The Role of Endoglin in the Resolution of Inflammation by Madonna Peter A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Immunology University of Toronto © Copyright by Madonna Peter 2012 The Role of Endoglin in the Resolution of Inflammation Madonna Peter Master of Science Department of Immunology University of Toronto 2012 Abstract Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation. ii Dedication To all of my family and friends, for their love, faith and constant support iii Acknowledgments I would like to thank my supervisor, Dr. Michelle Letarte, for giving me the opportunity to work in her lab. Through her guidance, support and encouragement, I have learned so much more than I could have ever imagined. Dr. Letarte’s dedication will be a constant inspiration in my life. I would also like to extend my gratitude to my supervisory committee members, Dr. Dana Philpott and Dr. Shannon Dunn, for their helpful suggestions and advice as well as allowing me to learn from members of their lab. I owe my deepest gratitude to Dr. Mirjana Jerkic from Dr. Letarte’s lab for taking the time to teach me as well as her constant support and being an amazing friend. I am also grateful to Dr. Guoxiong Xu for his guidance and training when I first started my graduate studies. I am indebted to my colleagues: Allison Gregory, Valentin Sotov, Daniela Ardelean and Niousha Ghamami for their helping hands and the unforgettable memories. Special thanks to Dr. Kaoru Geddes from Dr. Philpott’s lab for teaching me how to isolate lamina propria cells and to Flavia Lakschevitz from Dr. Michael Glogauer’s lab for showing me bone marrow neutrophil isolation. Last but not least, I am so thankful to my family and friends for their constant love and support. iv Acknowledgment of contributions Dr. Mirjana Jerkic Performed the myeloperoxidase (MPO) counts, western blots for MPO, Nox2 and Smad2 as well as the H2O2 measurement assay. This data was used to generate Figures 3.9, 3.11 A and C, 3.12 and 3.13 A and B. Dr. Christopher Waterhouse in Dr. Paul Kubes’ lab Performed the MPO activity assay and confirmed the MPO counts results. This data was used to generate Figure 3.11 A and B. Valentin Sotov Performed the superoxide generation assay in isolated bone marrow cells, which is represented in Figure 3.13 C. Niousha Ghamami, a summer student in our lab Helped to generate the B cell flow cytometry data. v Table of Contents Dedication ...................................................................................................................................... iii Acknowledgments .......................................................................................................................... iv Acknowledgment of contributions .................................................................................................. v List of Tables .................................................................................................................................. x List of Figures ................................................................................................................................ xi List of Figures (Continued) ........................................................................................................... xii List of Abbreviations ................................................................................................................... xiii Chapter 1 Introduction .................................................................................................................... 1 1 Introduction ................................................................................................................................ 1 1.1 Overview ............................................................................................................................. 1 1.2 Expression, structure and function of endoglin .................................................................. 1 1.2.1 Expression in cells and tissues ................................................................................ 1 1.2.2 Elucidating the structure of endoglin led to its identification as a TGF-β co- receptor ................................................................................................................... 3 1.2.3 Role in vascular development and regulation ......................................................... 4 1.2.4 Modulation of TGF-β superfamily signaling pathways .......................................... 6 1.2.5 Role in the immune system ..................................................................................... 9 1.3 Inflammation and its resolution ........................................................................................ 11 1.3.1 Cellular and molecular components of inflammation ........................................... 11 1.3.2 Role of TGF-β in resolution of inflammation ....................................................... 12 1.3.3 Activation of endothelial cells and immune-driven angiogenesis ........................ 13 1.3.4 Inflammatory bowel disease and the DSS experimental model ........................... 16 1.3.5 DSS-treated Eng+/- mouse model .......................................................................... 17 1.4 Inflammation and its resolution ........................................................................................ 19 1.4.1 Rationale ............................................................................................................... 19 vi 1.4.2 Hypothesis ............................................................................................................. 19 1.4.3 Specific Objectives ............................................................................................... 19 Chapter 2 Materials and Methods ................................................................................................. 20 2 Materials and Methods ............................................................................................................. 20 2.1 Mice .................................................................................................................................. 20 2.2 DSS-induced colitis .......................................................................................................... 20 2.3 Isolation of colonic lamina propria, splenocytes and bone marrow leukocytes ............... 21 2.3.1 Lamina propria ...................................................................................................... 21 2.3.2 Spleen .................................................................................................................... 21 2.3.3 Bone marrow ......................................................................................................... 22 2.4 Multi-color flow cytometry analysis ................................................................................. 22 2.5 Differential blood counts .................................................................................................. 24 2.6 RNA isolation and PCR array ........................................................................................... 24 2.7 Protein extraction from colonic and bone marrow tissues ................................................ 25 2.8 Cytokine/Chemokine profiles ........................................................................................... 25 2.9 Myeloperoxidase assays .................................................................................................... 26 2.9.1 MPO positive cells ................................................................................................ 26 2.9.2 MPO enzymatic activity ....................................................................................... 26 2.10 Western blots .................................................................................................................... 26 2.11 Superoxide and H2O2 assays ............................................................................................. 27 2.11.1 H2O2 assay in colonic samples .............................................................................. 27 2.11.2 Superoxide production by isolated bone marrow neutrophils .............................. 27 2.12 Statistical analysis ............................................................................................................. 27 Chapter 3 Results .........................................................................................................................