Translational Therapies for Immune-based Disorders Review Series
`
`Guest Editor: D. Pozo
`
`Systems biology approaches for the study of multiple sclerosis
`
`J. Cell. Mol. Med. Vol 12, No 4, 2008 pp. 1087-1093
`
`Francisco J. Quintana *, Mauricio F. Farez, Howard L. Weiner
`
`Center for Neurologic Diseases, Harvard Medical School, Boston, MA, USA
`
`Received: April 1, 2008; Accepted: May 10, 2008
`
`• Introduction
`• Genomics
`• Transcriptomics
`- Characterization of the MS lesion
`- Characterization of the immune response
`• Proteomics
`-
`Identification of new pathogenic processes
`
`Abstract
`
`- Role of environmental triggers in MS
`- Characterization of the autoimmune response:
`antibodies
`• Metabolomics
`• Integration of data from different ‘omics’ approaches
`• New experimental models
`• Conclusions
`
`Multiple sclerosis (MS) is a progressive neurological disease caused by an autoimmune attack to the central nervous system (CNS). MS
`is thought to result from a complex interaction between genetic and environmental factors. In this review, we analyse the contribution
`of genomics, trancriptomics and proteomics in delineating these factors, as well as their utility for the monitoring of disease progres-
`sion, the identification of new targets for therapeutic intervention and the early detection of individuals at risk.
`
`Keywords: multiple sclerosis • systems biology • autoimmunity
`
`Introduction
`Multiple sclerosis (MS) is an autoimmune disorder in which the
`central nervous system (CNS) is targeted by the dysregulated activ-
`ity of the immune system, resulting in progressive neurological
`dysfunction. A variety of symptoms characterize MS, among them
`are visual and motor problems, changes in sensation in the arms,
`legs or face and weakness. At the onset of the disease, 85–90% of
`the patients present a clinical course characterized by discrete
`attacks followed by periods of partial or total recovery (relapsing-
`remitting MS, RRMS); 10% of the patients present a slowly accu-
`mulating disability over time (primary progressive MS, PPMS). A
`total of 40% of the patients initially diagnosed with RRMS eventu-
`ally become progressive (secondary progressive MS, SPMS).
`The term MS refers to the scars (scleroses or plaques) that
`characterize the white matter of the brain and spinal cord of MS
`patients. The autoimmune attack that drives MS is thought to
`cause these scars, characterized by a perivascular infiltration by
`inflammatory cells (B and T lymphocytes among them) [1, 2]. In
`addition, demyelination, astrogliosis and axonal injury are also
`detected [1, 2]. Different mechanisms contribute to axonal dam-
`age, including the direct effects of pro-inflammatory cytokines,
`complement fixation, apoptosis, cell-mediated cytotoxicity and
`neurodegeneration [1, 2]. Pathological findings suggest that the
`
`relative contribution of each one of these processes in disease
`progression differs in each patient [3].
`The autoimmune response in MS targets components of the
`myelin sheaths surrounding neuronal axons, interfering with the
`neurons’ ability to conduct electrical signals and probably leading to
`their death. Several CNS proteins are targeted by the immune system
`in MS, among them are myelin oligodendrocyte glycoprotein [4],
`oligodendrocyte-specific protein [5], myelin basic protein [6],
`myelin-associated glycoprotein [7], 2⬘,3⬘-cyclic nucleotide 3⬘ phos-
`phodiesterase [8] and ␣␤-crystallin [9]. It is believed that the clinical
`symptoms that characterize MS result from the blockade in axonal
`transmission that follows axonal demyelination or axonal loss [1, 2].
`Epidemiological studies have suggested the contribution of sev-
`eral environmental factors to the susceptibility of a specific individ-
`ual to MS. Several viral, bacterial and parasitic infections have been
`classically linked to MS onset and progression [10–15], but no sin-
`gle environmental agent can be singled out as a ‘cause’ of MS.
`MS is heterogeneous in its rate of progression, clinical symp-
`toms, the specificity of the immune response and the pathology of
`the CNS lesions, reflecting the contribution of different factors to
`a pathogenic autoimmune response [16]. In this review, we
`analyse the contribution of genomics, trancriptomics, proteomics
`
`*Correspondence to: Francisco J. QUINTANA, Ph.D.,
`Center for Neurologic Diseases, Harvard Medical School,
`77 Ave. L. Pasteur HIM720, Boston 02115, MA, USA.
`
`Tel.: +617-525-5311
`Fax: +617-525-5305
`E-mail: fquintana@rics.bwh.harvard.edu
`
`© 2008 The Authors
`Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
`
`doi:10.1111/j.1582-4934.2008.00375.x
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`and metabolomics in delineating these factors, as well as their utility
`for the monitoring of disease progression, the identification of
`new targets for therapeutic intervention and the early detection of
`individuals at risk.
`
`Genomics
`
`MS is considered a complex genetic disease in which many poly-
`morphic genes have small effects on the overall disease risk, its
`severity, rate of progression and age of onset among several clin-
`ical outcomes. To date, the strongest chromosomal region linked
`to MS is the major histocompatiblity complex (MHC) locus on
`chromosome 6p21 [17–19]. In addition, several non-MHC candi-
`date loci have also been linked to MS [19], but it has proven diffi-
`cult to validate their association in independent studies. The diffi-
`culty in the identification of non-MHC genes associated to MS
`might reflect the genetic heterogeneity existing among MS
`patients, which results in different combinations of gene alleles
`leading to the same end phenotype. Nevertheless, polymorphisms
`in the ␣ chain of the IL-7 receptor (IL-7R␣) have been recently
`associated with MS [20–22]. These polymorphisms make only a
`small contribution to the genetic susceptibility to MS but are a sig-
`nificant step towards the identification of genetic determinants for
`MS outside the MHC locus. The IL-7R␣ allele associated with MS
`favours a relative decrease in the membrane-bound IL-7R␣ [21].
`IL-7 is produced by stromal cells in lymphoid tissues, its availabil-
`ity is controlled through its uptake by the membrane-bound IL-7R
`on T cells [23]. Thus, considering the positive effects that IL-7 has
`on lymphocyte survival and proliferation [23], the decrease in
`membrane IL-7R might result in increased levels of IL-7 available
`to fuel the inflammatory T-cell response in MS.
`The ␣ chain IL-2 receptor (IL-2R␣) gene has also been recent-
`ly linked to MS [22]. IL-2R␣ allelic variation has been previously
`associated to other autoimmune diseases such as type I diabetes,
`but at a different genomic position [24]. IL-2 is required for the
`development of regulatory T cells (Treg) [25, 26], and indeed,
`deficits in Treg activity characterize RRMS [27]; thus the IL-2R␣
`polymorphisms might be related to the immune dysregulation
`observed in MS. Notably, IL-2R␣-specific antibodies have shown
`promising beneficial effects for the treatment of MS on phase 2 clin-
`ical trials [28, 29]. Although the link between IL-2R␣ polymorphisms
`and MS is still awaiting further validation, the association of IL-7R␣
`and IL-2R␣ variants to MS supports the use of genome-wide stud-
`ies to delineate pathways contributing to disease pathogenesis.
`
`Transcriptomics
`
`Characterization of the MS lesion
`
`Large-scale studies of mRNA expression have been directed at
`characterizing either the lesion or the immune response in MS.
`Lock and coworkers found that ␣4-integrin was found to be
`
`elevated in MS lesions [30]. ␣4-integrin mediates the interaction
`of T cells with the endothelium in the inflamed CNS, a required
`step for the migration of the self-reactive T cells into the brain and
`spinal cord in MS [31]. Antibodies to ␣4-integrin reverse and
`reduce the rate of relapse in relapsing-remitting experimental
`autoimmune encephalomyelitis (EAE) an animal model for MS
`[32], and a humanized version of this antibody showed positive
`effects in the treatment of RRMS [33].
`In a separate study, the large-scale sequencing of non-normal-
`ized cDNA libraries derived from MS plaques revealed an increased
`expression of osteopontin (OPN) in the CNS of MS and EAE sam-
`ples [34]. The up-regulation of OPN levels in MS plaques [35] and
`in the circulation [36–38] of MS patients was replicated in inde-
`pendent studies, prompting the search for polymorphisms in the
`opn gene associated with MS. Although some controversy still
`remains [39], polymorphisms in the opnlocus have been associat-
`ed with increased levels of circulating OPN and the clinical course
`of MS [40]. To study the mechanism of action of OPN in MS, OPN-
`deficient mice were generated, which showed a reduced severity in
`EAE [34]. OPN-triggered signalling is thought to contribute to MS
`pathogenesis by increasing the pro-inflammatory phenotype and
`survival of pathogenic myelin-specific T cells [41]. In addition, OPN
`interacts with the ␣4-integrin and is also involved in cell migration
`into the inflamed CNS [42]. Neutralization of OPN with neutralizing
`antibodies results in the amelioration of EAE [43]. Thus, OPN is
`therefore an example of how results obtained in transcriptomics
`studies might lead to the identification of mechanisms of disease
`pathogenesis and new therapeutic targets for MS.
`
`Characterization of the immune response
`
`Transcriptional profiling has also been used to study the peripheral
`immune response in MS. Two limitations, however, should be kept
`in mind when using cDNA arrays for the analysis of the immune
`response in MS patients: First, these studies assume that changes
`in the peripheral immune system somehow reflect the immune
`response within the CNS. Second, the results of these studies are
`influenced by factors such as gender, age or changes in the relative
`proportion of different blood cell subsets that occur through the
`course of the disease. Nevertheless, two areas show significant
`progress in the transcriptional profiling of the immune response in
`MS: the follow-up of disease activity and the response to therapy.
`Follow-up of disease activity: Achiron and coworkers charac-
`terized the transcriptional activity in peripheral blood mononuclear
`cells (PBMC) from RRMS patients during the course of the dis-
`ease [44]. The authors identified a transcriptional signature asso-
`ciated to the relapse, that included genes involved in the recruit-
`ment of immune cells, epitope spreading and escape from
`immune-regulation. Although encouraging, these results should
`be validated using an independent set of samples and in longitu-
`dinal studies to assess their predictive value.
`Response to therapy: ␤–Interferon (␤–IFN) is widely used for
`the treatment of MS [45], however, biomarkers that would allow
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`the identification of patients that would benefit from treatment
`with ␤–IFN are still not available. Weinstock-Guttman and col-
`leagues used cDNA micro-arrays to study the effects of ␤–IFN
`therapy on monocyte-depleted PBMC [46]. They found significant
`changes in the expression of genes involved in the anti-viral
`response, ␤–IFN signalling and markers of lymphocyte activation.
`These studies provided a molecular description of the effects of
`␤–IFN on RRMS patients and were later on extended to identify
`transcriptional signatures associated to a favourable response to
`treatment with ␤–IFN [47]. Based on these observations,
`Oksenberg and coworkers constructed a classifier for the identifi-
`cation of MS patients likely to respond to treatment with ␤–IFN
`[48]. The work of Oksenberg and coworkers is remarkable for two
`reasons: First, it demonstrates that gene expression profiling can
`be helpful in the selection of therapeutic regimes for the manage-
`ment of MS. Second, it uses a technology (real-time PCR) acces-
`sible to clinical laboratories, facilitating the translation of their
`results into daily medical practice.
`
`Proteomics
`
`Proteomic studies in MS have been shown to identify new
`processes contributing to disease pathology and also, biomarkers
`for the early diagnosis and monitoring of MS patients.
`
`Identification of new pathogenic processes
`
`Recently a large proteomic study of MS lesions by Steinman and
`coworkers have identified tissue factor and protein C inhibitor
`expression within chronic active plaque samples, suggesting that
`the dysregulation of the clotting cascade contributes to MS patho-
`genesis [49]. The authors went on to investigate the potential ther-
`apeutic use of their findings on EAE, concluding that the coagula-
`tion cascade is an attractive therapeutic target in MS.
`
`Role of environmental triggers in MS
`
`Epidemiological studies suggest that environmental factors con-
`tribute to MS susceptibility. As a result, several groups are active-
`ly searching for microbial triggers for MS [10–15]. One of these
`putative triggers is the Epstein-Barr Virus (EBV) [50]. The link
`between EBV infection and MS has been recently strengthened by
`the work of Cepok and coworkers, who used protein expression
`arrays to characterize the antibody reactivity in the cerebrospinal
`fluid (CSF) of MS patients, most of those antibodies recognized
`EBV epitopes [51]. These results, together with the detection of
`EBV reactivation in active MS lesions [52], suggest that EBV might
`elicit an abnormal immune response in susceptible individuals
`that contributes to MS [53].
`
`J. Cell. Mol. Med. Vol 12, No 4, 2008
`
`Characterization of the autoimmune response:
`antibodies
`
`The autoimmune nature of MS suggests that the study of the
`immune response should be useful for the early diagnosis, prog-
`nosis and monitoring of MS patients. T cells are thought to make
`a major contribution to MS immuno-pathology [16], but the stan-
`dardized characterization of the T-cell response has proven diffi-
`cult in MS. Antibodies might also have a pathological role [54].
`Moreover, the activation of antibody-producing B cells is con-
`trolled by T cells, thus antibody response is thought to reflect the
`activity of the T-cell compartment [55]. Since it is easier to assay
`antibody reactivity than to follow antigen-specific T-cell respons-
`es, new technologies have been developed for monitoring the
`humoural response in MS patients and autoimmunity [56, 57].
`Antigen arrays have been shown to detect changes in the
`repertoire of antibodies reflecting the antigen spreading that
`accompanies EAE progression [58]. The information obtained
`about the antigen spreading was used to design tailored immuno-
`modulatory vaccines to control EAE [58]. Of note, these vaccines
`showed promising results in a phase 1/2 human clinical trial [59].
`Future experiments should study the antibody response in the
`serum of MS patients, searching for patterns of antibody reactivity
`that predict the progression of MS or the response to therapy, as it
`was shown for other autoimmune disorders, such as rheumatoid
`arthritis [60], autoimmune diabetes [57] and systemic lupus erythe-
`matosus [61]. Indeed, our own data suggest that antigen arrays
`might be used to identify antibody patterns linked to the different
`forms of MS and identify pathogenic mechanisms and therapeutic
`targets (F. J. Quintana et al., submitted). Thus, antigen arrays
`are promising platforms for the identification of patients at risk of
`developing MS, before the overt onset of the symptoms [57].
`
`Metabolomics
`
`The metabolome is defined as ‘the complete set of small-molecule
`metabolites (such as metabolic intermediates, hormones and other
`signalling molecules, and secondary metabolites) to be found within
`a biological sample, such as a single organism’ [62]. Although initial
`studies aimed at studying the metabolome in simple organisms like
`the yeast [63], the study of a limited subset of the human
`metabolome in health and disease is well underway [64, 65]. Several
`groups have undertaken the study of metabolomic aspects of MS.
`During the course of MS, macrophages and astrocytes pro-
`duce nitric oxide, a metabolite that is thought to contribute to sev-
`eral aspects of MS pathology such as the disruption of the
`blood–brain barrier, oligodendrocyte injury and demyelination,
`axonal degeneration [66]. Nitric oxide metabolites can be detect-
`ed in CSF, serum and urine of MS patients, and their levels seem
`to reflect the activity of inflammatory processes that contribute to
`the pathology of the disease [66–68].
`
`© 2008 The Authors
`Journal compilation © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
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`N-acetylaspartate (NAA) is only present in living mature neu-
`rons, thus decreases in NAA levels indicate neuronal loss.
`Magnetic resonance (MR)-based approaches have been success-
`fully used to measure the levels of NAA and other metabolites
`[69–72] in vivo, providing a novel non-invasive method for the
`acquisition of real-time data about the state of the CNS in MS. The
`power of this approach is highlighted by new data showing that
`the precise measurement of NAA levels by a combination of in vivo
`proton MR spectroscopic imaging with segmented, high-resolu-
`tion MR imaging can identify RRMS patients in their transition to
`the SPMS form of the disease [73]. Although preliminary, these
`results suggest that metabolomics might provide sensitive bio-
`markers to follow up changes in the neurodegenerative and
`inflammatory processes that contribute to MS pathogenesis [74].
`
`Integration of data from different
`‘omics’ approaches
`
`The combination of the data generated in transcriptomics and
`genomics studies can be an invaluable source of information and
`new hypotheses. Aune et al. compared the genes differentially
`expressed by lymphocytes in rheumatoid arthritis, systemic lupus
`erythematosus, insulin-dependent diabetes mellitus and MS, con-
`cluding that they are clustered within chromosomal domains in
`the genome [75]. Strikingly, they found that the chromosomal
`domains containing the genes differentially expressed in autoim-
`mune disorders could be mapped to disease susceptibility loci
`associated to those diseases by genetic linkage studies [75].
`These results suggest that the expression of disease-associated
`genes is co-regulated as a result of shared genetic regulatory ele-
`ments or local patterns of chromatin condensation. Recently,
`Baranzini and coworkers studied the genetic concordance
`between gene expression and genetic linkage in MS [76]. They
`first compiled the data on gene expression available for MS and
`EAE, and superimposed it with all the known susceptibility loci
`identified in MS and EAE. In their study, Baranzini and coworkers
`identified the MS susceptibility genes located in the MHC locus as
`overlapping with clusters of differentially expressed genes in MS
`and murine EAE. However, they could also identify an interesting
`region on chromosome X that might contribute to the sexual
`dimorphism observed in MS. The integration of the data generat-
`ed by different platforms, like transcriptomics, genomics and pro-
`teomics, is therefore likely to deepen our understanding of the
`mechanisms driving MS.
`
`New experimental models
`
`Screenings aimed at identifying genes or drugs controlling the
`immune response cannot be easily undertaken in mice because
`they are based on crossing, maintaining and screening large num-
`
`bers of animals, an expensive time-, space- and labour-intensive
`task; new experimental models are needed. Our current knowl-
`edge on innate immunity originated from pioneering studies that
`used flies and worms to carry out genetic studies and identify
`pathways controlling the response to microbes [77]. Invertebrates
`lack adaptive immunity, but the zebrafish (Danio rerio) harbours
`an adaptive immune system that resembles the mammalian
`immune system [78] and offers several experimental advantages
`for the study of pathways controlling vertebrate processes of inter-
`est. As part of our work on the zebrafish to identify pathways con-
`trolling immunity, we have characterized the zebrafish homologues
`of the transcription factors autoimmune regulator [79] and Foxp3
`[80, 81], pivotal for central and peripheral tolerance, respectively.
`Our work on zebrafish Foxp3 led us to identify the ligand-acti-
`vated transcription factor aryl hydrocarbon receptor (AHR) as a
`regulator of the expression of mammalian Foxp3 [82]. Upon acti-
`vation by its ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
`AHR interacts with its binding sites on the Foxp3 gene and stimu-
`lates its transcription. AHR activation by TCDD generates function-
`al Treg that inhibit the development of EAE by a transforming
`growth factor ␤1-dependent mechanism. Surprisingly, AHR acti-
`vation by an alternative ligand, 6-formylindolo[3,2-b]carbazole,
`interferes with Treg differentiation, boosts TH17 differentiation and
`worsens EAE. Thus, AHR regulates both Treg and TH17 differentia-
`tion in a ligand-specific fashion, constituting a unique target for
`therapeutic immuno-modulation. In addition, our findings suggest
`that the experimental advantages offered by the zebrafish can be
`exploited to characterize metabolic pathways controlling immunity in
`vertebrates and to identify new targets for therapeutic intervention.
`
`Conclusions
`
`How can we apply the information provided by genomics, tran-
`scriptomics and proteomics to the early diagnosis, prevention,
`monitoring and therapy of MS? A first step is the establishment of
`experimental models where biologic problems of interest can be
`investigated through genomic, transcriptomic and proteomic
`approaches simultaneously. The zebrafish, with its experimental
`advantages for the study of vertebrate-specific processes [78],
`might turn into a platform, where to identify pathways contribut-
`ing to MS pathology and therapeutic targets. Our findings on the
`control of the immune response by AHR support this view.
`Another step should be the adaptation of genomic, transcrip-
`tomic and proteomic technologies to a clinical setup. The person-
`al genome project, for example, aims at developing fast and reli-
`able methods to sequence the individual human genomes for
`US$1000 or less [83]. However, the data generated by these high-
`throughput approaches should be integrated to understand how
`the genomics, transcriptomics and proteomics of an individual
`influence each other. This would require the development of com-
`putational tools for the integration of networks and pathways into
`accurate quantitative models [84, 85]; with user interfaces aimed
`at facilitating its exploration and modification [86].
`
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`J. Cell. Mol. Med. Vol 12, No 4, 2008
`
`MS results from a complex dialogue between a susceptible
`individual and a fostering environment, a dialogue unique to each
`individual. The combination of genomic, transcriptomic and pro-
`teomic techniques might allow us to identify key elements in this
`dialogue to prevent, diagnose and cure MS.
`
`mental autoimmune encephalomyelitis (EAE), IL-7 receptor ␣ chain
`(IL-7R␣), IL-2 receptor ␣ chain (IL-2R␣), magnetic resonance (MR), major
`histocompatiblity complex (MHC), multiple sclerosis (MS), N-acetylaspartate
`(NAA), nitric oxide (NO), osteopontin (OPN), peripheral blood mononuclear
`cells (PBMC), primary progressive MS (PPMS), regulatory T cells (Treg),
`relapsing-remitting MS (RRMS), secondary progressive MS (SPMS).
`
`Abbreviations
`
`Acknowledgements
`
`2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), aryl hydrocarbon
`receptor (AHR), ␤–Interferon (␤–IFN), central nervous system
`(CNS), Cerebrospinal fluid (CSF), Epstein-Barr Virus (EBV), experi-
`
`FJQ is a recipient of a long-term fellowship from the Human Frontiers of
`Science Program Organization.
`
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