Regulation of Immunity to Visceral Leishmaniasis by Regulatory T cells (Tregs) and Hepatic Stellate Cells (HSCs) By Forough Khadem A Thesis submitted to the Faculty of Graduate Studies of the University of Manitoba in partial fulfillment of the requirements of the degree of Doctor of Philosophy Department of Immunology University of Manitoba Winnipeg Copyright © 2016 Forough Khadem ABSTRACT Leishmaniasis is a vector borne disease that spreads through the bite of an infected sandfly and is caused by the intracellular parasite-Leishmania. An estimated 10-15 million cases of leishmaniasis occur worldwide, presenting as one of the three forms: cutaneous (CL), mucocutaneous (MCL) or visceral leishmaniasis (VL). The occurrence of leishmaniasis is increasing due to global traveling, emergence of drug resistant species and Leishmania-HIV coinfection. Therefore, there is an urgent need for the development of new therapies or vaccines against leishmaniasis. Our laboratory previously showed that mice with an inactivating knock-in mutation in the p110δ gene (known as p110δD910A) are resistant to L. major (the causative agent of CL). Here, I demonstrate that signaling via the p110δ also regulates immunity to L. donovani (the causative agent of VL) resulting in hyper-resistance to experimental VL. This outcome is dependent on the impact of p110δ signaling on expansion and function of regulatory T cells (Tregs). I show for the first time that L. donovani can infect Hepatic Stellate Cells (HSCs) in vivo and in vitro and this infection leads to the production of cytokines that are known to induce Tregs. I also demonstrate that infection with L. donovani leads to dramatic expansion of HSCs in a PI3K-dependent manner, and this correlates with expansion of hepatic Tregs. I further show that L. donovani- infected HSCs can induce CD4+ T cells to become Tregs and this effect is dependent on p110δ signalling. Targeted depletion of HSCs during infection caused a dramatic reduction in liver Treg numbers and proliferation, which was associated with a more efficient parasite control. I also demonstrate that prophylactic and therapeutic administration of CAL-101 (a pharmacological I inhibitor of p110δ signalling) is associated with significant reduction in parasite burden, Treg numbers and cytokine production in both experimental models of VL and CL. More importantly, combination of CAL-101 with sub-therapeutic dose of Amphotericin-B leads to full cure from VL. Collectively, these results provide novel understandings into the mechanisms involved in the development and regulation of protective immunity against VL, which could have direct implications for immunotherapy and drug/vaccine development against leishmaniasis. II DEDICATION This thesis is dedicated to my “Parents”, “Mentors”, “Sisters”, “Friends” & “Partner in Life” for constantly reminding me that: “WHERE THERE IS A WILL, THERE IS A WAY” III ACKNOWLEDGEMENTS I would like to express my deepest and sincere appreciation to my supervisor and mentor, Dr. Jude Uzonna (or as we call him, Boss), for allowing me to become a part of his research team and for his limitless support, motivating guidance, continuous inspiration and for being a wonderful friend throughout this journey. His caring, honest, supportive, down to earth and magnificent personality and outstanding supervision and leadership has made this long, difficult and exhausting journey pleasant and unforgettable. I will be forever thankful for the knowledge and wisdom he has gracefully shared with me, for challenging me to become a critical thinker and for seeing the potential in me to develop as an Immunologist/scientist under his supervision. He has inspired me to pursue my goals and dreams with dedication and hard work, no matter what life throws at me and this has contributed substantially to my personal and professional successes. I can assert that he is the greatest supervisor/mentor any student could ever ask for. The completion of this study would not have been possible without his continuous guidance and nonstop support. I also extend my gratitude to my advisory committee members, Dr. Eftekhar Eftekharpour, Dr. Aaron Marshall and Dr. Neeloffer Mookherjee for their insightful inputs, constructive criticisms, and constant encouragement and support throughout these years. I also want to express my special thanks to my external examiner, Dr. Albert Descoteaux. This project was performed in collaboration with Dr. Matthew Wright, Dr. Abhay Satoskar and Dr. Yoav Keynan. I thank them for supporting my project and providing some research material. The friendly, healthy, supportive and family like lab environment has had great impact on the quality of research, success and productivity of both me and this project. Therefore, I want to show my appreciation to our kindhearted, competent and experienced technician, Ping Jia, for IV being a great help in most of the research done in this thesis. I appreciate Dr. Dong Liu, as his research project, defined the back bone of this Thesis. I am indebted to Dr. Ifeoma Okwor, Dr. Zhirong Mou and Helen Muleme, for their patience when teaching me new techniques and for being a great support team and fabulous friends. I am grateful to all the past and present members of the lab, Dr. Xiaoling Gao, Chukwunonso Onyilagha, Dr. Emeka Okeke, Dr. Shiby Kuriakose and Aida Feiz Barazandeh for being wonderful friends throughout the past years and facilitating this project significantly. I am forever grateful to the Department of Immunology support/administrative staff, Susan Ness and Karen Morrow, for not only taking care of the panicking adult side of me but making sure the inner kid side of me stays happy throughout these past years. I would also like to pass my thanks to all the past and present members of the Department of Immunology, particularly Dr. Kent HayGlass and late Dr. Redwan Moqbel for their encouragement and for teaching me how to be unique and disciplined. I am thankful to Dr. Xi Yang for his continuous support as the Department Head. I am grateful to Sandrine Lafarge, Carolyn Weiß, Mark Collister, Kanami Orihara, Sonia Charran, Ivan Landego and many more for their kind support and friendship and for making the department an enjoyable environment to learn and work in. I particularly thank Research Manitoba/MHRC and University of Manitoba Faculty of Graduate Studies for their financial support of this research project. I was also fortunate to receive many poster, travel and research awards of which I am grateful to all the funding organizations. I am extremely appreciative of Samantha Pauls and her family for being my family away from home and am thankful to my loyal and caring friend/sister, Farnaz Eftekhar, for checking on me and chatting with me every week nonstop in the past 26 (and counting) years. V My family has been a constant firm source of support in my life and there are basically no words to thank them enough! Thank you “Baba va Maman joone aziztar az janam” for always encouraging me to be unique, passionate, caring, strong, confident and independent. I am grateful for all the sacrifices you have made for me, for being proud of me even when I failed, for praying for me, for always having my back and for your words of wisdom. Dad, Thank you for being my hero and motivating me by being a perpetual example of a great researcher, educator and leader. Mom, thank you for being my biggest inspiration and for pushing me to do things that I thought were impossible to accomplish. Thank you both for staying up to date technologically to stay connected with me during the past years. I hope I can live a life that does justice to all that both of you have done for me. I know tolerating me as the annoying and motherly older sister has not been easy for my wonderful sisters, Faezeh and Farzaneh. You both have special corners in my heart and are the reason for my smiles. Thank you for being my strength and always being there beside me through thick and thin. Special thanks to Farzeneh, her family and my cute niece, Taranom. Seeing a picture or video of Taranom has never failed to cheer me up in the past two years. During the past adventurous decade, my partner in life, Hesam, has stayed beside me through laughs and cries, ups and downs, good times and bad. Although we have not been completely “immune” to all the “dangers” life has thrown our way, but together we have formed a great responsive “innate and adaptive immune system”. VI TABLE OF CONTENTS ABSTRACT .................................................................................................................................... I DEDICATION............................................................................................................................. III ACKNOWLEDGEMENTS ....................................................................................................... IV TABLE OF CONTENTS ......................................................................................................... VII LIST OF TABLES ....................................................................................................................... X LIST OF FIGURES .................................................................................................................... XI ABBREVIATIONS .................................................................................................................. XIV 1 CHAPTER 1: INTRODUCTION ........................................................................................ 1 1.1 Leishmaniasis