University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice Sheena Rose Baratono University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Developmental Biology Commons, Immunology and Infectious Disease Commons, and the Medical Immunology Commons Recommended Citation Baratono, Sheena Rose, "Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice" (2014). Publicly Accessible Penn Dissertations. 1204. https://repository.upenn.edu/edissertations/1204 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1204 For more information, please contact [email protected]. Toll-Like Receptor 9 and Interferon-Gamma Receptor Signaling Suppress the B Cell Fate of Uncommitted Progenitors in Mice Abstract TOLL-LIKE RECEPTOR 9 AND INTFERON-GAMMA RECEPTOR SIGNALING SUPPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE Sheena Baratono Edward M. Behrens Hyperinflammatory syndromes come in a variety of flavors with different immunogens and cytokines leading to variations in organ pathology. This inflammation can be driven by viral, autoimmune, or neoplastic conditions leading to profound and widespread organ pathology including hepatitis, fever, splenomegaly, cytokinemia and pan-cytopenias, including T and B lymphopenias. The developing immune cells in the bone marrow are known to express a variety of cytokine and inflammatory receptors yet the effects of signaling through these receptors on hematopoiesis is incomplete. Since many infectious and autoimmune syndromes result in sustained TLR9 stimulation, understanding the effects of TLR9 driven inflammation on hematopoiesis is important for both questions of pathogenesis as well as possible therapeutic interventions that might help to restore homeostasis. Utilizing in vitro and in vivo techniques, we demonstrate that B lymphopoiesis is inhibited during TLR9 driven inflammation from the Ly-6D+ CLP stage onwards with different effects inhibiting development at multiple stages across B cell development. We show that TLR9 signaling directly decreases in vitro B cell yields from CLPs, Pro and Pre B cells yet increases T cell yields. While TLR9 can inhibit B cell growth in vitro, using mixed bone marrow chimeras, we show in vivo that this TLR9 intrinsic effect is masked, likely due to other TLR9 induced inflammatory factors that also mediate decreases in B lymphopoiesis. This led us to demonstrate that IFN-γ also directly inhibits B cell differentiation in vitro as well as when induced by TLR9 in vivo. Microarray and RT- PCR analysis of Ly-6D- CLPs points to HOXa9 as an early B-cell directing transcription factor that is altered by TLR9 induced inflammation, as it and many of its known targets are decreased in Ly-6D- CLPs. Additionally EBF-1, another factor essential for initiation of the B cell program was transcriptionally decreased in Ly-6D- and Ly-6D+ CLPs. Our work demonstrates both cellular and molecular targets that lead to altered B-lymphopoiesis in sustained TLR9 driven inflammation that may be relevant in a number of infectious and autoimmune/inflammatory settings. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Immunology First Advisor Edward M. Behrens Keywords B cell, inflammation, interferon, lymphopoiesis, T cell, TLR9 Subject Categories Allergy and Immunology | Developmental Biology | Immunology and Infectious Disease | Medical Immunology | Medicine and Health Sciences This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1204 TOLL-LIKE RECEPTOR 9 AND INTERFERON-GAMMA RECEPTOR SIGNALING SUPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE Sheena Baratono A DISSERTATION in Immunology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation _____________________ Edward M. Behrens Associate Professor of Pediatrics Graduate Group Chairperson ______________________ David M. Allman Associate Professor of Pathology and Laboratory Medicine Dissertation Committee: David M. Allman, Associate Professor of Pathology and Laboratory Medicine Avinash Bhandoola, Senior Investigator National Cancer Institute Igor E. Brodsky, (Chair) Assistant Professor of Micorbiology Kathleen Sullivan, Professor of Pediatrics ACKNOWLEDGMENTS I have had an extremely rewarding learning experience throughout my graduate career. To start, I would like to thank my mentor, Edward Behrens, for all of his advice, guidance, and expertise. His flexibility to explore my interests and follow where the data leads, even if it’s not where you were expecting, have been invaluable to my development as a scientist. I would also like to thank all of the members of the Behrens and Koretzky labs for their help, support, and scientific advice over the past few years. I am especially grateful to Niansheng Chu and Lee Richardson for help with some of the experiments in this manuscript. I would like to thank Michael P. Cancro and David M. Allman for their critical review of our manuscript and Martha Jordan, Taku Kambayashi, Avinash Bhandoola, and Gary Koretzky for their support and helpful discussions. I am grateful for the scientific discussions and advice from various lab meeting groups I was a part of including the Cancro, Allman, and Laufer labs. Thank you to Igor Brodsky for chairing my thesis committee, and to the other members Dave Allman, Avinash Bhandoola, and Kate Sullivan. I would also like to thank Gary Koretzky for his guidance and support throughout the PhD. These individuals have allowed me to get the most out of my committee meetings and have guided my development as a young scientist. I would also like to thank the Immunology Graduate Group including the countless faculty and staff that have facilitated my training during my graduate career. ii I would also thank the NapCore at the Children’s Hospital of Philadelphia for their help running the microarray and technical support and the flow cytometry core at the University of Pennsylvania for technical help. Lastly, I would like to thank my family and friends for their support throughout graduate school. Their patience and support have enabled me to get through the roughest times of my training. I would like to give a special thanks to Dustin Shilling for his support and council throughout the PhD process, as well as for his encouragement and patience through the trails and excitements of graduate school. iii ABSTRACT TOLL-LIKE RECEPTOR 9 AND INTFERON-GAMMA RECEPTOR SIGNALING SUPPRESS THE B CELL FATE OF UNCOMMITTED PROGENITORS IN MICE Sheena Baratono Edward M. Behrens Hyperinflammatory syndromes come in a variety of flavors with different immunogens and cytokines leading to variations in organ pathology. This inflammation can be driven by viral, autoimmune, or neoplastic conditions leading to profound and widespread organ pathology including hepatitis, fever, splenomegaly, cytokinemia and pan-cytopenias, including T and B lymphopenias. The developing immune cells in the bone marrow are known to express a variety of cytokine and inflammatory receptors yet the effects of signaling through these receptors on hematopoiesis is incomplete. Since many infectious and autoimmune syndromes result in sustained TLR9 stimulation, understanding the effects of TLR9 driven inflammation on hematopoiesis is important for both questions of pathogenesis as well as possible therapeutic interventions that might help to restore homeostasis. Utilizing in vitro and in vivo techniques, we demonstrate that B lymphopoiesis is inhibited during TLR9 driven inflammation from the Ly-6D+ CLP stage onwards with different effects inhibiting development at multiple stages across B cell development. We show that TLR9 signaling directly decreases in vitro B cell yields from CLPs, Pro and Pre B cells yet increases T cell yields. While TLR9 can inhibit B cell growth in vitro, using mixed bone marrow chimeras, we show in vivo that this TLR9 iv intrinsic effect is masked, likely due to other TLR9 induced inflammatory factors that also mediate decreases in B lymphopoiesis. This led us to demonstrate that IFN-γ also directly inhibits B cell differentiation in vitro as well as when induced by TLR9 in vivo. Microarray and RT-PCR analysis of Ly-6D- CLPs points to HOXa9 as an early B-cell directing transcription factor that is altered by TLR9 induced inflammation, as it and many of its known targets are decreased in Ly-6D- CLPs. Additionally EBF-1, another factor essential for initiation of the B cell program was transcriptionally decreased in Ly- 6D- and Ly-6D+ CLPs. Our work demonstrates both cellular and molecular targets that lead to altered B-lymphopoiesis in sustained TLR9 driven inflammation that may be relevant in a number of infectious and autoimmune/inflammatory settings. v TABLE OF CONTENTS ACKNOWLEDGMENTS …………………………..….…………………………..….. ii ABSTRACT ……………………………………………….……………...……….…… iv TABLE OF CONTENTS ………………………………………………………......…. vi LIST OF TABLES ……………………………………………………….….……........ ix LIST OF FIGURES ……………………….….………….……………….…..…........... x CHAPTER 1 – Introduction……..……………………………….……….………........ 1 Diseases of Hyperinflammation ………………………………………….……...