University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Fall 2010 Dendritic Cell Migration and Traction Force Generation in Engineered Microenvironments Brendon Guenther Ricart University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemical and Biomolecular Engineering Commons, and the Molecular, Cellular, and Tissue Engineering Commons Recommended Citation Ricart, Brendon Guenther, "Dendritic Cell Migration and Traction Force Generation in Engineered Microenvironments" (2010). Publicly Accessible Penn Dissertations. 282. https://repository.upenn.edu/edissertations/282 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/282 For more information, please contact [email protected]. Dendritic Cell Migration and Traction Force Generation in Engineered Microenvironments Abstract Dendritic cells (DCs) are potent initiators of the adaptive immune response. Their trafficking omfr sites of inflammation ot lymphoid tissue is essential to their function. Exactly how dendritic cells integrate multiple chemotactic cues to organize an accurate migratory path is not fully understood. We first characterize DC random motility (chemokinesis) on extracellular matrix proteins in the presence of chemokines. Then, using a microfluidic device, we present both single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. We show that in counter gradients, CCL19 is 10 to 100 fold more potent than other chemokines CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells "home" to a central region in which the signals from multiple chemokines are balanced. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs. We extended this work to a combination of the microfluidic gradient generator and micropost array detectors to develop a novel method for probing traction forces during chemotaxis. We find DC migration is driven by short-lived traction stresses at the leading edge or filopodia. eW illustrate that spatiotemporal pattern of traction stresses can be used to predict changes in the direction of DC motion. Additionally, we determine the characteristic duration of local dendritic cell traction forces and correlate this duration with force. Overall, DCs show a mode of migration distinct from both mesenchymal cells and other leukocytes, characterized by rapid turnover of traction forces in leading filopodia. In this thesis, we extend the current understanding of DC motility to include signal integration and traction forces. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemical and Biomolecular Engineering First Advisor Daniel A. Hammer Keywords Dendritic cell, chemotaxis, traction force, microfluidic, cell motility Subject Categories Biochemical and Biomolecular Engineering | Biomedical Engineering and Bioengineering | Chemical Engineering | Molecular, Cellular, and Tissue Engineering This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/282 DENDRITIC CELL MIGRATION AND TRACTION FORCE GENERATION IN ENGINEERED MICROENVIRONMENTS Brendon Guenther Ricart A Dissertation in Chemical and Biomolecular Engineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 ______________________ Professor Daniel A. Hammer Dissertation Committee: Supervisor of Dissertation Professor Christopher A. Hunter, Pathobiology Professor John C. Crocker, C.B.E. Assistant Professor Casim A. Sarkar, C.B.E. ______________________ Assistant Professor Matthew J. Lazzara, C.B.E. Professor Raymond J. Gorte Graduate Group Chairperson DENDRITIC CELL MIGRATION AND TRACTION FORCE GENERATION IN ENGINEERED MICROENVIRONMENTS © Copyright 2010 by Brendon Guenther Ricart All Rights Reserved ii Acknowledgements At the end of my formal education, I don't know how to begin thanking the people who have brought me here. I will start with the two people who have known me longest. Thank you to my parents, Glenn Ricart and Patricia Guenther. From playing math games with me as a preschooler to trying to understand my thesis, you have never waivered in your support of my education. To the most important person in my life, Erin Ricart. You have been unyielding in your continuous love and support throughout this process. I appreciate the sacrifices you have made more than you know and I hope you view this thesis as your achievement as well. Marrying you overshadows any accomplishment contained in this text. I am grateful to my advisor, Daniel Hammer. The student-advisor relationship is crucial, and I could not have been paired with a more compassionate yet demanding mentor. You have always supported me, and the trust we have built has contributed greatly to my success. To my thesis committee: Dr. John Crocker, Dr. Chrisopher Hunter, Dr. Matthew Lazarra and Dr. Casim Sarkar. Our conversations lifted me out of ruts, put new tools in my hands, and opened my mind to new perspectives. To the members of the Hammer Lab, you have made this experience pleasantly bearable. My mentors, Risat Jannat and Natalie Christian, laid the groundwork for my thesis and iii guided me through the most trying period of my research. Each and every member of the Hammer Lab has enriched my experience in their own way. Thank you Randi, Aaron, Dalia, Jered, Josh, Kelly, Kevin, Laurel, Lauren, Lee, Neha, Nimil, Olga, Pam, and Steven. I would also like to thank my collaborators, Beena John, Michael Yang, Dooyoung Lee, Fiona Clarke and Debbie Klos Dehring. I could not have finished this work without you. I have had the blessing of forming some amazing friendships at Penn. Bob Meyer, Michael Beste, Calixte Monaste, Greg Robbins, Parag Shah and Ashley Vissing, you all know how special our bond has been over the last three years. To my other good friends, Alex, Andrew, Ben, Dan, Jeremy, Joel, Manash, Matt, Raynaldo and Tom, I look forward to a time when our paths cross again. Finally, I would like to thank the food trucks and cafés on Penn's campus. Frida's, El Rosa, Magic Carpet, Kim's Chinese, King's Wok, Fresh Fruit, MexiPhilly, The Pari Café, The Chem Café, Taco Bell and Potbelly's, your delicious and budget-friendly meals truly enhanced my graduate school experience. iv ABSTRACT DENDRITIC CELL MIGRATION AND TRACTION FORCE GENERATION IN ENGINEERED MICROENVIRONMENTS Brendon Guenther Ricart Professor Daniel A. Hammer, Advisor Dendritic cells (DCs) are potent initiators of the adaptive immune response. Their trafficking from sites of inflammation to lymphoid tissue is essential to their function. Exactly how dendritic cells integrate multiple chemotactic cues to organize an accurate migratory path is not fully understood. We first characterize DC random motility (chemokinesis) on extracellular matrix proteins in the presence of chemokines. Then, using a microfluidic device, we present both single and competing chemokine gradients to murine bone-marrow derived DCs in a controlled, time-invariant microenvironment. We show that in counter gradients, CCL19 is 10 to 100 fold more potent than other chemokines CCL21 or CXCL12. Interestingly, when the chemoattractive potencies of opposing gradients are matched, cells "home" to a central region in which the signals from multiple chemokines are balanced. These results provide fundamental insight into the processes that DCs use to migrate toward and position themselves within secondary lymphoid organs. We extended this work to a combination of the microfluidic gradient generator and micropost array detectors to develop a novel method for probing traction forces during chemotaxis. We find DC migration is driven by short-lived traction v stresses at the leading edge or filopodia. We illustrate that spatiotemporal pattern of traction stresses can be used to predict changes in the direction of DC motion. Additionally, we determine the characteristic duration of local dendritic cell traction forces and correlate this duration with force. Overall, DCs show a mode of migration distinct from both mesenchymal cells and other leukocytes, characterized by rapid turnover of traction forces in leading filopodia. In this thesis, we extend the current understanding of DC motility to include signal integration and traction forces. vi Table of Contents ACKNOWLEDGEMENTS.............................................................................................................................. III ABSTRACT .............................................................................................................................................. V LIST OF FIGURES ..................................................................................................................................... XIII LIST OF TABLES....................................................................................................................................... XVI CHAPTER 1: INTRODUCTION ...............................................................................................................1 MOTIVATION ...............................................................................................................................................2 ORGANIZATION OF THE THESIS ...................................................................................................................4