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Homeostasis of myeloid cells in the CNS and their roles in neuroinflammatory disease Ajami, Bahareh 2011

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HOMEOSTASIS OF MYELOID CELLS IN THE CNS AND THEIR ROLES IN NEUROINFLAMMATORY DISEASE  by  BAHAREH AJAMI  B.SC., The University of Tehran, 1999 M.SC., The University of Sydney, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2011  © Bahareh Ajami, 2011  Abstract  A key regulator of central nervous system (CNS) inflammatory responses is a highly specialized subset of tissue macrophages that reside in the CNS parenchymal and perivascular spaces known as “microglia”. Microgliosis is a common response to multiple types of damage within the CNS and is commonly characterized by an increase in microglial cells. What remains elusive, however, is the origin of cells involved in this phenomenon and whether the increase in the number of cells is due to local expansion or recruitment of myeloid progenitors from the bloodstream.  Here, we investigated the origin of microglia using chimeric animals obtained by parabiosis. We found no evidence of circulating myeloid cells recruitment under healthy conditions and in denervation or CNS neurodegenerative disease suggesting that microglia can respond to CNS trauma and degeneration by expanding in situ independently of the contribution of blood-derived myeloid precursors. Furthermore, I investigated the extent to which blood-derived myeloid cells contribute to the microglia population in conditions where other circulating blood-borne cells have access to the CNS, such as in multiple sclerosis, an autoimmune disease of CNS and its murine model experimental autoimmune encephalitis (EAE).  ii  Using a novel approach to specifically replace circulating progenitors without affecting CNS-resident microglia, we found a strong correlation between monocyte infiltration and progression to the paralytic stage of EAE. Inhibition of chemokine receptor-dependent recruitment of monocytes to the CNS blocked EAE progression suggesting that these infiltrating cells are essential for pathogenesis. Finally, we found that although microglia can enter the cell cycle and return to quiescence following remission, recruited monocytes vanish, thus not ultimately contributing to the resident microglial pool.  These findings collectively demonstrate that microglia constitute a unique myeloid cell population that are capable of long-term self-renewal within the CNS, and can respond to CNS trauma and degeneration by expanding in situ independently  of  the  contribution  of  blood-derived  myeloid  precursors.  Furthermore, two distinct subsets of myelomonocytic cells with unique roles in neuroinflammation and disease progression were identified under conditions where the blood-brain barrier is damaged and blood-derived leukocytes have access to the CNS parenchyma.  iii  Preface  Chapter 2 includes data published in: “Local self-renewal can sustain CNS microglia maintenance and function throughout adult life”. Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., Rossi, F.M. (2007). Nature Neuroscience. Dec; 10 (12):1538–43. B. Ajami participated in experimental design, performed all of the experiments, created all the figures, analyzed the data and wrote the manuscript. J. Bennett edited the manuscript; C. Krieger helped in the analysis of ALS experiments and the editing of the manuscript; W. Tetzlaff performed facial axatomy surgeries, helped in the design of experiments as well as in the editing of the manuscript; FM. Rossi designed and interpreted experiments, and edited the manuscript. Chapter 3 includes data that is currently in press for publication: “Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool”. Ajami, B., Bennett, J., Krieger, C., McNagny, K.M., Rossi, F.M. Nature Neuroscience. 2011. Advance Online Publication (AOP) on Nature Neuroscience's website on 31 July 2011. DOI number: 10.1038/nn.2887. B. Ajami designed and performed all of the experiments, created all the figures, analyzed data and wrote the manuscript. J. Bennett conducted the EAE induction and edited the manuscript; C. Krieger and K.M. McNangny edited the manuscript; F.M. Rossi designed and interpreted experiments, and edited the manuscript. This work was approved by the University of British Columbia (UBC) Animal Care Committee under Certificate Numbers: A09–0364, A09–0900 and A06–1487.  iv  Table of Contents Abstract............................................................................................................ ii Preface ............................................................................................................ iv Table of Contents ............................................................................................ v List of Tables ................................................................................................ viii List of Figures ................................................................................................ ix List of Abbreviations...................................................................................... xi Acknowledgements....................................................................................... xv Dedication .................................................................................................... xvii Chapter 1: Introduction................................................................................... 1 1.1  Overview ............................................................................................... 1  1.2  Myeloid Cells ......................................................................................... 3  1.2.1 Ontogeny........................................................................................... 3 1.2.1.1 The embryonic journey of hematopoietic stem cells ................... 3 1.2.1.2 The embryonic journey of myeloid cells ...................................... 6 1.2.2 Myeloid cell development in adults .................................................. 12 1.2.3 Transcription factors in myeloid development ................................. 17 1.3  The Mononuclear Phagocyte System.................................................. 18  1.4  Microglia: Resident Myeloid Cells of the CNS ..................................... 25  1.4.1 Ontogeny......................................................................................... 25 1.4.2 The role of microglia during CNS development ............................... 30 1.4.3 Microglia in adults ........................................................................... 32 1.4.4 Microglia: friends or foe? ................................................................. 37 1.5  Blood–Derived Macrophages: Aliens from Another Planet.................. 42  1.6  Thesis Objectives ................................................................................ 51  Chapter 2: Local Self-Renewal Can Sustain CNS Microglia Maintenance and Function Throughout Adult Life ........................................................... 53 2.1 Introduction ............................................................................................ 53 2.2 Material and Methods ............................................................................ 57 2.2.1 Mice................................................................................................. 57 2.2.2 Generation of chimeric mice by parabiosis...................................... 57 2.2.3 Generation of chimeric mice by irradiation and transplant............... 58 v  2.2.4 Facial nerve axotomy surgery ......................................................... 58 2.2.5 Generation of chimeric mice by parabiosis/irradiation ..................... 59 2.2.6 Peripheral blood analysis ................................................................ 59 2.2.7 Muscle preparation and analysis ..................................................... 59 2.2.8 Animal harvest and tissue preparation ............................................ 60 2.2.9 Immunohistochemistry .................................................................... 60 2.3 Results ................................................................................................... 61 2.3.1 Microglia are sustained by local proliferation rather than recruitment of myeloid progenitors from the peripheral circulation in a healthy CNS .. 61 2.3.2 An increase in microglia in response to denervation or neurodegenerative disease is the result of local microglial proliferation rather than the entry of blood-derived myeloid cells into the CNS............ 66 2.3.3 Irradiation alone is not sufficient to trigger the entry of circulating myeloid cells into the parenchyma following CNS injury .......................... 79 2.4 Discussion ............................................................................................. 83 Chapter 3: Infiltrating Monocytes Trigger EAE Progression but Fail to Contribute to the Resident Microglia Pool .................................................. 88 3.1 Introduction ............................................................................................ 88 3.2 Material and Methods ............................................................................ 92 3.2.1 Mice................................................................................................. 92 3.2.2 Generation of chimeric mice by parabiosis/irradiation ..................... 93 3.2.3 Generation of chimeric mice by irradiation and transplant............... 93 3.2.4 Isolation of c-Kit+ Lin− Sca-1+ (KLS) cells ........................................ 94 3.2.5 Transplantation ............................................................................... 94 3.2.6 Peripheral blood analysis ................................................................ 94 3.2.7 Induction of autoimmune experimental encephalitis ........................ 95 3.2.8 Animal harvest and tissue preparation ............................................ 95 3.2.9 Immunofluorescence of tissue sections........................................... 95 3.2.10 Quantification and statistics........................................................... 96 3.3 Results ................................................................................................... 97 3.3.1 Presence of infiltrating myelomonocytes in parabiotic mice correlates with progression to severe EAE ............................................................... 97 3.3.2 Microglia and circulating inflammatory cells are recruited with different kinetics ..................................................................................... 119 3.3.3 Blocking inflammatory cell infiltration prevents EAE progression .. 126 vi  3.3.4 The progeny of infiltrating blood-derived monocytes does not contribute to the CNS-resident cell pool ................................................. 129 3.3.5 Transplantation of hematopoietic stem cells, but not of lineage committed progenitors, leads to long-term contribution to microglia ...... 131 3.4 Discussion ........................................................................................... 136 Chapter 4: Conclusion ................................................................................ 140 4.1 Microglia: Ever-Changing or Reliably Self-Renewing .......................... 140 4.2 Towards an Understanding of the Functional Differences of Myeloid Cells and Microglia ............................................................................................. 147 Bibliography ................................................................................................ 154  vii  List of Tables Table 2.1 BM-derived microglial engraftment in axotomized facial nucleus after irradiation/transplantation .................................................................................... 72 Table 2.2 BM-derived microglial engraftment in mSOD1 spinal cords after irradiation/transplantation .................................................................................... 78  Table 3.1 Infiltration of blood-derived inflammatory cells increased as the disease progress to the paralytic stages (Figures 3.3, 3.7, 3.10) ................................... 108  viii  List of Figures Figure 1.1 Multiple sites of hematopoietic stem cells development in embryo ...... 5 Figure 1.2 Development of myeloid cells in embryos .......................................... 11 Figure 1.3 Proposed model of hematopoiesis ..................................................... 16 Figure 1.4 Origin of monocytes in adult mice ...................................................... 24 Figure 1.5 Functional plasticity of microglia ........................................................ 35  Figure 2.1 Blood chimerism in parabiotic and lethally irradiated/transplanted mice ............................................................................................................................ 62 Figure 2.2 Blood chimerism in parabiotic mice and contribution of GFP cells to different blood lineages ....................................................................................... 63 Figure 2.3 Microglia, unlike peripheral macrophages, are not replenished by bone marrow-derived progenitors throughout adult life ................................................ 64 Figure 2.4 Circulating progenitors do not contribute to the microgliosis that is induced by facial nerve axotomy in parabiotic mice ............................................ 70 Figure 2.5 Circulating cells significantly contribute to microgliosis in the axotomized facial nucleus following irradiation/transplantation, and to macrophage infiltration following muscle damage in parabiotic mice .................. 71 Figure 2.6 Damage-induced recruitment of circulating myelomonocytic cells is efficient in peripheral tissues of parabiotic animals ............................................. 73 Figure 2.7 Circulating progenitors do not contribute to the microgliosis induced by ALS in the spinal cord of parabiotic mSOD1 transgenic mice ............................. 77 Figure 2.8 Irradiation is not sufficient to trigger the entry of blood-borne microglia progenitors into the CNS ..................................................................................... 81  Figure 3.1 Efficient entries of partner-derived T-lymphocytes, but not myelomonocytic cells in the spinal cords of EAE-induced parabionts ................. 99 Figure 3.2 Irradiation and separation of parabiotic mice leads to peripheral blood chimerism in the absence of donor cell entry into the CNS ............................... 103  ix  Figure 3.3 Monocytic infiltration correlates with progression to paralytic stages of EAE................................................................................................................... 106 Figure 3.4 Blood-derived inflammatory cells are recruited to the diseased CNS and their increased number correlates with the disease progression ............... 107 Figure 3.5 The number of blood-derived inflammatory cells found in spinal cord sections does not correlate with the time between EAE induction and tissue analysis ............................................................................................................. 110 Figure 3.6 CD4+ lymphocytes are found in the spinal cord at all scores of EAE .......................................................................................................................... 114 Figure 3.7 Blood-derived infiltrating cells retain monocyte characteristics during progression to paralytic disease........................................................................ 116 Figure 3.8 Phenotype of infiltrating myelomonocytic cells at different stages of disease progression .......................................................................................... 117 Figure 3.9 Differentiation of infiltrating inflammatory monocytes to macrophages after entry into the CNS correlates with disease progression rather than time elapsed from EAE induction .............................................................................. 118 Figure 3.10 Kinetics of microglia expansion and blood-derived monocyte infiltration in EAE............................................................................................... 122 Figure 3.11 Proliferation of resident microglia during disease progression ....... 123 Figure 3.12 Endogenous microglia activation precedes the entry of blood-derived inflammatory cells in the CNS of EAE-induced mice......................................... 124 Figure 3.13 Endogenous microglia undergo apoptosis between clinical score three and four of EAE ....................................................................................... 125 Figure 3.14 Blocking monocyte infiltration prevents EAE progression .............. 128 Figure 3.15 Blood-borne inflammatory cell infiltration is transient ..................... 130 Figure 3.16 Sorting strategy for the isolation of c-Kit+ Lin- Sca-1+ (KLS) cells from bone marrow ............................................................................................. 133 Figure 3.17 Uncommitted stem/progenitor cells, but not myelomonocyticcommitted hematopoietic progenitors contribute to resident microglia in irradiated/transplanted recipients ...................................................................... 135  x  List of Abbreviations  AD  Alzheimer’s Disease  AGM  Aorta-gonad Mesonephros  ALS  Amyotrophic Lateral Sclerosis  ATP  Adenosine Triphosphate  BBB  Blood-brain Barrier  BM  Bone marrow  CCL2  Chemokine (C-C motif) Ligand 2  CCR2  CC-chemokine Receptor 2  CD  Cluster of Differentiation  CLP  Common Lymphoid Progenitor  CMP  Common Myeloid Progenitor  CNS  Central Nervous System  DC  Dendritic Cells  DNs  Dopaminergic Neurons  xi  E  EAE  Embryonic Days  Experimental Autoimmune Encephalomyelitis  EDTA  Ethylenediaminetetraacetic Acid  FACS  Fluorescence-activated Cell Sorting  FBS  Fetal Bovine Serum  GFP  Green Fluorescent Protein  GCV  Ganciclovir  GCF  Granulocyte Colony-stimulating Factor  GM-CSF  Granulocyte-Macrophage Colony-Stimulating Factor  GMP  Granulocyte-Macrophage Progenitors  HSC  Hematopoietic Stem Sells  HSV-TK  iNOS  Herpes Virus Thymidine Kinase  Inducible NO synthase  KA  Kainic Acid  KLS  c-Kit +/Lineage- /Sca-1+  LT-HSCs  Long-term Hematopoietic Stem Cells xii  LPS  Lipopolysaccharide  MCP-1  Monocyte Chemoattractant Protein-1  M-CSF  Macrophage Colony-Stimulating Factor  MEP  Megakaryocyte-Erythroid Progenitors  MPS  Mononuclear Phagocyte System  MPTP  1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine  NPCs  Neural Progenitor Cells  NO  NOS2  Nitric Oxide  Nitric Oxide Synthas-2  PBS  Phosphate-buffered Saline  PNS  Peripheral Nerve System  PD  Parkinson Disease  ROI  Reactive Oxygen Intermediates  SNc  Substantia Nigra pars Compacta  ST-HSC  Short-term Repopulating Hematopoietic Stem Cells  xiii  pSp  TGF-β  Para-aortic Splanchnopleura  Transforming Growth Factor-beta  Th1  T helper 1  Th 17  T helper 17  TLRs  Toll-like Receptors  TNF–  UTP  Tumour Necrosis Factor–  Uridine Triphosphate  xiv  Acknowledgements First and foremost I want to thank my husband, Manni Bojnordi, for his unlimited support throughout my PhD, his great sacrifices and his constant help in every possible way from helping with statistical analysis to staying late hours in the laboratory during experiments and in the past three years taking care of our daughter, Artina. You have always been my biggest support.  I also wish to thank my parents and my sisters for their belief, encouragement and support. Your constant support made you always close to me despite being far away.  I especially thank my advisor, Dr. Fabio Rossi for his support, guidance and encouragement throughout these years.  I wish to thank my supervisory committee Dr. Keith Humphries and Dr. Wolfram Tetzlaff for their guidance.  I am especially deeply grateful to Dr. Tetzlaff for always being very encouraging, supportive and understanding. It felt like you were my second supervisor. Also, to the members of Dr. Tetzlaff’s laboratory: Ms. Clarrie Lam, Mr. Jason Plemel and Mr. Joseph Sparling for their help and warm welcome in their lab and support especially during my preparation for the comprehensive exam.  xv  I would also like to thank Mr. Chih-Kai Chang for his help with parabiosis surgery in past two years.  I especially want to thank Dr. Mike Long and Mrs. Leslie So Alfaro for their friendships as well as personal support during the past few years; you guys are great friends.  On a personal note, I would also like to thank our “ mommy group”, very special women who I got to know after the birth of my daughter Artina: Jocelyn Laurenciano, Jennifer King, Jennifer Elliot, Tehilla Couzins, Barbara Arenson, Laura Plant, Dr. Colleen Brenner and Dr. Kim Bridget. I am grateful to their support and friendship through all the ups and downs during the past few years and looking forward to everlasting friendship.  This research was possible due to personal scholarships received from CIHR and MSFHR.  xvi  Dedication To my husband for his unconditional love, patience and for helping my dream becoming a reality – without you this endeavour could have never even been imagined.  To my father who defined for me how to be a strong person and fight for what I believe is right.  To my mother who showed me I should never let my dreams stay only “a dream” and who never stopped pursuing her own dreams.  To my beautiful daughter, “Artina”: every smile you gave me made this journey easier.  To loving memory of my grandfather, “baabaaii”: you would have been so proud of me today.  To loving memory of my brave and strong grandmother,”nanahaji”: you are always in my mind in hardest moment of my life. I miss you.  Finally, this thesis is dedicated to all those who suffer from Multiple Sclerosis.  xvii  Chapter 1: Introduction  1.1 Overview  The vertebrate central nervous system (CNS) is a highly complex and dynamic system with a limited regenerative capacity. Two cell types comprise the complex architecture of CNS tissue: neurons and glia. It was Rudolf Virchow who first observed the latter cell type, terming them “glia”, from the Greek word for glue, in response to their cementing function of holding neurons together. Although Virchow accurately described them as connective tissue he largely dismissed them as distinct cells. 50 years later, the Spanish neuroscientist Santiago Ramon y Cajal, who is often regarded as the father of modern neuroanatomy, described neurons as the building blocks of the central nervous system and called them the “first element”. Using gold impregnation, Cajal importantly divided glia into two groups: astrocytes (the “second element”) and a third element which were cells with small, round nuclei. Subsequently, using silver carbonate impregnation, Del Rio-Hortega, a student of Cajal, refined the composition of the third element, proposing it includes two distinct populations: oligodendrocytes, which he proposed have an ectodermal origin the same as astrocytes; and microglia, which he described as migratory phagocytic cells of mesenchymal origin. This discovery was heavily disputed by Cajal. In fact, until the early 1990’s, the existence of microglia as a “single distinct cell” was disputed in a leading textbook in the field of neuropathology (Graeber, 2010). The past two decades  1  have witnessed the emergence of research on microglia biology and their function in health and disease. Microglia are now firmly established as the immune cells of the CNS. Similar to macrophages in other tissues, they are key players of the central nervous system's innate immune response. In reaction to CNS insults, microglia become activated in a similar way to peripheral tissue macrophages, which involves alteration of their morphology, phenotype and increase in their numbers. This increase in cell number has been attributed to blood-derived inflammatory monocytes that infiltrate the CNS and differentiate into macrophages that are indistinguishable from microglia. To complicate matters further, some studies propose that blood–derived macrophages have a beneficial role in CNS damage whereas others believe they have a destructive role.  Unfortunately, the lack of a specific marker distinguishing CNS-resident microglia and peripheral blood-derived macrophages, as well as the absence of an animal model devoid of microglia, have hindered and complicated microglia research. Currently, most studies rely on bone marrow chimeras produced by irradiating mice to kill the endogenous bone marrow cells and transplanting labelled bone marrow cells to distinguish between CNS-resident microglia and blood-derived macrophages. However, this tool suffers from the artefact of the irradiation on the blood-brain barrier and the transplantation of progenitors in the bloodstream, which are absent under normal conditions (Davoust et al., 2008).  2  The overall objective of this thesis, therefore, is to explore the origin of the cell responsible for the maintenance of microglia in adult life and in a subset of neurodegenerative diseases, as well as to discover the role of infiltrating bloodderived myeloid cells in inflammatory diseases of the CNS.  1.2 Myeloid Cells 1.2.1 Ontogeny  Myeloid cells, which encompass monocytes, macrophages and granulocytes are the key mediators of innate immunity and the inflammatory response. They arise from hematopoietic stem cells (HSC) during embryonic and adult life. Hematopoietic stem cells are derived from the ventral mesoderm layer of the embryo after gastrulation and are the endless reservoir of mature blood cells during embryonic and adult life (Murry and Keller, 2008).  1.2.1.1 The embryonic journey of hematopoietic stem cells  In vertebrates, blood cell development (hematopoiesis) is accomplished in several anatomical sites during embryogenesis (Dzierzak and Medvinsky, 1995; Dzierzak and Speck, 2008; Galloway and Zon, 2003). The initial wave of blood production, which is called primitive or embryonic hematopoiesis, is first detected in the yolk sac of a mouse embryo at embryonic days (E) 7–8.5. The  3  hematopoietic progenitors in the yolk sac are transient and fail to reconstitute the hematopoietic system in an adult organism (Cumano et al., 2001).  Definitive or adult hematopoiesis, which is the second wave of blood production, occurs sequentially in an area surrounding the dorsal aorta, termed the aortagonad mesonephros (AGM) region from E10.5, and later on at E11.5, in the fetal liver. Finally, HSCs colonize the bone marrow around the time of birth (Mikkola and Orkin, 2006).The definitive adult stem cell refers to a stem cell that gives rise to the true adult hematopoietic system.  The relationship between primitive and definitive hematopoiesis is still debated. Initially, in 1970, it was suggested that once circulation is established, HSCs migrate from the yolk sac to the fetal liver, and thereafter, to the bone marrow, identifying the yolk sac as the source of the hematopoietic system (Moore and Metcalf, 1970). However, chimera studies in amphibians and avian species later suggested that the yolk sac and AGM are independent (Dieterlen-Lievre et al., 1988).  Currently, the AGM is being widely viewed as the principal site responsible for the establishment of definitive hematopoiesis (Cumano et al., 1996; Godin and Cumano, 2002; Medvinsky and Dzierzak, 1996; Muller et al., 1994). However, a recent study has challenged the dogma that the yolk sac lacks definitive hematopoietic stem cells, proposing that adult hematopoiesis originates  4  exclusively from the yolk sac and that the AGM region functions as an intermediate site for developing HSCs prior to their colonization of the liver (Samokhvalov et al., 2007) (Figure 1.1).  Figure 1.1 Multiple sites of hematopoietic stem cells development in embryo Disagreement exists about the relationship between primitive and definitive hematopoiesis. Two theories regarding this relationship exist: 1. “A” Suggests that embryonic or primitive hematopoiesis is independent from definitive or adult hematopoietic system development. In this model, embryonic hematopoiesis starts in the yolk sac. However, it is a transient event and does not contribute to the hematopoietic system in an adult organism. The definitive (adult) hematopoiesis starts independently in AGM and later on colonizes the fetal liver. Definitive hematopoietic stem cells seed the bone marrow around the time of birth and give rise to life-long hematopoiesis. 2. “B” Suggests that adult hematopoiesis originates exclusively from the yolk sac and the AGM region is an intermediate site for developing HSCs prior to their colonization of the liver and bone marrow.  5  1.2.1.2 The embryonic journey of myeloid cells  The earliest myeloid progenitors have been detected in the yolk sac during the initial wave of hematopoiesis before circulation has been established (Ferkowicz et al., 2003; Gordon et al., 1992; Palis et al., 1999). Explant cultures of mice also reveal cells with the potential to become myeloid progenitors in Para-aortic splanchnopleura (pSp), which is the prospective AGM region prior to the establishment of circulation (Cumano et al., 1996). Notably, at this stage of embryonic development myeloid progenitors derived from the yolk sac are limited to generation of short-term myeloid engrafting cells whereas cells from the pSpAGM, at this time, have the ability to generate long-term lymphomyeloid cells which can repopulate Rag2γc−/− immunocompromised recipients (Cumano et al., 2001; Cumano and Godin, 2001). Such cells appear in the yolk sac once the circulation is established. This suggests that AGM is the first anatomical site of de novo production for definitive myeloid progenitors, which seed the yolk sac and the placenta at a later time via blood circulation (Ottersbach and Dzierzak, 2005).  The final anatomic site that myeloid cells appear in during embryonic development is the fetal liver at mid-gestation. Around this time, definitive hematopoietic cells that have been produced in other tissues colonize the liver, which provides the microenvironment for long-term HSCs (LT-HSCs) to differentiate into common myeloid progenitors and common lymphoid progenitors  6  (Cumano and Godin, 2007; Houssaint, 1981; Johnson and Moore, 1975; Kumaravelu et al., 2002; Murayama et al., 2006). For the remainder of the fetal development period, the liver acts as the primary hematopoietic organ and the main site of HSC expansion and differentiation (Zhang and Rodaway, 2007).  Electron microscopy observations and immunostaining studies have revealed morphological and phenotypical details of myeloid lineage differentiation through their developmental journey in embryo.  Primitive myeloid cells that first appear in the blood islands of the mouse yolk sac exhibit characteristics of immature macrophages. These primitive myeloid cells are small and circular shaped with a large euchromatic nucleus and poorly developed microvilli and have a high proliferative capacity (Takahashi et al., 1989).  Significantly, it has been suggested that myeloid precursors in the yolk sac differentiate to fetal macrophages without following the intermediate monocyte pathway as is the case in adult macrophage development (Takahashi et al., 1989). Electron microscopy and culture studies by Naito and colleagues demonstrated the lack of peroxide activity in primitive macrophages in the yolk sac, a characteristic of immature macrophages and monocytes in liver and bone marrow (Naito et al., 1989; Takahashi and Naito, 1993).  7  Moreover, a significant body of literature, which used myeloid lineage specific markers in order to study different stages of macrophage maturation, supports the theory that primitive macrophages in the yolk sac bypass monocyte pathways. Primitive macrophages positive for the monoclonal antibody ERMP12, the marker for granulocyte-macrophage colony-stimulating factor (GMCSF) derived macrophages, and the monoclonal antibody ER-MP58, the marker for macrophage colony-stimulating factor (M-CSF) derived macrophages, were detected in the yolk sac prior to the establishment of circulation; however, no ERMP20 (Ly6C) positive cells, the surface marker for monocytes, were observed at this stage (Morioka et al., 1994). Additionally, the two S100 proteins, S100A8 (MRP-8) and S100A9 (MRP-14), whose expression is associated with monocytes and granulocytes in the process of macrophage differentiation from bone marrow precursors in vitro (Goebeler et al., 1993) and are potential indicators of myelopoiesis in the liver were not detected in cells originated from the yolk sac (Lagasse and Weissman, 1992; Lichanska et al., 1999; Passey et al., 1999a; Passey et al., 1999b).  On embryonic day (E) 10, these yolk sac primitive macrophages mature to fetal adult type macrophages with the development of intracellular organelles. After the yolk sac is connected to the cardiac system of the embryo via vatelline vessels, these primitive macrophages leave the yolk sac and colonize the liver and various other organs forming tissue-resident macrophage subpopulations (Takahashi et al., 1996).  8  The development of monocytes starts in the liver around embryonic day (E) 14 and their number increase each day. Later on around day 17, monocytes appear in the blood circulation for the first time. Interestingly, the number of circulation primitive macrophages subsides around the same time. Macrophages developed through intermediate monocyte pathways emerge in various fetal tissues around embryonic day (E) 18 and contribute to the tissue-resident macrophage population (Higashi et al., 1992; Izumi et al., 1990; Morioka et al., 1994).  The most convincing data demonstrating the existence of a monocyte independent pathway for primitive yolk sac-derived macrophages came from mice with a deficiency in the ets family transcription factor PU.1. PU.1 is a myeloid restricted transcription factor. Studies of mice carrying targeted disruptions of the gene encoding PU.1 suggest that PU.1 plays a pivotal role in hematopoiesis and a major role in myeloid lineage development (McKercher et al., 1996; Scott et al., 1994). Lethal PU.1-/- embryos suffer from anaemia and lack monocytes and most of the mature tissue macrophages in some organs such as the liver, spleen, thymus and lungs (McKercher et al., 1996; Scott et al., 1994).  A subsequent study on the PU.1-/- mice at early stages of development indicated no expression of this gene in primitive yolk sac macrophages until embryonic day (E) 11 when the hematopoiesis has already started in the liver. Moreover, the appearance of yolk sac-derived macrophages was unaffected by a mutation in  9  the PU.1 gene, and yolk sac-derived PU.1 independent phagocytes were maintained through embryonic development (Lichanska et al., 1999).  Based on these data, the following two-step scheme for the establishment of a myeloid population in developing embryo can be proposed. The first wave, which is identified as primitive macrophages, originates from precursors that appear in the yolk sac prior to the establishment of the cardiovascular system. These cells are PU.1 independent and do not develop through classical monocyte pathways. The contribution of these cells to tissue macrophages remains an open question. The second wave, which is identified as fetal or definitive macrophages, arises from multipotent hematopoietic cells during mid-late gestation. It develops through monocyte pathways and requires PU.1 for differentiation and maturation. It comprises a substantial population of macrophages in most of the adult and embryonic tissues (Figure 1.2).  10  Figure 1.2 Development of myeloid cells in embryos Two-step scheme of myeloid cells development in embryos: Step one: First, myeloid precursors are detected in the yolk sac. These cells develop to fetal macrophages without following the intermediate monocyte pathway. Step two: Later on in mid-late gestation, hematopoietic stem cells are developed to monocytes in the fetal liver. Monocytes leave the liver, circulate the blood and seed various organs where they develop to tissue resident macrophages.  11  1.2.2 Myeloid cell development in adults  Myeloid cells in adults arise from rare hematopoietic stem cells residing in the bone marrow of vertebrates (Orkin, 2000). In contrast to highly proliferative fetal hematopoietic stem cells, adult hematopoietic stem cells in the bone marrow are predominantly quiescent (Bowie et al., 2006; Ogawa et al., 2001; Passegue et al., 2005).  Adult hematopoietic stem cells have the ability to maintain their pool during the lifetime of an organism through a process of self-renewal as well as to lose their self-renewal capacity and differentiate into progenitors that eventually give rise to committed precursors of various lineages via several commitment steps (Rathinam et al., 2008).  Weissman and colleagues’ classical model of hematopoiesis suggests that the multipotent HSCs give rise to common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) (Akashi et al., 2000). The common lymphoid progenitors (CLPs) develop to committed progenitors that eventually give rise to T and B-lymphocytes. Common myeloid progenitors (CMPs) further develop to megakaryocyte-erythroid  progenitors  (MEP)  and  granulocyte-macrophage  progenitors (GMP). Erythrocyte and megakaryocytes mature from committed progenitors arising from MEP whereas GMP are the source of committed  12  progenitors that give rise to monocytes, mast cells, neutrophils and eosinophils (Reya et al., 2001) (Figure 1.3).  The mechanisms that delineate how the progeny of multipotent hematopoietic stem cells adopt one fate as opposed to another are extremely convoluted and have been a fundamental question in stem cell biology. Two hypotheses have evolved in relation to the mechanisms that determine the fate of hematopoietic stem cells during hematopoietic lineage development.  According to the stochastic theory, lineage commitment is the result of a cellautonomous or internally driven program. In contrast, instructive theory postulates that external cues such as growth factors drive the cells toward a particular fate.  In support of the stochastic model is the observation that individual hematopoietic stem cells and multipotent progenitors co-express a variety of lineage-specific transcription factors at low level (Miyamoto et al., 2002). These data suggest that lineage development is the consequence of stochastic fluctuation of transcription factors resulting in the selective activation and silencing of a set of genes (Akashi et al., 1997; Enver and Greaves, 1998; Fairbairn et al., 1993; Maraskovsky et al., 1997; Papayannopoulou et al., 2000; Suda et al., 1983; Ye et al., 2003) .  13  In favour of the instructive model, in vitro culture systems demonstrated that bipotential Granulocyte–macrophage colony forming cells (GM-CFCs) could differentiate towards macrophages when cultured with a macrophage colony stimulating factor (M-CSF) (Metcalf and Burgess, 1982; Metcalf et al., 1998).  Moreover, the ectopic expression of cytokines receptors in common lymphoid progenitors (CLP), which exclusively give rise to T and B-lymphocytes and natural killer cells under normal conditions, have demonstrated that when these cells were engineered to ectopically express L-2Rβ and cultured on bone marrow stroma in the presence of IL-2, they generated granulocytes and macrophages instead of only lymphoid cells (Borzillo et al., 1990; Kondo et al., 2000; Kondo and Weissman, 2000; Metcalf et al., 1998).  Taken together, these findings support the instructive theory as the model of lineage determinations. However, in vivo approaches using mice genetically deficient for cytokines or their receptors have provided strong evidence that lineage determination is not orchestrated by the cell extrinsic signals. For example, mice lacking GM-CSF have normal production of myeloid progenitors in their bone marrow (Stanley et al., 1994), and mice lacking granulocyte colonystimulating factor (G-CSF) as well as mice mutant for G-CSF receptor demonstrate a minor reduction in the number of myeloid progenitors in the bone marrow (Lieschke et al., 1994). Impaired terminal differentiation of granulocytes and increased apoptosis in neutrophil were the only significant phenotypes  14  detected as a result of mutation in the G-CSF receptor (Liu et al., 1996). The prevalent theory emerging from these data is that the lineage determination in the hematopoietic system is mainly a stochastic event; however, as the cells become more lineage restricted, extrinsic signals play an important role in supporting the survival, proliferation and maturation of committed cells (Lin et al., 1996; Liu et al., 1996; Semerad et al., 2002; Tenen et al., 1997; Wu et al., 1995).  Strikingly, however, a recent study has for the first time convincingly demonstrated that cytokines could instruct the progenitors fate (Sarrazin et al., 2009). Using knock-out mice for MafB, a highly expressed transcription factor in mature monocytes and macrophages, in a series of in vitro and in vivo experiments,  Sarrazin  and  colleagues  demonstrated  that  the  MafB-/-  hematopoietic stem cells have a myeloid progenitor repopulation advantage compared to wild-type hematopoietic stem cells. Furthermore, in MaFB-deficient hematopoietic stem cells increased expression of PU.1, the master transcription factor required for myeloid lineage differentiation was observed compared with wild-type hematopoietic stem cells after a short incubation with M-CSF. These data collectively suggest that the transcription factor MafB selectively inhibits the instructive role of M-CSF cytokine in myeloid lineage determination.  This new study has significantly advanced the long-lasting debate surrounding the permissive versus instructive role of cytokines on lineage commitment,  15  suggesting that some cytokines can, given a certain window of opportunity, “instruct” hematopoietic differentiation.  Figure 1.3 Proposed model of hematopoiesis Multipotent, self-renewing, long-term repopulating hematopoietic stem cells (LTHSC) first give rise to transiently reconstituting, short-term hematopoietic stem cells (ST-HSC). ST-HSCs in turn form common myeloid progenitors (CMP) and common lymphoid progenitors (CLP). CLP are the source of committed progenitors that eventually give rise to T and B lymphocytes. CMP further give rise to megakaryocyte-erythroid progenitors (MEP) and granulocyte-macrophage progenitors (GMP). MEP are the source of committed progenitors that eventually give rise to erythrocytes and megakaryocytes whereas GMP are the source of committed progenitors that eventually give rise to mast-cells, neutrophils, eosinophils and monocytes.  16  1.2.3 Transcription factors in myeloid development  The formation of myeloid cells is orchestrated by several transcription factors. PU.1 is the key transcription factor that induces myeloid commitment in immature multipotent progenitor cells and is required for generation of CMP in early myelopoiesis (Iwasaki and Akashi, 2007).  The antagonistic interaction between the transcription factors PU.1 and GATA-1 determines the myeloid versus erythroid fate from CMP along the myeloid development pathway. GATA-1 is a zinc finger transcription factor that is essential for development of the erythroid cells and megakaryocytes. Inhibition of GATA-1 by PU.1 via protein-protein interaction specifies the GMP cell fate (Huang et al., 2007; Nerlov and Graf, 1998; Rekhtman et al., 1999; Zhang et al., 1999). C/EBPα is another transcription factor critical for production of GMP from CMP (Radomska et al., 1998; Zhang et al., 2004). Interestingly, at the bipotent GMP stage, the expression level of PU.1 compared to C/EBPα is critical for monocyte over granulocyte fate. High levels of PU.1 expression by antagonizing C/EBPα favour the monocyte differentiation whereas low PU.1 levels support granulocyte production (Dahl and Simon, 2003; Dahl et al., 2003). Finally, MafB and c-Maf are the two master transcription factors required to drive monocyte fate in myeloid progenitors (Hegde et al., 1999; Kelly et al., 2000).  17  1.3 The Mononuclear Phagocyte System  The mononuclear phagocyte system (MPS), as described by Van Furth and colleagues, composes one of the major cells in the cellular arm of the innate immune system in most species.  Innate immunity is the first line of defence that responds quickly against any pathogens and tissue injuries. MPS encompasses the non-granulocyte population of myeloid cells, which includes circulating blood monocytes, tissue macrophages and dendritic cells (DC) in the body (Gordon, 1999; Steinman and Inaba, 1999). The twentieth century is marked by revolutionary discoveries regarding the development and differentiation of monocytes. A landmark study by Van Furth and Cohen, for example, using radioisotope labelling of blood cells, established monocytes as the precursors for tissue macrophages (van Furth and Cohn, 1968).  Monocytes constitute 10% of peripheral leukocytes in humans and 4% of peripheral blood leukocytes in mice under steady-state conditions (Rugh and Somogyi, 1969; Ziegler-Heitbrock, 2000). A clonogenic bone marrow precursor has recently been discovered downstream of the GMP, termed macrophage and dendritic cell progenitors (MDP) (Fogg et al., 2006). These biopotent progenitors differentiate into monocytes and common DC precursors (CDPs) but not granulocytes, defining them as direct precursors of monocytes in bone marrow.  18  Monocytes progress through a series of maturation stages defined by distinct morphological changes prior to their release in peripheral blood. MDP progenitors in the bone marrow give rise to monoblast, which mature to promonocytes and afterwards develop to monocytes, which are subsequently released to the peripheral blood circulation (Goasguen et al., 2009; Hume, 2008).  In the peripheral blood of mice, circulating monocytes are a population of nondividing cells with a short half-life between 24 and 60 hours (van Furth, 1989). Murine monocytes are distinguished by expression of the M-CSF receptor (CD115), CD11b and F4/80 antigens (Gordon and Taylor, 2005). Furthermore, all monocytes express the chemokine receptor CX3CR1, also called the fractalkine receptor, which binds to the chemokine CX3CL1.  Monocytes are a heterogeneous cell population in both humans and mice. Human blood monocytes have been divided into two subsets: CD14hiCD16- cells, which are called classic monocytes, and CD14+CD16+ that resemble tissue macrophages (Passlick et al., 1989). The construction of a mouse strain that has been engineered to harbour a targeted replacement of the gene encoding CX3CR1 with a copy of the gene encoding for “green fluorescent protein” (GFP) made it possible to discover the counterparts of human monocyte subsets in mice.  Murine  monocytes  are  composed  of  two  functionally  distinct  subpopulations with two different phenotypes. Their phenotypes have been distinguished based on the expression of CCR2, the receptor for monocyte  19  chemoattractant protein-1 (MCP-1), Ly6C, which is one of the two epitopes of myeloid differentiation antigen (Gr-1), and the level of expression for CX3CR1 based on GFP expression.  The Ly6C+CCR+CX3CR1low subpopulation is termed “inflammatory” monocytes. They correspond to the CD14hiCD16- classic monocytes in humans, which are also positive for CCR2 and express a low level of CX3CR1. The other major subset of monocytes (Ly6C–CCR–CX3CR1high) is known as the “resident” population, which corresponds to the CD14+CD16+ population in humans. The “inflammatory” population is short lived with a half-life of 24 hours. After adoptive transfer, this population is rapidly lost in healthy recipients and is not detected in any tissues. In contrast, following infection, this population is selectively recruited from the blood to inflamed tissues and lymph nodes and differentiates into macrophages and dendritic cells producing high levels of TNF-α and IL-1 during infection or tissue damage (Palframan et al., 2001; Serbina et al., 2008; Serbina and Pamer, 2006).  The “resident” monocytes are smaller and less granular (Geissmann et al., 2003; Sunderkotter et al., 2004) and have a longer half-life. Adaptive transfer experiments revealed that they home to various tissues under healthy conditions, therefore identifying this population as responsible for the replenishment of tissue-resident macrophages under homeostatic conditions. The definition of Ly6ClowCCR–CX3CR1high monocytes as the “resident” population against the  20  Ly6ChighCCR+CX3CR1low population as the “inflammatory” monocytes was put in doubt by recent intravital microscopy observations (Auffray et al., 2007). These observations revealed that under steady-state conditions, “resident” monocytes (Ly6ClowCCR–CX3CR1high) patrolled the endothelial surface up to several hours, with extravasation to peripheral tissues being a rare event in the absence of inflammation. Nevertheless, during infection with Listeria monocytogenes, intravital microscopy analyses show that “resident” monocytes (Ly6ClowCCR– CX3CR1high) invade the damaged site within an hour and are the main monocyte population for up to eight hours post infection preceding the “inflammatory” monocytes. During the initial phase of inflammation “resident” monocytes are the producers of TNFα, the major cytokine during inflammation. Moreover, gene expression analysis of “resident” monocytes, two hours after infection, revealed that these cells up-regulated the genes coding for inflammatory cytokines, lysozyme, complement, scavenger receptors and pattern recognition receptors such as TLRs and IgFc receptors. However, this inflammatory response is transient, and eight hours after the infection, Ly6ChighCCR+CX3CR1low cells or “inflammatory” monocytes are the main monocyte population in the site of infection responsible for production of inflammatory cytokines. These data suggest that, in response to inflammation, “resident” monocytes (Ly6ClowCCR– CX3CR1high) are actually responsible for the initial inflammatory reaction, which is followed by a secondary cascade of “inflammatory” monocytes.  21  A major body of work has attempted to address the developmental relationship between the resident and inflammatory monocyte subsets. Following in vivo depletion of all blood monocytes after administration of toxic liposome, Sunderkotter and colleagues were the first to study repopulation kinetics of both monocyte subsets. After depletion, Ly6Chigh monocytes were the first monocyte subset that repopulated the blood, followed three days later with the appearance of Ly6Clow monocytes. In addition, they identified a rare population of monocytes with an intermediate level of Ly6C expression. This study suggested that Ly6Chigh inflammatory monocytes act as precursors for resident monocytes via an intermediate population within the blood circulation. Furthermore, adoptive transfer experiments also confirmed that a resident monocyte subset develop from the Ly6Chigh population (Yrlid et al., 2006).  Intrabone cavity transfer of MDPs has also demonstrated the developmental relationship between the two different subsets of monocytes. Within bone marrow, MDPs differentiate into Ly-6Chigh before entering the blood. Following their egress from bone marrow to blood, they patrol the circulating blood for inflammatory stimuli for a short period of time. In the absence of any inflammation, the Ly-6Chigh subset shuttle back to the bone marrow where they convert to the Ly-6C  low  population prior to re-entering the blood (Varol et al.,  2007).  22  What is not clear at this point, however, is whether the MDPs can give rise to the Ly-6Clow population by bypassing the inflammatory monocyte subset. Antibodymediated depletion of Ly6Chigh monocytes over a prolonged period of two weeks, using intravenous injections of the RB6-8C5 antibody, did not affect the generation of a Ly-6Clow monocyte population, thus questioning inflammatory monocytes as the true precursors of the “resident” monocyte subset (Auffray et al., 2009) (Figure 1.4 ).  Monocytes migrate into peripheral tissues to contribute to the inflammatory process and to replenish the resident tissue macrophages. They are considered accessory cells that link innate and adaptive immune responses through antigen presentation to T cells (Auffray et al., 2009; Geissmann et al., 2003). Inflammation is an adaptive response to infection or tissue injury in order to restore tissue integrity. The inflammatory response involves activation of tissueresident macrophages as the first line of defence and subsequent recruitment of leukocytes including inflammatory monocytes from the blood. Neuroinflammation refers to the inflammatory response in the central nervous system to a wide variety  of  stimuli,  including  infectious  diseases,  autoimmune  or  neurodegenerative disorders, peripheral nerve damage or stress.  In the next section, I will shift focus to the myeloid cells involved in the CNS immune response.  23  Figure 1.4 Origin of monocytes in adult mice Hematopoietic stem cells (HSCs) in the bone marrow differentiate to common myeloid (CMP) and common lymphoid (CLP) precursors. CMPs give rise to granulocyte-macrophage progenitors (GMP). Macrophage and dendritic cell progenitors (MDPs) have recently been discovered as direct precursors of monocytes in bone marrow, downstream of GMPs, which differentiate into monocytes and common DC precursors (CDPs) but not granulocytes. MDPs give rise to monocytes. Two monocyte subsets, Ly- 6Chigh and Ly-6Clow, leave the bone marrow to enter the blood circulation. The Ly6Chigh population is termed “inflammatory” monocytes. Following infection, Ly6Chigh population is recruited from the blood to inflamed tissues and differentiates into macrophages. Ly6Clow is known as the “resident” monocytes. Under steady-state conditions, Ly6Clow patrol the endothelial surface and are responsible for the replenishment of tissueresident macrophages. The developmental relationship between the resident and inflammatory monocyte is not clear. It has been suggested MDPs differentiate into Ly-6Chigh before entering the blood. In the absence of any inflammation, the Ly-6Chigh subset shuttle back to the bone marrow where they convert to the Ly6C low population prior to re-entering the blood.  24  1.4 Microglia: Resident Myeloid Cells of the CNS  Microglia comprise 10% of the cells in the central nervous system. The study of microglia has been the centre of controversy for over a century. Their function, ontogeny, and even their very existence, have been debated extensively since the original descriptions appeared. While their existence is currently well accepted, disagreements regarding their origin, role and homeostasis during adult life continues. Several camps have evolved around each of these issues, making the field of microglia biology a particularly dynamic subject. In the following sections, I will examine these topics by presenting the controversial arguments surrounding each issue.  1.4.1 Ontogeny  Two conflicting schools of thought exist concerning the possible developmental origin of microglia. One hypothesis states that microglial precursors are derived from the neuroectodermal layer of the embryo similar to neurons, astrocytes and oligodendrocytes. The other hypothesis proposes that microglia are mesodermal in origin, developing outside the nervous tissue and subsequently invading the central nervous system during embryonic development. The lack of a specific cellular marker as well as the similar morphology of microglia and blood-derived macrophages had made it difficult to establish a conclusive theory regarding the origin of microglia. In 1932, Del Rio-Hortega, the “father of microglia”, postulated  25  that microglia originate from the migration of "embryonic corpuscles" from the pia mater, indicating that microglia had mesenchymal origins. Furthermore, he went on to propose blood monocytes as the exact progenitors for microglia based on their phagocytic ability. This initial observation has since been supported by a series of studies. Utilizing cytochemical and silver impregnation methods, Boya and colleagues demonstrated that in neonatal rats microglia derived from round acid phosphatase positive cells in the meninges, which then invaded the CNS parenchyma and transformed into ramified microglia (Boya et al., 1979). Furthermore, staining with isolectin B4 of Griffonia simplicifolia (GSA I-B4), a specific marker for macrophages, suggested that microglia have mesodermal origins similar to other tissue-resident macrophages (Boya et al., 1991; Boya et al., 1987; Streit and Kreutzberg, 1987).  The precursor cells that give rise to microglia have also been debated even by those scientists who agreed on the mesenchymal origin of these cells, some arguing that microglia originate from blood-derived monocytes and others arguing that microglia originate from primitive macrophages located in the yolk sac. Del Rio-Hortega originally described microglia as blood-derived monocytes. An electron microscopy study of perinatal rats identified amoeboid microglia similar to blood monocytes in the brain corpus callosum (Ling and Tan, 1974), thus supporting Del Rio-Hortega’s theory. Furthermore, radioautographic studies confirmed 3H-thymidine labelled amoeboid microglia resembling monocytes in the corpus callosum of rat brains around the time of birth, which subsequently  26  evolved to ramified microglia (Imamoto and Leblond, 1978). Using a cell tracking method, Ling and colleagues were the first group to demonstrate that monocytes enter into the developing brain and differentiate into microglia. Colloidal carbon particles were injected into newborn rats as well as adults. Carbon particles were observed in the amoeboid microglia cells in all newborn but not in adult animals. Since they did not observe any carbon particles in ventricular spaces, it was suggested that the amoeboid microglia in the developing brain of newborn rats is derived from carbon particles labelled monocytes that have entered the central nervous system and the particles have not been picked up locally (Ling, 1980).  These observations were followed by immunohistochemical studies using antibodies. Seminal work by Perry convincingly identified microglia labelled with the macrophage specific antigen F4/80 in adult and developing mouse brains (Perry et al., 1985). The F4/80 positive cells were closely associated with the blood vessels prior to their appearance in the brain parenchyma, leading to the speculation that the amoeboid microglia in developing brains originate from blood monocytes that primarily enter the white matter and, as time progresses, disperse through the white and grey matter developing a ramified morphology. Antibodies that mark monocytes were used in different species to confirm these findings (Cuadros et al., 1992; Ling et al., 1990; Paulus et al., 1992; Penfold et al., 1991; Robinson et al., 1986). However, microglia appear in the developing mouse neuroepithelium as early as embryonic day (E) 8 prior to both the establishment of vascularization in embryonic day (E) 9.5 and the development  27  of monocytes in embryonic days (E) 10–11 (Takahashi et al., 1989). This suggests that monocytes cannot, in fact, be regarded as the direct precursors for microglia during development.  A notable approach using chick-quail chimeras demonstrated that microglia are derived from primitive macrophage progenitors located in the yolk sac rather than circulating monocytes (Cuadros et al., 1993). Chimeras composed of chicken intraembryonic compartments and quail yolk sacs were analyzed before vascular connections had been established. Amoeboid microglia, identified by the QH1 antibody (Pardanaud et al., 1996), which recognizes quail hematopoietic cells, were found throughout the developing CNS suggesting they originated from the quail yolk sac (Cuadros et al., 1993; Cuadros et al., 1992). Serial sections taken at one-day intervals throughout development reveal that microglia precursors first enter the CNS from the meninges, by traversing the pial surface (Cuadros et al., 1993). After entering the CNS, microglia precursors first migrate parallel to the CNS surface, a process referred to as tangential migration. Then during a later phase known as radial migration, the cells gain access to deeper parts of the central nervous system (Cuadros and Navascues, 1998).  Alliot and colleagues supported this hypothesis in mice in vitro when they demonstrated the existence of microglia precursors in cell cultures derived from neural folds of eight-day old embryos which lack vascularization as this time, therefore dismissing invading monocytes as the precursors for microglia (Alliot et  28  al., 1999). In vivo experiments using lectin histochemistry (GSA I-B4) in the developing mouse brain confirmed that at embryonic day (E) 8, lectin labelled cells that were detected in the yolk sac at embryonic day (E) 7 start to penetrate the neuroepithelium and enter the mouse brain (Kaur et al., 2001).  In contrast to the mesodermal origin of microglia, in the early 1990’s Fedoroff and colleagues put forward the idea that microglia are derived from the neuroectoderm layer. Immunohistochemical studies from this group and others demonstrated that monoclonal antibodies such as lipocortin-1 labels both microglia and neuroepithelial cells thereby suggesting that they share the same origin (Fedoroff et al., 1997; McKanna, 1993a, b; Wolswijk, 1995). Furthermore, they reported the appearance of microglia in neuroepithelial cell cultures where microglia precursors expressing the Mac-1 antigen were eliminated prior to the cell culture (Hao et al., 1991). However, a caveat of this study is that based on Alliot’s findings, microglia acquire the Mac-1 epitope a few days later in culture, questioning the purity of the cell cultures that were used in these experiments (Alliot et al., 1991).  Finally, an elegant study in 2010, using a sophisticated lineage tracing technique, ended the eighty-year debate over the origin of microglia. In this study, the authors used a transgenic mouse line with a tamoxifen-inducible Cre recombinase under the control of the transcription factor Runx1, which is expressed in yolk sac myeloid progenitors in early embryonic life. They induced  29  the Cre recombinase activity via the injection of tamoxifen into pregnant females between days 7.25 and 7.50 after conception, that is, within a time frame where primitive hematopoiesis is restricted to the yolk sac. These experiments conclusively show that adult microglia are predominantly derived from primitive myeloid progenitors which are present in the yolk sac prior to embryonic day (E) 7.5 and invade the embryo through blood vessels between embryonic days (E) 8.5 and 9.5 (Ginhoux et al., 2010). Interestingly, they also demonstrated that unlike monocytes, microglia and yolk sac macrophages depend on colonystimulating factor-1 receptor (CSF-1R) but not its ligand CSF-1 for their development. Taken together, recent evidence strongly supports a primitive yolk sac myeloid progenitors’ origin of microglia.  1.4.2 The role of microglia during CNS development  Few studies have endeavoured to understand the function of microglia during CNS development. Several important activities, however, have been accredited to microglia during early embryonic life.  Phagocytosis in one of the major and the most well described roles of microglia in embryos (Streit, 2001). Numerous cells undergo apoptosis during the development of the nervous system due simply to an excess generation of cells (de la Rosa and de Pablo, 2000; Egensperger et al., 1996; Ferrer et al., 1990; Oppenheim et al., 1999). Microglia remove the apoptotic cells without generating  30  inflammation (Hume et al., 1983; Perry et al., 1985; Streit, 2001). Histological studies revealed that a single microglial cell is sufficient to perform removal of an apoptotic body. Furthermore, microglia are concentrated in regions with abundant cell death during development of the CNS (Ashwell, 1990, 1991; Perry et al., 1985).  It has been suggested that microglia can induce apoptosis of excess cells generated during development. Selective elimination of microglia in murine neonatal cerebellum slice cultures using toxic liposomes demonstrated a marked increase in the number of Purkinjie cells. Moreover, it was shown that microglia promote apoptosis in Purkinje cells by producing superoxide ions (Marin-Teva et al., 2004). The role of microglia in inducing programmed cell death has also been shown in the retina during development (Frade and Barde, 1998). Microglia have also been known to be responsible for synaptic pruning during the nervous system development. Synaptic pruning refers to the programmed loss of excessive or inappropriate synapses to form proper synaptic connections during the development of neurons (Rauschecker and Marler, 1987). Mice deficient in complement protein C1q, the initiating protein in the classical complement cascade, and the downstream complement protein C3, exhibit defects in CNS synapse elimination in the visual system (Stevens et al., 2007). Considering that microglia are the only cells in the CNS that express receptors for complement components (Gasque et al., 1998), this observation indicates that they are responsible for synaptic remodelling (Stevens et al., 2007).Microglia might also  31  promote vascularization in the CNS (Streit, 2001). Microglia are present in the developing CNS prior to vasculogenesis (Sorokin et al., 1992) and invading vessels are oriented towards microglia in experimental tissue implants (Pennell and Streit, 1997). Moreover, microglia have been recently shown to act as cellular chaperones that promote vascular anastomosis in the developing hindbrain of embryonic zebrafish following vascular endothelial growth factor (VEGF)-induced angiogenes (Fantin et al., 2010). The mechanisms for microglia mediated vascularization is unclear and the lack of a viable animal model devoid of microglia suggests that more needs to be done before concluding that microglia have a role in vascular development.  1.4.3 Microglia in adults  Kreutzberg introduced microglia as the “sensor of pathology” more than a decade ago, honouring them as the frontline defence in the CNS (Kreutzberg, 1996).  In a healthy CNS, microglia are highly ramified cells with a small soma and fine long cellular processes. Each microglia appears to occupy its own domain in a non-overlapping territorial manner that covers the entire CNS parenchyma (Raivich, 2005).  32  Ramified microglia with low levels of expression for key cell surface immune molecules such as MHC class II were known to be quiescent and called “resting” microglia with unknown functions under healthy conditions (Kreutzberg, 1996).  However, recent two-photon imaging microscopy of the cerebral cortex in live mice revealed that in the intact CNS, microglia continually sample and survey their microenvironment(Nimmerjahn et al., 2005). They carry out this dynamic surveillance of the CNS while constantly protruding and retracting processes, without movement of the soma. They scan the entire CNS parenchyma approximately every four to five hours. These landmark findings introduced the new concept of “surveillance” microglia challenging the previously accepted notion of microglia as “resting” in a healthy nervous system. Damage to the CNS results in the production of nucleotides such as adenosine triphosphate (ATP) that are released by injured cells. This leads to activation of purinergic receptors P2Y and connexin hemichannels on astrocytes and triggers further release of ATP from surrounding astrocytes, which activates microglia through the metabotropic purine receptor P2Y12 (Davalos et al., 2005; Haynes et al., 2006; Nimmerjahn et al., 2005) . Based on these findings, activation of microglia that was originally believed to only occur due to a disturbance of CNS homeostasis – since it is now recognized that microglia are constantly in motion and do not have any periods of inactivity refers to a shift of activity caused by injury (Hanisch and Kettenmann, 2007).  33  Microglia activation is identified by morphological changes and up regulation or de novo synthesis of cell surface receptors and increase in cell number (Kreutzberg, 1996). Based on morphological alterations of microglia in response to pathological conditions, Streit and colleagues introduced the concept of “functional plasticity” 20 years ago in the facial nerve axotomy paradigm (Streit et al., 1988). They demonstrated that, under normal circumstances, microglia display a ramified morphology, but, upon injury, they become activated which leads to hypertrophy of soma as well as retraction and shortening of processes. At this stage, the microglia are not phagocytic; however, if the stimuli persists and, for example, results in death of the neurons, they transform to an amoeboid phenotype and phagocyte the dead cells (Streit et al., 1988) (Figure 1.5 ).  34  Figure 1.5 Functional plasticity of microglia Ramified resting microglia (A) become activated upon most types of neuronal damage which results in activated microglia. Activated microglia (B) are ramified with stouter cell processes. If a neuron dies, activated microglia transform into macrophages (C) and phagocyte the dead cells.  Two different types of signals orchestrate the transition of microglia between surveillance and activated states. They are termed the “on” and “off” receptormediated signalling. Novel environmental signals such as microbial structures, unusual concentration of intracellular components and abnormal molecular formats of certain factors instigate the “on” signalling (Block et al., 2007; Hanisch and Kettenmann, 2007). These signals are recognized by an array of receptors in microglia. Toll-like receptors (TLRs) have been shown to recognize pathogen35  associated molecular patterns and release pro-inflammatory cytokines including IL-12, IL- 18, tumour necrosis factor– (TNF ) and nitric oxide synthase-2 (NOS2) (Olson and Miller, 2004).  Phagocytosis is one of microglia’s major duties that is highly regulated by the “on” signals. As previously discussed, damaged cells in the CNS produce and release nucleotides such as ATP, which signals microglia through the P2Y12 receptor and mediates the movement of their processes. Importantly, Koizumi and colleagues have recently discovered the receptor that mediates microglia phagocytic activity. They demonstrated that following neuronal cell death caused by an injection of kainic acid (KA) into mouse brains, the level of UTP is increased and P2Y6 receptors are up regulated in the activated microglia leading to phagocytosis. Collectively they proposed that damaged cells in the CNS produce both ATP and UTP Here, ATP acts as a “find-me” signal, facilitating microglia’s initial activation. In contrast, UTP conveys an “eat-me” signal, mediating the phagocytosis of the damaged cells later on (Koizumi et al., 2007) Activation of microglia can also be initiated by the disruption of constitutive signals that restrain microglia in a healthy CNS. These signals are called “off signals” and are mostly produced by neurons (Hanisch and Kettenmann, 2007). Neuronal inhibitory influence on microglia is mediated by several interactions: for example, CD200 expressed on the surface of neurons that binds to the CD200 receptor on the microglia surface (Barclay et al., 2002; Hoek et al., 2000). Microglia in CD200 knock-out mice exhibited the morphology of activated  36  microglia and had an accelerated response to damage compared to control animals (Hoek et al., 2000).  In addition, the chemokine CX3CL1, which is released by neurons, and its ligand CX3CR1, which is expressed on microglia, are involved in the communication between neurons and microglia (Biber et al., 2007). Deficiency in the chemokine receptor CX3CR1 exacerbated neuron loss in animal models of amyotrophic lateral sclerosis and Parkinson’s disease suggesting that the interaction between neurons and microglia play a role in modulating microglial activation (Cardona et al., 2006).  Activated microglia produce pro-inflammatory and anti-inflammatory cytokines, growth factors, chemokines and neurotrophins. Whether activated microglia have a detrimental or beneficial role in the central nervous systems is one of the most exciting and controversial topics in neuroimmunology. In the next section, I will discuss the current debates on the dual role of microglia in the CNS.  1.4.4 Microglia: friends or foe?  Microglia can be construed as a double-edge sword based on their different activities in the injured CNS. On the positive side, microglia are the major source of neurotrophic and growth factors that could be beneficial for neuronal function and survival and even assist regeneration. On the negative side, microglia, in  37  response to diverse stimuli, are able to produce a wide range of cytotoxic factors such as reactive oxygen species, nitric oxide (NO) or TNF- that can damage the CNS tissue.  Blinzinger and Kreutzberg suggested a regenerative role for microglia more than 40 years ago when they reported synaptic stripping in a facial nerve injury model. Following the axotomy of facial nerves outside of the CNS, microglia in the facial nucleus become activated. Activated microglia ensheath the damaged facial motor neurons and displace the afferent synaptic terminals from the surface of motor neurons. The displacement of afferent synapses results in the loss of excitatory input to the motor neuron, and through targeted delivery of growth factors from activated microglia the motor neuron is protected. This action on the part of microglia prevents the facial motor neurons from undergoing apoptosis and helps them to eventually regenerate their axons (Blinzinger and Kreutzberg, 1968).  Several studies have also suggested that microglia take part in neurogenesis. Walton and colleagues demonstrated that the presence of microglia or microgliaconditioned media is required for differentiation of neural stem cells into committed neuroblasts in vitro, suggesting that microglia provide instructive signals for neurogenesis (Walton et al., 2006). Furthermore, in vivo evidence supports a role for activated microglia in regulating neurogenesis with the anti– inflammatory cytokine, transforming growth factor-beta (TGF-) playing a key  38  role (Battista et al., 2006). In addition, transplantation of cultured microglia derived from neonatal rat brains into injured spinal cords enhanced neurite outgrowth suggesting a therapeutic potential of activated microglia (Rabchevsky and Streit, 1997).  However, the potential deleterious nature of microglia has also been revealed in various studies. Neurogenesis is inhibited in hippocampal neural progenitor cells (NPCs) when they are co-cultured with microglia activated by LPS, or exposed to an activated microglia conditioned medium. Pro-inflammatory cytokines such as IL-6 and TNF– are produced by activated microglia and can impair neurogenesis. Blocking IL-6 with an antibody successfully restored hippocampal neurogenesis in this study, indicating that activated microglia mediate their detrimental effect on adult brains by IL-6 (Monje et al., 2003).  Involvement of the parenchymal microglia in worsening disease severity has been shown in few CNS pathologies. In animal models of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), this issue was addressed by generating CD11b-HSV-TK mice, which expressed a lineage-restricted Herpes virus thymidine kinase (HSV-TK) suicide gene in myeloid cells. Administration of gancyclovir eliminates myeloid cells in these animals. In order to have selectively ablate microglia, and not all myeloid cells, chimeras with wild-type bone marrow were produced by irradiation and bone marrow transplantation. Gancyclovir treatment in these animals would then selectively ablate parenchymal microglia.  39  Following EAE immunization in these mice, the authors were able to demonstrate that the loss of microglia resulted in substantial improvement of clinical signs and a strong reduction of inflammatory lesions in the CNS (Heppner et al., 2005).  The major hallmark of Parkinson disease is the loss of dopaminergic neurons (DNs) of the substantia nigra pars compacta (SNc), which is accompanied by microglia  activation.  Administration  of  1-methyl-4-phenyl-  1,2,3,6-  tetrahydropyridine (MPTP) selectively induces the death of DA neurons and results in the development of Parkinson disease-like (PD) symptoms in mammals (Przedborski et al., 2001) . Increased levels of IFN- have been reported in the plasma of Parkinson patients. IFN- knock-out mice demonstrate attenuated microglia activation and subsequent decrease in loss of dopaminergic cells following MPTP treatment. This study suggests that microglia participates in the death of dopaminergic neurons and pro-inflammatory cytokine; IFN-γ regulates this action (Mount et al., 2007).  Microglia are shown to be actively involved in inherited forms of Amyotrophic Lateral Sclerosis (ALS) disease. Several studies have suggested that microglia contribute to motor neuron death in ALS. Don Cleveland group assigned a “noncell autonomous” function to microglia in transgenic mutant SOD1 mice, the animal model of familial ALS. Selective inactivation of mutant SODG37R using a CD11b-Cre transgene, that yields excision of “floxed” genes only in the myeloid lineage, prolonged the late stage of the disease significantly (Boillee et al., 2006). In agreement with this, replacement of mutant SOD1-expressing microglial cells 40  with wild-type microglia extended the survival in PU.1 knockout mice with mutation SOD1 and decreased motor neuron loss, pointing to a disease promoting role of microglia in this disease (Beers et al., 2006). However, ablation of fifty percent of reactive microglia in mSOD1 mice did not affect the rate of disease progression in mSOD1 mice (Gowing et al., 2008).  Collectively, these studies suggest opposite roles for microglia in the CNS. This functional dichotomy of microglia seems to be context-dependent, with both detrimental and beneficial characteristics, likely depending on their activation state. To make the matter more complicated, many CNS pathologies suffer from damage to the blood-brain barrier (BBB), which is believed to result in infiltration of peripheral blood-derived myeloid cells to the CNS. The lack of tools to distinguish between parenchymal microglia and blood-derived myeloid cells has created a considerable amount of controversy surrounding the role of infiltrating myeloid cells in different disease pathologies. In the next section, I will focus on blood-derived myeloid cells in the CNS. Furthermore, I will again re-examine the role of myeloid cells in CNS pathologies in which a heterogenic myeloid cell population of parenchymal microglia and blood-derived macrophages is believed to exist.  41  1.5 Blood–Derived Macrophages: Aliens from Another Planet  Traumatic injuries, inflammation and infections provoke an inflammatory response involving, for the most part, macrophages (Mosser, 2003; Mosser and Edwards, 2008). Macrophages have a remarkable plasticity that enable them to adopt different functional phenotypes upon exposure to a variety of microenvironmental signals (Van Ginderachter et al., 2006). In fact, depending on the particular cytokines and microbial products they encounter, macrophages develop divergent functional properties as either pro- or anti-inflammatory mediators. In both cases, they play a critical role in fighting against infection as well as promoting tissue healing.  Copying the Th1/Th2 nomenclature, macrophages have been classified into two groups: “classically activated” M1 macrophages and “alternatively activated” M2 macrophages (Gordon, 2003; Mantovani et al., 2003).  Classically activated macrophages are developed in response to the Th1 cytokine, IFN-, alone or in combination with a microbial trigger such as bacterial lipopolysaccharide (LPS) via toll-like receptors. Their activation leads to an inflammatory response and the release of pro-inflammatory cytokines such as IL1β, TNF-α, IL12 and IL6, as well as the production of high levels of oxidative metabolites such as nitric oxide (NO) and reactive oxygen intermediates (ROI). While M1 macrophages are essential for host defence – they eliminate  42  pathogens and inhibit tumour growth – they can also cause considerable further damage to the host tissue through the release of pro-inflammatory cytokines and mediators (Benoit et al., 2008; Bermudez and Young, 1988; Ding et al., 1988; Nathan, 1983; Nathan et al., 1983; Summersgill et al., 1992)  “Alternatively activated” M2 macrophages, by contrast, are involved in the resolution of inflammation. They are induced in the presence of Th2 cytokines. Based on the activating cytokines, M2 macrophages are further classified into three subpopulations: M2a, M2b or M2c (Benoit et al., 2008; Martinez et al., 2008) . M2a macrophages are induced in the presence of IL-4 or IL-13 and express high levels of immunosuppressive factors, such as IL-10 and arginase. Arginase diverts the arginine metabolism, which is oxidized to nitric oxide by inducible NO synthase (iNOS) a product of classically activated macrophages (M1), to generate ornithine, an amino acid that is important in collagen production and stimulates cell growth and differentiation (Hesse et al., 2001). This shift in arginine metabolism by alternatively activated macrophages fosters the wound healing potential of these cells (Curran et al., 2006; Mantovani et al., 2003; Mosser and Edwards, 2008; Shearer et al., 1997; Witte and Barbul, 2003).  M2b macrophages are induced by exposure to Immune complexes, Toll-like receptors or the IL-1 receptor. They release high levels of IL-10 and exert immune regulatory functions and provoke Th2 responses (Anderson and Mosser, 2002; Mantovani et al., 2004a; Mantovani et al., 2002; Mantovani et al., 2004b).  43  The M2c phenotype is induced by IL-10 and glucocorticoid hormones (Mantovani et al., 2004a). These macrophages produce high levels of immunosuppressive cytokines, IL-10 and the transforming growth factor β (TGF-β), and play a predominant role in immunosuppression, tissue remodelling and matrix deposition (Mantovani et al., 2004a; Mantovani et al., 2004b; Mosser, 2003). It is clear, then, that the initial phase of innate immune response is orchestrated by M1 macrophages that fight the invading organisms whereas the M2 macrophages are involved in the resolution of injury and restoring normal tissue homeostasis. However, a polarized macrophage population in the CNS is a complicated notion. The central nervous system is considered an immune-privileged site protected by the blood-brain barrier (BBB) that limits the access of circulating molecules and blood leukocytes (Carson et al., 2006a), making the CNS a unique environment with different stimuli mostly released by local cells such as neurons and astrocytes. As previously discussed, in injured central nervous systems, there is a correlation between reactive microglia and an increase in their cell numbers. Whether this increase in cell numbers in response to the CNS disease arises from a proliferation of resident microglia or is caused by the invasion of peripheral myeloid cells in response to the CNS disease is somewhat controversial and will be further explored both later in this chapter and in Chapter 2. A considerable body of literature in recent years has focused on this heterogeneous myeloid population in several CNS injuries. Much of this work has  44  attempted to differentiate resident microglia and blood-derived macrophages, placing these two populations of cells into categories of destructive or beneficial. Controversies have emerged from these studies as to whether immune responses in the CNS should be the therapeutic targets or, conversely, whether they should be interpreted as favourable and be enhanced to augment CNS repair.  Schwartz and colleagues have focused on the possible beneficial effects of blood-derived macrophages in various CNS pathologies and have demonstrated that an immune response that is properly controlled can facilitate repair and recovery in the CNS. Comparing peripheral nerve system (PNS) injury to an injury of the central nervous system, they suggested that the poor regenerative ability of the CNS is due to the low level of macrophage recruitment to the site of injury as well as an insufficient amount of phagocytosis (Hirschberg and Schwartz, 1995). Subsequently they claimed that transplantation of ex vivo activated autologous macrophages into transected rat spinal cords promotes axon regeneration and results in improvements in hind limb flexion (Rapalino et al., 1998). Based on this approach, ProCord, an activated macrophage cell therapy clinical trial in spinal cord injury patients within 14 days post injury, was initiated by Proneuron Biotechnologies Inc. Although the phase I clinical trials were completed, phase II studies were suspended with undisclosed reasons (Kigerl and Popovich, 2006; Knoller et al., 2005; Popovich and Longbrake, 2008).  45  Other studies, however, have indicated a different role for blood-derived macrophages in spinal cord injuries. The appearance of a large number of macrophages in a cat model of spinal cord injuries, independent of the severity of damage, was suggested to be responsible for a delay in demyelination (Blight, 1985). Furthermore, depletion of peripheral macrophages with liposomeencapsulated clodronate during a time when inflammation has been shown to be maximal after spinal contusion injury in rats resulted in an enhanced number of myelinated axons and reduced lesion cavitations (Popovich et al., 1999). Considering the depletion of peripheral macrophages in this study took place after the onset of microglia activation, an event that precedes macrophage invasion in spinal cord injuries, it has been suggested that blood-derived macrophages and not resident microglia play a major role in the secondary damage that follows the initial trauma in spinal cord injuries (Jones et al., 2005).  Recently, two independent studies touched on this debate, suggesting two distinct populations of blood-derived macrophages with pro- and antiinflammatory functions during spinal cord injuries (Kigerl et al., 2009; Shechter et al., 2009).  Using chimeric mice created with lethal irradiation and bone marrow transplants, Shechter and colleagues demonstrated that conditional ablation of blood-derived macrophages results in impaired functional recovery of the injured spinal cord. They observed that early after injury, between day four and day seven, the  46  recruitment of Inflammatory monocyte Gr1+/Ly6C+ appears to be important for tissue repair. These inflammatory monocytes differentiate into macrophages with an M2 phenotype and express anti-inflammatory cytokine IL-10 at the site of injury. Furthermore, impaired functional recovery in mice, following the ablation of circulating monocytes while microglia activity still exist, suggests that resident microglia and the invading macrophages have substantially different functions in the injured spinal cord. In fact, the authors show that activated microglia have a detrimental role which is restrained by blood-derived macrophages via secretion of IL-10 (Shechter et al., 2009).  Moreover, taking a different approach, the Popovich group also identified two distinct macrophage phenotypes following a spinal cord injury. Looking at gene expression analysis and tissues from the lesion site, they reported an equal presence of both M1 and M2 macrophages within the first three days post injury. However, the presence of M2 macrophages was transient and disappeared within three to seven days post injury. Unfortunately, this study did not discriminate between resident microglia and blood-derived macrophages when they examined the M1 and M2 phonotypes (Kigerl et al., 2009).  Although both studies seem in agreement over the existence of M2 macrophages in the early stage of the spinal cord injury, there is a discrepancy between their data. According to the Schwartz group, blood-derived macrophages with an M2 phenotype exist between day four and seven post injury. However, in this time  47  frame, according to the data from the Popovich group, the M2 phenotype is decreased and M1 macrophages are abundant. If direct manipulation of myeloid cell population towards the M2 phenotype is to be considered as a therapeutic option for spinal cord injuries, it is very important to determine the exact time frame that M2 macrophages are able to sustain themselves.  A similar dichotomy regarding the role of resident and blood-derived myeloid cells exists in neurodegenerative diseases, including Alzheimer’s disease (AD). Several studies using different approaches have attempted to address the distinct functions of infiltrating versus resident myeloid cells in Alzheimer’s disease (El Khoury et al., 2007; Malm et al., 2005; Simard et al., 2006; Stalder et al., 2005). Transplanting bone marrow cells from green fluorescent protein (GFP) transgenic mice into irradiated young or old AD model transgenic mice expressing mutant forms of APP (APP32 mice) or APP plus Presenilin-1 (APPPS1 mice) showed more GFP-positive myeloid cells in APP brains compared to the wild-type control. Moreover, the level of infiltration of blood-derived myeloid cells was significantly higher if the bone marrow transplants were performed prior to the formation of amyloid plaques (Malm et al., 2005). In an Alzheimer’s disease model, Simard and colleagues demonstrated significantly different roles for resident microglia and blood-derived macrophages. They ablated the bloodderived macrophages in the brain of APP–PS1 mice by crossing them with mice expressing an inducible myeloid-specific suicide transgene in which Herpesvirus Thymidine Kinase (HSV-TK) is regulated by CD11b (termed CD11b-HSVTK  48  mice). These mice were subjected to Intracerebroventricular delivery of ganciclovir (GCV) for 28 days when they were between 15–24 weeks of age, a time when pathology is present. The authors claim that this method of GCV treatment only affects new infiltrating blood-derived myeloid cells. Exaggerated plaque burden in these mice led to the conclusion that blood-derived macrophages are responsible for phagocytosis of amyloid plaques. These authors suggested that resident microglia struggle with efficient and effective clearance of amyloid deposits, and therefore require the recruitment of bloodderived myeloid cells. Delivery of the latter can, following this logic, therefore, be exploited as a new therapeutic approach for Alzheimer’s disease (Rivest, 2009; Simard et al., 2006).  Additional evidence by El Khoury and colleagues provided further support for the important role of blood-derived macrophages in the clearance of amyloid plaques. In this study, they crossed Tg2576 mice, an AD mouse model, with CCR2-/- mice, which lack the CC-chemokine receptor2 (CCR2) – the receptor specific for the Monocyte Chemoattractant Protein (MCP) family of chemokines. CCR2-/- mice suffer from impaired monocyte migration. Increased amyeloid plaque and a short life span were observed in tg2576/CCR2-/- mice suggesting a pivotal role for blood-borne myeloid cells in Alzheimer’s disease (El Khoury et al., 2007).  49  The cumulative conclusion from these observations is that blood-derived myeloid cells have a beneficial role in the pathogenic cascade of Alzheimer’s disease, and boosting the recruitment of blood-derived myeloid cells could denote a promising therapeutic target.  A recent report suggests that CNS-resident microglia do not play any role in amyloid plaque clearance. These investigators ablated brain resident microglia by crossing CD11b-HSVTK mice with a APP-PS1 transgenic mouse model of AD and administering ganciclovir (GCV) intracerebroventricularly for four weeks. Elegantly, the peripheral blood of these mice was replaced by wild-type bone marrow prior to GCV treatment. No effect on amyloid plaque formation and number were observed in these animals in the absence of microglia (Grathwohl et al., 2009). The authors did not make any clear comments regarding the bloodderived myeloid cells.  The discrepancy between the work by Grathwohl and colleagues and the study by Simard and colleagues rests on the assumption by the latter group that bloodderived myeloid cells infiltrate the CNS under pathological conditions.  Simard and colleagues, based on their initial observation in chimeric mice created by irradiation and bone marrow transplants, believe that blood-derived myeloid cells infiltrate the brain of AD mice and the administration of ganciclovir (GCV) only effects this population. Grathwohl and colleagues, on the other hand,  50  had a proper protocol in their study whereby the peripheral blood of the mice was replaced by wild-type bone marrow. In this way, the blood-derived macrophages were not affected by ganciclovir (GCV) treatment. Interestingly, based on the study by Grathwohl and colleagues, even if blood-derived myeloid cells do enter the CNS, they do not play any role in the clearance of amyloid plaques since neither amyloid plaque formation nor maintenance was affected in this study in which the wild-type blood-derived myeloid cells are unaffected by ganciclovir (GCV) treatment. There is a great deal of the controversy regarding the role of blood-derived myeloid cells and resident microglia, and much of this controversy stems from the burning question of whether the blood-derived cells are indeed recruited to the CNS in healthy adults and during different pathologies.  1.6 Thesis Objectives  As discussed above, it remains unclear whether blood-derived myeloid cells contribute to microglia population in the CNS, which is considered an immune privileged site with a limited exchange of blood cells and substances with peripheral blood. The inherent lack of specific cell-surface or enzymatic markers to distinguish blood-derived macrophages from resident microglia poses significant challenges in identifying to what extent blood-derived cells contribute to the normal turnover of microglia in the CNS. Our current knowledge of inflammatory cell infiltration into the normal adult CNS, under pathological circumstances, mostly stems from studies using bone marrow chimeras  51  produced by lethal irradiation and bone marrow transplantation which alter bloodbrain barrier function and introduce non-physiological numbers of progenitors into the circulation.  Therefore, the primary objective of my PhD dissertation is to determine the contribution of circulating myeloid cells to microglia in healthy and diseased central nervous systems. As described in chapter 2, using a parabiotic system which allows tracking of the fate of bone marrow precursor cells under homeostatic conditions and in the absence of lethal irradiation and intravenous bone marrow transplantation, I demonstrate that microglia in healthy adults or following degenerative (ALS) or traumatic (axotomy) tissue damage are capable of self-renewal and do not require any contribution from bone marrow-derived progenitors for their maintenance and function.  Infiltration of blood-derived leukocytes is a diagnostic hallmark of multiple sclerosis (MS) – a chronic inflammatory condition of the CNS. As described in chapter 3, using a modified parabiosis model, I reveal that blood-derived myeloid cells are responsible for the stepwise progression of the disease in Experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Furthermore, I demonstrate that the entry of these cells in the CNS of EAE mice is a transient event and does not contribute to the long-term resident population of myeloid cells in the CNS.  52  Chapter 2: Local Self-Renewal Can Sustain CNS Microglia Maintenance and Function Throughout Adult Life  2.1 Introduction  As disscused in the Introduction, microglia are the main inflammatory cells of the central nervous system (CNS), and play a critical role in the response to CNS damage (Davalos et al., 2005; Kreutzberg, 1996; Nimmerjahn et al., 2005).  Reactive microgliosis, defined here as an increase in number of microglia induced by disease, is a hallmark of essentially all CNS pathologies, including trauma, stroke, inflammation, autoimmunity and neurodegenerative diseases (Gonzalez-Scarano and Baltuch, 1999; Jensen et al., 1994; Lehrmann et al., 1997; McGeer et al., 1993; Ponomarev et al., 2005) . Yet, the mechanisms regulating microglia homeostasis remain elusive and whether microglia renew in situ or are replenished by precursors originating outside of the CNS is the subject of much controversy. This chapter, then, will expand on the analysis of research surrounding this debate – as introduced in Chapter 1 – and will add the results of my own work to determine whether microglia are capable of self-renewal.  Several lines of evidence support the notion that microglia are of hematopoietic origin suggesting that, similar to the vast majority of extramedullary 53  inflammatory/scavenger cells, they are in equilibrium with bone marrow-derived progenitors capable of reaching the CNS through the bloodstream (Hess et al., 2004). The extent to which bone marrow-derived cells contribute to microglia homeostasis and function has been investigated in bone marrow chimeras obtained by transplantation of lethally irradiated recipients. These studies reported that under homeostatic conditions a considerable percentage of microglia are replaced by donor derived cells (Biffi et al., 2004; Simard and Rivest, 2004). Furthermore, they suggested that the large increase in microglia observed upon CNS damage involved both the expansion of endogenous resident microglia and the acute, active recruitment of bone marrow-derived myeloid progenitors from the bloodstream (Flugel et al., 2001; Kennedy and Abkowitz, 1997; Priller et al., 2001; Simard et al., 2006) . These findings suggest that engineered BM cells could act as vehicles for gene delivery across the blood-brain barrier (BBB), providing the rationale for the development of therapeutic strategies (Asheuer et al., 2004; Biffi et al., 2004; Krall et al., 1994).  An alternative view is expressed by publications that, using similar methods, reported little or no contribution of circulating bone marrow-derived progenitors to the parenchymal microglia pool. These studies suggest that reactive microgliosis is mainly due to local expansion of resident microglia, supporting the notion that the CNS is a closed system with minimal cellular traffic across the BBB in physiological conditions (Hickey and Kimura, 1988; Hickey et al., 1992; Massengale et al., 2005). According to these findings, microglia maintenance 54  and function is independent of bone marrow-derived progenitors throughout adult life and the microgliosis triggered by a variety of pathogenic insults is the result of an enormous capacity of CNS-resident microglia for proliferation (Graeber et al., 1988).  In support of this second model, our own work in lethally irradiated bone marrow chimeric animals indicates that the microgliosis observed in the spinal cord of transgenic mice overexpressing mutant superoxide dismutase (mSOD1), an animal model of amyotrophic lateral sclerosis (ALS), is primarily due to an expansion of resident microglia and not to the recruitment of microglia precursors from the circulation, and bone marrow derived myeloid cells do not substantially replace the host microglial pool (Solomon et al., 2006).  A potential explanation to resolve the apparent discordance in these results may lie in the use of chimeric animals generated by irradiation and transplantation. In these models donor bone marrow-derived cells are traceable due to the expression of transgenic or genetic markers, thus allowing the quantification of their contribution to the microglial pool. However, two factors may confound the interpretation of these experiments. First, irradiation at the dosages commonly used for myeloablation leads to the temporary disruption of the BBB (Diserbo et al., 2002; Li et al., 2004b), potentially allowing the entry of circulating cells into the CNS that would normally not cross an intact BBB. Second, donor cells are routinely harvested by mechanically flushing whole bone marrow from bones 55  followed by intravascular injection into recipients. This could lead to an artificial persistence in the circulation of progenitors with the potential to cross the bloodbrain barrier and/or to differentiate along the microglial lineage. It is possible that these progenitors would not enter the bloodstream under physiological conditions.  To address this issue, we assessed whether circulating myeloid precursors are recruited to the CNS under physiological or pathological conditions in the absence of these experimental manipulations. To this end, we utilized chimeric animals obtained by parabiosis, which requires neither irradiation nor transplantation. We compared the extent of recruitment of circulating myeloid precursors to the CNS of parabionts with that observed in lethally irradiated mice that received a whole BM transplant. While we readily observed the recruitment of circulating myeloid precursors in lethally irradiated/transplanted mice, we found no evidence of recruitment from the bloodstream in parabiotic chimeras using two models of acute and chronic microglia activation, axotomy and neurodegeneration.  These findings conclusively demonstrate that, in adult animals, microglia can be maintained and function independently of bone marrow-derived progenitors throughout adult life. Furthermore, this observation, combined with the notion that CNS disease induces a massive local increase in the number of microglia (microgliosis), suggests that, similar to T-cells and Langerhans cells in the skin, 56  adult microglia are in fact, capable of self-renewal (Merad et al., 2002; Rossi et al., 2005).  2.2 Material and Methods  2.2.1 Mice  All animal experiments were approved by the University of British Columbia Animal Care Committee. Wild-type C57BL/6 and C57BL/6 mice expressing GFP ubiquitously from CMV-βactin hybrid promoter (a kind gift of I.L. Weissman) were bred in-house and maintained in a pathogen-free environment. Mice overexpressing the G93A mutant human transgene for CuZn superoxide dismutase (SOD1) were obtained from Jackson Laboratories and locally bred.These mice express 18-fold more SOD1 protein than the native level if the murine wild-type strain (Gurney et al., 1994; Li et al., 2004a).  2.2.2 Generation of chimeric mice by parabiosis  Female mice aged between four to six weeks old and at least 17 gr in weight were selected for parabiosis. Age- and weight-matched mice were housed together for at least one week prior to surgery. Parabiotic mice are prepared by creating a skin incision running along the side of the animal from the elbow to the knee on opposite sides of the mice to be paired. The mice were first joined with a 57  suture through the shoulder and thigh muscles, and next the inside faces of the skin flaps were juxtaposed and sutured. Complete blood sharing (an overall average of 44% GFP+ cells in the GFP- partner) verified between 10 and 15 days after surgery.  2.2.3 Generation of chimeric mice by irradiation and transplant  To generate irradiated/transplanted mice with comparable frequencies of GFP+ peripheral blood cells, lethally irradiated (1100 rads) C57BL/6 animals received intravenously a mixture of 1x106 GFP- and 1.5x106 GFP+ whole bone marrow cells.  2.2.4 Facial nerve axotomy surgery  The GFP negative partner in each pair of parabiotic mice was subjected to unilateral facial nerve transection (under isofluorane anesthesia) two weeks after parabiosis or five weeks post irradiation and transplantation as described earlier (Graeber et al., 1998). This results in ipsilateral whisker paresis and massive microgliosis in the facial nucleus as confirmed by immuno-histochemistry . Animals were sacrificed seven days after axotomy.  58  2.2.5 Generation of chimeric mice by parabiosis/irradiation  Two weeks post surgery, the GFP+ partner was shielded with lead and the pair subjected to a lethal dose of irradiation (1100 rads) using a gamma cell irradiator with a cobalt 60 source.  2.2.6 Peripheral blood analysis  Peripheral blood was obtained from the tail vein. Red blood cells were lysed by resuspending samples in ammonium chloride solution and incubating at room temprature for five minutes the white blood cells were resuspended in FACS buffer (Phosphate Buffered Saline) (PBS) supplemented with 2% FBS and 2mM EDTA). Analysis was accomplished using a FACS Calibur (Becton Dickinson), and flow cytometry data were analyzed using FlowJo (Treestar) analysis software.  2.2.7 Muscle preparation and analysis  Muscle damage was triggered by injecting notexin in the tibialis anterior of the GFP- partner of a parabiotic pair. Cells infiltrating the damaged muscle were analyzed five days after notexin injection. Damaged muscle was digested with Type IV Collagenase (400U/ml) for 20–45 minutes at 37°C.  59  2.2.8 Animal harvest and tissue preparation  Animals were injected with a lethal dose of avertin and monitored. Upon the loss of nociceptive reflexes, animals were perfused transcardially with 20 ml PBS /EDTA followed by 20 ml 4% paraformaldehyde in 0.1 M PBS at room temperature. The spinal cord was removed and the tissue was post-fixed for 24 hours in 4% paraformaldehyde at 4 °C and then cryoprotected in a 24% sucrose solution in PBS for 24 hours. Spinal cord tissues were embedded in OCT (Tissue-Tek) and frozen at -80°C.  2.2.9 Immunohistochemistry  After tissue processing, 20 μm thick cryosections were cut from the brain and spinal cords. Sections were allowed to thaw at room temperature, rehydrated in PBS for 2 h and incubated with blocking buffer (25% normal goat serum, 3% BSA, 0.3% Triton X-100) for another 1 h. FcγR was blocked by incubating the sections with 5-10 μg/ml purified anti-CD16/32 antibody on ice for 10 minutes. Primary antibody staining was carried on over night. Primary antibodies included: anti-Iba-1 as microglia marker (Wako), PECAM-1 (anti-mouse CD31) as an endothelial marker (BD Pharmingen). Secondary antibodies were either Alexa568- or Alexa647-conjugated goat anti rat (Molecular Probes). All sections were analyzed by confocal microscopy using Nikon C1 Laser Scanning Confocal  60  Microscope. Unless otherwise indicated, all images presented are maximum intensity projections of Z stacks of individual optical sections.  2.3 Results  2.3.1 Microglia are sustained by local proliferation rather than recruitment of myeloid progenitors from the peripheral circulation in a healthy CNS  To assess whether circulating microglia precursors are recruited to the CNS under physiologic conditions we induced peripheral blood chimerism by joining pairs of syngeneic mice in parabiosis. Surgically adjoined parabiotic mice establish a rich anastomotic circulation leading to complete peripheral blood exchange and blood chimerism by day 10 (Figure 2.1a, Figure 2.2). We joined C57BL/6 mice with transgenic partners expressing GFP in all hematopoietic cells except those belonging to the erythroid lineage. Five months later parabiotic pairs were harvested and analyzed. Microglia within the CNS were identified by staining with Iba-1 antibody, the marker for microglia and macrophages (Ito et al., 1998), and the contribution of circulating microglia precursors to the CNS pool was measured by assessing the frequency of GFP+ cells within the Iba-1+ population. Despite analyzing over 30 sections spanning the brain, brainstem and spinal cord of each animal, which contained in total over 2000 Iba-1+ cells, no GFP+ microglia were observed (Figure 2.3a–f). In contrast, colonization of the liver by partner-derived, Iba-1+ macrophages was readily detected in the same  61  animals (Figure 2.3g–i). Thus, microglia maintenance does not rely on the continuous influx of circulating myeloid progenitors from the bloodstream or bone marrow.  Figure 2.1 Blood chimerism in parabiotic and lethally irradiated/transplanted mice (a) Schematic diagram showing mice joined in parabiosis. Four groups of parabionts were generated by joining GFP+ mice with GFP– mSOD1 mice (ALS model) or with GFP– C57BL/6 partners that were subsequently left untreated (healthy) or were subjected to facial nerve axotomy. Right: representative fluorescent-activated cell sorting (FACS) plot showing the frequency of GFP+ in the spleen of a GFP– partner at the time of death. (b) Lethally irradiated C57BL/6 wild-type mice were competitively reconstituted with a mixture of syngeneic GFP+ and GFP– whole bone marrow cells. Right: representative FACS plot showing the frequency of GFP+ in the spleen of a GFP– partner at the time of death. The chimerism of parabiotic and irradiated animals was comparable throughout our experiments (average 48%).  62  Figure 2.2 Blood chimerism in parabiotic mice and contribution of GFP cells to different blood lineages (a) FACS plot showing the frequency of donor derived GFP positive cells in whole peripheral blood of a representative GFP negative partner at time of sacrifice. The average peripheral blood chimerism in the parabiotic mice generated for this study was 43.2% ± 10.7) . (b-d) GFP positive chimerism in specific lineages of circulating cells from the same animal. (b) Gr-1 expressing cells (granulocytes). (c) CD11b expressing cells (monocytes). (d) CD3 expressing cells (T-lymphocytes).  63  Figure 2.3 Microglia, unlike peripheral macrophages, are not replenished by bone marrow-derived progenitors throughout adult life (a-f) The images shown are representative of the results obtained in two parabiotic pairs joined for five months. Brain, brainstem and spinal cord sections were immunostained for the microglia marker Iba-1 (red) and the endothelial marker PECAM (blue). GFP, identifying partner-derived cells, is shown in green. Images shown are maximum intensity projections of stacks of confocal optical sections. No GFP positive cell also positive for Iba-1 was detected in any of the sections. Furthermore, the few GFP+ Iba-1- cells detected were closely associated with blood vessel endothelium. Scale bar: (a-c) 100 μm; (d-f) 50μm. (g-i) In stark contrast, partner-derived GFP+ cells that were also Iba-1 positive 64  were readily detected within the liver of the same animals. This supports the notion that, unlike microglia in the CNS, liver macrophages are in homeostatic equilibrium with bone marrow progenitors migrating through the blood stream. Scale bar: 50μm.  65  2.3.2  An  increase  in  microglia  in  response  to  denervation  or  neurodegenerative disease is the result of local microglial proliferation rather than the entry of blood-derived myeloid cells into the CNS  Active microglia precursor recruitment from the bloodstream across the BBB has been described in a variety of diseases (Ladeby et al., 2005). To assess whether the large increase in microglia that characterizes most CNS pathologies is dependent on progenitors generated in bone marrow, we investigated partnerderived cell contribution to microgliosis in parabiotic models of CNS disease of two different etiologies: facial nerve axotomy and amyotrophic lateral sclerosis. As controls we generated a set of irradiated/transplanted animals in which the frequency of GFP+ peripheral blood cells was comparable to that obtained by parabiosis (Figure 2.1b). Lethally irradiated C57BL/6 wild-type mice were competitively reconstituted with a mixture of syngeneic wild-type GFP+ and GFP– bone  marrow  mononuclear  cells.  As  expected,  two  weeks  after  irradiation/transplantation recipient mice had an average of 46% GFP+ cells in peripheral blood, and could therefore be directly compared with parabiotic mice.  The rodent facial motoneuron axotomy model represents one of the most widely used animal models to study the acute microglia reaction without mechanical disruption of the BBB in vivo (Graeber et al., 1998). In this model, the facial nerve is surgically severed after its emergence from the stylomastoid foramen, and its interruption leads to progressive atrophy and death of the motoneurons. Thus,  66  the primary insult takes place outside of the CNS, at considerable physical distance from the motoneuron cell bodies that reside in the brainstem. This leads to “synaptic stripping” whereby the motoneurons lose their afferent inputs, which involves acute local microgliosis in the absence of direct CNS trauma or mechanical disruption of the blood–brain barrier (Blinzinger and Kreutzberg, 1968; Graeber et al., 1998; Graeber et al., 1988; Moran and Graeber, 2004; Streit and Kreutzberg, 1988). To assess whether the resulting rapid microgliosis is dependent on recruitment of blood-borne progenitors, we generated parabiotic pairs (N=7) as well as irradiated/transplanted control mice as described above. Two weeks after establishment of parabiosis, the GFP– parabiotic partners were subjected to unilateral facial nerve transection. one week after the axotomy, the animals were sacrificed and the presence of partner-derived, GFP+ microglia infiltrating the axotomized facial nucleus was assessed. Despite the massive reactive microgliosis observed, no evidence of contribution from partner-derived microglia precursors was found in the axotomized facial nucleus (Figure 2.4a,b). Furthermore, the rare GFP+ Iba-1– cells observed in the axotomized facial nucleus were closely associated with blood vessels, suggesting that they may represent circulating cells adhering to the endothelium (Figure 2.4c–f). In contrast, in irradiated/transplanted control mice (N=3) that had been subjected to facial nerve axotomy five weeks after transplant, both endogenous CNS-resident as well as exogenous BM-derived parenchymal microglia (20.5% ±3.8%) were found (Figure 2.4g–l, Figure 2.5a–c, Table 2.1).  67  To verify that the lack of axotomy-induced recruitment of partner-derived circulating myelomonocytic cells in parabiotic mice is specific to the CNS and is not a characteristic of this model, we assessed the recruitment of macrophages to damaged muscle in the same animals that received a facial axotomy. We selected this cell type since macrophages and microglia are functionally and developmentally related and express a very similar set of genes (Sedgwick et al., 1991). Muscle damage was induced by injecting the snake venom-derived toxin, notexin, into the tibialis anterior muscle of the GFP– parabiont, and recruitment of partner-derived cells was analyzed based on Iba-1 and GFP expression (Corbel et al., 2003). As expected, a significant fraction (18.9%) of Iba-1 expressing macrophages infiltrating the damaged area was comprised of partner-derived cells (Figure 2.5d–i; Figure 2.6). Thus, in this model, damage-induced recruitment of circulating myelomonocytic cells is efficient in peripheral tissues but does not take place in the CNS if the BBB is intact.  68  69  Figure 2.4 Circulating progenitors do not contribute to the microgliosis that is induced by facial nerve axotomy in parabiotic mice (a) Collage of 52 confocal images showing the dorsal portion of the brain stem of a parabiotic mouse that was subjected to left facial axotomy. The microglia marker Iba-1 is shown in red and GFP, identifying partner-derived cells, in green. Arrowheads indicate rare green cells that were present in the area of the axotomized facial nucleus. No Iba-1 and GFP double-positive cell was observed in any of the seven parabiotic mice analyzed. Scale bar represents 500 μm. (b) Higher magnification of the axotomized facial nucleus in parabiotic mice. The arrowhead indicates a GFP+, Iba-1– cell found in the area of the axotomized facial nucleus. Scale bar represents 50 μm. (c) The few green cells found in the axotomized facial nucleus were located inside blood vessels. The endothelial marker PECAM is shown in red and GFP in green. Scale bar represents 100 μm. (d–f) Relationship between GFP-positive partner-derived cells, blood vessels and microglia in the axotomized facial nucleus. The rare GFP+ (green) cells were located inside of blood vessels (PECAM, blue) and were negative for Iba-1 (red). The inset in panel d shows a single optical section from the image stack. Scale bar represents 50 μm. (g–l) Bone marrow–derived microglia in the facial nucleus of irradiated/transplanted mice subjected to facial nerve axotomy. Arrowheads point to some of the donor-derived microglia. Single optical sections of a higher magnification of the area in the white box in panel i are shown (j–l). A ramified donor-derived (GFP, green) microglia positive for Iba-1 (red). s represent 100 μm for g–i and 50 μm for j–l.  70  Figure 2.5 Circulating cells significantly contribute to microgliosis in the axotomized facial nucleus following irradiation/transplantation, and to macrophage infiltration following muscle damage in parabiotic mice (a–c): Irradiated/transplanted mice containing approximately 50% GFP+ cells in the peripheral blood were subjected to facial nerve axotomy. A representative section of the axotomized facial nucleus stained with the microglia marker Iba-1 (c; red), and the endothelial marker PECAM (b,c; blue) is shown. Donor-derived GFP+ cells (green, arrowheads) with a typical ramified morphology are abundant in the area and are significantly contributing to the Iba-1+ microglial population. Scale bar: 50μm  71  (d–i): Macrophage precursors are able to reach and infiltrate damaged muscle through the circulation in parabiotic mice. Muscle damage was induced in the tibialis anterior muscle of GFP- parabiotic partners by injection of the snake toxin notexin. Three days later, macrophages infiltrating the area of damage were identified by staining with Iba-1 (red). GFP+ partner-derived cells constitute a significant proportion of the Iba-1 positive cells. Scale bars: (d-e) 100 μm; (g-i) 50 μm.  Table 2.1 BM-derived microglial engraftment in axotomized facial nucleus after irradiation/transplantation  Animal ID  Sections ID  Iba-1+ cells  GFP+/Iba-1+ cells  Percentage  a  228  47  20.6  b  186  36  19.4  c  194  38  19.6  a  210  35  16.6  b  228  54  23.7  c  209  61  29.1  a  190  35  18.4  b  193  34  17.6  c  208  40  19.2  1  2  3  Percentage of ramified GFP expressing microglial cells (Iba-1+) in the facial nucleus of mice transplanted with mixture of GFP+ and GFP- BM cells. Average: 20.5% ± 3.8.  72  Figure 2.6 Damage-induced recruitment of circulating myelomonocytic cells is efficient in peripheral tissues of parabiotic animals To exclude the possibility that a general defect in the recruitment of partnerderived inflammatory myelomonocytic cells may take place in parabiotic animals, we quantified partner-derived macrophages infiltrating notexin damaged muscle in parabiotic mice. (a) Inflammatory cells were identified as double positive for the myelomonocytic marker CD11b and the pan-hematopoietic marker CD45. (b) Shows the frequency of GFP+ in the CD11b, CD45 double positive subpopulation. Approximately 19% of the myelomonocytic inflammatory cells found in damaged muscle are partner-derived GFP+ cells.  73  Facial axotomy induces acute microgliosis lasting several weeks. However, it is possible that efficient recruitment of circulating cells to the CNS may require chronic, longer-lasting, damage-induced signals. Therefore, we investigated partner-derived cell recruitment in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disorder leading to loss of neurons in the cerebral cortex, brainstem, and spinal cord with strong activation and proliferation of microglia in these regions (Hall et al., 1998; Rowland and Shneider, 2001). A mouse model of this disease has been obtained by transgenic expression of a mutated form of Cu/Zn superoxide dismutase (mSOD1) (Rosen et al., 1993).  To investigate microglia progenitor recruitment from the bloodstream in ALS, we generated parabionts between healthy GFP mice and mice over-expressing mSOD1. Parabionts were surgically joined at ten weeks of age, a time that precedes the development of symptoms or microgliosis, and harvested and analyzed at a late stage of disease, when microgliosis is extensive (110–130 days old). Despite the massive reactive microgliosis induced by the disease in lumbar region of spinal cord, no evidence of a contribution from partner-derived (GFP+) microglia precursors was found (N=3). Furthermore, as was observed in the facial axotomy model described above, the rare GFP+ cells observed in the spinal cord were Iba-1 negative and closely associated with the endothelium of blood vessels (Figure 2.7a–e). In sharp contrast to these results, when irradiated/transplanted mSOD1 mice transplanted with wild-type GFP+ donor  74  bone marrow were analyzed, GFP+ parenchymal microglia were readily identified at a frequency consistent with previous reports (Figure 2.7f–k, Table 2.2). These data suggest that the timing of development of microgliosis does not influence the recruitment of microglia precursors from the bloodstream.  Taken together, these results indicate that the replacement of microglia by circulating precursors can be induced by the experimental manipulations associated with irradiation and transplantation but this replacement does not take place under physiological conditions.  75  76  Figure 2.7 Circulating progenitors do not contribute to the microgliosis induced by ALS in the spinal cord of parabiotic mSOD1 transgenic mice (a,b) Representative section through the spinal cord of an mSOD1 transgenic parabiotic mouse at disease end stage. No partner-derived (GFP+) cells expressing Iba1 were observed in any of the three animals analyzed. Scale bar represents 500 μm. (c–e) Higher magnification analysis of the rare GFP+ cells observed revealed their intravascular position. Scale bar represents 50 μm. (f–k) Contribution of GFP+, bone marrow–derived cells to microgliosis in the spinal cord of irradiated/transplanted mSOD1 mice. Arrowheads point to examples of cells expressing both GFP and Iba-1. Note that the vast majority of parenchymal GFP-expressing cells were also positive for Iba-1. Scale bars represent 100 μm for f–h and 50 μm for i–k. GFP is shown in green, PECAM in blue and Iba-1 in red for all panels.  77  Table 2.2 BM-derived microglial engraftment in mSOD1 spinal cords after irradiation/transplantation  Animal ID  Sections ID  Iba-1+ cells  GFP+/Iba-1+ cells  Percentage  a  233  60  20.8  b  193  59  30.6  c  250  57  22.8  a  122  35  28.7  b  263  58  22.1  c  258  66  25.6  a  272  58  21.3  b  193  46  23.8  c  201  42  20.9  1  2  3  Percentage of ramified GFP expressing microglial cells (Iba-1+) in spinal cord of mSOD1 mice transplanted with mixture of GFP+ and GFP- BM cells. Engraftment at disease endstage was quantified by counting all the microglial cells (Iba-1+) in a ventral quarter of each spinal cord. Average: 22% ± 3.5.  78  2.3.3 Irradiation alone is not sufficient to trigger the entry of circulating myeloid cells into the parenchyma following CNS injury  Two potential mechanisms could, alone or in combination, underlie the effect of the irradiation/transplantation procedure on microglia precursor recruitment. First, circulating cells that are not normally capable of migrating through an intact BBB may be able to cross it following its disruption by the irradiation dosage used for myeloablation. Second, cells with the potential to cross the BBB and differentiate into microglia may exist within adult bone marrow, but not normally be mobilized to the circulation. These cells would be mechanically extracted from the bone marrow and injected in the bloodstream of the recipient during transplantation. We asked whether disruption of the blood-brain barrier by irradiation is sufficient to allow microglia precursors into the CNS. To this end, parabiotic pairs of healthy GFP– and GFP+ mice were established as described. After one month of parabiosis, the GFP+ partner was placed into a lead container while the GFP– partner was left unshielded, both were irradiated at the same dosage used for myeloablation (Figure 2.8a). As a result, we expected that hematopoietic stem cells from the shielded partner, which are known to migrate through the circulation, would repopulate the bone marrow of the unshielded partner. This model has the advantage that only cells that can mobilize in response to the signals induced by irradiation damage will reach the circulation and migrate to the myeloablated partner. The GFP– myeloablated partners (N=3) were subjected to facial nerve axotomy five weeks after irradiation and analyzed after  79  one additional week. In these animals, up to 80% of bone marrow cells expressed  GFP,  suggesting  that  myeloablation  and  repopulation  by  physiologically migrating (Abkowitz et al., 2003; Wright et al., 2001b) hematopoietic stem cells was efficient (Figure 2.8b). Thus, in these parabiotic pairs essentially all nucleated cells generated in the bone marrow of each partner are GFP+. Furthermore, hypothetical GFP+ microglia progenitors would be produced by the bone marrows of both animals, overcoming any barrier to the free exchange of these cells between the parabiotic partners (Liu et al., 2007).  Despite the presence of an average of 78% GFP+ cells in the peripheral blood during the establishment of microgliosis following axotomy, none of the Iba-1+ cells observed at the level of the axotomized facial nucleus was partner-derived and the few GFP+ cells found in the area of damage appeared to be closely associated with vessels (Figure 2.8c–h). These data indicate that BBB disruption caused by irradiation alone is not sufficient to allow the entry of microglia precursors from the bloodstream into the CNS. They also strongly suggest that, unlike hematopoietic stem cells, microglia precursors do not physiologically leave the bone marrow and migrate through the bloodstream.  80  Figure 2.8 Irradiation is not sufficient to trigger the entry of blood-borne microglia progenitors into the CNS (a) Picture of the device used to shield the GFP+ parabiotic partner during the irradiation of the GFP– partner. (b) Up to 80% of the irradiated partner’s bone marrow cells expressed GFP at the time of harvesting, suggesting that myeloablation and repopulation by physiologically migrating cells was efficient. (c–h) Bone marrow–derived cells did not contribute to facial axotomy–induced microgliosis in irradiated parabiotic mice. Brain stem sections spanning the facial nucleus were stained with Iba-1 (red) and PECAM (blue). GFP is shown in green. Despite the intense microgliosis and the fact that an average of 78% of 81  peripheral blood cells were GFP positive in these mice, no bone marrow–derived microglia were observed, and the rare GFP+ cells in the area of damage appeared to be located in blood vessels. Scale bars represent 100 μm in c–e and 50 μm in f–h.  82  2.4 Discussion  Microglia are CNS-resident hematopoietic cells closely resembling tissue macrophages and act as the primary sensors of brain pathology. They are rapidly activated by most CNS insults and accumulate in large numbers at sites of injury. Consistent with macrophage recruitment to peripheral tissues, bone marrowderived progenitors have been proposed to reach the ailing CNS through the bloodstream and participate in disease-induced microglia expansion (Hess et al., 2004) (Flugel et al., 2001; Kennedy and Abkowitz, 1997; Krall et al., 1994; Priller et al., 2001; Priller et al., 2006). However, unlike tissue resident macrophages, CNS-resident microglia have been shown to respond to damage signals by rapidly entering the cell cycle and undergoing extensive local expansion (Graeber et al., 1988; Solomon et al., 2006). This has led to the distinction between local, CNS-resident microglia and infiltrating bone marrow-derived macrophages, with different specialized functions proposed for each population, despite the phenotypic and functional similarity of these two cell types (Graeber et al., 1998; Solomon et al., 2006). Importantly, all studies addressing the participation of circulating myeloid precursors to the microglia pool, or the role of infiltrating monocyte-derived macrophages in CNS disease, have relied on the establishment  of  peripheral  blood  chimerism  by  lethal  irradiation  and  transplantation of whole bone marrow. As explained above, here, we compared the results obtained using this traditional technique with those obtained in animal models in which peripheral blood chimerism was obtained by parabiosis.  83  Parabiosis has the advantage that it does not require experimental manipulations of the hematopoietic system, thus allowing the study of cell migration through the bloodstream in near-physiological conditions (Wright et al., 2001b). In contrast to what we observed in irradiated/transplanted animals, no contribution of bone marrow-derived cells to the microglia pool was detected in healthy parabiotic animals.  To rule out the possibility that the contribution of bone marrow cells to CNS microglia might only be revealed during microglia expansion, we induced microgliosis in parabiotic mice using two distinct models of CNS disease of different etiology. From each of these models, our data strongly indicate that in the absence of experimental manipulations of the hematopoietic system, microgliosis can result solely from the expansion of CNS-resident cells. The lack of recruitment of myelomonocytic progenitors from the bloodstream is specific to the CNS, as Iba-1+ tissue macrophages derived from circulating precursors were readily observed when muscle damage was induced in the same mice. In contrast with our findings, others have reported a low level of contribution of bone marrow-derived cells to CNS microglia in a single pair of mice parabiosed for seven months (Massengale et al., 2005). We could not reproduce this finding in two pairs joined for five months, despite using identical mouse strains. Furthermore, despite analyzing a total of 15 parabiotic pairs subjected to different types of CNS damage we found no evidence of participation of bone marrowderived cells to reactive microgliosis, strongly indicating that hypothetical newly  84  engrafted hematopoietic progenitors would not contribute to microglia function. Thus, our findings should prompt a careful reinterpretation of the literature describing the recruitment of circulating microglia precursors to sites of damage within the CNS.  Taken together these observations strongly suggest that the CNS microglia, under the conditions studied here, are a closed system and are not replenished by bone marrow-derived progenitors. The expansion potential of CNS-resident microglia is sufficient to provide enough progeny for the lifetime of the organism, both in health and disease. Thus, CNS-resident microglia can self-renew.  A potential limitation of the parabiotic model is that the frequency of partnerderived cells found in peripheral tissues is usually lower than the frequency of chimerism observed in the bloodstream (Donskoy and Goldschneider, 1992). Indeed, in our experiments, partner-derived cells represented only 20% of the macrophages recruited to damaged muscle. In theory, this could lead to an underestimation of the extent of recruitment of circulating microglia precursors to the CNS. However, we failed to observe partner-derived microglia even in animals that were subjected to irradiation while joined in parabiosis. In these mice, bone marrow cells from the non-irradiated partner efficiently colonized the bone marrow of the myeloablated partner, and the peripheral blood contained an average of 78% GFP+ cells at the time of analysis. These data exclude  85  incomplete blood mixing at the level of the CNS microcirculation as a basis of our results.  Another conclusion stemming from this study is that the reported endothelial disruption caused by irradiation is not sufficient to trigger the entry of microglia progenitors into the CNS. Yet, we readily observed partner-derived microglia in irradiated/transplanted animals at a frequency consistent with previous reports, indicating that cells with the potential to enter the CNS and generate microglia exist within bone marrow. However, these cells do not appear capable of spontaneously entering the bloodstream. Only when delivered to the circulation artificially do they reach the CNS and generate microglia. Thus these cells could be exploited as vehicles for gene delivery to the CNS, and their identification and isolation could benefit current strategies for the gene therapy of diseases such as lysosomal enzyme deficiencies. In a collaborative effort between Dr.Kreiger’s and our laboratory, we have previously shown that in transplanted/irradiated animals, bone marrow-derived microglia respond to damage by entering the cell cycle as efficiently as CNS-resident microglia (Solomon et al., 2006). Thus upon transplantation myelomonocytic progenitors give rise to a self-renewing population within the CNS but not within the bone marrow (Akashi et al., 2000), suggesting a strategy for the specific repopulation of the microglia compartment in the absence of bone marrow repopulation. The isolation of these progenitors and the implementation of such a strategy, which will be the subject of future work, may significantly reduce the risks associated with the transplantation of ex  86  vivo-engineered, long-term hematopoietic stem cells currently used to deliver genes to the CNS. As I have clearly demonstrated in this chapter, microglia are very much capable of self-renewal under hemostatic conditions, and in a subset of neurodegenerative diseases there is no entry of circulating cells into the CNS parenchyma. Questions regarding the contribution of blood-derived myeloid cells in conditions characterized by an inflammatory pathogenesis such as multiple sclerosis where other circulating cells have access to the CNS still, however, remain, and it is precisely these questions that the next chapter will shed light on.  87  Chapter 3: Infiltrating Monocytes Trigger EAE Progression but Fail to Contribute to the Resident Microglia Pool  3.1 Introduction  As discussed in the two previous chapters, within the central nervous system (CNS), immune functions are coordinated by microglia which monitor the tissue for injury or pathological changes (Nimmerjahn et al., 2005; Raivich, 2005). Alterations to CNS homeostasis leads to microgliosis, a process characterized by change in microglia morphology, cell surface antigen expression, proliferation and modulation of interactions with other immune cells (Streit et al., 1999).  Several reports have proposed that bone marrow (BM)-derived progenitors contribute both to microgliosis and microglia homeostasis based on experiments in which animals are lethally irradiated and their bone marrow is replaced with genetically labelled cells (Hess et al., 2004; Hickey and Kimura, 1988; Lawson et al., 1992; Priller et al., 2001). However, as discussed in chapter 2, our own recent work suggests that these reports must be interpreted with caution, as intravenous delivery of BM obtained by mechanical harvesting leads to altered dynamics of cell migration across the blood-brain barrier (BBB) (Ajami et al., 2007). Furthermore, recent work by Ginhoux and colleagous has confirmed that BM-derived cells do not enter the healthy CNS in the adult animal, and this research has also shown that microglia and blood-derived monocytes have  88  distinct embryonic origins, with microglia seeding the CNS early during embryonic  development  in the absence of  a contribution from adult  hematopoietic stem cells (Ginhoux et al., 2010). The active exclusion of bloodderived monocytes from the CNS during normal homeostasis, as well as in some neurodegenerative diseases, suggests that their role is fundamentally different from that of microglia and that, when mechanisms excluding blood-derived monocytes from the CNS parenchyma fail, monocyte recruitment into the CNS likely heralds a new phase of disease pathology.  Experimental autoimmune encephalomyelitis (EAE) is a well-studied murine model of the human disease, multiple sclerosis (MS), characterized by extensive infiltration of the CNS by inflammatory cells (Hickey, 1991). Initiation of EAE involves the activation of myelin-specific Th1 or Th17 cells, which in turn trigger the expansion of resident microglia as well as the recruitment of blood-borne myelomonocytic cells (Hickey et al., 1991; Swanborg, 1995). However, it is yet to be determined whether either of these events lead to further demyelination or, conversely, to neuroprotection by limiting T cell-induced damage (Steinman et al., 2002). In particular, the fundamental mechanisms leading to the distinct stages of relapsing and remitting disease, and the associated physical impairment, remains highly controversial.  Specific functional roles have been proposed for resident microglia and bloodderived monocytes/macrophages (Santambrogio et al., 2001; Ulvestad et al.,  89  1994). Experiments using BM chimeras suggest that the activation of resident microglia may represent one of the initial steps in EAE pathogenesis, preceding and possibly triggering the infiltration of blood-derived cells (Heppner et al., 2005; Ponomarev et al., 2005). In support of a role for monocytes and macrophages in the exacerbation of EAE, depletion of these cells using silica dust or liposomes containing dichloromethylene diphosphonate (Bauer et al., 1995) induces a marked suppression of clinical signs of EAE (Brosnan et al., 1981; Huitinga et al., 1990). Similarly, blockade or genetic deletion of CCR2, a chemokine receptor known to be involved in monocyte and T cell trafficking, prevents severe disease and can lead to faster remission (Fife et al., 2000; Izikson et al., 2000; Mildner et al., 2009). In addition, increases in circulating inflammatory monocytes have been shown to correlate with relapses in a murine model of EAE (King et al., 2009).  A key limitation to these transplantation experiments is that they lead to the artificial long-term replacement of a significant fraction of CNS microglia with donor BM-derived cells (Kennedy and Abkowitz, 1997). The presence of these donor-derived cells, which can expand locally in response to disease, as well as the absence of a specific marker for distinguishing microglia from blood-borne monocytes, has made it challenging to precisely determine the kinetics and relative importance of the entry of circulating monocytes and expansion of endogenous microglia.  90  It has been reported that lethal irradiation significantly alters the integrity of the BBB (Diserbo et al., 2002; Li et al., 2004b). Elegant work by Mildner and colleagus in which the head of recipient mice was shielded during myeloablation supports the notion that irradiation is necessary for the engraftment of microglia precursors in a BM transplant setting (Mildner et al., 2007). However,as disscused in chapter 2, our work indicates that while lethal irradiation is required for donor cells to reach the CNS, it is by itself not sufficient. Rather, harvesting of BM cells via flushing or crushing donor bone and subsequent introduction into the blood circulation is required in conjunction with irradiation in order for engraftment of infiltrating cells to occur (Ajami et al., 2007).  We have taken advantage of this finding to establish a new experimental approach based on a combination of parabiosis and irradiation which avoids the use of harvested BM, allowing the efficient replacement of bone marrow and peripheral blood cells in the complete absence of CNS repopulation. We combined this approach with transgenic labels to follow the entry of blood-borne monocytes into the CNS and their subsequent fate at specific stages of EAE progression and following remission. The results presented here reveal a dynamic interplay between blood-derived myelomonocytic cells and microglia and strongly support a causal link between monocyte invasion and disease progression. Indeed, we show that specifically blocking the entry of circulating inflammatory monocytes into the CNS prevented progression to severe disease. Additionally, we show that the invasion of the CNS by blood-borne  91  myelomonocytic cells is a transient event and that these cells do not provide a long-term contribution to the resident microglia pool. Our data identify the invasion of circulating monocytes into the CNS parenchyma as a major, outcome-altering event in EAE progression and suggest that therapeutic strategies specifically aimed at inhibiting the migration of monocytes rather than that of leukocytes in general would be efficacious.  3.2 Material and Methods  3.2.1 Mice  C57BL/6 mice expressing GFP ubiquitously under the control of CMV-βactin hybrid promoter (C57BL/6; GFP/ CD45.2) were a kind gift from Dr. I. Weissman. C57BL/6, CX3CR1GFP/+ and CCR2-/- mice were purchased from Jackson Laboratories. In the case of CX3CR1GFP/+, heterozygotes obtained by crossing homozygotes with C57BL/6 mice were used as donors in transplantation experiments. CCR2-/- C57BL/6 mice were bred in-house to the ubiquitous GFP strain. All mice were maintained in a pathogen-free facility, and all experiments were performed in accordance with the policies of the University of British Columbia’s Animal Care Committee.  92  3.2.2 Generation of chimeric mice by parabiosis/irradiation  Female mice aged between four to six weeks old and at least 17 gr in weight were selected for parabiosis. Age- and weight-matched mice were housed together for at least one week prior to surgery. Pairs of parabiotic mice were surgically generated as previously described. Two weeks post surgery, the GFP+ partner was shielded with lead and the pair subjected to a lethal dose of irradiation (1100 rads) using a gamma cell irradiator with a cobalt 60 source. Four weeks following irradiation, the parabiotic pairs were surgically separated.  3.2.3 Generation of chimeric mice by irradiation and transplant  Preparation of CX3CR1/GFP+ BM: six to ten weeks old CX3CR1GFP/+ female mice were used as BM donors. Donor mice were sacrificed with CO2 and their femurs and tibias removed. Marrow cavities were flushed with FACS buffer (Phosphate Buffered Saline (PBS) supplemented with 2% FBS and 2mM EDTA) using a 25-gauge needle attached to a syringe. Erythrocytes were lysed by resuspending samples in ammonium chloride solution and incubating at room temprature for five minutes.  Nucleated cells were suspended in FACS buffer and CX3CR1 expressing cells were sorted based on GFP expression. Sorts were performed on a FACS Vantage SE (Becton Dickenson). Cells were sorted in PBS.  93  3.2.4 Isolation of c-Kit+ Lin− Sca-1+ (KLS) cells  Bone marrow cells were isolated and stained on ice for 30 minutes with an antibody cocktail containing anti- CD3, Mac1, Gr1, Ter119, B220 antibodies directly conjugated to PE-Cy7 (lineage cocktail, BD Biosciences), c-kit−FITC or PE (clone 2B8, BD Pharmingen) and Sca1-APC (clone: D7, eBiosciences). Following staining, cells were sorted using a FACS Vantage or a FACS Aria (Becton Dickinson). Cells were sorted in PBS.  3.2.5 Transplantation  Immediately before transplantation four to six weeks-old C57BL/6 mice received a lethal dose of irradiation (1100 rads) as described above. Recipient mice received 1.5 × 106 sorted GFP+ BM cells from CX3CR1GFP/+ mice and/or 500 radioprotective KLS cells via the tail vein.  3.2.6 Peripheral blood analysis  Peripheral blood was obtained from the tail vein. Red blood cells were lysed as described and the white blood cells were resuspended in FACS buffer. Analysis was accomplished using a FACS Calibur (Becton Dickinson), and flow cytometry data were analyzed using FlowJo (Treestar) analysis software .  94  3.2.7 Induction of autoimmune experimental encephalitis  Mice were immunized by subcutaneous injection of the myelin peptide MOG35– 55 (200 µg) emulsified in complete Freund’s adjuvant containing 4 mg/ml Mycobacterium tuberculosis (Difco) three weeks after parabiosis or one week after separation. In addition, 200 ng of pertussis toxin (Biological Laboratories) was administered intravenously on the day of immunization and two days after.  3.2.8 Animal harvest and tissue preparation  Animals were injected with a lethal dose of avertin and monitored. Upon the loss of nociceptive reflexes, animals were perfused transcardially with 20 ml PBS /EDTA followed by 20 ml 4% paraformaldehyde in 0.1 M PBS at room temperature. The spinal cord was removed and the tissue was post-fixed for 24 hours in 4% paraformaldehyde at 4 °C and then cryoprotected in a 24% sucrose solution in PBS for 24 hours. Spinal cord tissues were embedded in OCT (Tissue-Tek) and frozen at -80°C.  3.2.9 Immunofluorescence of tissue sections  After tissue processing, 20 μm thick cryosections were cut from the lumbar region of spinal cords (L1-L5) resulting in approximately 500 sections per lumbar region for each mouse. Sections were examined using a Zeiss Axioplan 2  95  microscope equipped for epifluorescence (Carl Zeiss, Inc.) and the sections with most infiltrated cells (GFP+ cells) selected for immunofluorescence. Sections were allowed to thaw at room temperature, rehydrated in PBS for two hours and incubated with blocking buffer (25% normal goat serum, 3% BSA, 0.3% Triton X100) for another hour. FcγR was blocked by incubating the sections with 5–10 μg/ml purified anti-CD16/32 antibody on ice for 10 minutes. Primary antibody staining was performed overnight. Primary antibodies included: anti-Iba-1 as a microglia/macrophage marker (Wako), anti-mouse CD31 as an endothelial marker (PECAM-1, BD Pharmingen), T-cell marker anti-CD4 (clone L3T4, eBiosciences) and anti-BrdU (clone B44, BD). Secondary antibodies were Alexa568- or Alexa647-conjugated goat anti rat (Molecular Probes) and in the case of anti-BrdU, goat anti-mouse IgG1 Alexa568 (Molecular Probes). All sections were analyzed by confocal microscopy using a Nikon C1 Laser Scanning Confocal Microscope. Unless otherwise indicated, all images presented are maximum intensity projections of Z stacks of individual optical sections.  3.2.10 Quantification and statistics  Cells were manually counted in two diagonally opposite quadrants in the three sections with the most infiltrated cells (GFP+ cells) for each animal. All counts were blinded. All of the statistical tests were performed using Microsoft Excel X software.  96  The mean number of cells per quadrant counted in each group was calculated. Error bars in all figures represent the standard deviation. Unpaired t-tests (twotailed) were used for all statistical analyses. Linear regression analysis was performed using Microsoft Excel X software. When the data is presented as averages in the figures, the individual data points can be found in Table 3.1, and the related P values between each data set can be found in the legend to the same table.  3.3 Results  3.3.1 Presence of infiltrating myelomonocytes in parabiotic mice correlates with progression to severe EAE  To evaluate the kinetics of inflammatory cell infiltration and endogenous microglia expansion in EAE we needed to develop a method for tracking circulating cells without affecting the CNS environment or resident cells. First, we induced peripheral blood chimerism by surgically joining two syngeneic mice, one of which ubiquitously expressed GFP (Wright et al., 2001a). These parabiotic mice establish a rich anastomotic circulation, which quickly led to efficient peripheral chimerism9. Two weeks after parabiosis, the presence of GFP+ cells in the blood of the non-GFP partner was verified and EAE was induced in both partners by immunization with myelin peptides and adjuvant. Mice were monitored daily for  97  development of clinical signs of EAE (N=15). Two weeks after induction, the spinal cords of these animals were harvested.  Consistent with their key role in the pathogenesis of EAE, partner-derived GFP+ CD4+ T-lymphocytes were readily identified in the spinal cords of all animals (Figure 3.1). This confirmed that the extent of circulatory exchange in parabiotic pairs is sufficient for cells from one partner to reach the CNS of the other. A caveat of this model is that parabiosis places animals under increased levels of stress, a situation that is well known to dampen EAE progression (Bukilica et al., 1991; Mason, 1991). Although EAE developed in all 15 parabiotic pairs examined, we found that in ten of those pairs, EAE was initiated but failed to progress beyond clinical score two (hind limb weakness and loss of tail tone). Intriguingly, in animals where disease failed to progress to paralysis, we found no evidence of partner-derived macrophage (Iba-1+, GFP+) infiltration of the CNS despite the activation of resident microglia (Figure 3.1). Thus, while disease initiation leading to mild functional impairment can take place independently of blood-derived monocytes, a clear correlation exists between the presence of inflammatory infiltrates and EAE progression.  98  Figure 3.1 Efficient entries of partner-derived T-lymphocytes, but not myelomonocytic cells in the spinal cords of EAE-induced parabionts (a–e) Distribution of GFP+ partner-derived cells (green), CD4+ lymphocytes (blue), and Iba-1+ microglia in the spinal cord of a GFP- parabiotic mouse two weeks after immunization with myelin antigens. (a,b) Collage of maximum intensity projections of confocal image stacks showing a view of the entire spinal cord. The rare “yellow” appearing cells are due to the overlap of a green and a red cell present in different optical sections. Scale bars: 500μm. (c–e) Individual optical sections confirm that the majority of the GFP+ cells (green) stain for CD4 (blue) and that none of the GFP+ cells expressed Iba-1 (red). Scale bar: 50m. (f–h) Spinal cord sections were stained for the microglial marker Iba-1 (red) and 99  the endothelial marker PECAM (blue). Partner-derived GFP+ cells (green) were readily observed outside of the blood vessels in the spinal cords of parabiotic mice affected by EAE. However, none of the GFP+ cells was also positive for Iba1. Scale bars: 50μm.  100  To further explore the relationship between myelomonocytic infiltration and disease progression we modified our model so that mice would develop more severe disease. Parabiotic pairs of healthy GFP- and GFP+ mice were initially established (Figure 3.2a). Two weeks after the parabiosis surgery the GFP+ partner was placed into a lead container while the GFP- partner was left unshielded. The pair was then irradiated at the same dosage used for myeloablation (Figure 3.2a). Four weeks after irradiation, the peripheral blood of GFP- myeloablated partners contained an average of 79% GFP+ cells (Figure 3.2b, c). Consistent with our previous results and in contrast to data obtained using injected bone marrow cells, spontaneously migrating partner-derived cells did not colonize the CNS of irradiated animals, thus yielding chimeric mice in which hematopoietic cells throughout the body, but not CNS microglia, are GFP+ (Figure 3.2d).  Four weeks after irradiation, the partners were surgically separated and allowed to recover for two weeks (Figure 3.2a). Separated mice retaining over 60% GFP+ cells in their peripheral blood were identified and selected for EAE induction (Figure 3.2a). Thereafter, blood chimerism was consistently found to be stable, regardless of disease induction (Figure 3.2b).  101  102  Figure 3.2 Irradiation and separation of parabiotic mice leads to peripheral blood chimerism in the absence of donor cell entry into the CNS (a) Schematic representation of the experimental strategy. (P, parabiosis) Parabionts were generated by joining GFP+ and GFP– C57BL/6 mice. (I, irradiation.) Two weeks after surgery the GFP+ partner was shielded with lead, and the GFP– C57BL/6 partner was subjected to a lethal dose of irradiation. Average blood chimerism at this time point prior to irradiation was 55.7%. (C, chimerism) During the following four weeks, GFP+ cells migrate from the shielded partner to the irradiated one, and reconstitute its bone marrow. (S, separation) Four weeks post irradiation the parabionts were surgically separated. (E, EAE induction) Two weeks after separation, EAE was induced in the chimeric mice. Average blood chimerism at this time point was 79.6%. (b) Percentage of GFP+ cells in the blood of GFP– partners. The data presented in the plot correspond to the steps depicted in Figure 1a panel. For each data point, N=6. (c) Representative FACS plot showing the frequency of GFP+ cells in the blood of a GFP– partner four weeks after lethal irradiation. (d) Representative image of the spinal cord from healthy irradiated–separated parabionts not subjected to EAE induction (N=10 animals). Spinal cord sections were immunostained for the mature macrophage and microglia marker Iba–1 (red) and the endothelial marker PECAM (blue). GFP, identifying partner-derived cells, is shown in green. Images shown are maximum intensity projections of stacks of confocal optical sections. Note that no infiltration of GFP–positive cells was detected in healthy spinal cords. Scale bar: 500 μm.  103  Following EAE induction, mice were monitored daily for development of clinical signs and a group of six animals was harvested at each of the four clinical scoress, defined as follows: (1) loss of tail tone, (2) hind-limb weakness with gait abnormality, spasticity, and/or ataxia, (3) severe hind-limb weakness and partial paralysis, (4) complete hind-limb paralysis. This parabiosis/irradiation-/separation strategy allowed susceptibility to EAE, with 23 of the 24 mice that were not harvested for analysis at disease scores of one or two developing severe functional impairment.  The presence of infiltrating blood cells within the CNS parenchyma at each clinical score of EAE was measured by assessing the frequency of partnerderived GFP+ cells in spinal cord lesions. Blood-derived cells were already evident in the CNS parenchyma of animals with disease score of one and they increased in number as the disease progressed to score four (Figure 3.3a–f, Figure 3.4 and Table 3.1). The increase in infiltrating cell number correlated significantly with disease severity, rather than with the time lapsed since disease induction, suggesting that recruitment of blood-borne cells is linked to the tissue damage associated with EAE pathology, either as a cause or consequence (Figure 3.5, Figure 3.3b).  104  105  Figure 3.3 Monocytic infiltration correlates with progression to paralytic stages of EAE (a) Histogram representing the total number of GFP+ cells at different disease scores. To collect the data shown in graph a two diagonally opposite quadrants in each of three sections from the spinal cords of six animals per disease stage were counted. (b) Graph shows a strong correlation between total number of GFP+ cells and the progression of EAE disease. Data points represent individual animals. (c–f) Distribution of GFP+ blood-derived cells (green) and Iba-1+(red) microglia in the spinal cord at four clinical scores of EAE. Collages of maximum intensity confocal stacks projections showing a view of the entire spinal cord. Staining with endothelial marker PECAM (blue) illustrates the spatial relationship between blood-derived inflammatory cells (GFP+) and blood vessels. Outlined areas are provided in higher magnification as well as separated channels in Figure 3.4. Scale bars: 500 μm.  106  Figure 3.4 Blood-derived inflammatory cells are recruited to the diseased CNS and their increased number correlates with the disease progression (a–p) Higher magnifications of the boxed areas in Figure 3.3. The panels show increased cell number and changes in the morphology of GFP+ cells as the disease progress to score four. Iba-1:Red , GFP: green, PECAM: Blue. Scale bars: 50 μm.  107  Table 3.1 Infiltration of blood-derived inflammatory cells increased as the disease progress to the paralytic stages (Figures 3.3, 3.7, 3.10)  a  b  c  d  a  b  EAE Clinical Score  One  Two  Three  Four  Average total number of GFP+ cells  5.4 ±3.5  37.8 ±11.3  77.2 ±22.4  95.1 ±29.7  0  8.4±2.9  26.8±6.8  90.1±29.7  0  22.5±5.2  36.4±10.6  94.9±3.3  0  59±8.6  71±12.9  17±8.2  Average total number of macrophages (GFP+/Iba-1+) Mean Percentage of blood-derived macrophages (GFP+ / Iba-1+ ) Mean percentage of blood-derived monocytes (GFP+ / CD11b+/Iba-1-)  EAE Clinical Score  One  Two  Three  Four  Average total number of microglia (Iba-1+)  60.4 ±18.2  75.9 ±23.2  154.4 ±29.21  94 ±33.9  22.3±6.2  22.7±5.5  61.1±13.6  61.3±13.4  Mean Percentage of BrdU+ microglia (BrdU+/Iba-1+)  108  Table 3.1 Infiltration of blood-derived inflammatory cells increased as the disease progress to the paralytic stages (Figures 3.3, 3.7, 3.10) Two opposite quadrants in each spinal cord section were counted. The data represent average mean ± s.d. P values for each comparison point are as follow: (a) Average absolute number of GFP+ cells (Fig 3.3a). P value between stage one and two: less than 0.001; between stage two and three: less than 0.001; between stage three and four: less than 0.01. (b) Percentage of blood-derived monocytes (Fig 3.7a). P value between stage one and two: less than 0.001; stage two and three: less than 0.01; stage three and four: less than 0.001. (c) Percentage of blood-derived macrophages (Fig 3.7b). P value between stage one and two: less than 0.001; stage two and three: less than 0.001; stage three and four: less than 0.001. (d) Average absolute number of macrophages (Fig 3.10b). P value between stage one and two: less than 0.001; between stage two and three: less than 0.001; between stage three and four: less than 0.001. Activation of microglia precedes the infiltration of blood-derived inflammatory cells (Fig 3.10). Two opposite quadrants in each spinal cord section were counted. The data represent average mean ± s.d. P value for each comparison point is as follow: (a) Average absolute number of microglia (Fig 3.10b). P value between stage one and two: less than 0.01; between stage two and three: less than 0.001; between stage three and four: less than 0.001.  109  Figure 3.5 The number of blood-derived inflammatory cells found in spinal cord sections does not correlate with the time between EAE induction and tissue analysis  110  Evaluation of lineage-specific markers revealed that in mice with disease score of one most donor-derived cells were T-lymphocytes (Figure 3.6) and none were positive for myelomonocytic markers (Figure 3.7a, Figure 3.7c–f, Figure 3.8a). Recruitment of inflammatory monocytes (CD11b+, Iba1-) was first observed when animals reached a disease score of two (Figure 3.7a, 3.7g–j, Figure 3.8b) and cell numbers increased with disease progression to a score of three (Figure 3.7k–n, Figure 3.8c). Notably, donor-derived CD11b+ cells were initially found in proximity to the meninges suggesting that the entry of monocytes into the CNS is not the result of a generalized disruption of the BBB (Figure 3.3d, Figure 3.8b). Between clinical scores three and four most partner-derived GFP+ cells acquired expression of Iba-1 (Figure 3.7b), leading to a substantial reduction in CD11b+ Iba-1- cells found in the parenchyma and suggesting that infiltrating monocytes progressively differentiate into mature macrophages (Figure 3.7a,b, Table 3. 1).  The reduction in CD11b+ Iba-1- parenchymal cells and limited increase in GFP+ cell number between scores three and four (Figure 3.7a, Table 1) strongly suggests that at this stage the entry of new circulating monocytes into the CNS is greatly reduced compared to mice with disease score of two and three of the disease, despite the increase in functional impairment. In addition, the number of infiltrating macrophages correlated well with disease severity (R2= 0.89) but poorly with the time interval between disease induction and harvest (R2= 0.37, Figure 3.9). Therefore, a strong correlation exists between the extent of CNS infiltration by myelomonocytic cells and EAE disease progression, consistent with  111  that observed in the conjoined parabionts. Furthermore, most monocytes enter the CNS prior to the development of severe disease.  112  113  Figure 3.6 CD4+ lymphocytes are found in the spinal cord at all scores of EAE (a–l) Collages of confocal optical sections of the spinal cord of irradiated/separated parabionts at each clinical score of EAE. Macrophages/microglia are identified by staining for Iba-1 (red), CD4 staining is shown in blue and partner-derived cells are identified by GFP (green). Arrowheads indicate representative partner-derived lymphocytes. Scale bars: 100μm.  114  115  Figure 3.7 Blood-derived infiltrating cells retain monocyte characteristics during progression to paralytic disease (a) Graph showing the frequency of infiltrating cells (GFP+) displaying a monocytic phenotype (CD11b+ but Iba-1-) at each clinical score of disease. (b) Graph showing the frequency of infiltrating cells (GFP+) that express markers of mature macrophages (positive for Iba-1). (c–r) Representative high magnification images of spinal cord sections from each score of disease, stained for monocytic and macrophage markers. Lower magnification images showing the distribution of the cells across the entire section are available in Figure 3.8. Iba-1: Blue, GFP: green, CD11b: Red. (g–r) CD11b and GFP double positive cells with round morphology, representing blood-derived myelomonocytic cells, increase as disease progresses to paralytic stages. (o–r) At clinical score of four, however, the majority of blood-derived cells have changed morphology and acquired Iba-1 expression. Few monocytes are detected at this stage, suggesting cell influx is reduced relative to earlier stages of disease. Arrowheads point to representative monocytes. Scale bars: 50 μm.  116  Figure 3.8 Phenotype of infiltrating myelomonocytic cells at different stages of disease progression (a–d) Collages of maximum intensity projections of confocal image stacks showing a view of the entire spinal cord of mice at different clinical score of EAE. Scale bars: 500 μm. Higher magnification and split channel images of boxed areas at each time point are shown in Figure 3. Iba-1: Blue, GFP: green, CD11b: Red.  117  Figure 3.9 Differentiation of infiltrating inflammatory monocytes to macrophages after entry into the CNS correlates with disease progression rather than time elapsed from EAE induction (a) shows a strong correlation between the increase in numbers of blood-derived macrophages and the severity of EAE. In contrast (b) shows that the appearance of blood-derived macrophages does not correlate with time post-EAE induction.  118  3.3.2 Microglia and circulating inflammatory cells are recruited with different kinetics  Microglia activation is believed to play a key role in the regulation of CNS inflammation (Streit, 2002). To establish the temporal relationship between T-cell entry, microglia activation and infiltration of circulating monocytes we measured the incorporation of bromodeoxyuridine (BrdU) in Iba-1+ cells of the spinal cord during EAE progression. No BrdU incorporation was observed in the absence of EAE induction. BrdU+ microglia (Iba1+, GFP- cells) were already evident at disease score of one , a time in which no monocytic infiltrate is present in the CNS, confirming that activation of resident microglia is an early event in EAE pathogenesis (Figure 3.10a, Figure 3.10c–f, Figure 3.11a, Table 3.1). However, BrdU incorporation in microglia in the absence of infiltrating (GFP+) monocytes was also observed in three animals that were immunized but did not develop disease signs (Figure 3.12), suggesting that while microglia activation is required for EAE initiation (Heppner et al., 2005; Ponomarev et al., 2005) it may not be sufficient to trigger severe disease.  A significant increase in the frequency of proliferating microglia (P value < 0.001), as well as in the absolute number of Iba-1+ resident cells (P value < 0.001), was observed between mice with disease score of two and three (Figure 3.10a,b, Table 3.1). Since at these time points the majority of infiltrating monocytes have not differentiated (Figure 3.7a,b) the vast majority of Iba-1+ cells in the spinal  119  cord are derived from resident microglia, while CD11b+ Iba-1- monocytes are derived exclusively from circulating pools.  In contrast, between disease score of three and four, the fraction of BrdU+ microglia remained constant (Figure 3.10a, Table 3.1) and resident microglia number declined significantly in mice with disease score of four (P value < 0.001), concomitant with the generation of Iba-1+ macrophages from circulating precursors (Figure 3.10b). To further investigate the causes of the decline in cell number, we measured the rate of apoptosis in Iba-1+ GFP- cells by TUNEL staining. We observed a significant increase in TUNEL-positive microglia at disease score of four indicating that these cells are lost through apoptosis (Figure 3.13).  120  121  Figure 3.10 Kinetics of microglia expansion and blood-derived monocyte infiltration in EAE (a) Percentage of proliferating microglia (GFP-, BrdU+, Iba1+) during EAE progression. (b) Absolute number of resident microglia (GFP-, Iba-1+ cells) and blood-derived macrophages (GFP+, Iba-1+ cells) in the spinal cord parenchyma at different stages of disease. Microglia increase in number as disease progresses to a score of three and declines between scores of three and four, while the number of blood-derived macrophages continues to increase. (c–r) Representative high magnification images of spinal cord sections from each stage of disease, stained for BrdU and the macrophage marker Iba-1. Lower magnification images showing the distribution of the cells across the entire section are available in Figure 3.11. The presence of BrdU+, Iba-1+ cells at clinical score of one indicates that microglia activation takes place prior to inflammatory cell entry. GFP: green, BrdU: blue, Iba-1: red. Scale bars: 50 μm.  122  Figure 3.11 Proliferation of resident microglia during disease progression (a,b,c,d) Representative spinal cord sections of EAE-induced mice at four different clinical scores of the disease. Each panel shows a collage of maximum intensity projections of confocal image stacks showing a view of the entire spinal cord. A higher magnification and split channel images of boxed areas at each time point are shown in 3.10. GFP: green, BrdU: blue, Iba-1: red. Scale bars: 500 μm.  123  Figure 3.12 Endogenous microglia activation precedes the entry of bloodderived inflammatory cells in the CNS of EAE-induced mice (a,b,c) Confocal analysis shows the incorporation of BrdU in Iba-1+ cells in the absence of partner-derived GFP+ cells indicating that activation of resident microglia takes place prior to blood-derived inflammatory cell infiltration. Scale bars: 50 μm. Iba-1: Red. GFP: Green, BrdU: Blue.  124  Figure 3.13 Endogenous microglia undergo apoptosis between clinical score three and four of EAE (a–i) TUNEL staining in spinal cord sections of chimeric mice with score three and four of EAE disease. TUNEL positive (blue) endogenous microglia (Iba1+, GFP-) is readily detectable at score four (d–i) but not at score three (a–c) disease suggesting apoptosis as the possible mechanism for the observed decline in microglia number between the two stages. (g–i) Shows a higher magnification view of the boxed area in panel f. TUNEL: blue, GFP: green, Iba-1: red. Scale bar (a–f): 100 μm. Scale bar (g–i): 50 μm.  125  3.3.3 Blocking inflammatory cell infiltration prevents EAE progression  Our data establish a clear correlation between monocytic infiltration and disease progression. To clarify whether the two are causally linked and whether the lack of disease progression might be due to lack of monocytic infiltration, we used the parabiosis/irradiation/separation strategy to replace the peripheral blood and bone marrow of CCR2-/- animals with GFP+ CCR2+/+cells. The CCL2 (MCP-1) receptor, CCR2, is expressed at high levels on inflammatory monocytes and is required for the entry of these cells, but not T-cells, into the CNS. It has been previously shown that CCR2 is required for the development of severe EAE (Fife et al., 2000); whether this effect is due to infiltrating cells or to other cell types that express this receptor is still unclear. We found that CCR2-/- animals where the majority (86%) of circulating cells were replaced with GFP+ CCR2+/+ donor cells were fully susceptible to EAE and readily progressed to severe paralytic disease.  This  effect  correlated  with  the  restoration  of  blood-derived  myelomonocytic infiltration into the CNS. Conversely, we found that CCR2+/+ animals where the peripheral blood and bone marrow were replaced with cells from CCR2-/- mice expressing GFP ubiquitously had only mild EAE and never progressed beyond a disease score one (Figure 3.14). As expected, only rare GFP+ cells were detected in the CNS and none of these cells were positive for myelomonocytic markers (Figure 3.14d–g). Thus, interfering with the entry of myelomonocytic cells into the CNS successfully blocked EAE progression, clearly supporting a role for these cells in causing functional deficits.  126  127  Figure 3.14 Blocking monocyte infiltration prevents EAE progression The bone marrow and peripheral blood of CCR2+/+ animals were replaced with that of GFP-expressing CCR2-/donors (circles) using the parabiosis/irradiation/separation strategy. Conversely the peripheral blood and BM of CCR2-/- animals was replaced with GFP+ CCR2+/+ GFP+ cells (squares) using a similar experimental approach. EAE was induced in both groups. (a,b) Representative FACS plots showing the frequency of GFP+ cells in the blood of GFP– partners at the time of EAE induction. (c) Average clinical score for each group of animals (CCR2-/- to wild-type: N=10, wild-type to CCR2-/-: N=9). CCR2+/+ animals repopulated with CCR2-/- cells failed to progress beyond stage one disease, while blood repopulation with GFP+ CCR2+/+ cells restored the susceptibility of CCR2-/- animals to severe paralytic disease. Asterisks indicate a p value lower than P 0.01. (d,f) abundant GFP+ cells staining for the myelomonocytic markers Iba-1 (d) and CD11b (f) are found in spinal cord sections of CCR2+/+ animals reconstituted with GFP+ BM. (e,g) None of the donor cells found in spinal cord sections of C57/B6 mice reconstituted with GFP+ CCR2-/- express myelomonocytic markers. Iba1: Red, GFP: green. Scale bar: 50 μm.  128  3.3.4 The progeny of infiltrating blood-derived monocytes does not contribute to the CNS-resident cell pool  Our work, as outlined in chapter 2, suggests that unlike most peripheral macrophages, which are replenished by blood-derived precursors, CNS-resident microglia are capable of self-renewal in situ. However, here we show that in EAE circulating monocytes efficiently enter the CNS and give rise to mature macrophages that are phenotypically indistinguishable from resident microglia. Do these macrophages also acquire the ability to self-renew within the CNS, and thereby contribute long term to the microglia pool? To determine whether colonization of the CNS by blood-derived myelomonocytic cells is transient or sustained, we analyzed the spinal cords of three irradiated/separated parabiotic mice that were allowed to recover from severe EAE. In these mice, we found a dramatic reduction in parenchymal GFP+ cells compared to the numbers observed at the peak of disease (score of four). In addition, none of the remaining GFP+ cells were Iba-1+ (Figure 3.15) suggesting that the presence of blood-derived CNS myelomonocytic cells in the CNS parenchyma is a transient event.  Following significant expansion in the transition to paralytic disease a large fraction of resident microglia undergoes apoptosis, leading to a return of cell numbers to baseline. To determine whether all activated microglia are ablated after remission we labelled proliferating microglia in three irradiated/separated  129  parabiotic mice using BrdU during the week preceding maximum disability. Three months later, following complete remission, BrdU-labelled ramified microglia were clearly evident (Figure 3.15) suggesting that, unlike the progeny of infiltrating monocytes, expanded resident microglia can persist long term in the CNS.  Figure 3.15 Blood-borne inflammatory cell infiltration is transient (a) Collages of maximum intensity projections of stacks of confocal optical sections from the spinal cord of chimeric animals generated by parabiosis/irradiation/separation that recovered from EAE. Notice that the rare partner-derived cells (GFP+) still present did not stain with Iba-1. Yet, at this stage some of the endogenous microglia are still BrdU+ (blue, arrow heads), indicating that they returned to quiescence during or shortly after BrdU treatment. (b–e) Provide higher magnification of the white box in a panel. Arrows indicate BrdU label-retaining microglia (GFP-, Iba-1+, BrdU+). Iba1: Red, GFP: green, Brdu: blue. Scale bar a: 500 μm, Scale bar (b–e): 50 μm.  130  3.3.5 Transplantation of hematopoietic stem cells, but not of lineage committed progenitors, leads to long-term contribution to microglia  Transplantation of irradiated animals with whole BM is known to lead to the longterm persistence of donor-derived microglia in the CNS (Kennedy and Abkowitz, 1997; Krall et al., 1994). In contrast, our data indicates that monocytes normally found in the circulation are unable to engraft the CNS of healthy mice, and that even when specific pathologies trigger their infiltration, their presence is transient. A possible explanation for these conflicting results is that the BM cells mediating  long-term  CNS  engraftment  after  irradiation  are  immature  hematopoietic precursors that do not efficiently enter the circulation under normal circumstances. Immature cells in bone marrow belong to one of two functional groups: (1) multipotent progenitors endowed with varying abilities to self renew, generally contained within the lineage negative, c-Kit+, Sca-1+ (KLS) subset and (2) committed progenitors, which can generate all mature circulating cells belonging to specific hematopoietic lineages but are unable to extensively self renew. To test which of these subsets can permanently contribute to microglia in irradiated recipients, we generated a set of irradiated/transplanted chimeric animals  in  which  C57BL/6  mice  were  reconstituted  with  BM-derived  myelomonocytic progenitors from CX3CR1GFP/+ heterozygous donors. In this strain the coding sequence for one allele of CX3CR1, the receptor for CX3CL1 (fractalkine), is replaced with GFP resulting in marker expression in all myelomonocytic cells including BM progenitors, circulating monocytes, tissue  131  resident macrophages and microglia (Jung et al., 2000; Saederup et al., 2010). The CX3CR1+ fraction of BM comprises a non-self-renewing progenitor population that can transiently repopulate the peripheral blood of recipients. Cells positive for the transgenic marker were sorted from the BM of CX3CR1GFP/+ animals based on GFP expression and transplanted into lethally irradiated recipients. As these cells cannot generate erythroid cells or platelets, survival of the myeloablated mice was ensured by co-transplanting a radioprotective dose of GFP-, syngeneic KLS cells (Figure 3.16). As expected, FACS analysis confirmed that GFP+ cells persist in the peripheral blood of recipients until 14 weeks post transplant, after which they become undetectable (Figure 3.17a).  Two weeks after transplantation, at a time at which most animals had over 10% circulating GFP+ cells, the recipients were subjected to EAE induction. The contribution of blood-derived GFP+ monocytes to the CNS of EAE mice was evaluated at a disease score of four, approximately two weeks post-induction. At this time, abundant GFP+, Iba1+ donor cells were observed in the spinal cord (Figure 3.17b). A second evaluation after three months revealed only rare (less than one per section analyzed) donor-derived myelomonocytic cells regardless of whether mice had completely recovered from EAE or had developed permanent functional impairment (Figure 3.17c,d). In stark contrast, Iba-1+, donor-derived microglia were readily detected in the spinal cord sections of a parallel group of irradiated recipients that received GFP+ KLS cells (Figure 3.17e,f). Thus, while mature inflammatory macrophages can infiltrate the CNS and trigger EAE  132  progression, their presence in the CNS is transient. Only uncommitted stem/progenitor cells are capable of generating long-lived microglia in irradiated recipients.  Figure 3.16 Sorting strategy for the isolation of c-Kit+ Lin- Sca-1+ (KLS) cells from bone marrow (a–c) Plots showing the details of the gating strategy used to isolate KLS stem/progenitor cells by flow cytometry.  133  134  Figure 3.17 Uncommitted stem/progenitor cells, but not myelomonocyticcommitted hematopoietic progenitors contribute to resident microglia in irradiated/transplanted recipients (a) Percentage of GFP+ cells in the peripheral blood of irradiated mice (N=6) transplanted with the GFP+ fraction of bone marrow from CX3CR1GFP+ donors, representing myelomonocytic-committed progenitors. As expected, the frequency of donor-derived myelomonocytic cells in the blood declines rapidly during the first month following the transplant. (b) Spinal cord sections from mice transplanted with CX3CR1GFP positive cells and harvested at EAE clinical stage four (approximately two weeks after EAE induction) reveal significant myelomonocytic infiltration. The absence of donor derived cells in spinal cord sections from animals harvested two months after the peak of disease suggests that blood-borne cells within the CNS are transient and do not contribute longterm to the microglia pool, independent of whether the outcome of EAE was a permanent functional impairment (c) or full recovery (d). (e,f) Spinal cord sections from mice transplanted with KLS stem/progenitor cells from a donor ubiquitously expressing GFP and harvested two months after the peak of disease contain numerous donor -derived ramified microglia (GFP+, Iba1+). Iba-1: Red, GFP: Green, PECAM: Blue. Scale bar (b,c,d): 500 m. Scale bar (e,f): 50m.  135  3.4 Discussion  While adoptive transfer studies have established that T cells are both necessary and sufficient for induction of EAE (Carson et al., 2006b; Engelhardt, 2006), the cellular mechanisms that govern disease progression are still debated. Microglia and blood-derived myelomonocytes are both implicated in the development of EAE because of their capacity to present antigens, secrete pro-inflammatory cytokines (Benveniste, 1997) and participate in demyelination by phagocytosis of degraded myelin (Bauer et al., 1994). However, it is unclear if these two cell types play distinct roles in EAE pathogenesis.  In this chapter, we report a novel irradiation/parabiosis-based experimental model that can distinguish between blood-derived monocytes and CNS-resident microglia in the absence of bone marrow transplantation. Using this model we have revealed key functional differences between resident and infiltrating cells. During early disease, T cell infiltration and microglia activation are evident, even in animals that do not develop functional impairment. In contrast, the appearance of infiltrating monocytes correlates with significant disability, and impairing the CCR2-dependent recruitment of these cells prevents progression from very mild to severe disease. Thus, in EAE, the infiltration of monocytes, a cell type that is normally excluded from the CNS both in healthy animals and in a variety of diseases, may represent a pathogenic overreaction by the innate immune  136  system. A similarly deleterious role of monocytes has been recently reported in acute viral meningitis (Getts et al., 2008; Kim et al., 2009).  Numbers of resident microglia drop dramatically between mice with disease scores three and four, a time in which the number of infiltrating myeloid cells increases and coincides with functional impairment. Thus, our data suggest that monocytes and microglia respond differently to environmental stimuli regulating their survival. Finally, differences between the two cell types are also evident in their  ability  to  colonize  the  CNS.  Although  monocyte-derived  mature  macrophages are phenotypically indistinguishable from resident microglia, they are specifically and completely removed from the CNS. In contrast, detection of BrdU-retaining cells three months after disease remission demonstrates that microglia can enter the cell cycle, proliferate and then return to quiescence.  If circulating monocytes are unable to generate long-lived microglia, which cell type is responsible for their production in irradiated/transplanted animals? The results presented in this chapter show that the ability to generate microglia is restricted to uncommitted stem/progenitor cells within bone marrow. Interestingly, others have shown that hematopoietic stem/progenitor cells can generate myelomonocytic cells within peripheral tissues in response to Toll-like receptor signaling (Massberg et al., 2007; Nagai et al., 2006).  137  Thus, two distinct types of myelomonocytic engraftment occur in the CNS. The first is triggered by neuroinflammatory pathologies and transiently recruits inflammatory monocytes to affected areas. The second, observed in irradiatedtransplanted subjects, is dependent on hematopoietic stem/progenitor cells and can lead to permanent contribution to the microglial pool. This second type of engraftment may be exploited to deliver therapeutic gene products across the BBB.  Taken together, these observations lead to a three step model of EAE progression: (1) CD4+ T cells and endogenous microglia are responsible for disease initiation; (2) the mechanisms excluding blood-borne monocytes from the CNS break down, and (3) infiltrating blood-derived monocytes trigger, directly or indirectly, EAE progression to the severe paralytic stage. Once the disease reaches a score of four, the decline of microglia and disappearance of infiltrating myelomonocytic cells leads to remission and, depending on the extent of axonal and neuronal loss, possibly recovery (Wujek et al., 2002).  Blocking the migration of immune cells across the BBB has long been regarded as a likely therapeutic approach for treating CNS-directed autoimmune diseases (Luster et al., 2005). Understanding the pathological cascade of EAE will help us tailor more specific therapies for multiple sclerosis. For instance, blocking the homing of T-lymphocytes to the CNS using an antibody specific for α4 integrin suppresses EAE and reduces relapse rates in humans with multiple sclerosis by  138  66% (Miller et al., 2003; Yednock et al., 1992). Unfortunately, in a subset of patients this treatment leads to the reactivation of latent CNS viral infections and progressive  multifocal  leukoencephalopathy  (Langer-Gould  et  al.,  2005;  Steinman, 2005). Our observations here provide a rationale for a therapeutic strategy that specifically targets myelomonocytic cell entry, which might have potentially fewer side effects than existing therapies.  139  Chapter 4: Conclusion  Overall, my research has focused on elucidating how microglia, the myeloid cell population of the CNS, are maintained in healthy and damaged central nervous systems during adult life. I have demonstrated that blood-derived myeloid cells do not contribute to the microglia population under healthy conditions, nor following either degenerative (ALS) or traumatic injury (facial nerve axotomy). Additionally, in the case of autoimmune induced inflammation, I have shown that blood-derived myeloid cells enter the CNS and this entry plays a role in the progress of the disease to a paralytic stage. I have further demonstrated that this cell population – blood-derived myeloid cells – does not permanently contribute to the resident microglia population of the CNS.  4.1 Microglia: Ever-Changing or Reliably Self-Renewing  Similar to tissue resident macrophages in peripheral organs, microglia become rapidly activated in response to inflammatory or injurious stimuli in the CNS. Tissue resident macrophages and dendritic cells (DCs) are bone marrow-derived myeloid cells that originate from the common monocyte, macrophage and dendritic cell precursor (MDP). Historically, it has been believed that they circulate in the blood as monocytes and populate tissues as macrophages under steady-state conditions and in response to tissue damage and inflammation (Geissmann et al., 2010). This notion, however, has been challenged as being  140  too simplistic in recent years, especially in cases such as Langerhans cells (the dendritic cells of the epidermis) (Merad et al., 2002). In contrast to dendritic cells that are located in peripheral lymphoid organs and are constantly replenished by bone marrow-derived precursors, Langerhans cells are self-renewing and are maintained by local proliferation of precursors that are present in the skin. However, inflammation in the skin as a result of exposure to ultraviolet (UV) irradiation induces the recruitment of circulating bone marrow-derived precursors to the skin that are differentiated to Langerhans cells (Merad et al., 2002).  Significantly, my research, as presented in this dissertation, also introduces similar new aspects in terms of the mechanisms that are responsible for homeostasis of the microglia population.  In the CNS, the activation of microglia, the resident myeloid cells, is accompanied by an alteration in their morphology, surface phenotype, gene expression and an increase in cell number, a process termed microgliosis (Streit et al., 1999). The activation of microglia is intended to eliminate the invading pathogens, resolve inflammation and restore tissue homeostasis. However, microglia activation could also be a deleterious event due to the production of neurotoxic  molecules  and  proinflammatory  cytokines,  which  would  be  devastating for neurons that have limited regenerative potential. The increase in the number of microglia in response to injury (microgliosis) acts as a diagnostic marker of CNS pathology (Perry et al., 2010). A major body of literature has used  141  bone marrow chimeras, created by lethal irradiation and replacement of endogenous bone marrow with donor derived genetically labelled bone marrow, in an attempt to understand the contribution of blood-derived myeloid cells to microgliosis induced by various CNS pathologies (Kennedy and Abkowitz, 1997; Simard and Rivest, 2004). Bone marrow chimeric animals are a valuable tool for distinguishing between microglia and blood-derived macrophages, which are morphologically and phenotypically indistinguishable. Furthermore, utilizing bone marrow chimeras, researchers have endeavoured to delineate the individual roles that are played by CNS-resident microglia and blood-derived macrophages given different types of damage to the CNS (Heppner et al., 2005; Ponomarev et al., 2005; Simard et al., 2006). These studies revealed that the increase in microglia numbers in damaged CNS is due to the infiltration of blood-derived myeloid cells that enter the CNS and differentiate into macrophages.  However, irradiation complicates the interpretation of these data due to its significant effects on both the integrity and the increased permeability of the blood-brain barrier (Diserbo et al., 2002; Yuan et al., 2003) (Wilson et al., 2009). Moreover, transplantation of wild-type bone marrow cells in lethally irradiated SOD1G93A mice, a model of ALS disease, performed in a collaborative effort between Dr.Kreiger’s and our laboratory, revealed that the contribution of bloodderived macrophages to microgliosis induced by this disease is small – only 20% (Solomon et al., 2006). We demonstrated that even though the recruitment of circulating bone marrow-derived cells increases as the disease progresses, the  142  frequency of blood-derived versus resident microglia remained the same (Solomon et al., 2006). These data raised the possibility that microgliosis may primarily be due to the expansion of CNS-resident cells.  Therefore, as described in chapter 2, we used the parabiotic mouse model to investigate the extent to which bone marrow-derived precursors contribute to the resident microglia population within the CNS parenchyma under normal conditions and during both chronic (ALS model) and acute (axotomy) models of CNS microgliosis. Parabiotic mice, which consist of two surgically attached congenic mice that share a common blood circulation, provide a physiological model for tracking the contributions of circulating bone marrow-derived precursors to peripheral tissues without the need for lethal irradiation and transplantation of the donor cells from the bone marrow.  While studies using bone marrow chimeric mice revealed that circulating bone marrow-derived precursor cells populate the CNS and differentiate to microglia thus contributing to the pool of parenchymal microglia (Simard and Rivest, 2004), we found no evidence of bone marrow-derived microglia in the CNS parenchyma of healthy parabiotic mice.  Previous studies in bone marrow chimeric mice demonstrated that transection of the facial nerve leads to a rapid increase in the number of microglia around the injured motor neurons of the facial nucleus, consisting of infiltration of both bone  143  marrow-derived myeloid progenitors that had differentiated into microglia as well as resident microglia (Flugel et al., 2001; Priller et al., 2001). The authors of these studies concluded that the entry of the blood-derived myeloid cells at the site of injury in this model where the blood-brain barrier disruption is absent is due to the signals from damaged neurons (Priller et al., 2001). However, our data from parabiotic mice revealed that the massive microgliosis observed in these animals following facial axotomy takes place in the absence of any contribution from blood-derived myeloid cells and relies exclusively on the CNS-resident microglia population.  Comparably, we demonstrated similar results in the transgenic animal model of amyotrophic lateral sclerosis (ALS) where, unlike previous reports from bone marrow chimeric mice, no recruitment of bone marrow-derived cells was revealed in microgliosis triggered by the disease. Conventional irradiated-transplanted chimeras were created parallel to these experiments that showed the engraftment of infiltrating blood-derived myeloid cells in the CNS in both disease models. These findings were corroborated by an independent study by Mildner and colleagues, published in the same issue of Nature Neuroscience journal as ours. In this study, bone marrow chimeras were generated by shielding their head from the irradiation field thus avoiding confounding effects associated with irradiation on cranial vasculature (Mildner et al., 2007). Similar to our data, the authors demonstrated that blood-derived myeloid cells do not contribute to parenchymal microglia in protected brains under normal conditions nor do they  144  contribute to microgliosis induced by chronic inflammation (cuprizone induced corpus callosum demyelination) and acute inflammation (facial nerve axotomy). However, blood-derived myeloid cells were still detected in unprotected irradiated parts of the CNS such as the spinal cord (Mildner et al., 2007).  Taken together these two studies suggest that irradiation is required for circulating blood-derived myeloid cells to enter the CNS and differentiate to parenchymal microglia. We subsequently demonstrated that lethal irradiation alone is not sufficient to entice the entry of the blood-derived myeloid cells to the CNS. Modifying the parabiotic model by lethally irradiating the parabiotic recipient while the GFP+ mouse was shielded, we demonstrated that following the facial nerve axotomy, no contribution of blood-derived myeloid cells was found in microgliosis around the injured motor neurons of the facial nucleus. This experiment, together with Mildner’s data as referred to above, where the bloodderived myeloid cells were identified in the spinal cord but not the protected brain of bone marrow chimeric mice, suggests that irradiation is necessary but not sufficient, and that transfer of bone marrow progenitors into the blood stream (transplantation) combined with irradiation is required for the recruitment of blood-derived myeloid cells across the blood-brain barrier and differentiation to a parenchymal microglia population.  Mildner and colleagues further identified the “inflammatory” subpopulation of monocytes characterized by expression of Ly6ChighCCR+ as the direct precursors  145  of parenchymal microglia that infiltrates the brain after irradiation and contributes to microgliosis evoked by inflammation (Mildner et al., 2007).  Here, our results, complemented by the Mildner study, suggest that the contribution of blood-derived myeloid cells – as reported by previous studies – to CNS microglia in healthy adult mice, ALS and traumatic damage is likely the consequence of irradiation and transplantation. This observation of the influx of peripheral myeloid cells into the CNS of bone marrow chimeric mice likely reflects an artefact of: 1. the damage by irradiation to the blood-brain barrier and 2. the non-physiological presence of bone marrow cells into the circulation by a bone marrow transplant that would not otherwise leave the bone marrow.  Ultimately, this research provides evidence that the parenchymal microglia population is sustained by the proliferation of resident cells in the healthy CNS, and it is the proliferation of resident microglia rather than the invasion of bloodderived myeloid cells that accounts for the increase in microglia cell numbers in response to a variety of CNS lesions.  As discussed in chapter 1, a recent study using runx1-(ER)-Cre and a YFP reporter to label extraembryonic yolk sac macrophages, indicates that adult microglia originate during early embryonic life from primitive myeloid precursors in the yolk sac (Ginhoux et al., 2010).  146  The cumulative conclusion from this study and our data described in chapter 2 is that microglia represent a distinct myeloid population that are established from primitive myeloid progenitors during the early-embryonic period and self-renews throughout adult life independent of any contribution from the periphery.  Significantly, a number of researchers, based on the data derived from using bone marrow chimeric animals, have suggested that blood-derived myeloid cells infiltrating the CNS have beneficial consequences, and have proposed that enhancing this immune response could be used as a therapeutic approach (Schwartz and Shechter, 2010; Shechter et al., 2009; Simard et al., 2006) While these findings may be valid, caution should be taken, and the entry of peripheral myeloid cells to the brain and spinal cord in different CNS pathologies should be re-evaluated in the light of our data prior to the design of any therapy based on this phenomenon.  4.2 Towards an Understanding of the Functional Differences of Myeloid Cells and Microglia  The exclusion of the central nervous system from the peripheral immune cells by the blood-brain barrier under physiological conditions is disturbed in the case of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). The essential pathological character of MS and EAE entails recruitment of inflammatory immune cells from the periphery, leading to a  147  cascade of local inflammatory reactions within the CNS that result in myelin destruction (Sanders and De Keyser, 2007).  Thus, the final specific aim of my research was to identify the role that bloodderived myeloid cells exert when they gain access to the immune privileged CNS parenchyma.  The cumulative data on EAE suggest that while autoreactive CD4+ T cells have the ability to initiate the disease, infiltrating myeloid cells and resident microglia likely play an essential role in the mechanism leading to demyelination and axonal damage within the CNS (Benveniste, 1997).  However, distinct roles for these two cell types have been difficult to establish due to a lack of any specific markers. Based on our data described in chapter 2, in healthy central nervous systems and some neurodegenerative diseases, it is essential to distinguish peripheral from CNS-resident myeloid cells to fully comprehend the protective or deleterious roles for each of these populations, individually, in the pathogenesis of different CNS diseases. Researchers have attempted to dissect distinct functional roles played by resident microglia and blood-derived myeloid cells in EAE through extensive studies using bone marrow chimeric mice (Becher et al., 2001; Heppner et al., 2005; Mildner et al., 2009; Ponomarev et al., 2005).  148  Heppner and colleagues, for example, created transgenic mice that express the thymidine kinase suicide gene under the control of the CD11b promoter (CD11bHSVTK). Creating bone marrow chimeric mice where only microglia expressed CD11b-HSVTK, they demonstrated that ablation of microglia following treatment with gancyclovir (GCV) during EAE delays disease onset, reduces disease severity and attenuates demyelination and inflammation in the CNS (Heppner et al., 2005). Furthermore, experiments using bone marrow chimeric mice have also illustrated that activation of resident microglia represents one of the initial steps in the development of EAE disease, suggesting that these cells are essential for development of the disease and promote the infiltration of blood-derived cells (Ponomarev et al., 2005).  The importance of infiltrating blood-derived myeloid cells in the exacerbation of EAE disease has also been shown in bone marrow chimeric mice, in which CCR2+ Ly-6Chi, the “inflammatory” subset of monocytes, were identified entering the inflamed CNS and playing a pathological role in EAE disease (Mildner et al., 2009).  Despite attempts to understand the role of individual myeloid cells in EAE disease, the relative influence of resident microglia versus recruited bloodderived myeloid cells in disease progression remained poorly understood.  149  These bone marrow chimeric experiments suffer from experimental confounds associated with irradiation and transplantation procedures as described in chapter 2. This experimental setting which leads to replacement of bone marrow and circulating cells with either genetically marked or knockout cells permits concomitant replacement of a significant percentage of the resident microglia cells even in the absence of any CNS damage (Simard and Rivest, 2004), which precludes the ability to draw a solid conclusion if the entry of myeloid cells into the CNS is merely a consequence of the CNS pathology or technical limitations. Therefore, in order to evaluate the causal role of CNS-resident microglia versus invading blood-derived myeloid cells in the development and progression of EAE disease, we utilized the irradiated-parabiosis model, in which the peripheral blood and bone marrow of the irradiated parabiotic recipient is replaced by GFP+ cells from the non-irradiated partner in the complete absence of colonizing the CNS parenchyma. This novel approach, as outlined in chapter 3, enables us to establish the relative importance of CNS-resident microglia versus infiltrating blood-derived myeloid cells in the progression of EAE disease.  Activation of resident microglia was evident as one of the first steps of the inflammatory cascade within spinal cord tissues of mice induced with EAE, whether or not these mice went on to develop the disease. Indeed, we observed activation of microglia in animals that failed to develop any signs of the disease. The CD4+ T–lymphocytes were identified at the onset of the disease correlating with the first sign of EAE. Activation of microglia prior to infiltration of CD4+ T–  150  lymphocytes, as it was observed in mice that were induced for EAE but failed to develop any signs of the disease, could lead to speculation that microglia activation triggers the entry of T-lymphocytes leading to the initiation of the disease. However, it is difficult to draw such a definitive conclusion, since the activation of microglia at this stage could also be due to the EAE induction protocol including the effect of pertussis toxin. Nonetheless, the evidence of microglia activation along with the presence of T-lymphocytes in the onset of EAE, as it was revealed at score one disease, suggest that CNS-resident microglia are important for the initiation of EAE disease.  On the other hand, infiltration of blood-derived myeloid cells – the other myeloid cell population involved in EAE – was correlated with disease severity. The entry and accumulation of blood-derived myeloid cells in the CNS was quantitatively associated with the spectrum of EAE severity.  Furthermore, prevention of the infiltration of blood-derived myeloid cells to the CNS by deletion of CCR2, a chemokine receptor known to be involved in monocyte trafficking, results in halting disease progress. This observation proves that there is a causal relationship between the infiltration of circulating cells and disease progression, and the entry of blood-derived myeloid cells is not the consequence of tissue damage.  151  Together, these data suggest that endogenous microglia activation is required for the initiation of the disease and precedes the entry of blood-derived cells. The responsibility for the progress of EAE disease, however, lies with blood-derived myeloid cells.  Importantly, we also identified that microglia and infiltrating blood-derived myeloid cells adopt distinguishably different fates within the CNS following inflammation. Resident microglia proliferate and undergo apoptosis within lesions during EAE disease and then they return to quiescence following remission and survive long term in order to restore myeloid homeostasis in the CNS. However, bloodderived myeloid cells are short lived in the CNS: they disappear following disease remission and do not contribute to the resident myeloid population of the CNS.  The results presented in this thesis, then, address a fundamental issue in myeloid cell biology in the CNS. They demonstrate that microglia constitute a unique myeloid cell population that self-renews and is separated throughout life from circulating blood-derived myeloid cells in healthy central nervous systems and with potentially different functional abilities and roles from those of bloodderived myeloid cells.  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