CEACAM3: Specific Innate Immunity by Anna Sintsova A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Anna Sintsova (2015) Anna Sintsova CEACAM3: Specific Innate Immunity Doctor of Philosophy Department of Molecular Genetics University of Toronto 2015 Abstract The bacterial species Neisseria gonorrhoeae remains one of the most prevalent bacterial causes of sexually transmitted infections. Gonorrhea is typically characterized by a painful purulent discharge and inflammation, the outcome of overzealous recruitment of neutrophils, phagocytic immune cells highly specialized in the detection and elimination of microbial pathogens. Although molecular mechanisms of gonococcal pathogenesis have been studied in great detail, the causes for the excessive inflammation and immunopathology that follow infection are still not clear. The human neutrophil-restricted innate immune receptor CEACAM3 has been previously shown to promote opsonin-independent uptake and killing of N. gonorrhoeae by neutrophils. In the course of my thesis work, I have established that the role of CEACAM3 is not restricted to the neutrophils’ direct engulfment and killing of gonococci, since it also drives a vigorous inflammatory response that typifies gonorrhea. By carrying the potential to mobilize increasing numbers of neutrophils, CEACAM3 represents a tipping point between the protective and pathogenic outcomes of N. gonorrhoeae infection. My biochemical profiling of gonococcal-infected, genetically modified neutrophils revealed that CEACAM3 engagement triggers a Syk-, PKCδ-, Bcl10-MALT1-, and Tak1-dependent signaling cascade that leads to the activation of an NF-κB-dependent transcriptional response, with consequent production of pro- inflammatory cytokines. I also showed that CEACAM cross-linking on neutrophils signals in synergy with TLR4, significantly amplifying the response to LPS, suggesting that CEACAM3 acts as an integral part of a complex innate immune detection network. Finally, I co-led a study that characterized Opa protein variant expression among a collection of clinical specimen-derived N. gonorrhoeae, and then assessed their CEACAM binding profile. This study suggested that mucosal infection in humans selects for binding to epithelial-expressed CEACAMs but against CEACAM3 binding in vivo. Together, my studies implicate a role for CEACAM3 as a host- adaptive innate receptor that promotes gonococcal detection and elimination in the context of natural infection, but also reveal potential pathogenic consequences of CEACAM3 activation. ii Acknowledgments I would like to thank Dr. Scott Gray-Owen for his guidance and his patience. His constant support and encouragement over the last 6 years have made me the scientist I am today. If I learned nothing else, I hope that I will take away with me his enduring optimism and enthusiasm for all things science. I would like to thank my supervisory committee members, Dr. Philpott, Dr. Glogauer, and Dr. Egan, who were always there to keep me on my toes, and whose invaluable ideas greatly helped to shape and guide this project. I would also like to thank all the members of the Gray-Owen lab, past and present, who are a truly amazing group of people, and who made 6 years seem like a very short time indeed. I want to especially thank Dr. Sarantis, who taught me the ropes of neutrophil work and Epshita Islam who was always there through all the highs and the lows, and kept me sane. I would like to thank my family for their (sometimes wavering) understanding of why I chose to spend a whole decade of my life at the University of Toronto and their (never wavering) love. I would also like to thank my husband, who has recently taken over Epshita’s responsibility of telling me “We’re going to be all right”, and is doing a stellar job. iii Table of Contents Table of Contents ........................................................................................................................... iv List of Tables .................................................................................................................................. ix List of Figures ................................................................................................................................. x 1 General Introduction ................................................................................................................... 1 1.1 Neisseria gonorrhoeae: clinical manifestation and molecular mechanisms of disease ...... 1 1.1.1 Clinical Manifestation ............................................................................................. 1 1.1.2 Immune responses to gonococcal infection ............................................................. 2 1.1.3 Neisseria gonorrhoeae pathogenesis ....................................................................... 3 1.2 CEACAM family of Receptors ........................................................................................... 3 1.2.1 CEACAM1 .............................................................................................................. 4 1.2.2 CEACAM5 and CEACAM6 ................................................................................... 5 1.2.3 CEACAM3 .............................................................................................................. 5 1.2.4 CEACAMs as microbial receptors .......................................................................... 6 1.3 Neutrophil responses to bacterial infection ......................................................................... 9 1.3.1 Neutrophil lifecycle ................................................................................................. 9 1.3.2 Neutrophil-mediated pathogen elimination ........................................................... 10 1.3.3 Neutrophil-derived cytokines and regulation of inflammation ............................. 12 1.3.4 Intimate interactions between N. gonorrhoeae and neutrophils ............................ 13 1.4 Molecular mechanisms of innate immune response and phagocytosis ............................. 14 1.4.1 Fc receptor-mediated phagocytosis ....................................................................... 14 1.4.2 Dectin-1 mediated immunity ................................................................................. 16 1.5 CEACAM3: specific innate immunity .............................................................................. 17 1.5.1 CEACAM3 mediated phagocytosis ...................................................................... 17 1.5.2 CEACAM3-driven bacterial elimination .............................................................. 18 iv 1.6 Summary ........................................................................................................................... 19 2 Global analysis of neutrophil responses to Neisseria gonorrhoeae reveals a self- propagating inflammatory program .......................................................................................... 23 2.1 ABSTRACT ...................................................................................................................... 24 2.2 INTRODUCTION ............................................................................................................. 25 2.3 MATERIALS AND METHODS ...................................................................................... 27 2.3.1 Ethics Statement .................................................................................................... 27 2.3.2 Animals ................................................................................................................. 27 2.3.3 Reagents and Antibodies ....................................................................................... 27 2.3.4 Bacterial Strains .................................................................................................... 27 2.3.5 Primary Neutrophil Isolation ................................................................................. 28 2.3.6 Human CEACAM Expression in CEABAC Neutrophils ..................................... 28 2.3.7 Whole Cell Phosphorylation Assays ..................................................................... 28 2.3.8 Bacterial infections for immunofluorescence microscopy .................................... 28 2.3.9 PMN Killing Assays .............................................................................................. 29 2.3.10 Oxidative Burst and Degranulation Assays ........................................................... 29 2.3.11 PMN Gene Array Experiments ............................................................................. 30 2.3.12 Cytokine Measurements ........................................................................................ 30 2.3.13 Chemotaxis Assay ................................................................................................. 30 2.3.14 Air Pouch ............................................................................................................... 31 2.3.15 Neutrophil Depletion ............................................................................................. 31 2.4 RESULTS .......................................................................................................................... 32 2.4.1 CEACAM-humanized transgenic mouse neutrophils respond to N. gonorrhoeae 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