Advances in Basic and Translational Science Research in Multiple Sclerosis

Advances in Basic and Translational Science Research in Multiple Sclerosis

Salim Chahin, MD, MSCE

University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania

The revolution in our understanding and treatment of multiple sclerosis (MS) continues as we learn more about the genetics and pathways of this chronic, progressive disease. This paper reviews our current understanding of the immunobiology of MS and the influence of the environment and genetics on its pathogenesis and neuropathology. Speakers at the 66th Annual Meeting of the American Academy of Neurology emphasized advances in basic science research and their translation to clinically useful applications. Their presentations included descriptions of the complex interplay of environmental and genetic contributions to disease susceptibility, recent findings in epidemiology and neuropathology, essential immunologic mechanisms involved in MS, and the immunologic effects of current disease-modifying treatments.

Salim Chahin, MD, MSCEMultiple sclerosis (MS) is a chronic heterogeneous disease with a complex epidemiology and interacting environmental and genetic risk factors. Unraveling epidemiologic and immunologic factors that influence the course of the disease and understanding the pathologic substrates for neurodegeneration have produced great advances in MS therapeutics.

During the 66th Annual Meeting of the American Academy of Neurology, experts in the neuropathology, immunology, and epidemiology of MS presented the latest advances in basic and translational science research into this disease.

NEUROPATHOLOGY OF MS
Based on a presentation by Josa M. Frischer, MD, PhD, Department of Neurology, Mayo Clinic, Rochester, Minnesota.

Although MS has been considered an inflammatory, demyelinating disease of the white matter in the central nervous system (CNS), studies over the past decade have demonstrated that it possesses a much more complex pathology involving dynamic changes in MS plaques, heterogeneity in the immunopathogenesis of white-matter lesions, and several likely interacting etiologies for neuronal and axonal loss and disease progression.1

Similar to clinical progression in MS, lesion pathology changes over time and evolves during the early versus chronic phases of the disease. Several processes drive the formation of plaques, including inflammation, myelin breakdown, oligodendrocyte injury, axonal loss, and remyelination.1

Table 1MS Plaque Types and Stages of Demyelinating Activity
Acute active plaques are characterized by relative axonal preservation and massive infiltration by macrophages containing myelin debris.1 These myelin debris products disappear at different rates, allowing for more accurate pathologic timing of the demyelinating event. Early and late active and inactive demyelination differ in the presence or absence of certain myelin degradation products within macrophages, which stain differently in histologic specimens (Table 1).1 The presence of minor myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG), indicates early active demyelination, whereas the presence of larger, major myelin proteins (proteolipid protein and myelin basic protein) without the minor proteins indicates a late active lesion.

In contrast to acute plaques, smoldering demyelinating lesions are characterized by an inactive center surrounded by a rim of activated macrophages and microglia, with few lesions containing myelin degradation products. Lastly, inactive plaques are completely demyelinated; there is substantial loss of axons and oligodendrocytes, and shadow plaques are sharply circumscribed regions that represent remyelinated areas.1,2

Relationship of Plaque Type to Disease Duration and Clinical Course
Acute active plaques are the pathologic substrate of clinical attacks and are seen most frequently in acute or fulminant MS (relapsing-remitting MS [RRMS]) and secondary progressive MS (SPMS) with relapses. Smoldering and inactive plaques, in contrast, are more predominant in patients with primary progressive MS (PPMS) or SPMS without relapses.1 The frequency of shadow plaques is similar in RRMS and progressive MS beyond 1 year of disease duration.1,2

Remyelination
The hallmark of remyelinated plaques is the presence of thinly myelinated axons with short intermodal distances.1 As mentioned previously, shadow plaques are the result of complete remyelination. Interestingly, older remyelinated plaques show an almost normal myelin thickness and may be difficult to distinguish from normal white matter.1,3

The extent of remyelination depends, at least partially, upon the availability of oligodendrocyte precursor cells (OPCs) and the pro- or anti-inflammatory balance within each plaque.1,2,4 Several hypotheses for remyelination failure and variation among patients with MS have been explored,2,4 including:

  • Areas of remyelination are preferential sites for new inflammation, and repeated demyelinating insults may exhaust the source of OPCs.
  • Axonal injury and loss may prevent appropriate interactions between axons and oligodendrocytes, thus inhibiting remyelination.
  • The dense glial scar that forms within lesions may act as a barrier that prevents OPC migration and remyelination.
  • Age-dependent loss of trophic support from microglia also may contribute to atrophy and prevent remyelination.

Heterogeneity of Early Active MS Plaques
The variation in clinical, genetic, and radiographic features and response to treatment among patients with MS partially may be due to underlying pathologic heterogeneity.1 Work at the Mayo Clinic has shown that MS lesions can be classified into four immunopatterns based upon specific myelin protein loss, plaque extent and topography, oligodendrocyte destruction, remyelination, immunoglobulin deposition, and complement activation.2,5

These four distinct immunopatterns may differ among patients but are similar within each patient in early active MS (Table 2).1 Patterns I and II show close similarities to T-cell–mediated and T-cell– plus antibody-mediated experimental autoimmune encephalomyelitis (EAE). Patterns III and IV are highly suggestive of a primary oligodendrocyte dystrophy, reminiscent of virus- or toxin-induced demyelination.1,5

Table 2

Other studies have challenged the concept of pathologic heterogeneity, arguing that it is dependent on the age of the lesions (and the duration of disease) and not on the patient. Breij et al6 showed that no lesion heterogeneity existed among different patients. They concluded that the initial heterogeneity of demyelinating lesions in the earliest phase of MS lesion formation might disappear over time, as different pathways converge in one general mechanism of demyelination.

Recently, Metz et al7 demonstrated that 95% of 22 pathologic cases showed persistence of all major immunopathologic patterns in tissue sampled at different times. This observation suggests that pathologic heterogeneity persists in early active MS lesions.

Pathologic Substrate of Disease Progression
Several overlapping factors contribute to neuronal and axonal loss and ultimate disease progression in MS.1 How these factors interact needs further exploration, but studies, especially over the recent years, have expanded our knowledge about some of these factors and their contribution to disease progression.

Axonal pathology. Axonal injury is now a well-recognized feature in MS that correlates with disability and disease progression.8 Proposed mechanisms of axonal damage in MS include the influence of damaging inflammatory and immune processes, lack of trophic support from damaged myelin and oligodendrocytes, repeated demyelination within the lesion, and chronic mitochondrial failure.1,8

Axonal degeneration is not a late phenomenon in MS. It begins early in the disease and seems to correlate with the degree of inflammation.8 Axonal damage remains clinically silent until a threshold of axonal loss is reached and the compensatory capacity is surpassed, resulting in irreversible, progressive neurologic disability and heralding the secondary progressive phase of the disease.8

Mitochondrial injury, oxidative stress, and iron accumulation. Mitochondrial injury may play an important role in neurodegeneration.9 It likely is triggered by reactive oxygen and nitrogen species that mediate mitochondrial dysfunction10 and result in a state of histotoxic hypoxia, leading to oligodendrocyte and neuronal damage10 and, ultimately, neuronal and axonal loss.9–11 This mechanism may be the etiology behind pattern III lesions12 and may be involved in the neurodegeneration that results in chronic progression clinically.9

Recently, iron deposition has been hypothesized to play a role in neurodegeneration in MS.1,13 In liberated form, ferrous iron ions may generate reactive oxygen species, which are toxic to the mitochondria.13 Hametner and others13 showed an age-related increase in iron in the white matter of healthy individuals and MS patients with a short disease duration. However, in chronic MS, there was a significant, age-corrected decrease in iron concentration in the normal-appearing white matter that corresponded to disease duration.13 Furthermore, extracellular accumulation of iron was seen in active MS lesions. Thus, iron may contribute directly or indirectly—through mitochondrial damage—to disease progression and degeneration.13,14

Cortical pathology. Previously thought to be unaffected in MS, cortical involvement is now a recognized phenomenon15–17 that correlates with physical and cognitive impairment.15 Cortical pathology can occur early in the MS disease process as a result of classically demyelinated lesions or neuronal loss following retrograde degeneration from white-matter lesions.15,18 Furthermore, meningeal inflammatory infiltrates may contribute to the gray-matter pathology in MS.18 In a cohort of patients with early-stage MS, cortical demyelinating lesions were frequent, inflammatory, and strongly associated with meningeal inflammation.18

On the basis of their locations, three types of cortical lesions have been identified,1,19 as follows:

  • subpial lesions extending from the pial surface into the deeper cortical layers, which are common in chronic MS;
  • intracortical lesions—small perivascular lesions confined to the cortex; and
  • leukocortical lesions involving both the gray and white matter at the gray-white matter junction.

Cortical lesion pathology is similar to that of white-matter lesions in that there are well-demarcated areas of demyelination with loss of oligodendrocytes and axons.1,17 However, cortical lesions are less inflammatory, devoid of lymphocytes and macrophages, and, as mentioned previously, driven partly by meningeal infiltrates.1,15,17,19,20

Interestingly, certain cortical areas in the brain are more affected than are others in MS, including the cingulate gyrus, insular cortex, temporobasal cortex, and hippocampus.16 In fact, hippocampal demyelination may be frequent and extensive in MS.21

Does neurodegeneration occur independently of inflammation? Neurodegeneration may occur independently of inflammation in patients with MS.1 However, it is more likely that neurodegeneration occurs on an inflammatory background.1,22 Recent magnetic resonance imaging (MRI) and pathology studies have shown that cortical lesions, despite being more common in SPMS, are already present in early disease, and these early cortical lesions can be highly inflammatory.15,19 These findings suggest that cortical demyelination in early MS is inflammatory and argue against a neurodegenerative process at this early stage.19 The process by which inflammation leads to degeneration is not completely understood, but the two are not likely to be independent.

A lot has been learned this past decade about gray- and white-matter pathology in MS.1 White-matter MS plaques are heterogeneous among patients, especially early in the disease course, and their appearance changes with disease progression.1,7 Cortical and gray-matter involvement is now recognized, and often early, phenomenon1,15,19 in MS that plays an integral role in disease progression.1 Further exploration of the nature of cortical involvement in MS and the complex relationship between degeneration and inflammation and the role of the mitochondria and oxidative stress will lead to better treatments for MS patients.

EPIDEMIOLOGY AND GENETICS
Based on a presentation by Emmanuelle Waubant, MD, PhD, Professor of Neurology, University of California at San Francisco School of Medicine, San Francisco, California.

The epidemiology of MS may hold the key to complex mechanisms that contribute to the risk of MS and to disease progression. Epidemiologic studies have uncovered several genetic and environmental factors that place individuals at an increased risk of developing MS. Recent studies in pediatric MS have added a trove of information about development of the disease in young patients. Challenges persist, however, in understanding the contribution that genetic and environmental factors make to the underlying mechanisms, pathogenesis, and progression of MS.

Epidemiology
MS is a relatively common disease in Europe, the United States, Canada, New Zealand, and parts of Australia.23 Among white, non-Hispanic individuals, the lifetime risk of MS is about 1:400.23 The risk of MS tends to be lower among Hispanic, black, and Asian populations,24 although it may be increasing in the non-Hispanic black population.25

Several large studies have evaluated the role of the environment in MS development.24 The incidence of MS follows a latitude gradient,24,26 in which the risk is low in the tropics and increases in frequency with increasing latitude in both hemispheres.26 This latitude gradient was generally considered to be influenced by genetic factors, but an environmental role cannot be ignored.24 People who migrate from high-risk regions to areas of low risk acquire a lower incidence of disease in a graded, age-related fashion: The younger the age at migration, the lower the risk.27,28 Thus, an environmental exposure early in life (before age 15 years) could be important to determining MS risk.27,28

A marked attenuation of the latitude gradient, however, has been observed in the United States and Europe.29–31 The disappearance of the gradient in the United States is probably due to an increased incidence of MS in the southern portion of the country. In contrast, a strong latitude gradient persists in the southern hemisphere (Australia and New Zealand).32,33 This difference between the northern and southern hemispheres is interesting, but no clear explanation has been set forth.24

Furthermore, studies suggest a rising incidence of MS in certain populations, especially among women.34,35 Orton et al35 reported an increase in the ratio of females to males in the risk of developing MS in Canada. Their findings, along with other large cohort studies, suggest that the increase in affected females to males is independent of access to care and improvements in diagnosis and that the incidence of MS actually may be rising among women.34,35 The authors further suggested that this increase among woman may have an environmental role, with environmental-genetic interactions potentially contributing to the risk of developing MS.34,35

Known environmental risk factors. Three environmental factors—infection with the Epstein-Barr virus (EBV), low levels of vitamin D, and cigarette smoking—likely are related to a greater risk of MS development.24 Some of these risk factors follow a geographic distribution that resembles the latitude gradient observed in MS epidemiology24 and help to explain the potential source of this gradient in MS incidence.

Infection with EBV and other viruses. EBV is the most studied, possibly infectious agent linked to MS development; it consistently has shown an association with MS risk.24 Infection with EBV in early life is common and usually asymptomatic. If the primary infection occurs later in life, an acute febrile illness known as infectious mononucleosis may result.36 The geography and epidemiology of infectious mononucleosis are strikingly similar to those of MS,37 and the risk of MS is two to three times higher in individuals with a history of infectious mononucleosis.24,38 Older age at EBV infection (manifested as infectious mononucleosis) is a risk factor for MS.24 Several pediatric MS studies also have shown an association between MS and EBV.39 Some investigators have reported the presence of EBV in the meninges of MS patients, whereas others have not been able to reproduce these findings.24

Some investigators have proposed that this association between EBV and the risk of MS can be explained by the "hygiene hypothesis,"40 which suggests that good hygiene in childhood, and not EBV infection, is the common link between infectious mononucleosis and MS. Levin et al,41 however, showed that in a large prospective study, only 10 cases of MS developed among EBV-negative individuals; in each case, symptoms developed months after serologic evidence of EBV infection was found.

Waubant et al39 have shown that remote cytomegalovirus (CMV) infection in children is associated with a lower risk of developing MS. This finding is supported by a study showing that CMV infection is negatively associated with adult-onset MS.42 Remote infection with herpes simplex virus (HSV-1) has a more complex effect. Although remote infection with HSV-1 has not been shown to increase or decrease the risk of developing MS, a strong interaction between HSV-1 status and expression of HLA-DRB1 has been observed, further supporting a gene-environment interaction related to MS.39

Table 3 summarizes the risk of developing MS in children39 and adults42 depending upon infectious findings.39,42

Table 3

Vitamin D and sunlight exposure. The geography of MS also correlates with the degree and duration of sunlight exposure,43–45 which is the primary source of vitamin D in most populations. Although no clear causal relationship has been established, vitamin D is hypothesized to play an immunomodulatory role in several diseases,24 and evidence is accumulating on the role of vitamin D in the pathogenesis of MS.24,46,47

Longitudinal research, such as the Nurses' Health Study, has shown that the risk of MS is 40% lower among woman who reported taking vitamin D regularly.46 Munger and others47 reported a 62% lower risk of MS among non-Hispanic white individuals who had high serum levels of vitamin D. These and other studies support the importance of vitamin D sufficiency in adolescence and young adulthood and possibly also in childhood and even in utero.24

Further evidence of the possible effects of sun exposure (and vitamin D) on the risk of MS comes from a North American twin study,48 which found that concordance rates among monozygotic twins born in the United States were twice as high as those for twins born elsewhere. Further, the average age at diagnosis for northern-latitude twins was independent of ancestry but earlier than for southern-latitude twins. Additionally, obesity, which is known to be associated with lower levels of vitamin D, was associated with a twofold increase in the risk of MS.24

Exactly how vitamin D affects the risk of developing MS and disease progression remains under investigation. Interestingly, Ramagopalan et al49 identified a vitamin D response element (VDRE) in the HLA-DRB1 promoter region, the main susceptibility locus for MS. This finding, although not yet reproduced, suggests a possible direct interaction between HLA-DRB1 and vitamin D.

Vitamin D may also contribute to the disease course.24 Mowry et al50 showed that a 10 ng/mL increase in hydroxyvitamin D levels resulted in a 34% decrease in subsequent relapse rate among patients with pediatric-onset MS. Ascherio et al51 showed that among patients with MS treated with interferon β-1b, a low vitamin D level early in the course of the disease was a strong risk factor for long-term relapse and disease progression. Several ongoing clinical trials are testing the association between vitamin D and disease progression and attempting to establish causality. The link between vitamin D and MS will be clearly established only in clinical trials. In fact, a recent systematic review of the literature on vitamin D suggests that the low vitamin D levels seen in inflammatory and autoimmune diseases may result from the inflammatory process rather than contributing to the inflammation.52

Cigarette smoking. Cumulative evidence supports the association between cigarette smoking and the risk of MS. The risk of MS is 50% higher in people who always smoked than among those who never smoked.53–55 In a longitudinal study, MS risk was about twofold higher among women who smoked ≥ 25 pack-years than among those who never smoked.53 In patients with an established MS diagnosis, cigarette smoking also was associated with more rapid disease progression.56

Other environmental factors. Among the large number of environmental exposures that have been investigated in the pathogenesis of MS,24 few factors other than vitamin D level and cigarette smoking have shown a confirmed association. Recently, sodium and increased dietary salt intake have been implicated in the pathogenesis of the disease, since sodium chloride levels may play a role in modulation of the immune system and the development of autoimmune diseases such as MS via induction of pathogenic T helper 17 (Th17) cells.57,58

Heredity. The genetic contribution to the susceptibility of developing MS is suggested by several studies.24,59 A high degree of heritability has been established in studies of twins, siblings, and adoptees.24,60 The risk of MS in monozygotic twins is 25% and in dizygotic twins, 5%.60 Furthermore, having a sibling with MS increases the risk 20- to 40-fold when compared with people who have no relatives affected by the disease.60

The genetic association in MS likely results from multiple interacting polymorphic genes, with each exerting a small-to-moderate effect on the overall risk.59 The strongest genetic risk of MS is conferred by the HLA-DRB1*1501 allele,24 which has a 14%–30% frequency in populations of countries with a high risk of developing MS. This allele increases the risk of disease by an average of three times in heterozygous carriers and six times in homozygous individuals.24,59 Other HLA-DRB1 alleles also are associated with a high MS risk.59 However, the exact mechanism(s) by which the DRB1 gene influences susceptibility to MS remains unclear.24 Genome-wide association studies (GWAS) have identified the contribution of over 110 non-HLA genes, many of which code for proteins involved in the immune response.59,61

Environmental/Genetic Interactions
MS is likely caused by a complex interaction between multiple genes and environmental factors, "the relative importance of which varies by person, time, and location."24 Despite recent advances in the study of MS genetics, about 80%–90% of patients with MS have a negative family history,24,62,63 suggesting a possibly dominant role of the environment and genetic-environment interactions. Examples of such an interaction include twin studies suggesting a role for geography and latitude in increased concordance of monozygotic twins,48 the interaction between HSV-1 status and HLA-DRB1,39 and vitamin D response element and HLA-DRB1.49 Recently, Mechelli et al,64 based upon aggregate analysis of GWAS data in MS, proposed a multifaceted approach to gene-environment interactions as triggers of MS. Their results support a causal role of the interaction between EBV infection and the products of MS-associated gene variants. The authors recommend future, more expansive research to study these interactions.64

Epigenetics
In the context of genetic susceptibility, the effect of environmental risk factors on MS pathogenesis could be explained by epigenetic modifications.59,65,66 Several observations suggest a role for epigenetics in MS, including results from twin studies reporting a concordance rate of 30% (and not 100%) in monozygotic twins and an increase in MS risk if the mother (more so than the father) has MS.24,66

These epigenetic changes influence gene expression without altering the DNA sequence and include processes such as DNA methylation, histone modification, microRNA (miRNA)-associated post-transcriptional gene silencing, and heterochromatin formation.65,66 Evidence on the potential influence of miRNA on disease immunology will be discussed in detail later in this article.67

The Challenges Ahead
Very few breakthroughs in mitigating the risk of MS onset or progression have been made despite our growing knowledge of several genetic and environmental risk factors for the disease. This failing is likely due to the heterogeneity of risk factors among individuals and the direct versus indirect effect of some of the environmental risk factors. However, Ascherio and colleagues24 proposed that some risk factors may be addressed now: vitamin D supplementation and smoking cessation are immediately available interventions that may reduce the risk of MS and disease progression.24

NEUROIMMUNOLOGY OF MS
Based on a presentation by Michael K. Racke, MD, Professor of Neurology and Neuroscience, The Ohio State University College of Medicine, Columbus, Ohio.

Evidence from MS pathology and epidemiology studies helped establish MS as an autoimmune, inflammatory disorder that has the target antigen or antigens located in the CNS myelin.68 Experts in neuroimmunology may have witnessed the greatest advances in uncovering disease pathophysiology; such research has been the driving force behind the development of therapeutic options in MS.68–70

T-Cell Involvement
Well-established animal models of MS, such as EAE, have demonstrated that autoreactive, myelin-specific T lymphocytes may work toward demyelination in the CNS.69–71 The mechanisms of T-cell involvement in demyelination remain under investigation, but several discoveries have helped shape our understanding of T-cell involvement in MS.

In human studies, the frequency of myelin-reactive T cells is very similar in MS patients and healthy individuals, but qualitative differences in the response mediated by these cells exist between patients and healthy individuals.72,73 Notably, myelin-specific T cells have a memory or activated phenotype when compared with the naïve phenotype seen in healthy individuals.72,73

Flow-cytometry techniques performed on neuroantigen-specific CD8+ T cells ex vivo have also demonstrated significant differences in cytokine production and chemokine receptor expression in patients with MS, suggesting that these cells have a greater pro-inflammatory phenotype in MS patients.74,75 Both CD4+ and CD8+ T cells have been implicated in the pathogenesis of MS. Myelin-specific CD8+ T cells obtained from patients with RRMS exhibited a reduction in regulatory capacity during a relapse,74 but this suppressed capacity seemed to return to normal as the patient recovered from the relapse. This and other works highlight the importance of evaluating both CD4+ and CD8+ T-cell responses in MS.74,75

Identifying the target antigen of the T-cell response has been the focus of many studies. In a clinical trial on altered peptide ligands in MS, Bielekova et al76 reported a significant increase in T cells responding to a particular component of myelin basic protein (MBP 83-99) in those patients with increased clinical or radiologic activity. These observations support the hypothesis of a potential direct response of T cells against myelin antigens and their role in MS pathogenesis.76

T-cell phenotypes in MS. Several teams of investigators have pioneered work exploring and clarifying the roles of different myelin-specific T cells phenotypes. Myelin-specific T cells from MS patients produce cytokines (interferon γ) consistent with a T helper 1 (Th1) response,77 whereas myelin-specific T cells from healthy individuals produce cytokines consistent with a T helper 2 (Th2) response.75 A different subset of T cells, Th17 cells, may also contribute to inflammation in MS.78 Interleukin (IL)-23 is an essential cytokine that results in survival of IL-17–producing T cells,79 which are believed to be important in encephalitogenicity.79 In fact, microarray studies on MS lesions have shown an increased expression of IL-17, suggesting that it may contribute to the development of inflammation and demyelination.80 The differentiation of encephalitogenic Th1 and Th17 cells may differ among MS patients and is influenced by several transcription factors.81–85 These differences may also explain why interferon therapy may not be effective in some patients.

B-Cell and Humoral Involvement
Several studies have demonstrated increased levels of B cells, plasma cells, and antibodies in the cerebrospinal fluid (CSF) of MS patients.86 Cepok et al86 showed that plasmablasts were present in high numbers in the CSF and correlated with radiologic disease activity. These plasmablasts were also responsible for the elevated immunoglobulin G (IgG) synthesis observed in the CSF of MS patients. The number of CSF plasmablasts strongly correlated with intrathecal IgG synthesis and inflammatory parenchymal disease activity on MRI. Thus, these plasmablasts are likely the main effector B-cell population involved in ongoing active inflammation in MS.

Similar to the T-cell studies in MS, work is being done to identify target antigens for the antibody response in MS. Srivastava et al87 identified an antibody specific for KIR4-1 that could bind to glial cells in a subgroup of MS patients. KIR4-1 is an inward-rectifying potassium channel located on astrocytes and oligodendrocytes. This antibody was present in 47% of patients with MS and fewer than 1% of patients with other neurologic disorders; however, neuromyelitis optica (NMO) was not included in this analysis.87 The authors suggested that KIR4-1 might be a target of the antibody response in a subset of patients.87,88 Recently, Kraus et al89 also found serum antibodies to KIR4-1 in most children with acute demyelinating disorders but not in children with other diseases or healthy controls, confirming the potential role for KIR4-1 as an important target for autoantibodies in children as well as adults.

Interestingly, the KIR4-1 channel co-localizes with the aquaporin 4 (AQP4) channel,88 the antigen targeted in NMO, raising the possibility that KIR4-1 could be targeted in NMO patients who are NMO IgG-negative.88 In fact, antibodies to KIR4-1 and AQP4 share a similar characteristic—they are rarely detected in the CSF.88 Additional work is needed to clarify the presence of the KIR4-1 antibodies in disorders such as NMO, to identify other potential targets in MS, and to incorporate this and other findings in developing more specific therapeutic interventions.

Evidence of the role of B cells in MS pathogenesis also comes from successful therapeutic trials that have targeted B cells. Cross et al90 showed that rituximab, an anti-CD20 monoclonal antibody that depletes CD20+ B cells, could benefit MS patients who do not respond to first-line treatment. CSF analysis showed that 90% of CD19+ cells in the CSF were depleted, and the population of T cells also was reduced. These findings suggest that one of the mechanisms through which B cells may play a role in MS is the subsequent recruitment of T cells into the CSF. Ocrelizumab, a humanized version of rituximab, is being evaluated in several ongoing clinical trials as a potential therapy for both RRMS and progressive MS.

Epigenetics and the Role of miRNA
As mentioned previously, several epigenetic pathways may influence the effect of environment and genes in the pathogenesis of MS.59,65,66 Recently, miRNA has emerged as a leading epigenetic mechanism in MS via regulation of gene expression and T-cell activation.65,66 These miRNAs can be influenced by both genetic and environmental factors, making them attractive for study in MS.91,92 The miRNAs are small RNA molecules (19–24 nucleotides long) that bind to the 3ʹ UTR (untranslated region) target of mRNAs and inhibit translation or induce mRNA degradation, thus altering gene expression.91,92 Studies of mononuclear cells obtained from the peripheral blood of MS patients have demonstrated miRNA dysregulation.93,94

Guerau-de-Arellano and colleagues67 studied miRNA expression in highly purified naïve CD4+ T cells from MS patients. Use of naïve CD4+ T cells is advantageous in such studies because these cells have not been activated, allowing the investigators to examine how miRNA influences the differentiation of T cells into pro-inflammatory phenotypes in MS. Three miRNAs (miR-128, miR-27a/b, and miR-340) are increased in naïve T cells from MS patients. These miRNAs suppressed Th2 differentiation by targeting Bmi-1 (a molecule that promotes survival of the transcription factor GATA3 that drives Th2 differentiation) and IL-4 expression and setting the stage for pro-inflammatory Th1 autoimmune responses.

Another miRNA (miR-29b) that targets interferon γ and another transcription factor called T-bet are overexpressed in memory T cells of MS patients. T-bet levels are higher in MS patients than they are in healthy controls,95 and they influence CD4+ differentiation and encephalitogenicity. The expected increase in miR-29b should result in reduced levels of T-bet and interferon γ, but the opposite happens—T-bet levels actually were higher in MS patients, a discrepancy that can be explained if this miRNA were dysregulated in MS.

Smith and colleagues95 activated memory CD4+ T cells from MS patients and healthy controls and found that levels of miR-29b increased in healthy controls but decreased in patients with MS. Thus, although resting memory CD4+ T cells appear to be primed to regulate a Th1 response in MS patients by controlling T-bet and interferon γ levels (via miR-29b), this regulatory mechanism fails upon activation. This failure allows for the high T-bet expression observed in MS CD4+ T cells and promotes effector functions associated with the CNS pathology present in patients with MS.67,95

These results add to our understanding of T-cell regulatory mechanisms and illustrate the biologic significance of miRNAs in MS susceptibility.67 Additional work is under way to identify other regulatory and epigenetic pathways in the immune system that might serve as potential therapeutic targets for patients with MS.

MECHANISMS OF ACTION: CURRENT AND FUTURE MS THERAPIES
Based on a presentation by Lawrence Steinman, MD, Professor of Neurology and Neurological Sciences, Pediatrics, and Genetics, Stanford University School of Medicine, Stanford, California.

Nine agents are currently approved by the US Food and Drug Administration (FDA) for the treatment of RRMS, including interferons, glatiramer acetate, natalizumab, fingolimod, teriflunomide, and dimethyl fumarate. Several other therapies for RRMS are in advanced stages of clinical development or nearing FDA approval. Little progress, however, has been made in the treatment of progressive MS.

The mechanism of action of a therapeutic agent is sometimes well known and at other times may be complex and little understood. Knowing a drug's mechanism of action provides valuable insight into not only how a drug works but also its risk profile and side effects. Table 4 summarizes the mechanisms of action of FDA-approved drugs for treating RRMS.

Table 4

FDA-Approved Treatment Options
Type 1 beta interferons.
Interferons (specifically, interferon β-1a and β-1b) were among the first therapies approved for treating MS. Despite being available for two decades, the mechanism of action of these drugs in MS is still not well understood.96 In vitro, interferons impact intracellular signaling, but the mechanism of action in vivo is much more complex and likely involves more than one pathway.96 Several potential mechanisms have been proposed,97–99 some of which are on the genetic level. Attempts to identify molecular biomarkers of the therapeutic effect of interferons in MS100 so far have been unsuccessful.

We have gained insight into the mechanism of action of interferons from their effect in NMO. Once again, the two types of inflammatory pathways in MS are the Th1 pathway (driven by interferon γ) and the Th17 pathway (with its signature cytokine IL-17).77,78 Both interferon γ and IL-17 are found in large amounts in MS lesions.80 CSF levels of IL-17 are higher in NMO patients than in MS patients. IL-17 may recruit and activate granulocytes through several cytokines.101 Treating NMO with interferon β not only is ineffective but may have devastating consequences, including an increased relapse rate, severity, and antibody levels.101–113 This worsening of clinical status in the context of treatment with interferon β observed in NMO and in some patients with MS has occurred when the Th17 immune response is prominent.101,104

Glatiramer acetate. Several mechanisms of action have been proposed for how glatiramer acetate works in MS. In animal studies, an anti-inflammatory monocyte called M2 is elicited in response to glatiramer acetate exposure,105 resulting in increased secretion of certain anti-inflammatory cytokines, such as IL-10, and an overall shift from the Th1 and interferon γ response to a Th2-modulated pathway.105

Natalizumab is a humanized monoclonal antibody to α4 integrin, the main homing molecule on lymphocyte surfaces that binds to vascular adhesion molecule VCAM1 on endothelial cells and mediates lymphocyte transmigration across the blood-brain barrier.106,107 Natalizumab interferes with this step and impedes both T- and B-cell entry into the CNS. The mechanism of action of natalizumab explains its efficacy but also highlights the risks associated with its use—namely, infection with the JC virus resulting in progressive multifocal leukoencephalopathy (PML).

It is unclear why PML is the only opportunistic infection associated with natalizumab use.106,107 Studies to stratify the risk of PML in natalizumab-treated patients have identified three risk factors: exposure to the JC virus, duration of treatment, and prior exposure to immunosuppressive therapy or chemotherapy.108

Fingolimod is a sphingosine phosphate (SP) receptor agonist. Several subtypes of SP receptors exist. The S1P1 subtype is present primarily on immune cells and neural cells. Through its agonist effect on S1P1 receptors, fingolimod causes the S1P1 receptor to be internalized, resulting in a pharmacologic antagonist effect. The drug effectively traps lymphocytes in the lymph nodes and impedes their egress.109 This accounts not only for its anti-inflammatory activity but also the potential for opportunistic infections to occur in patients being treated with drug.

Fingolimod has agonist activity toward other SP receptor subtypes, including S1P4, S1P5, and S1P3. The latter subtype is found on cardiac cells; modulation of these receptors is associated with bradycardia, a known complication of fingolimod use.109

Teriflunomide is the active metabolite of leflunomide, an agent approved by the FDA to treat rheumatoid arthritis. Teriflunomide has a distinct profile and a complex mechanism of action. It interferes with pyrimidine synthesis through reversible inhibition of dihydroorotate dehydrogenase and decreases lymphocyte proliferation and activation toward key myelin antigens in MS.110

Dimethyl fumarate and its primary metabolite, monomethyl fumarate, have shown efficacy in RRMS,111,112 likely through cytoprotection from oxidative stress. A potential mechanism of neurodegeneration in MS is the effect of oxidative stress and reactive oxygen species. Dimethyl fumarate may protect against neuroinflammation, neurodegeneration, and oxidative stress through activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) antioxidant pathway.113 Other potential mechanisms of action that have been explored include induction of IL-4–producing CD4+ Th2 cells, generation of type II dendritic cells that produce IL-10 instead of IL-12 and IL-23, suppression of pro-inflammatory cytokines, and direct inhibition of pro-inflammatory pathways.111,112,114

Therapies in Advanced Stages of Development
Alemtuzumab.
This monoclonal antibody targets CD52 receptors and depletes T and B lymphocytes and monocytes mainly via antibody-dependent cytotoxicity and complement-mediated lysis.115 This depletion can be long-lasting. Alemtuzumab may drive a sort of immune programming. When cells repopulate, they tend not to have the same autoimmune response; in 30% of cases, new autoimmune diseases, notably immune thrombocytopenia, occur. The risk of this new autoimmunity is associated with increased levels of IL-21.116,117

Ocrelizumab. Whereas rituximab is a chimeric monoclonal antibody against CD20+ cells, ocrelizumab is a recombinant humanized antibody designed to selectively target CD20+ B cells, resulting in antibody-dependent, cell-mediated cytotoxicity. In early clinical trials, it has shown efficacy in MS.118

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Dr. Chahin is a Multiple Sclerosis Fellow in the Department of Neurology at the University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.

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