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Central Nervous System Immune Inflammation L.M. Healy, H. Touil, V.T.S. Rao, M.A. Michell-Robinson, J. Antel McGill University, Montreal, QC, Canada

R.O. Weller University of Southampton, Southampton, United Kingdom

O U T L I N E 3.1 Introduction

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3.3 Immune Regulation within the Central Nervous System 3.3.1 Microglia

3.2 Regulation of Immune Trafficking to and from the Central Nervous System30 3.2.1 Blood–Brain Barrier 30 3.2.2 Choroid Plexus/Cerebrospinal Fluid/Draining Vein Trafficking 31 3.2.3 Cell Trafficking-Related Therapeutic Interventions 33 3.2.4 Immune Trafficking at Specialized Central Nervous System Sites 33 3.2.5 Antibody/Immunoglobulin Access to the Central Nervous System 34

Translational Neuroimmunology in Multiple Sclerosis http://dx.doi.org/10.1016/B978-0-12-801914-6.00003-9

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Microglia/Microglia-Directed Therapy38





3.3.2 Astrocytes 3.3.3 Central Nervous System Compartment-Directed Immunomodulatory Therapies

3.4 Conclusion

38 39 40

References40

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© 2016 Elsevier Inc. All rights reserved.

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3.1 INTRODUCTION In this chapter on regulation of inflammation within the central nervous system (CNS), we consider both those mechanisms that regulate access of constituents of the systemic immune system into the CNS and those that regulate the activities of the cells that have accessed the CNS. The latter will focus on the role of endogenous glial cells, microglia and astrocytes, that can directly mediate effects linked to the immune system. The properties of these glial cells are themselves responsive to signals derived from the CNS environment and/or from the infiltrating exogenous immune cells and products. It is now well recognized that immune surveillance is ongoing within the CNS under physiologic conditions. In context of multiple sclerosis (MS), we ask what changes in immune regulation at the level of the CNS contribute to the acute and chronic inflammatory activity that characterizes the disease. Attention will be drawn to therapeutic agents and approaches that have successfully or unsuccessfully been used to modulate these processes in MS or its experimental models. Keep in mind that modulating properties of both infiltrating immune cells (adaptive and innate) and endogenous glial cells will also impact their neural cell protection and repair processes.

3.2  REGULATION OF IMMUNE TRAFFICKING TO AND FROM THE CENTRAL NERVOUS SYSTEM Understanding the dynamic processes whereby cells of the immune system access the CNS under physiologic conditions and during the course of CNS-directed inflammatory disease has greatly advanced with development of techniques to label systemic immune cells and follow their trafficking patterns by live imaging techniques.1–7 Such studies allow evaluation of trafficking when selected molecules associated with either immune, endothelial, or glial cells are genetically deleted or blocked by administration of specific antibodies or pharmacologic agents. The dominant experimental animal approach used to investigate cell trafficking and used as a model of MS is experimental autoimmune encephalomyelitis (EAE), which involves active immunization with neural autoantigens and/or adoptive systemic transfer of autoreactive lymphocytes considered to play a distinct role in inducing disease.

3.2.1 Blood–Brain Barrier A traditional view has been that trafficking of immune cells from the systemic to the CNS compartment during the course of MS or in EAE requires passage of such cells across the blood–brain barrier (BBB) at the level of postcapillary venules.1–7 Contributing to the BBB are specialized endothelial cells that interact with each other; such interaction is dependent on an array of proteins, belonging to the tight junction (claudins, occludin, and junctional adhesion molecules) and adherens families of molecules (reviewed in Ref. 5). The endothelial cells are separated from the parenchyma by two basement membranes—vascular and parenchymal (glia)—between which is a potential perivascular space. The endothelial barrier-regulating molecular interactions are responsive to molecules provided via astrocytes whose end-feet contact and cover the parenchymal basement membrane of the BBB. Astrocyte-derived molecules including sonic hedgehog (SHH) and angiotensin II induce signaling cascades that regulate

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endothelial cell–cell interactions promoting a competent BBB.8,9 As an example, SHH is shown to stimulate netrin1 expression on human BBB endothelial cells, resulting in increased tight junction protein expression.10 This regulatory pathway is responsive to inflammatory conditions reflecting an attempt to restore homeostasis. Conversely, production of proinflammatory molecules by activated astrocyte molecules would enhance permeability of the barrier. Immune cell transmigration across most endothelial barriers is dependent on a sequence of molecular steps that begin with selectin-dependent rolling (see later comment regarding cerebral microvessels) leading to firm adhesion that results from interaction with integrins; the latter are activated by chemokines produced by the cellular constituents (glia and endothelial cells) forming the BBB (reviewed in Refs 1,4). Such transmigrating cells enter the perivascular space formed between the vascular and parenchymal basement membranes; chemokinerelated signals, specifically CXCL12, which is constitutively expressed by BBB endothelial cells, promote their retention in this space.1 Transmigration of cells across the parenchymal barrier and into the actual tissue involves actions of matrix metalloproteinases on this barrier.11 As will be discussed, these molecular sequences have been and continue to be targets of therapeutic interventions.

3.2.2 Choroid Plexus/Cerebrospinal Fluid/Draining Vein Trafficking Cerebrospinal fluid (CSF) in the subarachnoid space (SAS) is largely separate from interstitial fluid (ISF) in the brain parenchyma. Pia mater separates the SAS from the brain and from vessels entering and leaving the surface of the brain.12,13 The SAS does not exhibit the same immunological privilege as the brain parenchyma (as discussed later); CSF drains to cervical lymph nodes from the SAS predominantly through channels that traverse the cribriform plate of the ethmoid bone and join nasal lymphatics14–16 (see Fig. 3.1). Traffic of antigen-presenting cells has been observed along this route of lymphatic drainage17 (Fig. 3.1). Other routes for lymphatic drainage of CSF via dural lymphatics and cranial nerve roots have been described.18 There are no traditional lymphatic vessels in the brain. Lymphatic drainage of ISF from the brain is along very narrow 100-nm-wide basement membranes in the walls of cerebral capillaries and between smooth muscle cells in the tunica media of cerebral arteries16,19 (see Fig. 3.1). Although this route allows lymphatic drainage of fluid and solutes, it is too narrow for the trafficking of antigen-presenting cells to regional lymph nodes19; this feature may contribute to immunological privilege in the brain.20,21 As arteries enter the cerebral cortex at the surface of the brain, there is no clear perivascular space between the artery wall and brain tissue.13 However, CSF does enter the brain from the SAS and tracers pass along the outer aspect of cerebral arteries into the brain parenchyma,22,23 where mixing of CSF with ISF is mediated by aquaporin 4.22 This route has been termed convective influx23 or the glymphatic system.22 A mixture of CSF and ISF with brain metabolites passes out of the brain alongside veins to the CSF.22 It is not yet clear whether the route alongside veins allows traffic of antigen-presenting cells from the brain parenchyma into the CSF. The convective influx/glymphatic system is separate from the rapid periarterial lymphatic drainage system that is a direct link between ISF and cervical lymph nodes16,24 (see Fig. 3.1). An emerging concept regarding access of immune cells under physiologic conditions (immune surveillance) and at the initiation of an autoreactive CNS-directed immune response implicates trafficking across a blood/CSF barrier.2,3 This is referred to as a two-wave model.

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FIGURE 3.1  Diagram showing the largely separate pathways of lymphatic drainage of interstitial fluid (ISF) and cerebrospinal fluid (CSF) from the brain. (1) Blood flows into the brain along branches of the carotid (and vertebral) arteries (red) and forms a network of capillaries (red) that are the sites of the blood–brain barrier through which nutrients enter brain tissue. ISF and solutes drain from the brain along perivascular pathways (curved blue line) in the walls of capillaries and in the tunica media (brown) and adventitia (light blue) of arteries to cervical lymph nodes. (2) Blood flows into postcapillary venules (blue) from which lymphocytes and monocytes (***) enter the brain by receptor-mediated mechanisms, gather in perivenular and perivenous spaces, and migrate into brain tissue. (3) CSF is formed in the ventricles (yellow), flows into the subarachnoid space from which it drains via nasal lymphatics to cervical lymph nodes or into venous sinuses through arachnoid villi or granulations. Antigen-presenting cells (**) traffic with CSF to cervical lymph nodes by this route. The convective influx/glymphatic system is not shown in this diagram. Modified from Fig. 3.1 in Weller RO, Galea I, Carare RO, Minagar A. Pathophysiology of the lymphatic drainage of the central nervous system: implications for pathogenesis and therapy of multiple sclerosis. Pathophysiology. 2010;17:295–306. doi:10.1016/j.pathophys.2009.10.007 and reproduced with permission.

Trafficking begins at the level of the choroid plexus, where the endothelial cells lack the full molecular complement of junctional proteins found with parenchymal endothelial cells.5 This allows immune cell movement into the CSF. The SAS contains competent antigen-presenting cells including dendritic cells.25 Studies of MS cases illustrate that potential disease-relevant antigens such as myelin components can be identified in these meningeal spaces, providing a means to induce and sustain a potentially disease-relevant immune response.26 A sustained meningeal response in MS is postulated to result in proinflammatory molecules penetrating into the underlying gray matter and result in subpial demyelination; the latter is not observed in other meningeal inflammatory conditions including chronic meningitis and lymphomas.27 As mentioned, there is no perivascular space around penetrating arteries, raising the issue

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of how cells trafficking via the choroid/SAS pathway would reach the perivascular space around small venules that are the site of penetration of the BBB. Immune cells when present in the brain under physiologic conditions are usually found between the basement membranes of glia limitans and walls of venules and veins.

3.2.3 Cell Trafficking-Related Therapeutic Interventions Optimal therapeutic interventions directed at immune cell trafficking would aim to target molecules that enhance trafficking of putative cell types mediating a pathologic response while sparing migration of cells most involved in physiologic surveillance, which may themselves serve to regulate immune activity. Selection of therapeutic strategies aimed at specific members of the families of molecules that regulate trafficking will need to consider both the individual subpopulations of immune cells and the different migration sites (choroid/meninges vs BBB) being targeted. In the EAE model, blocking E- and P-selectins that are expressed in leptomeningeal and choroid plexus vessels but not in the parenchymal microvessels does not inhibit lesion formation. This suggests that CSF accumulation of immune cells is not necessary for lesion development.5 Preclinical studies in the EAE model of monoclonal antibodies and small molecules directed at adhesion molecules have often produced inconsistent results, likely reflecting the complexity of the migration process and the limitations of single-antigen/single-strain models.28–31 Natalizumab, which recognizes VLA-4, whose partner ligands are vascular cell adhesion molecule (CAM) and fibronectin, is now an approved therapy for relapsing MS although its use is limited by the risk of development of progressive multifocal leukoencephopathy (PML).32 The small molecule firategrast that also targets VLA-4 was found to reduce disease activity in phase II clinical trials, albeit to a lesser extent than observed in the natalizumab trials.33 Concerns regarding development of PML have limited evaluation of efalizumab, a monoclonal antibody recognizing LFA-1, whose partner ligand is intercellular adhesion molecule.34 An expanded array of adhesion molecules participating in the cell trafficking process continue to be identified including activated leukocyte CAM, melanoma CAM, and Ninjurin-1 (reviewed in Ref. 5) with evidence of selectivity for regulating migration of specific leukocyte subtypes to the CNS as exemplified by mucosal vascular addressin cell adhesion molecule regulation of Th17-cell–endothelial-cell interaction.35–37 Further to be considered is that the overall state of activation of the immune system may be impacted by multiple variables that may be operative under different pathologic and experimental conditions. Examples in the EAE model include use of adjuvants or pertussis that acts on the BBB or whether animals are housed under clean or dirty conditions. Variables in the clinical situation include intercurrent viral and bacterial infections and the status of the microbiome. These issues call attention to the role of innate immune cells including neutrophils and innate lymphocytes in initiating CNS inflammatory responses.38–40

3.2.4 Immune Trafficking at Specialized Central Nervous System Sites Cell passage into the CNS can occur relatively more directly at distinct sites where the BBB is less developed such as the area postrema.41,42 Studies of myeloid cells in chimeric models indicate macrophage entry through these less protected areas with subsequent migration

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through the parenchyma.42 Access of cytokines, chemokines, and other products of systemic inflammation such as endotoxins, to the CNS from the systemic compartment also will occur more readily through sites less protected by the BBB such as the circumventricular organs; as reviewed by Sankowsi et al., such inflammatory mediators can interfere with neuronal and glial cell well-being, leading to a break of balance in brain homeostasis.43 Cognitive and behavioral manifestations commonly resulting from such occurrences during acute infections including anorexia, malaise, depression, and decreased physical activity are collectively referred to as sickness behavior.

3.2.5 Antibody/Immunoglobulin Access to the Central Nervous System In context of MS and its therapies, a specific issue to consider is the capacity of antibodies to pass from the systemic compartment into the CNS. The role of antibodies in MS lesion pathogenesis continues to be defined including the relative contribution of blood-derived versus intrathecally produced immunoglobulin (Ig). In neuromyelitis optica, most of the target-specific antibody (antiaquaporin A) would seem to be derived from a systemic source.44 High titers of anti-interferon (IFN)β antibodies can develop in some patients receiving this therapy, raising the question as to whether these could access the CNS in sufficient amounts to impact endogenous production of IFNβ by astrocytes and disrupt IFNβ-dependent signaling pathways.45 Of further concern is the use of systemic delivery routes for monoclonal antibodies aimed at modulating inflammation or promoting repair within the CNS. Similar consideration may be given to therapy with whole Ig molecule preparations (I-V Ig) and Fc receptor fractions.46

3.3  IMMUNE REGULATION WITHIN THE CENTRAL NERVOUS SYSTEM In this chapter, the focus is on the role of endogenous CNS cells, microglia and astrocytes, although acknowledging the role of T and B lymphocytes within the meningeal, perivascular, and parenchymal compartments. In the case of MS, where blood-derived macrophages access the CNS, their relative role compared to microglia as innate immune cells mediating regulatory and effector functions also needs to be considered; similar recognition should be given to dendritic cells. Further to be recognized is the bidirectional effect these cells have on each other and with other endogenous neural cells, neurons, and oligodendrocytes (see Fig. 3.2).

3.3.1 Microglia Microglia are demonstrated to be a mesodermal cell lineage distinct from blood-derived monocytes/macrophages, arising during primitive hematopoiesis in yolk-sac blood islands.47–51 Microglia are estimated to make up 6–18% of neocortical cells in the human brain.52,53 They are long-lived cells that are not replaced by blood-derived cells under usual conditions.54–57 If depleted using a small molecule inhibitor of colony-stimulating factor-1 (CSF-1) receptor, microglia cells will be repopulated, implicating presence of progenitor populations.58 Presence of microglia may also inhibit access of blood-derived myeloid cells to the

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FIGURE 3.2  Cellular interactions regulating microglia functional properties within the central nervous system under physiologic and pathologic conditions. Physiologic conditions: Maintenance of quiescent state of microglia by interaction of specific surface molecules with regulatory signals derived from adjacent cells (neurons, astrocytes) and extracellular matrix. Interactions include CX3CR/CX3CL1, SIRP1a/CD47, M-CSFR/M-CSF or IL-34, CD200R/ CD200, and TREM-2. Pathologic conditions: Response of microglia to signals derived from injured and dying cells (neurons, oligodendrocytes), astrocytes, and infiltrating immune cells or their products. Responses to dying/injured cells include MerTK-dependent phagocytosis of apoptotic cells expressing phosphatidyl serine on their surface and of myelin breakdown products; P2Y12-dependent migration in response to release of ATP/ADP by injured cells, and TLR-dependent inflammasome activation by molecules (danger-associated molecules, HMGB, and IL-33) released by cells undergoing necrosis. Astrocytes and infiltrating immune cells released molecules that can modulate activation/polarization state of microglia.

CNS parenchyma as shown by irradiated chimeric rodent studies where blood-derived macrophages populate the brain only after ablation of microglia.59 At least in the human system, microglia unlike macrophages do not appear to be dependent on signals delivered by CSF-1 for survival through its protein tyrosine kinase receptor, CSF-1R.60 Gene sequencing studies have defined a unique molecular signature of microglia compared to blood-derived myeloid cells under homeostatic conditions.58 For mice, the in situ homeostatic signature is best defined in microglia maintained in transforming growth factor (TGF)β. Human microglia present a similar distinct profile compared to blood monocytes as assessed using cells isolated from surgical samples of noninflamed adult human brain and maintained under basal culture conditions for 2–4 days.61 Analysis of microglia immediately upon isolation from normal human brain will be needed to define their physiologic homeostatic profile and what culture conditions best model these. As discussed later, the molecular

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signature of microglia in situ and in vitro, as well as that of blood-derived macrophages, is responsive to external stimuli. This property would be analogous to T cells that have distinct genetically determined lineage markers, CD4 and CD8 T cells, but where an array of their functional properties is dependent on exposure to specific external signals, for example, selective cytokines that induce Th1, Th2, Th17/Treg phenotypes. The gene sequencing studies also identify distinct profiles of expression of microRNAs that would support activation of inflammatory responses (eg, Mir-155) or favor a state of quiescence (eg, Mir-124) that acts by restricting Sp1 (PU.1) and CSF-1 expression.62 The functional phenotypes of microglia (and macrophages) continue to be defined based on exposing these cells in vitro or in vivo to defined combinations of molecules. A continuum of phenotypes may exist, especially under actual complex in vivo conditions as described in a later section. Classically activated (GM-CSF, endotoxin, proinflammatory cytokines) myeloid cells have been characterized by their rounded amoeboid morphology, which is consistent with a state of hyperactivity (reviewed in Ref. 58). These cells, often referred to as being of the M1 phenotype, express surface markers such as costimulatory molecules CD80 and CD86, the chemokine receptor CCR7, and increase the expression of HLA-DR. In addition, they produce reactive nitrogen species and proinflammatory cytokines such as IL-12,60 and express the microRNA-155 that is a master regulator of proinflammatory genes via its effects on suppressor of cytokines secretion −1. In contrast, cells generated by activation with macrophage colony-stimulating factor (M-CSF) and anti-inflammatory cytokines (IL-4 and IL-13) and conveniently referred to as M2, possess a typical elongated morphology, express cell surface markers including the scavenger receptor CD163, C-type lectins CD206 and CD209, and produce anti-­inflammatory cytokines such as TGFβ1 and IL-10.63–66 M2 cells are noted to be more efficient than M1 cells with regard to phagocytosis of opsonized targets.67 We observe M2-polarized human microglia to be more efficient at phagocytosing myelin compared to either M1 microglia or any polarized macro­ phage phenotype.68 When comparing functional immune-related properties of M1- and M2-polarized human microglia, we found that the M1 cells are more capable of supporting proliferation of naïve T cells in a mixed lymphocyte reaction69 and in producing effector molecules, especially tumor necrosis factor, which will induce injury of oligodendrocyte progenitor cells.70 The M2 phenotype has been further subclassified according to specific activation stimuli, with M2 cells generated as described earlier (IL-4 and IL-13) being referred to as M2a, M2b are generated by using immune complexes and lipopolysaccharide, and M2c using IL-10 and TGFβ.71 M2b cells are described as continuing to express M1-associated costimulatory molecules and produce nitric oxide, but also secreting anti-inflammatory cytokines such as IL-10. M2c macrophages, originally termed deactivated, are associated with immune suppression and tissue remodeling. Miron et al. ascribed the remyelination promoting effects of microglia following lysolecithin-induced demyelination in mice to production of activin A by cells having acquired an M2 phenotype.72 The signals that innate immune cells are likely actually to encounter within the CNS can be considered under the concept of “stranger” and “danger” signals. The former refer to molecules such as endotoxins produced, expressed, and released by exogenous agents, specifically bacterial infections. There is now recognition that comparable molecules, referred to as alarmins or danger-associated molecules, can be released by endogenous cells whose surface

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membranes are being ruptured (cell necrosis) under inflammatory or injury conditions (see Fig. 3.2).73 Mitochondria are a particular source of such products, reflecting their ancestral bacterial origin.73 These stimuli would favor generation of a proinflammatory phenotype in myeloid cells. Toll-like receptors would serve as a common receptor family for these molecules. Danger signals arise as a consequence of cell injury or death and include surface molecules such as phophatidyl serine expressed by cells undergoing programmed cell death and from cell-type-specific constituents such as myelin and iron that would be released from damaged oligodendrocytes in the case of MS. Iron is observed to promote a rapid switch from the M2 to M1 phenotype.74 In specific context of the inflamed environment of MS lesions, we need to consider signals derived from the immune constituents present in the lesions. Previously mentioned was the potential for specific crystallizable fragment (Fc) components of Ig to interact with the corresponding Fc receptors expressed on the myeloid cells. We have previously shown that supernatants derived from Th1- and Th2-polarized cells will respectively induce an M1- or M2-biased phenotype on human microglia.69,75 The Th2 cells were induced using glatiramer acetate, an agent that does not access the CNS, providing an example of how systemic immune-modulatory therapy may have an indirect effect on innate immunity within the CNS. Adding further to the complexity in concluding how the array of interactive environmental signals will produce a net effect on microglia/macrophage properties in MS is the status of specific receptors on the myeloid cells and their ligands expressed by endogenous neural cells, both neurons and astrocytes, whose interactions regulate the functional properties of the myeloid cells (see Fig. 3.2). These include triggering receptor expressed on myeloid cells-2 (TREM2), whose ligand remains uncertain and that modulates phagocytosis76; SIRP-1 (previously known as CD172a), which interacts with its ligand CD47 that is expressed by neurons and astrocytes, to induce a “do not eat me” signal77,78; CD200R, which interacts with CD200 that can be soluble or expressed on the neuron surface; and CX3CR1 (known as fractalkine receptor), which binds to its soluble ligand CX3CL1. Both CD200R and CX3CR1 once activated mediate a resting phenotype of the microglia. M-CSFR response to M-CSF or IL-34 enhances macrophage although not necessarily human microglia survival.60 The molecular profiling studies of microglia referred to previously include identification or confirmation of a number of surface molecules that are linked to functional responses of the microglia and thus could be potential therapeutic targets.79,80 In this regard, however, we need to consider how their expression is modulated, depending on the polarization state of the cells and whether the distinct differences with macrophages are maintained under all such polarization conditions. In animal model studies, we can bypass the challenge of distinguishing microglia and macrophages by use of genetic labeling techniques and by tracking each of the populations under all the selected test conditions. Such studies have delineated differences in participation of macrophages and microglia in their contributions to myelin destruction and myelin clearance, respectively.81 To date no unique natural lineage marker exists to distinguish microglia and macrophages with relative expression of CD45 and the fractalkine receptor being the most commonly used.82,83 Our studies of expression of the purinergic receptor P2Y12 on human microglia and macrophages illustrate this complexity. This receptor is responsive to ATP/ADP that we and others have shown guide migratory responses of microglia, whereas macrophages are more

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responsive to chemokine signals.84 P2Y12 is more highly expressed on microglia compared to monocytes and macrophages under basal culture conditions and further increases under M2 conditions in vitro and in situ as illustrated by its expression in parasite-infected brain samples.84 In contrast, P2Y12 is downregulated under M1 conditions including the regions of active MS lesions.84 We also observe that expression is upregulated on macrophages under M2 conditions. Thus application as an in situ marker would be complicated, depending on the polarization state of the cells as would be any prediction regarding targeting this receptor for therapeutic purposes (ie, regulating migration of myeloid cells). Microglia/Microglia-Directed Therapy Because these cells are the dominant cell type in both acute and chronic active MS lesions and are contributors to the changes in the normal-appearing white matter, these cells are recognized as potential targets to reduce tissue injury and enhance repair. As mentioned, an indirect approach would be via modulation of properties of T cells that infiltrate the CNS and interact with the myeloid populations present in the meningeal or parenchymal compartments of the CNS.85 Using ganciclovir treatment of CD11b-HSVTK transgenic mice to ablate either microglia or blood-derived macrophages, Heppner et al. concluded that microglial paralysis inhibits the development and maintenance of inflammatory CNS lesions in EAE86; establishing potential effects in progressive aspects of MS is limited by lack of animal models. Current clinical trials using parasite-associated molecules to deviate the immune response in an M2 direction are not CNS selective.87 The identification of the molecular signature of microglia and macrophages and linking them with functional properties raises the potential to modulate functions via targeting specific molecules. These would include migration (P2Y12, chemokine receptors), phagocytosis (MerTK, TREM2), production of inflammatory molecules (mir-155), and production of trophic molecules (activin A).82

3.3.2 Astrocytes These cells can also be considered as contributing to enhancing or inhibiting the injury and repair processes ongoing in MS at all stages of disease evolution. Mayo et al. found that the depletion of reactive astrocytes during the acute phase of disease resulted in a significant worsening of EAE, whereas astrocyte depletion during the progressive phase led to a significant amelioration of disease.88 To be noted is that there is significant topographic heterogeneity in the distribution of astrocytes in the CNS.89 We observe relative differences in microRNA expression between astrocytes laser dissected from gray and white matter of noninflamed adult human tissue sections, a potential contributor to the differences in inflammatory reactivity seen in white and gray matter MS lesions.90 Astrocytes can contribute to multiple steps in the cascade of events related to immune cell entry into the CNS and formation of a new lesion in MS, with much direct evidence coming from studies using the EAE model. Already mentioned is their contribution to maintaining the integrity of the BBB. Astrocytes can express molecules such as major histocompatibility complex (MHC) class II required for participation in antigen presentation although the consensus would be that they are less competent than myeloid cells.91 Astrocytes are also sources of chemoattractants for immune cells.92

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Astrocytes can also play a direct and indirect role in actual tissue injury. We have observed that soluble products released by human fetal brain-derived astrocytes when exposed to supernatants derived from Th1-activated T cells will be cytotoxic to oligodendrocyte progenitor cells, an effect that could be inhibited using an anti-CXCL10 antibody.70 An example of indirect effects is provided by the observation that IL-15 released by astrocytes can induce expression of NGKG2 molecules (NKG2D and C) on T cells resulting in enhanced cytotoxic capacity of CD8 T cells and allowing CD4 cells to acquire promiscuous (non-MHC-restricted) cytotoxic capability including those of oligodendrocytes.93 The functional contribution of astrocytes in the EAE model is further illustrated by studies showing that preventing activation of astrocytes by sphingosine-1-phosphate (S1P) by genetically depleting S1P-receptors on astrocytes results in amelioration of EAE.94 Using a gene array screening approach in the progressive NOD EAE, Mayo et al. identified overexpression of B4GALT6, which codes for a LacCer synthase in the progressive phase of the disease.88 B4GALT6 expression and LacCer levels are increased in MS CNS lesions. Inhibiting B4GALT6 suppressed local CNS innate immunity and neurodegeneration in this model. The suppression linked genes comprised of interferon-sensitive response elements and NF-κB response elements. Conversely, astrocytes can play a protective role by serving as scavengers of reactive oxygen species and removing excess glutamate from the microenvironment (reviewed in Ref. 95).

3.3.3 Central Nervous System Compartment-Directed Immunomodulatory Therapies The therapeutic era in MS evolved from use of therapies that targeted constituents of the systemic immune system. Continuing therapeutic objectives include more selectively intervening in immune trafficking to the CNS and directly modulating immune reactivity within this compartment. Initial success with anti-VLA4-directed antibody therapy provided proof of principle that targeting lymphocyte/monocyte trafficking would interrupt new lesion formation in MS. As the molecular mechanisms are defined that regulate trafficking through the now-recognized two-wave model and that may distinguish molecules (adhesion, chemoattractant) used for trafficking by distinct immune cell subsets, more disease-selective therapies will be developed. The CNS compartment-directed approach would have relevance to reducing the extent of initial tissue injury, inhibiting ongoing injury, and promoting repair. Such therapies would seem to require access of the agent to this compartment. Initial emphasis has been on development of conventional drugs that can achieve such access. Two approved agents for treatment of relapsing forms of MS can access the CNS with in vitro and EAE-related data suggesting effects on both microglia and astrocytes. Fingolimod is shown to inhibit the S1P-induced proinflammatory responses of astrocytes to S1P while concurrently inducing signaling pathways that result in inhibition of intracellular calcium release and nitric oxide production.94,96,97 However, clinical trials up to 2015 have shown that this agent impacts on the relapsing but not progressive phases of the disease as measured by clinical criteria and magnetic resonance imaging indices of tissue loss. Dimethyl fumarate (DMF) is demonstrated to have both anti-inflammatory- and antioxidant-inducing effects on astrocytes in vitro and in experimental models.98 These effects can also be reproduced in vitro using adult human CNS-derived microglia.99 However, the in vitro effects are reproduced using DMF rather than

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by monomethyl fumarate (MMF) even though the MMF receptor HCAR-2 is present on these cells.99 Oral DMF is rapidly metabolized to MMF, leaving open the question of its mechanism of action within the CNS. The receptors on microglia that regulate their functional responses, including migration, phagocytosis, and production of immune regulatory and effector molecules, provide potential drug-able targets as do the receptor–ligand interactions that regulate overall cell activation. For astrocytes, potential exists to inhibit production of inflammatory mediators and upregulating protective processes as exemplified by the studies cited regarding effects of fingolimod and inhibition of lac ser production. Ongoing efforts are aimed at developing additional therapies that will effectively access the CNS compartment. Successful experimental approaches include use of regulatory microRNAs, specifically mir-155 to reduce inflammation.80 Gene therapy approaches using myeloid cells are already in use for enzyme replacement purposes in inherited disorders (leukodystrophies).100

3.4 CONCLUSION This chapter emphasizes the need to understand the precise pathogenic mechanisms underlying MS at each stage of disease evolution including both the neurobiologic and immunologic aspects so that novel therapies or combinations thereof can be developed and optimally utilized.

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