The good and the bad of neuroinflammation in multiple sclerosis.

Handbook of Clinical Neurology, Vol. 122 (3rd series) Multiple Sclerosis and Related Disorders D.S. Goodin, Editor © 2014 Elsevier B.V. All rights reserved

Chapter 3

The good and the bad of neuroinflammation in multiple sclerosis 1

MATTHIAS NAEGELE1 AND ROLAND MARTIN2* Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center for Molecular Neurobiology Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2

Neuroimmunology and MS Research, Neurology Clinic, University Hospital, Zurich, Switzerland

INTRODUCTION Inflammation is a characteristic response of tissue to a wide range of stimuli, defined centuries ago by the cardinal signs of redness (rubor), swelling (tumor), heat (calor), pain (dolor), and loss of function (functio laesa). Molecularly, it represents both cause and effect of complex immunologic cascades. Inflammation can be acute, when the cardinal signs and cells of the innate immune system such as granulocytes predominate, but can also take a chronic course, in which lymphocytes and monocytes are seen more commonly. On the one hand, a wellregulated inflammatory response is usually beneficial for its host and essential to deal with systemic threats such as infection, trauma, and malignancy. On the other, an excess of inflammation is pathogenetically involved in many chronic diseases of modern medicine today. Concerning the field of neurology, inflammation is found in a wide range of disorders, including brain infection, hypoxic and traumatic brain injury, brain malignancies, multiple sclerosis (MS), Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Hunot and Hirsch, 2003; Hagberg and Mallard, 2005; Minghetti, 2005; Lakhan et al., 2009). In MS, autoimmune inflammation is considered a central pathogenetic component and is widely seen as the causative event, which leads to neurologic dysfunction. Based on this perception, most of the effective agents available today aim to reduce inflammation. Some are relatively unspecific immunosuppressants with potentially deleterious long-term side-effects. Others act in an immunomodulatory manner, and we are just beginning to understand their complex mechanisms of action. As insight into the molecular pathways

of MS expands, it becomes clear that inflammatory responses also encompass regulatory elements. They may provide a significant counterbalance to inflammation and could become new therapeutic targets in the future. This chapter focuses on the neuroinflammatory network of MS. It has to be kept in mind that the disease also possesses a considerable neurodegenerative component, especially during the later, progressive stages of the disease (Trapp and Nave, 2008; Frischer et al., 2009; Lassmann, 2010). Although there is increasing evidence that neurodegeneration is a secondary event following inflammation, a primary involvement in disease pathogenesis cannot be excluded (Lassmann, 2010). Finding effective treatments which address the neurodegenerative part of the disease is an emerging field in both clinical and basic research. To illustrate the complexity of neuroinflammation in MS, this chapter will first provide a brief overview of the disease. After that, the immune privilege of the CNS will be discussed; it is important to understand the immunologic pathways leading to neurologic dysfunction. Then, intrinsic regulatory and regenerating elements are mentioned and finally a short summary of present and future therapeutic strategies will be given. In parallel, when applicable, insights from clinical research will be integrated into the respective sections of the chapter.

A BRIEF OVERVIEW OF MULTIPLE SCLEROSIS MS is an inflammatory, demyelinating and neurodegenerative disorder of the central nervous system (CNS), affecting over 2 million people worldwide (Dutta and

*Correspondence to: Prof. Dr. med. Roland Martin, University Hospital Zurich, Neurology Clinic, Neuroimmunology (nims) and MS Research, Frauenklinikstrasse 26, 8091 Zurich, Switzerland. Tel: þ41-(0)-44-255-1218, Fax: þ41-(0)-44-255-4507, E-mail: roland. [email protected]

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Trapp, 2007). After decades of research on both the human disease and its animal model, experimental autoimmune encephalomyelitis (EAE), it is considered an autoimmune disease mediated by CD4 þ T cells, which orchestrate a multifactorial attack on myelin sheaths, oligodendrocytes, axons, and neurons (Sospedra and Martin, 2005). The etiology of the disease is not fully understood and appears to involve both genetic and environmental factors (Fig. 3.1) (Goodin, 2009).

Regarding its epidemiology, MS – like many other autoimmune diseases – more commonly affects women. This imbalance has increased in the last 20 years, in parallel to the apparent overall increase in MS prevalence. Several underlying factors, such as the influence of sex hormones, are currently discussed (Schwendimann and Alekseeva, 2007; Eikelenboom et al., 2009). Geographically, there is a gradient of increasing prevalence towards the poles on both hemispheres, with highest

Fig. 3.1. A multifactorial model of multiple sclerosis (MS). Risk factors (red) raise susceptibility (S) to disease manifestation (dashed line). They consist of a strong genetic component (G) and additional environmental events (E). Suspects are cells and single molecules, which are thought to be overall deleterious. Victims are targets of the suspects, with myelin sheaths and oligodendrocytes thought to be the primary target of the immune reaction. The central image depicts the main mechanisms of damage in MS: Extensive demyelination and myelin degradation, attack on oligodendrocytes, apoptotic oligodendrocytes, axonal channelopathy and transection, and eventually neurotoxicity. Guardians are cells and single molecules, which are thought to contain the suspects and repair their damage. Protective factors (green) are associated with a lower susceptibility to disease manifestation: People without strong genetic risk factors (e.g., HLA-DR15) are unlikely to develop MS. Certain environmental conditions could lower susceptibility to MS in people with genetic risks. ?, Relatively disputed compared to the other factors; AA, some of the putative myelin autoantigens (Sospedra and Martin, 2005); APC, antigen-presenting cell; bact., bacterial; BBB, blood–brain barrier; bolt, damaging event (see effector phase); CNS, central nervous system; DCs, dendritic cells; IFN, interferon; IL, interleukin; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; NK, natural killer; PLP, proteolipid protein; Reg., regulatory (see regulatory elements); ODC, oligodendrocyte; OPCs, oligodendrocyte precursor cells; ROS, reactive oxygen species; SNPs, single nucleotide polymorphisms; t, time; “Th117,” IFN-g and IL-17 coexpressing CD4 þ T cells.

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS 61 rates found in Northern Europe, North America, and patients present with progressive neurological deterioraSouth Australia. This observation might be explained tion from the beginning, termed primary progressive MS by the distribution of genetic susceptibility alleles and (PPMS) (Miller and Leary, 2007). Additionally, less fretheir interaction with certain environmental factors such quent demyelinating disorders are known, which are paras exposure to sunlight and regional infectious backtially reminiscent of MS, i.e., acute disseminated ground (Noseworthy et al., 2000). encephalomyelitis, neuromyelitis optica and Marburg’s There is unequivocal evidence that genetic factors are disease (Hu and Lucchinetti, 2009). the most important etiologic components determining Early MS lesions are characterized by focal infiltradisease manifestation and expression. While monozytion of lymphocytes and monocytes into regions of the gotic twins share a concordance rate of 20–30%, the brain or spinal cord, breakdown of the blood–brain barabsolute risk of disease manifestation in first-degree relrier (BBB), and various degrees of demyelination, atives of patients is around 2–5%, compared to 0.1% in remyelination, and axonal loss. Inflammatory lesions the general population (Sadovnick et al., 1988, 1993). can be visualized by magnetic resonance imaging Similar to other autoimmune diseases, MS is associated (MRI) and are about 10 times more frequent than with distinct human leukocyte antigen (HLA) class II periods of acute clinical worsening (Stone et al., 1995). genes (Jersild et al., 1973). The HLA-DR15 haplotype Classically, lesions are found in the white matter of appears to be the most prominent risk factor, accounting the brain. However, evidence indicates that the gray matfor 14–50% of the genetic susceptibility (Ebers et al., ter is also affected (Geurts and Barkhof, 2008), which 1996; Hafler et al., 2005). Although it has been raises the question whether MS affects only distinct difficult to identify further genetic determinants in the regions or the entire CNS (Chard and Miller, 2009). As past, recent genomewide association studies clearly mentioned above, a considerable neurodegenerative identified further risk alleles (Hafler et al., 2007). This component exists, and this is responsible for most of supports the view of MS as a complex polygenic disease the neurologic deficits at later stages of the disease with multiple quantitative trait loci jointly contributing to (Trapp and Nave, 2008). Depending on the relative domits etiology. Among the most significant associations are inance of inflammatory and neurodegenerative aspects single nucleotide polymorphisms in the interleukin 7 in MRI measurements, patients can be separated into and interleukin 2 receptor alpha chains (IL-7Ra, four different clinical subgroups (Bielekova et al., 2005). IL-2Ra). Emerging studies have begun to explore their In the last years, detailed and systematic studies shed functional associations with disease pathogenesis new light on the pathology of MS lesions. Active demy(Booth et al., 2005; Gregory et al., 2007; Maier et al., elinating lesions are immunopathologically character2009; Hoe et al., 2010). ized by the presence of myelin-laden macrophages Correlations with several environmental factors have (Breij et al., 2008) and the upregulation of major histobeen established, such as the amount of exposure to suncompatibility complex (MHC) class II molecules by maclight, vitamin D deficiency, and bacterial and viral infecrophages and microglia (Bo et al., 1994). According to tions, in particular Epstein–Barr virus (EBV) (Marrie, the composition of cellular and humoral immune com2004; Milo and Kahana, 2010). However, a clear causalponents as well as oligodendrocyte and myelin damage, ity for any of the factors could not be determined so far Lucchinetti et al. (2000) divided active lesions into four (Pender, 2009; Stewart, 2009; Beretich and Beretich, distinct immunopathologic patterns (IP I–IV). All pat2010). In fact, the disease might be the consequence of terns are characterized by the presence of infiltrated T a highly heterogeneous and multifactorial cascade cells and macrophages. While signs of remyelination (Goodin, 2009). (shadow plaques) are regularly seen in patterns I and Apart from the genetic and environmental backII, patterns III and IV present with a lack of remyelinaground, MS also shows heterogeneity in other aspects. tion, extensive loss of oligodendrocytes, and signs of oliIn regard to the clinical presentation, patients are divided godendrocyte apoptosis. The most common pattern into three subgroups (Lublin and Reingold, 1996). among MS patients is IP II ( 58%). It is found in Eighty percent of patients follow a disease course patients of RRMS, SPMS, and PPMS disease course which is characterized by episodes of acute clinical worsand is distinguished from pattern I (15%) by higher ening (relapses), followed by intervals of clinical remisplasma cell numbers and a prominent deposition of sion (relapsing-remitting MS, RRMS) (Noseworthy immunoglobulins and complement proteins at sites of et al., 2000). However, as the disease progresses, many myelin destruction. Pattern III (26%) presents with RRMS patients enter a chronic stage, identified by an extensive oligodendrocyte apoptosis-like pattern IV, continuous worsening of neurologic function in the but myelin loss seems to be preferentially restricted to absence of relapses (secondary progressive MS, SPMS) myelin-associated glycoprotein (MAG). IP IV, the rarest (Weinshenker et al., 1989). Around 10–15% of the pattern (1%), is only found in PPMS cases and shows

62 M. NAEGELE AND R. MARTIN extensive oligodendrocyte degeneration without evilast, there is evidence that all three axes are impaired dence of complement deposition and MAG loss. in MS and contribute to pathogenesis. Lucchinetti et al. (2000) stated that lesion patterns differ between patients (interindividual heterogeneity), but are homogeneous within multiple active lesions from THE BLOOD–BRAIN BARRIER the same patient (intraindividual homogeneity). HowThe BBB is a specialized structure formed by the blood ever, this perception has been contested by other invesvessels of the CNS and hinders not only immune cells, tigators (Barnett and Prineas, 2004; Breij et al., 2008). but also other potentially harmful factors such as pathIn addition, it has been proposed that pathologic patterns ogens or xenobiotica from accessing the parenchyma differ in early disease stages but later converge into one (Daneman and Rescigno, 2009). Molecularly, it consists single immunopathologic pattern (heterogeneity in time) of specialized tight junction-interconnected endothelial (Bielekova et al., 2005). Further studies will hopefully cells, associated pericytes, a surrounding basal lamina, clarify the current pathologic disputes (Lassmann and an outer layer of astrocyte endfeet, which ensheath et al., 2007; Hu and Lucchinetti, 2009). the vessels (Kacem et al., 1998; Allt and Lawrenson, 2001). Directly associated with this complex are differTHE IMMUNE PRIVILEGE OF THE ent immune cells, including perivascular macrophages CENTRAL NERVOUS SYSTEM and mast cells (Abbott, 2000). Introduction The filtering function is mainly performed by the endothelial cells themselves. They prevent uncontrolled Immune privilege is the capacity of certain organs to paracellular transport by their lack of fenestrations and limit immune reactions upon introduction of antigens. their tight intercellular connections mediated by occluAt first sight, this capacity appears to be detrimental dins and other tight junction proteins (Bradbury, 1984). for host survival by creating an immunological “blind This renders brain capillaries 50–100 times tighter than spot” which exposes the host to infection. However, a peripheral microvessels and molecules larger than growing body of evidence suggests that immune priviapproximately 600 Da are effectively excluded from lege is not constituted by the simple suppression of all the brain unless active transport mechanisms exist. immune responses. Rather, it appears to be the conseTight junctions are of particular importance: They sepquence of selectively downregulating those immune arate the luminal from the abluminal part of the endoresponses that are able to inflict the most collateral injury thelial cell (“fence function”). However, this separation to innocent bystander tissues, while other, more benefiis not absolute and upon appropriate stimuli they allow cial, components are preserved (Niederkorn, 2006; selective paracellular transmigration (“gate function”) Mellor and Munn, 2008). Four organs seem to benefit (Wolburg et al., 2009). The gatekeeper function of particularly from this state: The brain, the eye, the testis, endothelial cells is not restricted to paracellular pathand the pregnant uterus. The need for immune privilege ways but also includes transcellular routes. Endothelial of the CNS can teleologically be explained by its limited cells elaborately control transcellular transport of both capacity for regeneration and extraordinary role in evocells (by only expressing low amounts of adhesion mollutionary survival. Its privileged state becomes evident ecules in their unactivated state) and molecules (by by the virtual absence of peripheral lymphocytes, granbeing equipped with an array of xenobiotica-exporting ulocytes, and dendritic cells in the healthy state and transport proteins) (Dallas et al., 2006). Apart from the presence of specialized resident immune cells, most their intrinsic function, endothelial cells strongly internotably mesodermal microglia (Hickey and Kimura, act with cells and molecules of their perivascular envi1988; Perry and Gordon, 1991; Hickey et al., 1992; ronment. Danger signals from perivascular cells are Dowding and Scholes, 1993; Ling and Wong, 1993). sensed by the endothelium. It responds with an upreguCNS immune privilege represents the product of sevlation of adhesion molecules and secretion of chemoeral anatomic, physiologic, and immunoregulatory fackines, which subsequently recruit circulating tors. The three most important features are: (1) the leukocytes to the site of injury (Piccio et al., 2002; long-appreciated physical and molecular separation of Schiltz and Sawchenko, 2003). the CNS and the peripheral compartment by the BBB; There are some sites of the CNS where the BBB is (2) alterations in the “afferent immunological axis,” physiologically less tight even in an uninflamed state i.e., pathways of the adaptive immune system involved and these include the liquor-producing choroid plexus in antigen recognition and transport; (3) suppression and the sensory and secretory circumventricular organs. of the “efferent immunological axis,” i.e., T-cell effecThese regions may constitute alternative routes for leutor functions, by the abundant presence of both soluble kocytes to gain CNS access in immunologically mediated and membrane-bound immunoregulatory factors. At

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS 63 disorders such as MS and EAE (Ransohoff et al., 2003; as microglia and perivascular macrophages (Hatterer Reboldi et al., 2009). et al., 2006). Indeed, exogenous antigens are insuffiAs leukocyte trafficking across the BBB is considciently transported from the parenchyma into peripheral ered a critical step in MS pathogenesis, blocking transenlymphoid organs, eliciting either no or very delayed dothelial migration represents a feasible therapeutic peripheral immune reactions (Stevenson et al., 1997; approach in today’s clinical practice. This is highlighted Matyszak and Perry, 1998). However, once inflammaby the efficacious monoclonal antibody natalizumab, an tion within the CNS is established, peripheral lymphoid inhibitor of the a4-integrin (VLA4), which is involved in organs and cells become involved in immune responses. lymphocyte transmigration into the CNS (Engelhardt This is supported by the presence of dendritic cells in and Kappos, 2008). Apart from the a4-integrin, many inflamed CNS tissue (Matyszak and Perry, 1996), and other molecules, i.e., selectins (E-, L-, P-selectin), vascumyelin antigen-bearing macrophages in cervical lymph lar cell adhesion molecules (ICAM-1, -2, and VCAM) nodes of EAE monkeys and MS patients (de Vos and other integrins (LFA-1, MAC-1), are involved in et al., 2002). the process of BBB transmigration (Petri et al., 2008). Knockout and blocking studies in rodent models have THE EFFERENT IMMUNOLOGICAL AXIS provided insight into how each of these molecules is Once activated T cells have breached the BBB and involved in the sequential steps of the cascade (Ransohoff et al., 2003). entered the CNS, they face an inhospitable microenviLack of transendothelial migration (i.e., natalizumab ronment. One of the first inhibitory factors is Fas ligand, treatment) or deficits in peripheral immunity (i.e., which is expressed on all cells of the healthy CNS acquired immunodeficiency syndrome) can lead to (Bechmann et al., 1999) and upregulated upon inflamopportunistic infections of the CNS, most notably promation (Choi and Benveniste, 2004). It can induce apogressive multifocal leukoencephalopathy (PML). Their ptosis of T cells independently of their antigen specificity (Bauer et al., 1998; Flugel et al., 2000). emergence shows that active immunosurveillance by The second obstacle is that T cells usually require peripheral leukocytes is needed to maintain immunity against certain CNS pathogens. In support of this view, local antigen-specific restimulation until they can fully it is widely appreciated that activated CD4 þ T cells easexert their effector function. Since T cells cannot recogily cross unactivated brain endothelium (Wekerle et al., nize soluble antigens, they depend on APCs, which pro1987; Hickey et al., 1991). Even activated T cells, which cess antigens, present them on MHC class I and II are not specific for CNS antigens, commonly crawl molecules, and provide co-stimulatory signals. As part inside CNS vessels and, in low numbers, accumulate in of the immune privilege of the CNS there is only a minimal expression of MHC class I and almost no expresmeningeal compartments, where they are able to comsion of MHC class II molecules within the healthy municate with perivascular professional antigenpresenting cells (APCs) (Bartholomaus et al., 2009). This CNS (Perry, 1998). Although MHC class I molecules supports the hypothesis that CNS immunosurveillance is are generally present on all nucleated cells, neurons do carried out by memory T cells, which regularly leave not express them under physiologic circumstances, probmeningeal vessels of subarachnoidal or choroidal comably to protect themselves from lysis by virus-specific partments to scan resident perivascular APCs for their cytotoxic T lymphocytes (Turnley et al., 2002). corresponding antigen (Ransohoff, 2009). Recently, Apart from the expression of Fas ligand, T cells face further inhibitory signals from microglia, astrocytes, the chemokine CCR6/CCL20 axis was found to define and neurons. In inflammatory contexts, including CSF-homing lymphocytes, which may be involved in EAE and possibly MS pathogenesis (Reboldi et al., EAE, microglia express B7-H1, a homolog of the co2009). stimulatory molecule B7, which negatively regulates Tcell activation and cytokine production (Magnus et al., 2005). In addition, microglia upregulate indoleamine THE AFFERENT IMMUNOLOGICAL AXIS 2,3-dioxygenase, an enzyme which modulates tryptoThe “afferent immunological axis” includes all prophan metabolism and thus generates an anticesses involved in the first recognition of antigens, from inflammatory microenvironment (Kwidzinski et al., phagocytosis by APCs, transportation, to secondary lym2005). Astrocytes, the most abundant glial cells in the phoid organs and presentation to T cells via HLA molebrain, secrete anti-inflammatory cytokines such as transcules. In the healthy state, the afferent axis appears forming growth factor (TGF)-ß (Meinl et al., 1994). Furheavily suppressed within the CNS parenchyma. This ther, they are able to inhibit antigen-specific T cells by notion is supported by the lack of classical dendritic cells cell–cell contact, e.g., by utilizing CTLA-4 molecules and the presence of non-emigrating resident APCs such (Gimsa et al., 2004). In the context of EAE, TGF-ß is

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commonly upregulated during CNS inflammation (Logan et al., 1992). Apart from regulating T cells, TGF-ß inhibits microglia activities by downregulating their MHC class II molecules (O’Keefe et al., 1999). This is of particular significance in prion diseases, where microglia play a central role in mediating inflammation (Boche et al., 2006). Besides TGF-ß and other leukocyte cytokines, a number of neuropeptides, such as VIP, CGRP, a-MSH, and somatostatin, are known to have anti-inflammatory properties (Niederkorn, 2006).

Compromising CNS immune privilege During MS, the CNS immune privilege is severely compromised, and unfortunately, little is known about the initial steps, which allow peripheral leukocytes to establish inflammation within the CNS. The inciting event could be a subclinical infection with an as yet unknown pathogen or, as another possibility, death of an oligodendrocyte, fragmentation of myelin, and subsequent activation of CNS resident cells. Such activation could lead among other events to the upregulation of MHC molecules. For instance, astrocytes challenged with interferon (IFN)-g in vitro upregulate MHC class II molecules and gain capability to present antigen to T cells (Fontana et al., 1984; Wong et al., 1984; Sedgwick et al., 1991). Indeed, astrocytes, microglia, and endothelial cells express higher amounts of MHC and co-stimulatory molecules (CD80 and CD86) within MS lesions (De Simone et al., 1995; Windhagen et al., 1995; Issazadeh et al., 1998; Prat et al., 2000; Zeinstra et al., 2000). Astrocytes have been shown to express the proinflammatory cytokine IL-17 within lesions (Tzartos et al., 2008). Therefore, although overall anti-inflammatory in the physiologic state, resident cells can switch over to a proinflammatory phenotype in inflammatory conditions such as EAE and MS. Most of the evidence suggests that this involvement is only a secondary event following leukocyte infiltration (see section below). However, some investigators proposed a primary involvement of resident cells, in particular astrocytes and microglia, in disease pathogenesis (De Keyser et al., 2003).

THE INFLAMMATORY CASCADE OF MULTIPLE SCLEROSIS Introduction This section describes the chronology of a developing MS lesion: (1) autoreactive CD4 þ T cells are activated in the periphery, transmigrate through the BBB into the CNS, and are locally reactivated by APCs; (2) cytokine-releasing CD4 þ T cells and other successively recruited and activated immune cells establish the inflammatory lesion; (3) within the proinflammatory

milieu, activated effector mechanisms mediate myelin, oligodendrocyte, and axon damage, leading to neurologic dysfunction. In parallel, activated immunomodulatory elements begin to limit inflammation and initiate repair, which often results in at least partial remyelination and clinical remission (Fig. 3.1). The presented order might facilitate understanding of the complex inflammatory cascades. However, one should keep in mind that most of the pathogenetic insight derives from the animal model EAE. In this model, the autoimmune background of CNS damage is artificially induced, and its mechanisms have been examined in detail. There are convincing overlaps between EAE and MS. For instance, some of the treatments that are used in MS have previously been shown to be efficacious in EAE, e.g., glatiramer acetate, mitoxantrone, and natalizumab (Steinman and Zamvil, 2006). Yet, owing to difficulties in translating several findings to the human system and the above-mentioned heterogeneity of MS, it cannot be excluded that other, currently unappreciated factors play more important roles. Corresponding to the diverse genetic and environmental background, it is even possible that pathogenetic cascades differ between individuals. Therefore, the presented order, although it is based on current knowledge, should be approached with caution. In addition, less is known about the immunologic mechanisms in the progressive disease patterns (SPMS, PPMS). The failure of many therapeutics that are efficacious in RRMS, but not in SPMS and PPMS, suggests profound differences.

INITIATING PHASE The predominating role of myelin-specific CD4 þ T cells The thymus negatively selects high-avidity autoreactive T cells. This process prevents such cells from populating the peripheral immune repertoire and plays a crucial role in preventing autoimmunity (central tolerance). However, our adaptive immune system needs to be capable of mounting an immune response to practically any antigen in order to be protective. Therefore, it is no surprise that low-avidity autoreactive T cells, that cross-react with foreign antigens in a higher avidity, regularly escape this selection process (Jones and Diamond, 1995; Steinman, 1996). T cells reactive to myelin antigens, including myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG), are found in the peripheral blood of virtually every healthy individual (Burns et al., 1983; Martin et al., 1990; Trotter et al., 1998; Hellings et al., 2001). During the last two decades a number of laboratories have characterized myelin-specific T cells and it appears that those from MS patients have a higher avidity, state of

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS activation, and potency to induce inflammation than myelin-specific T cells from healthy individuals (Bielekova et al., 2004). These findings indicate that autoreactive T cells of MS patients have been activated or, in general, have undergone changes that lead to a break of self-tolerance. Due to the overall low prevalence of MS, one can assume that disease only develops when precise genetic and environmental susceptibilityraising elements converge in one individual. Apart from a priori defects in thymic selection and maturation (Hug et al., 2003; Haas et al., 2007; Chiarini et al., 2010), two possible tolerance-breaking mechanisms have been proposed. Both can involve an infectious trigger, and it is possible that they act in conjunction with each other (von Herrath et al., 2003): Molecular mimickry as the antigen-specific event and bystander activation as an antigen-unspecific component. The molecular mimickry hypothesis has been coined following the observation that similarities between peptides from pathogens (foreign) and autoantigens (self) can result in cross-activation of autoreactive T cells (Libbey et al., 2007). Numerous studies have been performed to identify cross-reactive peptides, especially with the putative autoantigen MBP (Sospedra and Martin, 2005). However, while the existence of molecular mimickry is well established, it is less clear if it primarily serves a role in maintaining the T-cell repertoire, i.e., is entirely physiologic, or is also involved in initiating autoimmune diseases. The evidence for the latter is still scarce, and further investigation is clearly needed. Apart from the above-mentioned specific crossreactivity with foreign antigens, it is also possible that autoreactive T cells are activated by a non-specific inflammatory event. This mechanism is termed bystander activation and could partly explain increased relapse rates after bacterial (Rapp et al., 1995; Metz et al., 1998; Correale and Farez, 2007) and viral (Andersen et al., 1993; Granieri et al., 2001) infections. Bystander activation could be induced by an increase in inflammatory T-cell-stimulating cytokines and chemokines, the activation of T-cell receptors (TCRs) by superantigens (Brocke et al., 1993), the modulation of T cells by Toll-like receptor-mediated danger signals (Hoebe et al., 2004), or the facilitation of epitope spreading by persistent viral infections (Miller et al., 1997; Horwitz et al., 1998). These mechanisms may act in parallel, synergistically raising the probability of tolerancebreaking events. However, it should not be neglected that inflammatory or infectious contexts not only act on CD4 þ T cells, but likely modulate many other important components involved in MS, such as dendritic cells, the BBB, B cells, CD8 þ T cells, and cells of the innate immune system (e.g., mast cells).

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In addition to the hypotheses of molecular mimickry and bystander activation, several other findings support the crucial importance of myelin-reactive CD4 þ T lymphocytes in MS pathogenesis: ●

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CD4 þ T cells, among other cells, are found within CNS lesions and the cerebrospinal fluid (CSF) of MS patients. In addition, cells which can be attracted and activated by CD4 þ T cells, such as macrophages and B cells, are found within lesions (Barnett and Prineas, 2004). MS is clearly associated with susceptibility-raising HLA-DR and HLA-DQ molecules. These molecules are primarily responsible for presenting processed antigen to the TCRs on CD4þ T cells. Mice transgenic for human HLA-DR or HLA-DQ molecules are susceptible to EAE (Das et al., 2000; Kawamura et al., 2000). Furthermore, mice which express both MS-associated HLA-DR and MS patient-derived TCRs develop spontaneous EAE (Madsen et al., 1999; Quandt et al., 2004). MHC class I molecules, on the other hand, which are recognized by CD8þ cytotoxic T lymphocytes, only show weaker genetic associations (Sospedra and Martin, 2005). EAE can be induced not only by immunizing susceptible animals directly with myelin proteins together with adjuvant, but also by adoptively transferring activated myelin-specific CD4 þ T cells into naive mice (Ben-Nun et al., 1981). This adoptive transfer of EAE has so far not been replicated with antibodies and only under certain circumstances with CD8 þ T cells (Huseby et al., 2001; Sun et al., 2001). Finally, the probably most convincing single evidence, that myelin-reactive T cells are principally able to induce inflammatory demyelination in humans, derives from a clinical trial (Bielekova et al., 2000). Here, a specifically modified MBP (83–99) peptide (altered peptide ligand, APL) was injected into patients to shift the T-cell repertoire into a more tolerogenic state. But instead of inducing tolerance, three patients developed exacerbations, as shown by increased contrast-enhancing lesions. In two of them it could be linked clearly to the APL, as, preceding the relapses, MBP (83–99)specific T cells expanded up to 2000-fold and increased in avidity and cross-reactivity to the APL.

Activated CD4 þ T cells pass the blood–brain barrier Once naive CD4 þ T cells have been primed by APCs in peripheral lymphoid organs and differentiated into effector CD4þ T cells, they re-enter the circulation. A local reactivating signal, such as a repeated stimulation

66 M. NAEGELE AND R. MARTIN by antigen:MHC class II complexes or a highly inflamElevated serum MMP-9 levels correlate with matory cytokine environment, would enable them to gadolinium-enhancing lesions in RRMS and SPMS exert their effector function fully. Myelin as an isolator patients (Rosenberg, 2005). Treatment of rats with and trophic structure for axons is a highly abundant MMP inhibitors was found to inhibit BBB disruption component of the CNS. Therefore, it is likely that (Yang et al., 2007). Finally, apart from paracellular CNS-resident APCs such as perivascular phagocytes migration, which likely plays an important role once phagocytose myelin debris and present myelin peptides the lesion has been established, T cells can also extravaon HLA class II molecules. Perivascular phagocytes sate transcellularly. This process requires an active are positioned in strategic proximity to CNS microvesdeformation of the endothelial cell, involving large sels and were found to have characteristics of both macplasma membrane protrusions. Its relevance in MS rophages and dendritic cells (Bartholomaus et al., 2009). and EAE in vivo is currently not clear (Petri et al., 2008). Hence, myelin-reactive T cells first need to pass the BBB to reach their reactivating APCs (Wekerle, 1993). InacActivated CD4 þ T cells are reactivated by tive and intact brain endothelial cells usually pose a sigperivascular phagocytes nificant obstacle for naive CD4 þ T cells (Wekerle, As soon as autoreactive CD4 þ T cells arrive at the CNS, 1993). However, activated CD4 þ T cells have several they begin to scan local perivascular APCs for their corBBB-breaching features. They secrete proinflammatory responding peptide–MHC class II complex (Hickey and cytokines such as IFN-g and tumor necrosis factor Kimura, 1988; Goverman, 2009). This process has been (TNF)-a, which upregulate several endothelial adhesion visualized in meningeal microvessels using intravital molecules, including ICAM-1 and VCAM-1 (Petri microscopy (Flugel et al., 2001; Bartholomaus et al., et al., 2008). Indeed, serum levels of IFN-g and TNF-a 2009). Upon cell contact with an activated and and leukocytes expressing TNF-a mRNA are elevated antigen-loaded perivascular APC, CD4 þ autoreactive during relapses (Rieckmann et al., 1994; Ozenci et al., T cells are strongly reactivated, as seen by an acute upre2000). Corresponding to the increase in endothelial adhegulation of proinflammatory cytokines (i.e., IFN-g, ILsion molecules, CD4þ T cells express higher amounts of 17, and TNF-a), proteases (MMP-9), and chemokines their integrin counterparts, LFA-1 and VLA-4 (Baron (CCL5) (Bartholomaus et al., 2009). This reactivation et al., 1993). While selectin-mediated rolling plays an appears to enable them to infiltrate the parenchyma. Corimportant role in peripheral organs and meningeal brain responding with parenchymal infiltration, first clinical vessels of EAE mice (Bartholomaus et al., 2009), parensymptoms arise, i.e., limb paralysis and weight loss. chymal transmigration seems to be more dependent on The peak of clinical symptoms of the EAE mice is synVLA-4-mediated adhesion (Engelhardt, 2008). The chronous with the peak of parenchymal MBP-specific Timportance of VLA-4 and LFA-1 can be observed in real cell invasion (Bartholomaus et al., 2009). Very limited time with intravital microscopy studies (Bartholomaus information about the sequence of events exists in et al., 2009): Before MBP-specific CD4 þ T cells extravMS. However, observations from the APL trial regardasate into the CNS, they crawl inside the vessels for loning the timing of inflammatory CNS lesions versus the ger periods of time, likely searching for an appropriate expansion and contraction of peripheral MBP-specific exit point. Upon blockade of LFA-1 and VLA-4, an T cells suggest that the order is similar (Bielekova almost immediate detachment of CD4þ T cells is et al., 2000; Muraro et al., 2003). observed. After CD4 þ T cells have bound to the endothelium, Debate about the disease-defining CD4 þ they can transmigrate via either a transcellular or a paraT-cell subset cellular route (Komarova and Malik, 2010). To extravasate paracellularly, tight junction proteins need to be Once a naive CD4 þ T cell has experienced both successmodulated (Engelhardt and Wolburg, 2004). In MS, ful TCR peptide–MHC ligation and APC-mediated cothere is evidence that activated autoreactive CD4 þ T stimulation, it differentiates into an effector T cell. This cells and activated monocytes secrete elevated amounts differentiation depends on polarizing stimuli mainly of proteolytic enzymes, such as matrix metalloproteiprovided by the APC and eventually leads to differentinases (MMPs) (Sindern, 2004). Apart from mediating ation into a more or less distinct T-helper subset (Th). direct CNS tissue damage, these proteases can degrade These subsets are defined by the expression of certain tight junction proteins and thereby cause BBB disruption transcription factors and effector cytokines and and tissue edema (Yang et al., 2007). Correspondingly, guide the immune response into the direction expression of MMP-2, -3, -7, and -9 is increased in which is necessary to clear infection (Table 3.1). For EAE and MS lesions and higher levels of MMP-9 are approximately one decade the Th1 subset was considered found in the CSF during relapses (Leppert et al., 1998). the main culprit in MS. However, recent developments in

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS

67

Table 3.1 Main T-helper subsets* Main polarizing cytokines

T-cell subset

Main transcription factors

Main effector cytokines

Suspected physiologic and pathologic roles Cellular immunity Clearance of intracellular pathogens Autoimmunity Humoral immunity Clearance of certain extracellular pathogens Allergy Tissue inflammation Clearance of certain extracellular pathogens Autommunity Tolerance/immune suppression Autoimmunity

IL-12 IFN-g

Th1

T-bet STAT1 STAT4

IFN-g

IL-4 IL-33

Th2

GATA3 STAT6

TGF-ß þ IL-6{ (IL-23 important for stabilization)

Th17

RORgt RORa STAT3

TGF-ß þ RA{

Treg

Foxp3 STAT5

IL-4 IL-5 IL-10 IL-13 IL-17A (IL-17) IL-17 F IL-21 IL-22 TGF-ß IL-10 IL-35

Adapted from Jetten (2009) and Palmer and Weaver (2010). *Note that a number of other T-helper subsets have been discovered in the last years, i.e., TFH, Tr1, or Th9 cells (Palmer and Weaver, 2010). In addition, there is evidence for an intensive T-helper plasticity, i.e., between Th1 and Th17 cells (Jetten, 2009; Lee et al., 2009; Palmer and Weaver, 2010). Therefore, this list is meant to provide only a general overview. { Other polarizing conditions are possible and they appear to differ between mice and humans (Annunziato and Romagnani, 2009; Korn et al., 2009). { Not fully understood; other combinations appear possible. IL, interleukin; IFN, interferon; TGF, transforming growth factor; RA, retinoic acid.

defining additional Th subsets have led to a more complicated picture. Four main findings fuel this debate: 1.

2.

The inhibition or knockout of Th1-related polarizing and effector cytokines and receptors, i.e., IFN-g signaling, yielded highly controversial results. Mice were still susceptible to EAE, partly with a more exacerbated disease course (Ferber et al., 1996; Willenborg et al., 1996; Becher et al., 2002; Gran et al., 2002; Zhang et al., 2003). Nevertheless, a clinical trial performed over 20 years ago revealed increased relapse rates upon intravenous administration of IFN-g to MS patients, suggesting an involvement of the IFN-g axis in MS pathogenesis (Panitch et al., 1987). Unfortunately, this trial was not accompanied by mechanistic studies regarding the expansion of Th1 cells, and therefore only limited conclusions can be drawn. In 2005, Th17 cells were discovered (Harrington et al., 2005; Park et al., 2005), a subset characterized by IL-17 (IL-17A) secretion. Now, Th17 cells are known for being prominently involved in a number of putative autoimmune diseases, including psoriasis, inflammatory bowel disease, rheumatoid

3.

arthritis, lupus, and eventually MS (Korn et al., 2009; Crome et al., 2010). In the early phase of their discovery, several pieces of evidence provoked the hypothesis that not Th1 but Th17 cells are the real initiators of EAE and possibly MS (Cua et al., 2003; Brucklacher-Waldert et al., 2009). However, similar to Th1-lineage cytokines, modulation of Th17related cytokines, such as IL-17A, IL-17 F, and IL22, yielded conflicting results in EAE (Komiyama et al., 2006; Kreymborg et al., 2007; Haak et al., 2009). In MS lesions, IL-17 is upregulated (Lock et al., 2002) and expressed not only by CD4 þ T cells, but also by CD8 þ T cells and astrocytes (Tzartos et al., 2008). Adoptive transfer EAE experiments showed that T-cell encephalitogenicity cannot be restricted to a single T-cell phenotype. Both Th1 and Th17 cells are independently able to induce CNS autoimmunity (Kroenke et al., 2008; Luger et al., 2008; Steinman, 2008). Other subsets, such as Th2 and Th9 cells, also appear to induce EAE under certain circumstances (Lafaille et al., 1997; Jager et al., 2009). Interestingly, dependent on the T-cell subset, distinct patterns of EAE histology, CNS chemokine profile, and response to selective cytokine

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M. NAEGELE AND R. MARTIN inhibition are observed (Kroenke et al., 2008). For future trials and research into these questions will solve instance, anti-IL-17-antibody treatment ameliorated our current puzzles. only Th17- but not Th1-induced EAE (Kroenke et al., 2008). ESTABLISHING PHASE 4. A subpopulation of CD4 þ -T cells in MS patients Following CNS entry and local reactivation, myelinwas found to produce both IFN-g and IL-17, hence specific T cells begin to establish an inflammatory site. exhibiting characteristics of both Th1 and Th17 cells This is realized by direct cell contact-mediated activation (“Th117 cells”) (Durelli et al., 2009; Kebir et al., of resident immune cells (i.e., perivascular APCs, micro2009; Edwards et al., 2010). Th117 cells are like glia, astrocytes) and indirect cytokine-mediated promo“pure” Th17 cells elevated during relapses tion of an inflammatory environment, which attracts compared to states of remission, migrate more effiperipheral leukocytes. At this stage, inflammatory pathciently through human brain endothelium than IL-17 ways seem to diversify greatly. Therefore, it is difficult or IFN-g single expressors, and are also found to be to attribute the presence of certain proinflammatory involved in EAE (Durelli et al., 2009; Friese and mediators within MS lesions to CD4 þ T cells alone. It Fugger, 2009; Murphy et al., 2010). is more likely that these factors are increasingly proAccording to currently available evidence encephaloduced by other resident cells, i.e., microglia, astrocytes genicity cannot be attributed to single effector cytokines (De Keyser et al., 2003), and mast cells (Sospedra and such as IFN-g and IL-17. T-cell cytokines may act in a Martin, 2005), and recruited peripheral leukocytes, complex synergistic and redundant network, substituti.e., activated macrophages and CD8 þ T cells. ing for each other in artificial situations such as experiThe following inflammatory components are seen mental knockout and therapeutic inhibition (Yang et al., within active MS lesions: 2009). While data on T-cell subset end products are controversial, their lineage transcription factors appear to be 1. Presence of activated cytokine-secreting resident crucial (Bettelli et al., 2004; Yang et al., 2008, 2009). In cells such as CCR6 þ and MHC class II-expressing EAE, the Th1-related transcription factor T-bet appears astrocytes and MHC class II-expressing microglia to be more important than the Th17-related factor and mast cells (Sospedra and Martin, 2005). RORgt (O’Connor et al., 2008; Yang et al., 2009). In 2. Presence of activated cytokine-secreting perRRMS, Th17 and Th117 but not “pure” Th1 cells are eleipheral leukocytes, most notably activated macrovated during relapses (Durelli et al., 2009; Kebir et al., phages (Barnett et al., 2006), CD8þ cytotoxic 2009; Edwards et al., 2010). T cells (Friese and Fugger, 2009), CD4 þ T Interestingly, both Th17 and Th117 cells express low cells (Sospedra and Martin, 2005), gd T cells levels of T-bet along with higher levels of RORgt. This (Wucherpfennig et al., 1992), and antibodysuggests that our stringent classification into distinct producing plasma cells (Lucchinetti et al., 2000). T-cell “subsets” may not represent the situation 3. Antero- and retrograde activation of the BBB. Actiin vivo. Indeed, evidence indicates an immense plasticity vated cerebral endothelial cells express higher of these cells (Annunziato and Romagnani, 2009; Peck amounts of adhesion molecules and activation and Mellins, 2010). This might represent a loophole receptors and secrete proinflammatory cytokines out of the current controversy: Autoreactive effector and chemokines. Subsequently, recruitment and T cells might initially have a T-bet phenotype, which entry of further leukocytes into the inflammatory enables effective BBB transmigration. Once arrived in site are facilitated. Once the impairment of the the CNS, their IL-17-producing RORgt phenotype domBBB reaches a certain threshold, i.e., by extensive inates inflammatory cascades due to IL-23-mediated staMMP-mediated tight junction damage, it is detected bilization by local reactivating APCs (Li et al., 2007). by gadolinium contrast accumulation on postconIndeed, in EAE, IL-23 appears to be similarly important trast T1-weighted MRI. to T-bet and RORgt (Cua et al., 2003; t Hart et al., 2005). 4. Alterations in numerous intercellular signaling However, in RRMS, inhibition of both IL-23 and IL-12 by pathways within lesions (Table 3.2). There is suffithe monoclonal anti-p40 antibody ustekinumab did not cient evidence that not only cytokines and chemosignificantly reduce gadolinium-enhancing lesions and kines, but also other pathways such as arachidonic clinical relapse rates (Segal et al., 2008). The failure acid signaling cascades, are involved in pathogenemight be explained by the antibody’s inability to reach sis (Whitney et al., 2001). However, it is currently the CNS compartment (Steinman, 2010). Eventually, if unknown how they interact within the complex only one conclusion should be drawn, then it is “not to microenvironment of an MS lesion (Kunz and jump to conclusions” (Steinman, 2008). Hopefully, Ibrahim, 2009).


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Table 3.2 Selected cytokines, chemokines, and chemokine receptors which appear to be significantly altered in active lesions (mostly measured in SPMS patients due to the lack of specimens and biopsies from younger RRMS patients) and during RRMS relapses in CSF and peripheral blood) *

Compartment

Change

Active lesion

General abundance (quantification of extra- and intracellular mRNA/protein, e.g., whole-tissue-microarrays)

Cell-specific abundance (quantification of cell-associated mRNA/protein, e.g., by labeling tissue with antibodies)

Osteopontin IFN-g IL-17A TNF-a, TNF-ß/LT, TNFR, IL-1ß, IL-2, IL-6, IL-11a, TGF-ß, SMAD6 (TGF-ß signaling), IL-5, aB-crystallin SCF, G-CSF, M-CSF, IGF-1, FGF-12

IFN-g-expressing cells IL-17A-expressing cells IL-17R-expressing cells IL-23-expressing cells Cells expressing various chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CXCL10) or corresponding receptors (e.g., CCR1, CCR2, CCR5, CCR6, CXCR1, CXCR3) IL-4-expressing cells IL-10-expressing cells TGF-ß-expressing cells

Extracellular abundance (quantification of soluble protein, e.g., by ELISA)

Cell-specific abundance (quantification of cell-associated mRNA/protein e.g. by RT-PCR, FACS) TNF-a expressing cells IFN-g expressing cells IL-17A expressing cells Cells expressing various chemokine receptors (CCR1, CCR2, CCR3, CCR5, CCR6, and CXCR3)

Compartment

Change

CSF during relapse

Osteopontin IFN-g TNF-a GM-CSF CXCL10, CXCL13

IL-10, TGF-ß CCL2 Osteopontin IL-1ß, IL-6 CXCL8, CXCL13 MMP-9

Blood during relapse

CCL20

TNF-a-expressing cells TNF-ß/LT-expressing cells IFN-g-expressing cells IL-17A-expressing cells IL-23-expressing cells CXCL8-expressing cells IL-10-expressing cells TGF-ß-expressing cells

*Source: Cannella and Raine, 1995; Correale et al., 1995; Carrieri et al., 1998; Franciotta et al., 2001; Lock et al., 2002; Werner et al., 2002; Ishizu et al., 2005; Rosenberg, 2005; Sospedra and Martin, 2005; Ysrraelit et al., 2008; Festa et al., 2009; Kalinowska and Losy, 2009; Kunz and Ibrahim, 2009; Vogt et al., 2010. The list is far from complete and only illustrates the complexity of cytokine and chemokine changes. It is not sorted by putative importance of single cytokines due to the lack of studies comparing all the cytokines and chemokines at once. There is evidence that the cytokine and chemokine composition differs in silent lesions and the chronic progressive stages of the disease (SPMS, PPMS) (Lock et al., 2002; Ukkonen et al., 2007; Ysrraelit et al., 2008). SPMS, secondary progressive multiple sclerosis; RRMS, relapsing-remitting multiple sclerosis; CSF, cerebrospinal fluid; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription polymer chain reaction; FACS, fluorescence-activated cell sorting. (lesion), cytokines/chemokines/cell is more abundant within the MS lesion than in healthy parenchyma; (CSF/blood), cytokines/chemokines/ cell is more abundant in CSF/blood during relapse than during remission.

70 5.

M. NAEGELE AND R. MARTIN Alterations in numerous intercellular signaling pathways within the CSF and serum (Table 3.2), most likely deriving from both the lesions and activated leukocytes in circulation. Longitudinal studies revealed that certain proinflammatory cytokines are upregulated in the serum preceding and during relapses, while others are increased during states of remission (Rieckmann et al., 1994; Correale et al., 1995). However, apart from the presence of oligocloncal bands (see below), currently no other biomarker, e.g., cytokine or chemokine, is used to detect relapses. The main reason is their minute quantity and hence difficulty of detection in peripheral blood. Eventually, it is possible that neurological symptoms and MRI enhancement arise at a time when acute serologically measurable reactions have already subsided. This is supported by a previous study, which found no significant correlation of serum IFN-g, IL-4, IL-10, and lymphotoxin with MRI data (Kraus et al., 2002).

Two components putatively involved in the initiating and establishing phase of EAE and MS have recently received increased attention: One is osteopontin (OPN), a protein initially associated with bone metabolism. Interestingly, it is one of the most abundant gene transcripts within acute MS lesions (Chabas et al., 2001) and is also elevated in plasma around the time of relapse (Steinman, 2009a). Studies showed that the VLA-4 integrin binds to OPN, thus increased levels of OPN within lesions could attract VLA-4-carrying cells (Steinman, 2009a). This might explain the efficacy of the VLA-4-blocking antibody natalizumab, apart from its direct inhibitory effect on leukocyte BBB transmigration. Administration of OPN induced EAE relapses (Hur et al., 2007), OPN–/– mice had an ameliorated EAE course (Jansson et al., 2002), and inhibition of OPN with antibodies attenuated acute EAE (Steinman, 2010). Thus, it has been proposed to inhibit OPN in MS (Steinman, 2010). A cell type gaining more recent attention in MS research are gd T cells (Sospedra and Martin, 2005; Wohler et al., 2010). gd T cells are T cells which carry gd TCRs and therefore differ from the more common aß TCR T cells. They are thought to be involved in the very first phase of pathogen recognition by orchestrating acute innate immune responses (Martin et al., 2009). Interestingly, gd T cells are found within MS lesions (Wucherpfennig et al., 1992), carry Toll-like receptors, and are able to express CCR6 and produce IL-17 upon appropriate stimuli (Martin et al., 2009). Future studies will need to determine whether autoreactive gd T cells contribute to pathology in MS.

EFFECTOR PHASE Once autoreactive T cells have established the lesion (“legislative part”), effector cascades mediating the direct damage to myelin, oligodendrocytes, axons, and eventually neurons come to the fore (“executive part”). Although there is in vitro evidence that CD4 þ T cells can mediate direct damage themselves, i.e., by granzyme Bmediated killing of neurons (Kebir et al., 2007), it is more likely that other cells, such as macrophages, microglia, B cells, and CD8 þ T cells, as well as their effector molecules, are the actual myelin-damaging factors. This is based on the following observations: First, in acute lesions, macrophages and microglia are much more abundant than lymphocytes. Second, within lymphocytes, CD8 þ T cells are more abundant than CD4 þ T cells deep in the parenchyma (Lucchinetti et al., 2000; Friese and Fugger, 2009). Third, axonal damage appears to correlate better with the number of phagocytes (macrophages and microglia) and CD8 þ T cells than with CD4 þ T cells (Friese and Fugger, 2009). Pathological correlates of acute damage within lesions are loss of myelin, reduced numbers of oligodendrocytes with the remaining in apoptotic states (Barnett and Prineas, 2004), high numbers of myelin-laden macrophages, widespread axonal damage (e.g., axonal transaction (Trapp et al., 1998)), and hypertrophic astrocytes (Frohman et al., 2006; Hu and Lucchinetti, 2009).

Phagocytes Phagocytes are responsible for phagocytosing invading pathogens or debris from damaged host tissue. With respect to MS pathogenesis, infiltrating activated macrophages and activated microglia are probably most important. In acute lesions, macrophages and microglia outnumber lymphocytes by at least 10–20 times (Barnett et al., 2006). Chronic and progressive lesions are characterized by diffuse microglial activation (Compston and Coles, 2008). Possible CNS-damaging effector mechanisms of phagocytes are: The release of proteolytic tissue-degrading enzymes (i.e., MMP-2 and 9), the secretion of apoptosis-inducing mediators (i.e., TNF-a), the extensive generation of nitric oxide, and reactive oxygen species (ROS) and antibody-dependent cell-mediated cytotoxicity (Sospedra and Martin, 2005). EAE experiments support an important role of macrophages: When they are depleted shortly before disease flare-up, Lewis rats develop a markedly ameliorated EAE course (Huitinga et al., 1990). However, while macrophages appear to be important at the peak of inflammatory lesions, there is evidence that extensive damage already occurs before their appearance within lesions.

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS 71 Barnett and Prineas (2004) studied pathologic specicomplement deposition and axonal injury and exacermens of acute MS and noticed that oligodendrocytes bate EAE (Mathey et al., 2007). are already gone or apoptotic before myelin-laden It has been proposed that the extensive release of self macrophages are found within lesions. This suggests antigens within chronic inflammatory sites further that other factors predominate in the very early phase impairs tolerance and promotes autoantibody generaof CNS damage and macrophages rather exert a tion (Wu et al., 2001). This might explain the occurrence debris-clearing role once tissue damage has occurred of antibodies against certain extracellular and intracellu(Barnett et al., 2006). Putative suspects are activated lar non-myelin antigens such as aB-crystallin (Celet microglia, which appear to be involved from the very et al., 2000; Steinman, 2009b), contactin-2 (Derfuss acute to chronic stages of the disease: Investigators et al., 2009), neurofilaments (Sjogren et al., 2001), and studying acute Marburg’s disease sections found prothe proteasome (Mayo et al., 2002). In neuromyelitis found microglial activation within “pre-demyelinating” optica, antibodies against aquaporin 4 are found in the pattern III lesions, which are characterized by mild majority of patients and their existence within the serum edema, slight axonal injury, and the absence of overt is clinically used to discriminate this disorder from condemyelination (Marik et al., 2007). ventional MS (Paul et al., 2007). A number of recent findings go beyond the autoreactive antibody hypothesis (Franciotta et al., 2008; Pender, B cells and antibodies 2009): Investigators found ectopic lymphoid follicles The presence of oligoclonal bands (OCBs) within the within the meninges in two-thirds of analyzed SPMS CSF is a long-known (Kabat et al., 1942) and wellbrain specimens (Serafini et al., 2004). These structures established diagnostic finding in MS (McDonald et al., harbor B cells and plasma cells (Serafini et al., 2004), lie 2001; Polman et al., 2005). OCBs are bands of oligocloin close proximity to subpial lesions, and correlate with nal immunoglobulins, mainly immunoglobulin G (IgG), early onset of disease (Magliozzi et al., 2007). Intriguwhich are separated based on differences in isoelectric ingly, EBV-infected B cells and plasma cells were points (Rammohan, 2009). The existence of these bands found within these structures (Serafini et al., 2007). within the CSF, but not within the serum, is a strong indiTherefore, it has been hypothesized that a persistent cator of intrathecal antibody synthesis and, interestingly, EBV infection of B cells within active lesions and ectopic is found in nearly all patients with clinically definitive follicles contributes to MS pathology (Lublin and MS (Zeman et al., 1996). Intrathecal antibodies are Reingold, 1996; Franciotta et al., 2008). However, earlier mainly produced by plasma cells (terminally differenti(Serafini et al., 2007) and subsequent studies (Peferoen ated B cells), and hence an involvement of B cells in the et al., 2010; Willis et al., 2009) could not reproduce the pathogenesis of MS has long been suspected. However, findings of Serafini et al. Whether homing of EBVinsight into the role of B cells and their intrathecal antiinfected B cells into the CNS is a primary event remains bodies has been hindered by the difficulty in determining pure speculation (Serafini et al., 2007). Nevertheless, an their specificity (McFarland and Martin, 2007). involvement of EBV in MS pathogenesis is supported by B-cell involvement in MS pathogenesis can be maniother investigations (Franciotta et al., 2008): Intrathecal fold (Sospedra and Martin, 2005): On the one hand they antibodies were found to be specific for EBV proteins could directly stimulate and activate autoreactive T cells (Cepok et al., 2005). Corresponding with previous assoin their function as APCs. On the other, autoreactive ciation studies (Lublin and Reingold, 1996), recent invesantibodies bound to CNS tissue could either act as opsotigations found that increased antibody responses to the nins, facilitating Fc receptor-mediated phagocytosis and EBV protein EBNA1 correlate with MRI severity and cytotoxicty, or activate the complement system, promotpredict the conversion of clinically isolated syndrome ing further chemotaxis and cell damage. The latter is to clinically definitive MS (Hoffmann et al., 2010). In supported by prominent complement and antibody depoaddition to an altered B-cell axis, EBNA1-specific T cells sition within active demyelinating pattern II lesions from MS patients were found to cross-react more fre(Lucchinetti et al., 2000). quently with myelin than other MS-unrelated antigens However, although myelin-reactive antibodies have (Lunemann et al., 2008). been found in MS patients, their role and specificity Finally, the putative involvement of B cells in MS has are still highly controversial (Sospedra and Martin, been strengthened by a recent phase II randomized con2005). For some of them, there is evidence of a trol trial (Bourdette and Yadav, 2008): Rituximab is a disease-exacerbating role. For instance, antibodies monocloncal anti-CD20 antibody and on two-times against neurofascin, a myelin and neuronal adhesion administration leads to a near-complete (> 95%) and protein found at the nodes of Ranvier and other comsustained (up to 48 weeks) depletion of peripheral partments of the myelin–axon unit, were found to cause CD19 þ B cells (Bourdette and Yadav, 2008). In RRMS

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patients, a two-dose administration of rituximab leads to a quick (within 12 weeks), sustained (up to 48 weeks), and highly efficacious (91 relative reduction) reduction in gadolinium-enhancing lesions compared to placebo (Bourdette and Yadav, 2008). Interestingly, immunoglobulin levels were not significantly altered in number, which is supported by the absence of CD20 on antibody-producing plasma cells (Franciotta et al., 2008). This suggests that it is not the reduction in potentially deleterious autoantibodies, but other functions of B cells, e.g., antigen presentation, that is responsible for the high efficacy of rituximab.

CD8 þ T cells The role of CD8þ T cells in MS and autoimmunity in general has long been a neglected issue, probably owing to the predominance of the CD4 þ T cell presenting HLA class II alleles as the major risk factors in several autoimmune diseases, including MS (Friese and Fugger, 2009). Now, more evidence is available pointing towards an important role of CD8 þ T cells in MS (Sospedra and Martin, 2005; Friese and Fugger, 2009). One striking feature of MS lesions is that CD8 þ T cells are regularly found to be more abundant than CD4þ T cells, contrasting the CD4þ:CD8þ ratio of about 3:1 to 6:1 in the CSF and 2:1 in the peripheral blood (Friese and Fugger, 2009). One possible pathogenic role of CD8þ T cells within the lesions could be the recognition of myelin antigens on oligodendrocytes and subsequent CD8 þ -mediated cytotoxicity (Sospedra and Martin, 2005). Although the associations of HLA class I alleles with MS are weaker, HLA-A*0301 was found to double the risk for MS in an HLA-DR2-independent fashion (Fogdell-Hahn et al., 2000; Burfoot et al., 2008). Certain epitopes of myelin proteins (e.g., MBP, PLP, and others) appear to be HLA class I-restricted (Sospedra and Martin, 2005). CD8þ T cells were found to be oligoclonally expanded within lesions, CSF, and peripheral blood, indicating an involvement in antigen-specific responses similar to CD4þ T cells (Jacobsen et al., 2002; Friese and Fugger, 2005). Apart from mediating direct cell contact-mediated cytotoxicity, they could promote the inflammatory and CNS toxic microenvironment by participating in the IL-17 cytokine axis: In a diabetes model, CD8 þ T cells expressed IL-17A and IL-22 upon IL-23 stimulation and became pathogenic upon adoptive transfer (Ciric et al., 2009). IL-17-expressing CD8 þ T cells are also observed within active lesions, along with equal amounts of IL-17-expressing CD4þ T cells (Tzartos et al., 2008), suggesting that local IL-23 or another IL-17-promoting factor acts on both cells. Nevertheless, the current notion from EAE experiments is that CD8þ lymphocytes still require CD4þ T cells before becoming truly encephalitogenic (Friese and Fugger, 2009). Finally, it should be noted that, parallel to regulatory CD4þ T cells, a

population of regulatory CD8 þ T cells exists, which might exert an anti-inflammatory role within lesions (Friese and Fugger, 2009).

Cytotoxic cytokines Direct cell contact-mediated cytotoxicity is likely not the only effector mechanism. Within lesions a wide range of soluble proinflammatory molecules can be found, i.e., cytokines, chemokines, arachidonic acid metabolites, proteolytic enzymes, antibodies, complement proteins, and ROS. While their accumulation provides further positive feedback for invading cells, some of the molecules might mediate a substantial proportion of CNS damage. Regarding abundant cytokines within lesions, several, including TNF-a and lymphotoxin, were found to be toxic for both neurons and oligodendrocytes in vitro (Selmaj and Raine, 1995; Zhang et al., 2005; McCoy and Tansey, 2008). Others, such as IL-1ß and IL-6, appear to be more pleiotropic in regard to their direct toxicity (Sallmann et al., 2000; Vela et al., 2002). One of the most prominent cytokines putatively involved in direct damage is TNF-a. Overexpression of TNF-a within the CNS of mice leads to spontaneous demyelination and TNF-a inhibition was found to suppress EAE manifestation (Probert et al., 1995; Selmaj and Raine, 1995). To achieve neurotoxicity, TNF-a synergizes with other factors such as glutamate excess and IFN-g (Andrews et al., 1998; Zou and Crews, 2005). Although the findings strongly support a deleterious role of TNF-a, the exacerbating effect of TNF-a inhibitors in MS patients indicates the opposite (van Oosten et al., 1996; Group, 1999). If TNF-a inhibitors reached the CNS, there is evidence that they might have impaired important regulatory mechanisms: In a cuprizone-induced demyelinating model, TNF-a–/– mice exhibited an initially delayed but otherwise indistinguishable demyelination after 5 weeks (Arnett et al., 2001). Interestingly, after cuprizone withdrawal, TNF-a–/– mice had significant problems in remyelination. The pleiotropic effect of TNF-a appears to be mediated by different receptors: The investigators showed that TNFR1 signaling mediated demyelination, whereas TNFR2 signaling was crucial for successful remyelination (Arnett et al., 2001). Therefore, TNF-a inhibitors might have impaired remyelinating and inflammation-terminating cascades within lesions. If the pleiotropy of cytokines is a common phenomenon, therapeutic strategies will be further complicated, given that demyelination and remyelination often occur simultaneously within a single patient. Hopefully, future trials which target other presumably deleterious cytokines (i.e., IL-17A, IL-17 F, OPN) will clarify whether single-cytokine modulation is a feasible therapeutic strategy in MS.


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Complement

Ion overload

The complement system is a soluble protein cascade of the innate immune system leading to chemotaxis, opsonization, and cytolysis by the formation of protein pores, also called membrane attack complexes (MAC). It can interact with adaptive immunity, i.e., cytolytic cascades can be activated by cell-bound antibodies. There is substantial evidence that complement activation plays a role in MS (Kulkarni et al., 2004; Ingram et al., 2009), also seen by the prominent deposition of complement proteins in type II lesions (Lucchinetti et al., 2000). The complement protein C5 plays a critical role in initiating MACs. Eculizumab, a monoclonal antibody against C5, was found to reduce attack frequency and disability progression in neuromyelitis optica (Pittock et al., 2013).

CNS inflammation impairs the myelin–axon unit not only through clear immunologic cascades but also by disrupting its crucial electrophysiologic balance. In MS, electrophysiologic disruption of oligodendrocytes and neurons can occur at many levels, but one central converging pathway is the intracellular overload of calcium and sodium. An excessive accumulation of these ions leads to protease overactivation, cytoskeleton disruption, mitochondrial dysfunction, energy failure, oxidative stress, and eventually axonal and neuronal degeneration (Arundine and Tymianski, 2003; Demaurex and Scorrano, 2009). One contributing factor is glutamate excitotoxicity, defined by a glutamate-mediated overactivation of NMDA and AMPA receptors, which are permeable for calcium and sodium ions. Two factors synergize in causing glutamate accumulation and subsequent excitotoxicity within inflammatory areas: One is the increased glutamate production by activated cells such as lymphocytes and macrophages (Piani et al., 1991), the other is its impaired clearance by resident cells such as astrocytes (Korn et al., 2005). The relevance of glutamate excitotoxicity is supported by therapeutic intervention: Treatment of mice with AMPA antagonists substantially ameliorated EAE and increased oligodendrocyte and axonal survival (Pitt et al., 2000). In addition, a small clinical trial with the agent riluzole, a glutamate signaling inhibitor used for amyotrophic lateral sclerosis, was able to reduce slightly cervical cord atrophy and T1 MRI lesion load in PPMS patients (Kalkers et al., 2002). Another ion overloading pathway is the compensatorily increased expression and redistribution of voltage-gated sodium channels along demyelinated axons (Waxman et al., 2004). In an attempt to regulate the subsequently increased sodium ion influx, sodium-calcium exchangers reverse their transport, i.e., export sodium and import calcium ions. The accumulation of intracellular calcium again leads to toxicity. Supporting this hypothesis, sodium channel blockers such as flecainide and phenytoin preserved axons in EAE models (Bechtold et al., 2004; Lou et al., 2005). Recent research has also focused on non-selective cation channels such as TRPM4. Blockade of TRPM4 with the antidiabetic drug glibenclamide was found to ameliorate EAE (Schattling et al., 2012). Local inflammation is characterized by tissue acidosis, i.e., the overabundance of protons. There is evidence that acidosis itself is linked with axonal degeneration. Neurons express the proton-gated acid-sensing ion channel 1 (ASIC1). Interestingly, ASIC1–/– mice have a markedly ameliorated EAE course and reduced axonal degeneration compared to wild-type mice (Friese et al., 2007). Investigations revealed that local acidosis within inflamed CNS tissue can principally mediate the opening of ASIC1 channels, which

Oxidative stress Another important pathogenetic factor in MS and other neurodegenerative disorders is oxidative stress (GilgunSherki et al., 2004; Halliwell, 2006; Gonsette, 2008). Oxidative stress occurs when ROS excessively oxidate DNA, lipids, and proteins and antioxidative mechanisms fail to intervene. Excessive oxidative damage impairs proteosomal and mitochondrial function and induces cell death (Halliwell, 2006). The mammalian brain appears to be especially sensitive to oxidative damage, possibly due to its high basal oxygen consumption (and subsequent oxygen radical formation) (Halliwell, 2006). Inflammation is regularly associated with oxidative stress, as ROS are physiologically used to eradicate intra- and extracellular pathogens. In MS, activated macrophages and other cell types are able to produce substantial amounts of these mediators and thereby damage oligodendrocytes (Qin et al., 2009) and neurons (Demaurex and Scorrano, 2009). In addition, intrinsic oxidative stress induced by glutamate excitotoxicity contributes to pathology (Demaurex and Scorrano, 2009). Several factors indicate that oxidative damage is a common phenomenon in EAE and MS lesions, i.e., presence of oxidized lipids (Qin et al., 2007), carbonylated cytoskeletal proteins (Smerjac and Bizzozero, 2008), and the indirect detection of free radicals (Qi et al., 2007; Lassmann, 2008). Overexpression of radical scavenger proteins within mice significantly reduced long-term tissue damage in a chronic EAE model (Qi et al., 2007). Thus, antioxidative therapeutic approaches might be beneficial in MS and other neurodegenerative diseases. Unfortunately, up to this point many of the agents which are successful in animal models yielded only very marginal results in the human system (Halliwell, 2006). Hopefully, further trials will elucidate whether antioxidative agents are able to slow down disease progression in MS and other neurodegenerative disorders (Gilgun-Sherki et al., 2004).

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subsequently promote calcium- and sodium-mediated neurotoxicity (Friese et al., 2007). Amiloride, an ASIC blocker, was found to be neuroprotective in EAE and nerve explants, suggesting that ASIC blockade might be a feasible strategy in reducing neurodegeneration in MS (Friese et al., 2007). Altogether, in addition to the previously mentioned elements, ion overload significantly contributes to the toxic damage of oligodendrocytes and neurons. Therapeutic modulation of ion channels may delay neurodegeneration in MS and other chronic disorders of the CNS.

THE REGULATORY CASCADE OF MULTIPLE SCLEROSIS The previous section only covered our current understanding of proinflammatory mechanisms in MS. However, it is very likely that antagonizing elements play an equally important role by moderating the extent of inflammation and initiating steps leading to remission. Similar to polymorphisms in proinflammatory pathways, variable defects in these opposing components might define the heterogeneity of MS. In general, they can be classified into regulatory elements, which prevent inflammation and myelin damage, and repairing elements, which mediate remyelination and regeneration after damage has occurred. It is likely that they highly overlap and accompany each other. Therefore, this separation should not be considered absolute.

Regulatory and preventive elements The immune system, similarly to every other complex biological system, is characterized by agonists and antagonists, which hold the system in perfect balance at states of rest. Therefore it is not surprising that CD4þ T cells not only develop into proinflammatory effector subsets (Th1, Th17, etc.), but also into distinct anti-inflammatory lineages. Probably the most important anti-inflammatory CD4þ T cells are regulatory T cells or Tregs (Vignali et al., 2008). Tregs are generally defined as being CD4þ and CD25high and characterized by their transcription factor, Foxp3. They are either naturally generated in the thymus (nTregs) or induced upon appropriate stimuli (iTregs). Their function is to inhibit effector T cells, e.g., by direct cell–cell contact, or by secreting inhibitory cytokines, such as TGF-ß, IL-10, and IL-35 (Vignali et al., 2008). Interestingly, they seem to be reciprocally related to Th17 cells since they share TGF-ß for differentiation, but differ in other polarizing cytokines (Korn et al., 2009). There is accumulating evidence that functional Foxp3 þ Tregs are crucial for preventing autoimmunity (Kim et al., 2007; Wing and Sakaguchi, 2010). Correspondingly, data indicate that their function is impaired in EAE and MS (Venken et al., 2010). In MOG- and

complete Freund’s adjuvant-induced EAE, myelinspecific Foxp3þ Tregs were found to accumulate within the CNS, but failed to inhibit effector T cells (Korn et al., 2007). The authors suggested that, once tolerance has been broken, early accumulation of IL-6 and IL-17 within inflammatory lesions inhibits Treg function. Increase in IL-10 within the CNS preceded clinical remission of mice, which is produced by Tregs but also effector T cells. Higher serum IL-10 mRNA levels are also seen in RRMS patients during phases of remission (Rieckmann et al., 1994), suggesting that IL-10producing cells are involved in terminating inflammation. However, what causes the shift from relapse to remission remains an enigma. Apart from their role during relapses, Tregs from RRMS patients appear to be generally impaired. They have a reduced capacity to suppress anti-CD3-activated effector T cells, possibly due to a diminished Foxp3 expression (Viglietta et al., 2004; Venken et al., 2008a). Additionally, reduced numbers of recent thymic emigrating Tregs were found in the peripheral blood of MS patients, suggesting alterations in the thymic generation of T-cell subsets (Haas et al., 2007; Venken et al., 2008b). However, whether these alterations have genetic causes or are just the consequence of the inflammatory environment in MS patients is currently unknown (Venken et al., 2010). Apart from CD4 þ T cells, regulatory lineages have also been reported for other cell types and there is evidence for their impairment in MS (Costantino et al., 2008). Interestingly, higher frequencies of several regulatory lineages are associated with certain immunomodulatory treatments, suggesting that part of their efficacy is mediated by these subsets (Venken et al., 2010). For instance, glatiramer acetate appears to induce regulatory CD8 þ T cells which are able to kill CD4 þ T cells (Karandikar et al., 2002; Tennakoon et al., 2006). In addition, glatiramer acetate induces certain type II monocytes, which are able to shift the T-cell repertoire towards protective Tregs and Th2 cells in EAE (Weber et al., 2007a). Likewise, there is evidence for a regulatory role of the natural killer (NK) cell lineage in both EAE and MS: NK cell depletion exacerbated EAE and NK cells were found to be qualitatively and quantitatively impaired in MS patients (Sospedra and Martin, 2005). Correspondingly, the effective anti-CD25 antibody daclizumab expanded a regulatory CD56high NK cell subset in MS patients (Bielekova et al., 2006). Eventually, even gd T cells can exert regulatory functions: gd T-cell-deficient mice are unable to recover from EAE because they are needed to induce Fas/Fas ligandmediated apoptosis of encephalitogenic T cells during the phase of remission (Ponomarev and Dittel, 2005). Most of these regulatory lineages secrete IL-10, which appears to be a key anti-inflammatory cytokine in MS.

THE GOOD AND THE BAD OF NEUROINFLAMMATION IN MULTIPLE SCLEROSIS 75 Indeed, IL-10–/– mice develop severe EAE and overex(see section on establishing phase, above), administration pression of IL-10 induces EAE resistance (Bettelli of recombinant aB-crystallin or a corresponding analog et al., 1998). MS patients have lower numbers of might help to dissolve relapses and reduce CNS inflammaIL-10-secreting peripheral mononuclear cells, and IFNtion (Ousman et al., 2007; Steinman, 2009b). ß treatment resets IL-10 production to the levels of In short, regulatory leukocytes, their products, and healthy controls (Ozenci et al., 2000). In vitro, IL-10 proresident anti-inflammatory mechanisms are capable of tects oligodendrocytes from lipopolysaccharide and restraining CNS autoimmune responses. It is likely that IFN-g-mediated cell death (Molina-Holgado et al., many other yet unknown cytokines and signaling path2001). IL-10 secretion itself appears to be moderated ways act together in a complex network to achieve this by other cytokines, such as IL-27. This cytokine belongs goal. Future studies will hopefully provide greater to the same family as IL-6 and IL-12 and is produced by insight into how we could stimulate and use these pathactivated dendritic cells and infiltrated macrophages ways therapeutically to achieve anti-inflammatory and (Batten et al., 2006). Similar to IL-10, IL-27 appears to neuroprotective effects. have a crucial role in restraining autoimmunity within the CNS: IL-27 deficiency leads to severe EAE and IL27 treatment suppresses its effector phase (Batten Repairing and remyelinating elements et al., 2006; Fitzgerald et al., 2007). Interestingly, IL27 induces effector CD4 þ and CD8þ T cells to secrete The aftermath of an acute relapse leaves behind an area IL-10 and inhibits their IL-17 secretion (Stumhofer et al., purged of oligodendrocytes and rich in demyelinated 2006; Fitzgerald et al., 2007). Although this supports the and damaged axons. Initially, remyelination was relevance of anti-inflammatory cytokines, translation of regarded as a rare event. However, it is now clear that, these findings into the clinical setting has yielded disapto a certain extent, remyelination occurs frequently pointing results so far: In small phase I trials, administrawithin inflammatory lesions, as evidenced by prominent “shadow plaques” in pattern I and II lesions (Lucchinetti tion of IL-4, IL-10, or TGF-ß had no significant influence et al., 2000). Although less is known about the precise on the MS course, again suggesting that circulating cytokines, similar to antibodies, fail to reach the CNS (Wiendl molecular mechanisms, remyelination is thought to be and Hohlfeld, 2002). Alternatively, it is possible that orchestrated by a complex network of neurotrophins, these cytokines are only effective when secreted within cytokines, and chemokines, which recruit oligodendrothe immunologic synapse during direct cell–cell contact. cyte precursor cells (OPCs) from the vicinity of the lesion Most of the factors described above relate to the and induce their differentiation into myelinating oligointrinsic capability of the peripheral immune system in dendrocytes (Franklin and Ffrench-Constant, 2008). Although inflammation in MS leads to demyelination, restraining its overactivation. This physiologic mechaevidence suggests that it may also be required for nism limits excessive (bystander) damage to the body’s own tissue. However, it should not be forgotten that reseffective remyelination (Hohlfeld, 2007). Supporting ident inhibitory mechanisms of the immunoprivileged evidence derives from animal models: In a chronic CNS also contribute to the repression of damaging elenon-inflammatory demyelinating model, remyelination ments (see section on the immune privilege of the central only occurs when an inflammatory stimulus is given in nervous system, above). parallel (Foote and Blakemore, 2005). Similar results One molecule involved in resident counter-regulatory have been obtained by other investigators (Setzu et al., 2006). This suggests that acute inflammation in nonmechanisms is aB-crystallin (CRYAB). It belongs to a remyelinating situations generates stimulatory signals family of proteins carrying a common heat shock protein domain (HSP20), which enables them to inhibit protein for OPCs to initiate remyelination. These stimulatory aggregation when induced under cellular stress signals might derive from both resident and infiltrating (Steinman, 2009b). aB-crystallin has gained attention in immune cells: One the one hand, they produce putative recent years due to three observations: First, it appears oligodendrocyte-attracting chemokines (Omari et al., to be the most abundant gene transcript in early MS lesions 2005). On the other, they secrete cytokines and neurotro(Chabas et al., 2001). Second, it appears to be one imporphins, which might provide the right microenvironment for OPC differentiation (Hohlfeld, 2007). This notion tant target of T-cell- and antibody-mediated autoimmune might explain the remyelinating and pleiotropic effects responses in MS (van Noort et al., 1995; Ousman et al., 2007). Third, the protein appears to perform significant of TNF-a, IL-1ß, MMPs, and other molecules abundantly neuroprotective and anti-inflammatory effects in the brain secreted in the acute phase of MS (see section on (Steinman, 2009b). Indeed, injection of recombinant aBcytotoxic cytokines, above) (Yong, 2005). Some investicrystallin resolved ongoing paralysis in various models gators go even further by proposing the existence of a of EAE (Ousman et al., 2007). In parallel to OPN inhibition “protective autoimmunity” (Schwartz and Kipnis, 2005).

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This hypothesis is supported by the capability of autoreactive T cells to protect local axons and neurons from degenerating after traumatic spinal cord injury (Schwartz et al., 1999). Correspondingly, naturally occurring autoreactive IgM antibodies were found to bind to antigens on specific CNS cells, activating intracellular repair pathways (Wright et al., 2009). Investigators in support of this view advocate to abandon unspecific immunosuppression in MS (i.e., azathioprine, corticosteroids, certain monoclonal antibodies) and instead prefer more specific immunomodulatory therapies (i.e., glatiramer acetate) (Schwartz and Kipnis, 2005; Glezer et al., 2007; Yong and Rivest, 2009). Our understanding of regulatory and remyelinating cascades has significantly improved throughout the years. Certainly, many questions remain. It is unclear why some lesions present with a sharp edge, where demyelinated axons lie in direct neighborhood to myelinated and intact tissue (Frohman et al., 2006). The presence at the lesion border of inhibitory molecules, which impair OPC repopulation, has been suggested. However, the underlying cause of this inhibitory zone is yet unknown. Before we can begin to modulate successfully without impairing these complex biologic cascades, a much greater understanding of intrinsic immunoregulation and remyelination is needed.

important role in improving the quality of life of patients. Beyond symptomatic approaches and treatment of acute relapses, several disease-modifying therapies are now available with more approaching approval or being in late-stage clinical testing. The two first-line agents are IFN-ß and glatiramer acetate. Clinical trials have revealed that they are able to reduce annual relapse rates, slightly delay expanded disability status scale progression and improve MRI parameters (Goodin, 2008). However, due to the general lack of long-term follow-up trials, their influence on long-term disease outcome is as yet unclear (Wiendl and Hohlfeld, 2009). Immunomodulatory or immunosuppressive therapy in general only seems to be efficacious in the relapsing-remitting phase of the disease, where inflammation is thought to dominate events (Table 3.3). In addition, recent trials with IFN-ß suggest that starting treatment as early as possible has a beneficial impact on disease course (Goodin and Bates, 2009). Unfortunately, once the secondary progressive stage has been reached, therapeutic possibilities diminish, with only some (e.g., IFN-ß, mitoxantrone) showing modest effects. Besides, up to today no single treatment has shown significant efficacy in PPMS patients (Hartung and Aktas, 2009). The failure to treat PPMS and SPMS might have a lot to do with our current inability to treat neurodegenerative disorders in general. In short, five main therapeutic developments are currently underway:

THERAPEUTIC APPROACHES

1.

To this day, MS, like most autoimmune diseases, cannot be cured. Therefore, symptomatic therapy (e.g., management of bladder dysfunction and spasticity) plays an

The understanding of the current first-line diseasemodifying agents, IFN-ß and glatiramer acetate, has improved significantly. For instance, both

Table 3.3 Selected available and promising disease-modifying therapies for relapsing-remitting multiple sclerosis (November 2013)

Group

Therapy

Proposed mechanism of action

First line

Interferon-ß

● Complex (i.e., inhibition of

Glatiramer acetate

●

Dimethyl fumarate

●

Teriflunomide

●

Fingolimod

● ● ●

BBB transmigration and Th17-cells, promotion of regulatory lineages) Complex (promotion of regulatory lineages) Not clear (immunomodulation) Pyrimidine synthesis inhibitor Impairs T-cell activation S1P-receptor modulator Inhibits lymphocyte emigration from lymphoid organs

Comments ● Moderate efficacy ● Good side effect profile ● Neutralizing antibodies ● ● ● ● ● ● ● ● ●

Moderate efficacy Good side effect profile Moderate efficacy Oral agent Moderate efficacy Risk of hepatotoxicity Oral agent High efficacy Risk of infections and cardiac side effects ● Oral agent


77

Table 3.3 Continued

Group

Therapy

Second line

Natalizumab

Proposed mechanism of action

Comments

● Anti-CD49d antibody ● Inhibits BBB

● High efficacy ● Risk of progressive

transmigration

Promising agents (phase III)

Mitoxantrone

● Immunosuppressive agent ● Induces lymphopenia

● ●

Azathioprine

● Immunosuppressive agent ● Induces lymphopenia

Rituximab/Ocrelizumab

● ● ● ●

● ● ● ● ● ● ●

Daclizumab

Alemtuzumab

Anti-CD20 antibody Depletes B-cells Anti-CD25 antibody Induces regulatory NK-cells ● Anti-CD52 antibody ● Depletes lymphocytes

multifocal leukoencephalopathy High efficacy Risk of cardiomyopathy and secondary leukemia Moderate efficacy Risk of malignancies Oral agent High efficacy Side effect profile unclear High efficacy Side effect profile unclear

● High efficacy ● High risk of B-cell

mediated autoimmunity Laquinimod

● Not clear

AHSCT

● Reconstitution of the

● Oral agent

(immunomodulation) Promising alternative approaches

immune system

● High efficacy ● Risk of mortality (1–2%)

around the procedure Note: The listed efficacies and side-effects represent the general impression from phase II to IV trials and may not reflect the overall risk–benefit profile of the drug (Berger and Houff, 2009). NK, natural killer; AHSCT, autologous hematopoietic stem cell transplantion.

2.

3.

agents appear to induce certain regulatory lineages which inhibit the proinflammatory elements of the pathogenic cascade, i.e., Th17 cells (Weber et al., 2007a, b; Venken et al., 2010). Evidence also points towards neuroprotective properties, i.e., the induction of neurotrophins by glatiramer acetate (Weber et al., 2007b). Similar to other autoimmune disorders, monoclonal antibodies gain importance in the clinical therapy of MS (Table 3.3) (Lutterotti and Martin, 2008). They are potent agents, but the rise of serious long-term side-effects such as opportunistic infections (i.e., PML) would counsel for a better understanding of their efficacy and risk profile (Berger and Houff, 2009). Fingolimod was the first oral agent to be approved by the FDA in 2010 and since then, dimethyl fumarate and teriflunomide have followed. Compared to injection therapeutics, they have the ability to improve patient compliance. However, some of the agents have a worse side-effect profile than

4.

5.

IFN-ß and glatiramer acetate. Therefore, more careful risk-benefit analyses and on-drug monitoring strategies are required for these agents. Antigen-specific therapies are in preclinical and clinical development. However, until now, most of them have yielded highly efficacious results in animal models, but upon transfer from bench to bedside unfortunately failed their high expectations (Lutterotti et al., 2008). To achieve widespread success with antigen-specific therapy, it is likely that we require an even greater understanding of the individual genetic, environmental, and pathogenetic components of MS. Another major issue in MS therapy is the treatment of its neurodegenerative component. It is now clear that neurodegeneration can to a certain extent be prevented by successfully treating the inflammatory aspect, as seen by the moderate neuroprotective effects of glatiramer acetate and other immunomodulators (Khan et al., 2005). However, up to now, no single convincing agent has evolved. Treating and understanding neurodegeneration will be a

78


Table 3.4 Some of the crucial challenges multiple sclerosis research has to face in the next decade Etiology

Immunology

Clinic

Identify further genetic components and functionally associate them with the pathogenetic cascade Identify further environmental components and functionally associate them with the pathogenetic cascade Link the heterogeneic findings from pathologic studies with genetic, environmental, and immunologic phenotypes Further outline the role of CD4 þ T cells, CD8þ T cells, gd T cells, B cells, Epstein–Barr virus infection, and the innate immune system in pathogenetic cascades of multiple sclerosis Determine whether cytokines and chemokines are indeed highly redundant and pleiotropic in pathogenesis Improve animal models to reflect the human disease more closely Find ways to transport protein-based pharmaceuticals successfully into the central nervous system (or to find small molecules which achieve similar results) Find efficient and safe ways to immunomodulate selectively without impairing general host immunity (antigen-specific therapies) Find ways to prevent, anticipate, and cure the adverse effects of monoclonal antibodies (e.g., progressive multifocal leukoencephalopathy) Identify further biomarkers which predict disease outcome and expression and guide therapeutic approaches Find ways to prevent and treat neurodegeneration

major future challenge, not only for MS but for neuroscience research in general.

CONCLUSIONS Our understanding of MS has advanced greatly over the past decades. It is becoming increasingly clear that CD4þ T cells are involved in a complex pathogenetic inflammatory network with both resident and peripheral cells. In addition, evidence strongly suggests that regulatory and regenerating elements play crucial roles in modulating disease manifestation and expression. These findings should in the future enable us not only to find appropriate drug targets within inflammatory cascades, but also build on knowledge about intrinsic regulatory elements toward novel therapies. However, many challenges remain (Table 3.4). Connecting the many “datapoints” to a coherent pathogenetic model and filling in the gaps as well as developing efficacious anti-inflammatory treatments remain important goals in MS research. There is no question that numerous novel approaches have already been identified, and a number of these agents have successfully entered phase II or III, or are even close to approval. Important aspects in the continuing quest for a better understanding of the disease are: to pay particular attention to investigating which data from animal models are applicable to humans, and to integrate findings from epidemiologic, genetic, environmental, pathologic, immunologic, imaging and clinical studies, and dissect disease heterogeneity in MS. Our current

understanding of MS heterogeneity already suggests that targeting a single mechanism will not be of benefit for all MS patients. Perhaps, in the near future, we will begin to characterize patients by their etiologic (genetic polymorphisms, environmental triggers) and pathogenetic (biomarkers, MRI data, immunologic phenotype) factors before initiating an individualized, multimodal, efficacious, safe and cost-effective treatment.

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