typically known for their applications for bone and cartilage regeneration. Additionally, MSCs have shown to have strong immunomodulatory properties, acting on both adaptive(Ma et al. 2014), and innate immune cells, including monocytes (J. Kim and Hematti 2009). Consequentially, MSC cell therapy have been proposed as an alternative new treatment for autoimmune diseases, such as rheumatoid arthritis (Luque-Campos et al. 2019). However, studies examining the interactions between MSCs and monocytes have commonly used magnetic cell separation methods or a monocytic cell line (Melief, Schrama, et al. 2013; Choi et al. 2011; Vallés et al. 2015). Therefore, this study investigated the immunomodulatory effects of MSCs or their conditioned media on healthy and RA monocytes, using a whole blood-based assay, which provided a more physiologically relevant setting. All MSCs used in co-culture experiments (IP006,Y201 and Y202 MSCs) conformed to the International Society of Cellular therapy phenotypic criteria for MSCs (Dominici et al. 2006). For the detection of intracellular tumour necrosis factor (TNF) and Interleukin 6 (IL-6) pro-inflammatory cytokine production via flow cytometry, healthy and RA monocytes were activated with lipopolysaccharide (LPS) and treated with Brefeldin A for 6 hours, using the whole blood assay. Results demonstrated that the addition of MSCs to healthy control monocytes, significantly reduced intracellular TNF and IL-6 levels. An even greater inhibition of TNF and IL-6 was observed when MSC CM was used instead of MSCs. Thus, for the treatment of early RA patients’ response, bloods were treated with MSC CM only. This treatment showed statistically significant inhibition of TNF and IL-6 levels in activated monocytes, with the greatest potency being displayed in the immunosuppressive abilities of IP006 MSC CM treatment of established RA patients’ bloods, from multi drug resistant patients. In conclusion, MSCs and to a greater extent MSC CM showed potent immunosuppressive effects on monocytes in health and in both early and established RA. These results provide further evidence for a non-cell contact mechanism of MSC action and support the development of novel therapies for RA treatment. iii Abbreviations ACPA – Anti-citrullinated protein antibody APCs – Antigen presenting cells ASC – Adipose derived multipotential stromal cells BD – Becton Dickinson bDMARD – biological disease modifying antirheumatic drug BM – Bone marrow BM MSCs – Bone marrow derived multipotential stromal cells CDAI – Clinical disease assessment index CCP – Cyclic citrullinated peptide CM – Conditioned media csDMARD – conventional synthetic disease modifying antirheumatic drug CTLA-4 – Cytotoxic T-lymphocyte associated protein 4 DAMPs – Damage-associated molecular patterns DC – Dendritic cell DMARDs – Disease modifying antirhuematic drugs DPBS – Dulbecco's phosphate buffered saline ELISA – Enzyme – linked immunosorbent assay FBS – Fetal bovine serum FSC – Forward scatter GM-CSF – Granulocyte colony-stimulating factor GvHD – Graft versus host disease HGF – Hepatic growth factor HLA – Human leukocyte antigen HSP – Heat shock protein IDO – Indoleamine 2,3-dioxygenase IFN-β – Interferon- beta IFN- – Interferon gamma IL – Interleukin ISCT – International society of cellular therapy LPS – Lipopolysaccharide iv M-CSF – Macrophage colony-stimulating factor MCP-1 – Monocyte cehmo-attractant protein 1 MFI – mean/median fluorescence intensity MHC – Major histocompatibility complex miRNA – Micro RNA mRNA – messenger RNA MSC – Multipotential stromal cell NF- B – Nuclear factor kappa B NSAIDs – Nonsteroidal anti-inflammatory drugs OA – Osteoarthritis P – Cell culture passage number PADs – peptidyarginine PAMPs – Pathogen-associated molecular patterns PBMC – Peripheral blood mononuclear cells PBS – Phosphate-buffered saline PE – Phycoerythrin PerCP – Peridinin chlorophyll protein-cyanine 5.5 PFA – Paraformaldeyde PGE2 – Prostaglandin E2 RA – Rheumatoid arthritis S-MSCs – Synovial derived MSCs SSC – Side scatter STAT – signal transducer and activator of transcription TCR – T cell receptor TERT –Telomerase reverse transciptase TGF-β – Transforming growth factor beta TLR – Toll like receptor TNF – Tumour necrosis factor TSG-6 – Transforming necrosis factor-inducible gene 6 UCB – Umbilical cord blood v Table of Contents Acknowledgements ............................................................................................................ ii Abstract .............................................................................................................................. iii Abbreviations ..................................................................................................................... iv List of figures ..................................................................................................................... ix List of tables ....................................................................................................................... xi Chapter 1 – Introduction ..................................................................................................... 1 1.1 Rheumatoid Arthritis .................................................................................................... 1 1.1.1 Pathogenesis ........................................................................................................ 4 1.1.2 Genetics of RA ...................................................................................................... 5 1.1.3 The role of leukocyte populations in RA ................................................................ 7 1.1.4 Treatment of RA – biologics in RA, alternative therapies, new targets for therapy of RA ........................................................................................................................... 10 1.1.5 Key cytokines in RA: TNF and IL-6 ..................................................................... 15 1.2 Multipotent stromal cells (MSCs) ............................................................................... 18 1.2.1 Bone Marrow derived MSCs (BM-MSCs) ............................................................ 20 1.2.2 Adipose derived MSCs (ASCs) ........................................................................... 21 1.2.3 Synovial MSCs (S-MSCs) ................................................................................... 21 1.2.4 MSCs as a therapy ............................................................................................. 22 1.3 Monocytes/Macrophages ..........................................................................................