MECHANOBIOLOGY OF LEUKOCYTE ADHESION by BRYAN LAUCK BENSON Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisor: Alex Yee-Chen Huang, MD, PhD Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2019 Case Western Reserve University School of Graduate Studies We hereby approve the dissertation1 of Bryan Lauck Benson candidate for the degree of Doctor of Philosophy George Dubyak, PhD Committee Chair Justin Lathia, PhD Clive Hamlin, PhD Umut Gurkan, PhD Richard Ransohoff, MD Alex Huang, MD, PhD August 16th, 2018 1We certify that written approval has been obtained for any proprietary material contained therein. Acknowledgements This thesis represents the contributions of many people, amongst whom I am just one minor player. My parents Lauck and Mary Lynn gave me unconditional love and support my entire life, and gave me the resources and freedom to build things at home. This ob- session with building is evident in the microfluidic devices. My grandmother LeGrace and uncle Lloyd supported me with perspective and encouragement in navigating academia. Rachael took me in as a college freshman with no experience, and skillfully bal- anced promoting and nurturing my love of science with instilling rigor and discipline. She guided me from complete newcomer to the conclusion of an independent research product and continues to be a valued mentor today. Joaquin introduced me to a style of science using careful reading of the literature leading to wild ideas, and pairing a Zenlike approach to repetition with unbridled intellectual curiosity. This remains my style today, and is what I try to instill in my own students. The Shipman Society and Nerd Herd breathed vitality into my college experience and pushed me to achieve more. I am forever thankful to Kim Elliott who advocated for me to join that group. Steve Allen, fellow Shipmate and future Co-PI, introduced me to the idea of the MSTP and gave me the example to follow in pursuing this training program. The CWRU MSTP took a chance with a Psychology undergraduate wanting to become a translational biomedical researcher, and my MSTP class made this whole iii process fun, mixing "the couch that lived" silliness with peer role models of excellence. Cliff Harding has been a steadfastly excellent MSTP director, a needed voice of reason, and strong representative for us to the broader the university community. Kathy, Crista, and Jane have kept me shipshape and Bristol fashion throughout this voyage. Richard courageously accepted me into his lab after just a brief, unscheduled meeting. He models the approach I one day hope to have the necessary wisdom and patience to emulate: accepting the extreme complexity of biology, taking each constituent piece in turn to give it full attention, and rejecting generalizations. At times he also has taken on the difficult but necessary role of scientific spiritual advisor, for which I am very grateful. Richard surrounded himself and filled his lab with excellent people. Among these many excellent scientists, I have special thanks for Roo, Anna, LiPing, and Bunny for teaching me bench science from the ground up and remaining friends. Kate, likewise, was a critical friend in need, informal advisor, and did much to keep my head afloat. Birgit conspired with me to start the microfluidics investigation, taught me cell culture technique (about which I remain admittedly somewhat stuck up, superior, and persnickety), and gave me unending enthusiastic encouragement. She has remained a staunch supporter and loyal friend throughout, protecting me from unhelpful negative influences while also being singularly unafraid to give needed honest constructive feedback. Saurabh Vyawahare provided an excellent microfluidics bootcamp course that ultimately enabled this crazy project. Without this course and his later technical assistance, none of this could have come together. iv Judy Drazba patiently introduced me to the wonderful worlds of confocal and multiphoton microscopy, two cornerstones of every phase of this project. Alex took me into the lab when he already had four other graduate students, and even let me keep my ongoing project. He gave me the resources and independence to grow as a researcher in my own right, even when it would have benefited him to guide me in other directions. Above all, he exemplified grace under pressure through multiple stressful situations, a quality I greatly admire and hope to achieve with much practice. Those other four students: AT, Fred, Dixon, and Deb, were welcoming and kind almost to a fault, taught me a new work ethic, and slowly (feet kicking) dragged me into the world of T lymphocyte biology. I look forward to collaborations with them in the future, or just saying "I knew them when." Lifer Huang Lab members Jay, Saada, and Dave are trusted confidants, helped keep me sane within the sometimes chaotic lab environment, and provided much needed guidance and help on countless occasions. Peter was a much needed comrade during the peak of my hours in lab and pushed me to stay on task, while also getting me onto the ski hill. Joseph and Steven guided me through the worlds of synthetic and molecular biol- ogy, taught me Gibson cloning and CRISPR, are genuinely good people, and are fun and loyal friends outside of lab despite our mutually impossible schedules. Anna HB kept me going many times when I considered quitting, and beyond being a trusted friend is also an excellent collaborator and scientific sounding board. The project also owes almost its whole existence to her and her mentor Diana graciously allowing our extended use of their treasured microscope. v Graham in Boston gave valuable input and camaraderie as part of the "Beebs" team. Brooke, Suzanne, and Mari brought in a fun collaboration and also offered some much needed eleventh hour emotional support. Susi exhibited saintlike patience with two rounds of fellowship grant applications with late-breaking changes. Kathy M exuded both professionalism and cool by sub- mitting that late-breaking application from a beach via smartphone. Christy kept me on track within my degree program, with similar patience in asking my neurotic questions. Alan and Neil took interest in my work, supported my morale, and are true academics who refreshingly enjoy just talking about science and medicine. Justin, Umut, and Clive gave valuable project, meta-project, and life advice and will hopefully remain collaborators in the future. George was an unfailing pillar of support, especially when times got tough, and an ideal committee chair. He also on more than one occasion provided last-minute needed reagents after 5 on Friday. Collaborators and consultants Tracy Handel, Rafick Sekaly, Jim McGrath, Pulak Nath, Lance Munn, and Derek Abbott kept the direction of the project in bounds and provided valuable advice and reagents. Dr. Rae-Grant introduced me to the clinical world of MS treatment and gave valuable pointers on what \you research guys" should be doing. The MSTP, CTSTP, Neurodegeneration T32 with Wenquan and Xiongwei, and the NINDS, especially Jim Koenig, believed in me and the project enough to give essential support. vi Libby, my first student at a time when I knew very little, contributed substantially to our understanding of CXCL12 expression by BBB endothelial cells. Candi worked tirelessly to open a new front on PIEZO1 biology in sickle erythrocytes, which we hope to revisit when our understanding of PIEZO1-integrin relationships is more complete. Ben was a great help in the early days of the microfluidic adhesion experiment quan- tification as we scaled up into more and more devices around a circular manifold. Hannah brought fresh life into the project, and did tremendous service by finding the one decent PIEZO1 antibody, out of a field of over a dozen, and by generating the PIEZO1 expression lentiviral vectors. Lucy followed me down the rabbit hole of trying (unsuccessfully) to procedurally generate complex biomimetic microfluidic networks using scaling laws, and more practically developed a streamlined approach to generating devices and performed much of the immunofluorescence staining opti- mization. Luis took over the monotony of creating new PIEZO1 truncation mutants when I had to take a step back for family, and by rights should take most of the credit for identifying the critical interaction domain. Ali pushed me to work at a simply ridiculous pace during the final crunch of dissertation writing and data collection, and accomplished the incredible task of completing a figure using CRISPR knockouts in primary human T cells within a span of about three weeks - something we had been trying for two years. Each of these students taught me a great deal about how to be a good mentor, and are also to be thanked for putting up with my unorthodox mentorship style. Susan and Mike took many trips out to Cleveland to vitally support the final stretch with a new baby. That new baby Arthur was always ready with a smile and vii gave this all a renewed sense of purpose. Finally, my partner in crime and unfailingly patient wife Anna quite simply gave me the strength (and sometimes the necessary push) to get up every morning and try again. viii Table of Contents Acknowledgements iii List of Figures xiii Abstract xiv 1 Leukocyte trafficking: a clinical problem 1 1.1 Selectins and selectin ligands . 4 1.2 Integrins . 8 1.3 Chemokines and chemokine receptors . 11 1.4 Clinical implications . 14 2 The environment of leukocyte adhesion is microfluidic 17 2.1 Chemokines and convection . 20 2.2 Intravascular signals . 25 2.3 Leukocyte intravascular crawling . 30 2.3.1 Neutrophils . 31 2.3.2 Monocytes . 32 2.3.3 Lymphocytes . 32 2.3.4 Conclusions . 34 3 Leukocyte adhesion requires erythrocyte-driven forces 35 3.1 Abstract . 35 3.2 Introduction .