UNIVERSITY of CALIFORNIA RIVERSIDE Control
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UNIVERSITY OF CALIFORNIA RIVERSIDE Control of Cyst Burden in the Brain During Chronic Toxoplasma gondii Infection A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Cell, Molecular, and Developmental Biology by John Philip Nance Jr. June 2012 Dissertation Committee: Dr. Emma H. Wilson, Chairperson Dr. Monica J. Carson Dr. Neal L. Schiller Copyright by John Philip Nance Jr. 2012 The Dissertation of John Philip Nance Jr. is approved: ______________________________________________________________ ______________________________________________________________ ______________________________________________________________ Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS My deepest appreciation and respect go to my advisor Dr. Emma Wilson for all of the direction, advice, and freedom to pursue this project over the last four years. She has encouraged me from the beginning to present my research to my peers at UCR and at various conferences and this has produced multiple collaborations that my research and development as a scientist have benefited from. I could not imagine a better mentor both personally and professionally and I will always be grateful to her for this training opportunity. I am thankful to Dr. Neal Schiller for providing me with my first rotation here at UCR and generously providing me with knowledge and advice as a committee member throughout my graduate career. I will always be grateful to Dr. Monica Carson for serving as a committee member and mentor to me for the past four years. Dr. Carson has constantly expected the best from me and I have greatly benefitted from her knowledge, honesty, advice, and humor. I wish to thank my labmates, Shahani Noor, Clement David, and Kathryn McGovern for all of the help, support, and fun over the years. One could not ask for a better group of colleagues to work with on a daily basis. None of this would have been possible without the support, encouragement, and love from my family. To Aimee, Jessica, and Emma: Words cannot express my gratitude for your sacrifice. I love and appreciate you more than you can imagine! iv DEDICATION To My Wife, Aimee v ABSTRACT OF THE DISSERTATION Control of Cyst Burden in the Brain During Chronic Toxoplasma gondii Infection by John Philip Nance Jr. Doctor of Philosophy, Graduate Program in Cell, Molecular and Developmental Biology University of California, Riverside, June 2012 Dr. Emma H. Wilson, Chairperson As an immune-privileged site, the brain represents a particularly challenging area when it comes to controlling infection and inflammation. Toxoplasma gondii provides us with a model of a chronic infection in the CNS that represents an efficient and balanced response from the healthy immune system. The effector functions of immune cells coupled with the ability to effectively migrate to and within the brain to sites of infection is essential in controlling the replication of the parasite in the brain. Impairment of this immune response results in parasite reactivation and fatal encephalitis, as seen in AIDS patients. Immune-compromised patients are currently subject to lifelong drug therapies that do not result in cyst clearance, presenting a constant danger of reactivation. Therefore, the identification of new mechanisms of cyst control is particularly important as it may lead to new and effective therapies. This dissertation examines two novel mechanisms of parasite control in the CNS during chronic infection. Previously published data has shown that a reticular network of fibers is present in Toxoplasma infected brains and absent in naïve brains which T cells migrate along. vi SPARC (secreted protein acidic and rich in cysteine) is a multifunctional glycoprotein that has been shown to be involved in the production and maintenance of similar fiber networks. Here, data will be presented demonstrating upregulation of SPARC in the brain following infection and association of this molecule with a seminal fibrous network in the CNS as well as with cysts and migrating cells. Furthermore, a requirement for SPARC in protective immunity and matrix assembly will be demonstrated in vivo, using infection studies with SPARC-null mice and live imaging in the brain. The exact mechanisms and cells involved in the clearance and control of cysts from the brain remain poorly defined. Data presented here will demonstrate the presence of a population of alternatively activated macrophages (AAMϕ) in the infected brain that maintain control of the cyst burden through the secretion of the chitolytic enzyme, acidic mammalian chitinase (AMCase). This mechanism is demonstrated in vitro by both chemical and genetic inhibition of the enzyme and the requirement for protective immunity is shown through infection of AMCase-null mice. These mechanisms of parasite control during chronic T. gondii infection represent two novel areas of study that provide hopeful therapeutic targets in combating this chronic infection. vii TABLE OF CONTENTS List of Figures.................................................................................................................... ix Chapter One Introduction 1.1 Background..................................................................................................2 1.2 Materials and Methods...............................................................................15 1.3 Figures and Legends ..................................................................................24 Chapter Two SPARC is Required for Efficient Cell Migration and Protective Immunity in the CNS During Chronic T. gondii Infection 2.1 Abstract......................................................................................................35 2.2 Introduction................................................................................................36 2.3 Results........................................................................................................40 2.4 Discussion..................................................................................................51 2.5 Figures and Legends ..................................................................................56 Chapter Three Macrophage-derived Acidic Mammalian Chitinase is Required to Control Cyst Burden in the Brain During Chronic T. gondii Infection 3.1 Abstract......................................................................................................67 3.2 Introduction................................................................................................68 3.3 Results........................................................................................................71 3.4 Discussion..................................................................................................85 3.5 Figures and Legends ..................................................................................90 Chapter Four Conclusions and Perspectives..................................................................108 Reference .........................................................................................................................112 viii LIST OF FIGURES Figure 1-1 Worldwide seroprevalence of the parasite Toxoplasma gondii.................25 Figure 1-2 The life cycle of T. gondii. ........................................................................27 Figure 1-3 The protective immune response to T. gondii. ........................................29 Figure 1-4 The multifaceted role of the matricellular protein SPARC.. .....................31 Figure 1-5 Macrophage phenotype and function. ......................................................33 Figure 2-1 Expression of SPARC in the CNS during chronic T. gondii infection......57 Figure 2-2 SPARC-null mice have no defect in cytokine production during acute T. gondii infection..................................................................................................................59 Figure 2-3 SPARC-null mice have no defect in immune cell migration to the brain during chronic infection.....................................................................................................61 Figure 2-4 SPARC-null mice have higher parasite burden in the brain and increased cellular accumulation in perivascular spaces. ...................................................................63 Figure 2-5 SPARC-null mice have compromised reticular fiber network in the brain during chronic T.gondii infection. .....................................................................................65 Figure 3-1 Expression of CXCR3 and its ligands during chronic T. gondii infection. ...........................................................................................................................................91 Figure 3-2 CXCR3 is required for efficient macrophage migration to the CNS during chronic infection.. ..............................................................................................................93 Figure 3-3 Alternatively activated macrophages in the brain during chronic T. gondii infection. ............................................................................................................................95 ix Figure 3-4 Macrophages produce active chitinase in the CNS in response to the presence of cysts. .............................................................................................................97 Figure 3-5 Chitinase expressing AAMΦ are in close association with cysts