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Endocytosis, Phagocytosis, and Innate Immune Responses: a Dissertation University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2010-07-13 Endocytosis, Phagocytosis, and Innate Immune Responses: A Dissertation Christine A. St. Pierre University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Cells Commons, Environmental Public Health Commons, Hemic and Immune Systems Commons, Immunology and Infectious Disease Commons, Investigative Techniques Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons, Pharmaceutical Preparations Commons, and the Therapeutics Commons Repository Citation St. Pierre CA. (2010). Endocytosis, Phagocytosis, and Innate Immune Responses: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/0zzv-kd21. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/488 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. ENDOCYTOSIS, PHAGOCYTOSIS, AND INNATE IMMUNE RESPONSES A Dissertation Presented By Christine A. St. Pierre submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY July 13, 2010 Biomedical Sciences ii ENDOCYTOSIS, PHAGOCYTOSIS, AND INNATE IMMUNE RESPONSES A dissertation presented by Christine A. St. Pierre The signatures of the Dissertation Defense Committee signifies completion and approval as to style and content of the Dissertation Robert W. Finberg, M.D., Thesis Advisor Evelyn A. Kurt-Jones, Ph.D., Member of Committee Silvia Corvera, M.D., Member of Committee Katherine Fitzgerald, Ph.D., Member of Committee Amy Hise, M.D., M.P.H., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Alan Rothman, M.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Immunology and Virology Program July 13, 2010 iii ACKNOWLEDGEMENTS I would first like to thank my mentor Dr. Robert Finberg for allowing me to perform my graduate research in his laboratory. I have grown immensely as a scientist under his direction and will be forever grateful for his guidance, support, and humor throughout my graduate career. I would like to thank Dr. Evelyn Kurt-Jones, whom I consider my co-mentor, for helping me to grow not only as a scientist but also as a scientific writer. She has provided me with countless hours of scientific discussion and allowed me to be a part of several grant-writing processes. I would also like to thank all the members of the Finberg/Kurt-Jones lab for all their support throughout the years. I would especially like to thank Dr. Jennifer Wang, Dr. Shenghua Zhou, and Melvin Chan for their assistance in experimental design and many scientific discussions. I would like to thank my fellow graduate student, Ryan Nistler, for all his help in optimizing experiments and for being a great friend. I would like to thank my collaborators, Dr. Ryan Hayward in the Department of Polymer Science and Engineering at UMass Amherst, and Dr. David Ayers in the Department of Orthopedics here at UMass Medical School, for allowing me to take part in really exciting projects and I look forward to future collaborations with them. I would like to thank Dr. Donna Ambrosino and her team and MassBiologics for all her help and support during my first years as a graduate student. She introduced me to several projects, and trusted me to work with her team when I had very little experience. iv I would like to thank my current and former committee members, Dr. Eicke Latz, Dr. Katherine Fitzgerald, Dr. Silvia Corvera, Dr. Evelyn Kurt-Jones, Dr. Alan Rothman, and my outside member Dr. Amy Hise, for all their comments, suggestions, and support of my work. They have assisted me in moving my projects into a direction that I am proud of. Finally, I would not be where I am today if it weren’t for the love and support of my family and friends. They have always, and continue to believe in me in all that I do, and I am forever grateful. I will always cherish the friendships I have made in graduate school, and wish them all the best in their scientific careers. Last, but not least, I would like to thank Dr. Michael Vaine for making my last three years of graduate school the best years of my life. It has been great to be able to share all of my experiences with someone, especially a fellow scientist. He has pushed me to become a better person, both at home and professionally, and I love him dearly for that. v ABSTRACT In this dissertation, the roles of endocytosis and phagocytosis pathways in a variety of clinically relevant scenarios were examined. These scenarios include antibody-mediated internalization of cell surface proteins, titanium wear-particle uptake in failed joint replacements, and polymeric microparticle uptake and immune responses for drug delivery or adjuvant use. The use of antibodies specific for cell surface proteins has become a popular method to deliver therapeutics to target cells. As such, it is imperative to fully understand the ability of antibodies to mediate internalization and endosomal trafficking of the surface protein that it recognizes, so that drug delivery can be optimized. By comparing the internalization and endosomal localization of two different antibody- bound proteins, the transferrin receptor (TfR) and rabies G, we have found that there is a specific antibody-mediated internalization pathway that occurs when an antibody binds to a cell surface protein. Interestingly, the internalization pathway induced by antibody binding is different than that seen with recycling receptor internalization after ligand binding. This may have broad implications for the future development of antibody-based therapeutics. Joint replacement failure is a major clinical problem. Studies have indicated that a large amount of metal and polyethylene wear debris is found in the synovial membrane and tissue surrounding failed replacements. Through examination of the immune response following uptake of titanium particles, our results suggest that titanium wear-particle induced inflammation and subsequent joint replacement failure vi may be due to activation of the NLRP3 inflammasome, leading to increased IL-1β secretion and IL-1 associated signaling. These findings introduce IL-1 as a target for potential therapeutics for patients exhibiting significant inflammation. Polymeric microparticles have been widely used in a variety of therapeutic applications, including drug delivery and vaccine adjuvants. It is essential to understand the ability of such particles to either activate or inhibit an immune response following uptake. Through comparison of particles with varying surface morphology, we have determined that particles with regions of high surface curvature (budding) are more immunogenic than particles with low surface curvature (spherical). Budding particles were more rapidly phagocytosed and induced higher levels of the inflammasome-associated cytokine, IL-1β, when exposed to mouse macrophages. Additionally, budding particles induced a more rapid neutrophil response in vivo, when compared to spherical particles. These findings have broad implications for the development of future targeting vehicles for delivery of vaccines, drugs, proteins, and siRNA therapeutics. vii TABLE OF CONTENTS Page Signature Page.................................................................................................................. ii Acknowledgements......................................................................................................... iii Abstract............................................................................................................................. v Table of Contents........................................................................................................... vii List of Figures................................................................................................................. xi Abbreviations................................................................................................................ xiv Copyright Notice......................................................................................................... xviii Chapter I: Introduction A. Internalization pathways............................................................................................. 1 Phagocytosis pathways......................................................................................... 2 Clathrin-depdendent receptor-mediated internalization...................................... 5 Role of actin in clathrin-mediated internalization............................................... 6 B. Endosomal compartments........................................................................................... 7 Early endosomes................................................................................................... 9 Recycling endosomes.........................................................................................
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