Investigation of the human immune receptors CEACAM3 and CEACAM4 Dissertation Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Julia Delgado Tascón An der Universität Konstanz des Fachbereichs Biologie Konstanz, Oktober 2015 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-306516 Tag der mündlichen Prüfung: 05.11.2015 Vorsitzender und mündlicher Prüfer: Herr Professor Dr. Bürkle 1. Referent und und mündlicher Prüfer: Herr Professor Dr. Hauck 2. Referent und und mündlicher Prüfer: Herr Professor Dr. Tschan, Universität Bern A mi familia Acknowledgements I would like to express my special gratitude to my advisor Prof. Dr. Christof Hauck. His patient guidance and enthusiastic encouragement during these four years of PhD were a crucial aid to my process. I’m very thankful for his willingness and for granting me with his time in search for valuable and constructive suggestions during the planning and development of this research work. This certainly allowed me to grow as a person and as a scientist. I would also like to thank my committee members: to Prof. Dr. Mario Tschan for giving me his academic support at this last phase of my PhD thesis, and to Prof. M.Dr. Alexander Bürkle for his wise advices accompanied with Spanish greetings along this time. My thanks are extended to every member of the AG Hauck as well. To Anne, Susana, Petra and Claudia: thank you very much for your technical and personal guidance during these years. I’m also thankful to my fellow colleagues for countless ‘Kaffeepausen’ full of jokes, nice discussions, and delicious vegan cakes. I specially thank Nina, Arnaud, Lexi, Chris, Yong and also our former colleagues Alexa, Naja, Timo and Thomi for the nice times ‘inside-out’ of the lab. Thank you for your motivations in the right moment and for the party time we had together. I’m very grateful with Lisa for her support and co-work during every hard time we had with the mice. I’m also thankful to Annette for the FRET analysis in the Tec manuscript, and to Dr. Joachim Hentschel for his assistance when employing the electron microscope. Thanks to the Center of Molecular Biology of Inflammation (ZMBE) for the generation of the transgenic CEACAM3 mice, and thanks as well to my lovely Nora H. for her time and help in the formatting process of this thesis. Special thanks to my beloved family and friends. This has been a wonderful life experience and words cannot express how grateful I am! Gracias totales! Table of contents Table of contents Summary ..................................................................................................................... 11 Zusammenfassung .................................................................................................... 13 1 General Introduction ..................................................................................... 15 1.1 Innate (natural) Immunity .......................................................................... 15 1.1.1 Host defense against infection .................................................................... 17 1.1.2 Response of phagocytic cells to infection .................................................. 21 1.2 Human CEACAM family ........................................................................... 24 1.2.1 Granulocytes CEACAMs ........................................................................... 26 1.2.2 CEACAM3: Phagocytic Hem-ITAM receptor .......................................... 28 1.2.3 CEACAM4: Orphan receptor of the CEACAM family ............................. 30 2 Aims of the study ............................................................................................. 32 3 Chapter I: Tec kinase contributes to CEACAM3-initiated Hem- ITAM signaling to promote bacterial phagocytosis and destruction by human granulocytes ......................................................... 34 3.1 Summary .................................................................................................... 35 3.2 Introduction ................................................................................................ 35 3.3 Material and Methods ................................................................................. 37 3.4 Results ........................................................................................................ 43 3.5 Discussion .................................................................................................. 56 3.6 Supplemental figures .................................................................................. 62 3.7 Acknowledgments ...................................................................................... 64 4 Chapter II: Generation of a humanized mouse model for in vivo analysis of CEACAM3 .................................................................................. 65 4.1 Abstract ...................................................................................................... 66 4.2 Introduction ................................................................................................ 66 4.3 Material and Methods ................................................................................. 69 4.4 Results ........................................................................................................ 77 4.5 Discussion .................................................................................................. 93 4.6 Acknowledgments ...................................................................................... 95 5 Chapter III: The granulocyte orphan receptor CEACAM4 is able to trigger phagocytosis of bacteria .................................................. 97 5.1 Abstract ...................................................................................................... 98 5.2 Introduction ................................................................................................ 98 5.3 Material and Methods ............................................................................... 100 5.4 Results and Discussion ............................................................................. 107 5.5 Conclusion ................................................................................................ 119 5.6 Supplementary figures .............................................................................. 120 9 Table of contents 5.7 Acknowledgments .................................................................................... 123 6 Chapter IV: Finding bacterial ligand of the orphan receptor CEACAM4 ...................................................................................................... 124 6.1 Abstract .................................................................................................... 125 6.2 Introduction .............................................................................................. 125 6.3 Material and Methods .............................................................................. 127 6.4 Results ...................................................................................................... 133 6.5 Discussion ................................................................................................ 139 6.6 Acknowledgments .................................................................................... 142 7 Concluding remarks .................................................................................... 143 8 Declaration of author’s contributions ................................................... 152 List of publications ................................................................................................ 154 8.1 Publications part of this thesis or ongoing to be submitted ..................... 154 8.2 External publications ............................................................................... 154 Abbreviations ........................................................................................................... 155 References ................................................................................................................. 157 10 Summary Summary The innate immune system provides an immediate response to infection which can be activated via different recognition molecules allowing specialized immune cells to recognize but also to phagocytose and eliminate a wide range of microbes. Human granulocytes express a peculiar receptor, the carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3). This membrane protein is responsible for initiating the opsonin-independent recognition, phagocytosis and killing of a limited set of human- specific gram-negative bacteria. CEACAM3 harbors a hemi-immunoreceptor tyrosine- based activation motif (Hem-ITAM) which after phosphorylation by Src family Kinases serves as a docking site for SH2-domain containing proteins. This study provides the first evidence of a specific role of Tec kinase in the host innate immune response of neutrophils against bacterial infection. Upon phosphorylation of the Hem-ITAM of CEACAM3, the SH2 domain of Tec kinase directly binds to the phosphorylated Hem-ITAM and gets recruited to the site of bacterial phagocytosis. Functional experiments showed that Tec kinase is essential and maximizes Hem-ITAM signaling for efficient CEACAM3-mediated bacterial uptake and actin reorganization as well as contributing in the clearance of CEACAM-binding bacteria such as Neisseria gonorrhoeae.