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The Emerging Oral Pathogen, Filifactor Alocis, Disrupts Neutrophil Functions to Enhance Survival and Dysregulate Inflammation

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The Emerging Oral Pathogen, Filifactor Alocis, Disrupts Neutrophil Functions to Enhance Survival and Dysregulate Inflammation University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 5-2020 The emerging oral pathogen, filifactor alocis, disrupts neutrophil functions to enhance survival and dysregulate inflammation. Irina Miralda Molina University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Bacterial Infections and Mycoses Commons, and the Oral Biology and Oral Pathology Commons Recommended Citation Miralda Molina, Irina, "The emerging oral pathogen, filifactor alocis, disrupts neutrophil functions to enhance survival and dysregulate inflammation." (2020). Electronic Theses and Dissertations. Paper 3386. Retrieved from https://ir.library.louisville.edu/etd/3386 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. THE EMERGING ORAL PATHOGEN, FILIFACTOR ALOCIS, DISRUPTS NEUTROPHIL FUNCTIONS TO ENHANCE SURVIVAL AND DYSREGULATE INFLAMMATION By: Irina Miralda Molina B.S., University of Louisville, 2014 M.S., University of Louisville, 2017 A Dissertation Submitted to the Faculty of the School of Medicine of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology and Immunology Department of Microbiology & Immunology University of Louisville School of Medicine Louisville, Kentucky May 2020 Copyright 2020 by Irina Miralda Molina All rights reserved THE EMERGING ORAL PATHOGEN, FILIFACTOR ALOCIS, DISRUPTS NEUTROPHIL FUNCTIONS TO ENHANCE SURVIVAL AND DYSREGULATE INFLAMMATION By Irina Miralda Molina B.S. University of Louisville, 2014 M.S. University of Louisville, 2017 A Dissertation Approved on April 15, 2020 By the following Dissertation Committee: Silvia M. Uriarte, Ph.D. Matthew B. Lawrenz, Ph.D. James E. Graham, Ph.D. Pascale Alard, Ph.D. Jan Potempa, Ph.D. ii DEDICATION This dissertation is dedicated to my amazing family, Abner2 and Carmen2 Miralda, who I try to make proud with every breath I take, and to those who live defiantly to follow their passion. “You may say I’m a dreamer, but I’m not the only one,” John Lennon. iii ACKNOWLEDGEMENTS I would first like to thank my mentor, Dr. Silvia Uriarte for taking a chance on me as an undergraduate student with zero lab experience. In the past 8 years I have come to admire you for your enthusiasm, curiosity, fearlessness, and kindness. Words will never capture my gratitude to you for believing in me, encouraging me, advocating for me, and giving me the confidence to pursue a career in science. We are the perfect example of the quality of student that can evolve from a loving and supportive lab environment and I look forward to spoiling my future students the way you have done with me! Besides my advisor, I would also like to thank my committee members, Dr. Pascale Alard, Dr. Jan Potempa, Dr. James Graham and Dr. Matthew Lawrenz. Your enthusiasm, support and guidance has been invaluable for the completion of this dissertation. I would specifically like to highlight Dr. Lawrenz, who acted as my department co-mentor. This department and its’ students are so fortunate to have you. Thank you for listening, understanding and guiding me in the toughest times of my dissertation. I would also like to give a special thank you to my other co-mentor, Dr. James Graham, who opened his lab for me to earn the microbiology part of this degree. I always cherished your insight and mentorship, and I will always miss our Friday meetings. Thank you to Dr. Russell Salter for offering an avenue to connect with diversity students in STEM. Attending the SACNAS meetings will forever be one of my fondest memories. iv A huge heartfelt thank you to the past and present members of the Uriarte lab family. Chris and Max, thank you for your being amazing lab mates and contributing to my project. To the brilliant Dr. Aruna Vashishta, thank you for everything. Our collaboration and friendship is one of the things I will cherish the most from my time in graduate school. To our close collaborators (and their labs), Dr. Richard Lamont and Dr. Juhi Bagaitkar, without your knowledge, expertise, and this project would not have been possible. Juhi, thank you for always taking time out of your busy schedule to teach me techniques and give me too many ideas for experiments. Your passion for science and learning is infectious, and I look forward to seeing your career skyrocket. I would also like to say a heartfelt thank you to Terri Manning who is an incredible person and has tolerated me being a neutrophil tyrant for the past year. Similarly, I would like to thank all of the amazing past and present members of the Kidney Disease Program, who have helped me grow up as a scientist and as a person. I cannot begin to express my appreciation to my many friends, who have lent their joy during the successess and their support during the difficult times. It really does take a village, and I’m so glad they were mine. Finally, it is difficult to describe the debt of gratitude I feel for my loving family. Your work ethic, tenacity, and passion inspire me every day to be the best version of myself. May this achievement be the first of many to validate the sacrifices you have made to see me succeed. v ABSTRACT: THE EMERGING ORAL PATHOGEN, FILIFACTOR ALOCIS, DISRUPTS NEUTROPHIL FUNCTIONS TO ENHANCE SURVIVAL AND DYSREGULATE INFLAMMATION Irina Miralda Molina April 15, 2020 Periodontitis is an irreversible, chronic inflammatory disease where pathogenic microbial communities accumulate in the gingival crevice. Advances in culture- independent techniques have facilitated the identification of new bacterial species in periodontal lesions, such as the Gram-positive anaerobe, Filifactor alocis. Neutrophils are a major component of the innate host response, and the outcome of their interaction with F. alocis may be a determinant of oral health. Neutrophil functions typically protect the host against periodontal disease, oral pathogens have adapted to evade or disarm neutrophil microbicidal functions while promoting mechanisms that drive inflammation, which also provides a source of nutrients for growth. This study has two main goals: to determine how F. alocis interferes with microbicidal mechanisms to survive in neutrophils and to examine how F. alocis contributes to the chronicity of periodontitis by promoting inflammation. This dissertation characterizes human neutrophil global gene expression during infection with F. alocis (Chapter 2). Challenge of human neutrophils with F. alocis resulted in the differential expression of genes involved in multiple neutrophil effector functions vi such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. F. alocis challenge also affected the expression of components from the TNFα and MAPK kinase signaling pathways. This resulted in transiently dampened p38 MAPK activation by secondary stimuli TNFα. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on priming of the respiratory burst response, or the delay of apoptosis by TNFα. Since this modulatory effect was characteristic of viable F. alocis only, this is an F. alocis’ mechanism to control neutrophils and their functional responses. I also examined neutrophil functional mechanisms in bone marrow neutrophils from TLR2-/- mice (Chapter 3). I found that compared to WT, TLR2-/- neutrophils were more efficient at killing F. alocis, but the increase in killing capacity was not due to a difference in phagocytosis or production of ROS. Instead, it was due to an increase in fusion of myeloperoxidase granules with the F. alocis phagosome. Meanwhile, F. alocis promotes inflammation by inducing the release of pro-inflammatory cytokines through TLR2 signaling. The TLR2 adaptor protein, MyD88, was also critical for cytokine production, but the events downstream of MyD88 follow non-canonical signaling. Apoptotic pathways were also showed significant changes in mRNA levels when neutrophils are challenged with F. alocis. Neutrophils had prolonged lifespans and higher functional capacity than unchallenged cells (Chapter 4). Despite their viability, F. alocis- challenged cells were significantly efferocytosed by macrophages, which developed a pro- inflammatory profile (Chapter 5). Collectively, this data confirms that F. alocis interferes with TLR2 signaling to promote its survival within neutrophils and promotes chronic vii inflammation by extending neutrophil lifespan and delaying the resolution of inflammation. viii TABLE OF CONTENTS DEDICATION .................................................................................................................. iii ACKNOWLEDGEMENTS .............................................................................................. iv ABSTRACT ...................................................................................................................... vi LIST OF FIGURES ........................................................................................................... xi LIST OF TABLES .........................................................................................................
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