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Hypothesis:Vitamin D Epstein-Barr interaction increases MS risk – Sept 2011

Expert Review of Neurotherapeutics - - - - - (PDF is attached at bottom of this page)
September 2011, Vol. 11, No. 9, Pages 1221-1224 , DOI 10.1586/ern.11.97
Giulio Disanto; University of Oxford, Oxford, UK
Ute Meier; Blizard Institute, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London, UK
Gavin Giovannoni; Blizard Institute, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London, UK
Sreeram V Ramagopalan; University of Oxford, Oxford, UK and Queen Mary University of London, Blizard Institute, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London, E1 2AT, UK. s.ramagopalan at qmul.ac.uk

Multiple sclerosis (MS) is a complex immune-mediated disorder of the CNS, which results from a combination of genetic and environmental factors and their interactions (1). Although genome-wide association studies have discovered numerous genetic variants predisposing to MS, the environment exerts a greater influence on susceptibility (2). That nurture is fundamental in MS has been known for decades. Monozygotic twins are, at most, 30% concordant, and familial MS risk is significantly influenced by location and season of birth and the childhood environment (1,2). Growing evidence lends strong support to vitamin D deficiency and Epstein–Barr virus (EBV) infection as being key environmental risk factors.

The data supporting a role for vitamin D in the development of MS are overwhelming. The worldwide prevalence of MS positively correlates with latitude (3). Essential for the production of vitamin D, ultraviolet B (UVB) light radiation is the factor that most likely mediates such a correlation, and this is especially clear in national studies from France and England (2,4). Vitamin D intake significantly decreases the risk of MS, and vitamin D levels inversely correlate with risk of MS later in life (5,6). Furthermore, CYP27B1, the gene encoding 1?-hydroxylase (the enzyme that activates vitamin D) is associated with MS susceptibility (7). In addition, vitamin D can also influence MS course. A recent prospective investigation of a large cohort of MS patients has demonstrated that vitamin D status is inversely associated with disease activity over the subsequent 6 months (8).

Functional evidence comes from studies showing that both the vitamin D receptor (VDR) and CYP27B1 genes are expressed in an exceptionally broad range of immune cells, including those that are known to play a central role in MS, such as Th1 and Th17 subsets, FOXP3+ regulatory T cells and B cells (9,10). This extremely pleiotropic hormone is able to influence cellular function and proliferation through a fine regulation of gene expression. Using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq), our group has demonstrated the presence of 2776 different vitamin D responsive elements (VDREs) through the entire genome bound by the VDR in B cells. Notably, genetic loci associated with MS are strikingly enriched for VDR binding sites (11).

However, vitamin D is not the only environmental factor implicated in MS. EBV is a B-lymphotropic human DNA herpes virus associated with lymphoproliferative and immune disorders (12). Although more than 90% of the general population appears to encounter EBV at some point during their life, several lines of evidence highlight its role in the pathogenesis of MS. Large independent studies have shown that nearly all MS patients have been infected with EBV. Furthermore, both high anti-EBV antibody titers and a history of infectious mononucleosis (IM) increase the risk of developing MS (1,13).

Whether such a strong association between MS and EBV is actually causal has been, and still is, debated. For example, the elevated risk of MS after IM could arise from a common genetic susceptibility to MS and IM. However, this suggestion can be refuted since the HLA-DRB1*1501 class II allele, the main genetic risk factor for MS, is not associated with the development of IM (14). Similarly, the hypothesis that good hygiene during childhood may predispose both to MS and to a later contact with EBV, and therefore IM, should be equally rejected given the observation that EBV-negative individuals (likely to be exposed to the highest levels of hygiene) have the lowest risk of MS (13). Pediatric MS represents a valuable tool for the identification of environmental risk factors. As a consequence of early disease onset, less time is available for potential confounders, and thus any associated factor is more likely to be truly causative. In a recent investigation of a large cohort of pediatric MS patients, both EBV positivity and high EBV antibody titers predicted conversion to MS independently of HLA and vitamin D status, although not all pediatric patients were infected with EBV (15).

While it is possible that vitamin D and EBV may be influencing MS risk through independent mechanisms, another hypothesis is also plausible, which is that these two factors may be biologically interacting to increase the risk of MS. This idea is supported by our recent observation that a statistical interaction between IM prevalence and UVB light radiation could explain 72% of the variance in MS prevalence across England (4). How this interaction may take place at the molecular level remains largely unknown, but there are a number of potential explanations.

First, it is plausible that vitamin D deficiency influences the immune response to EBV. Vitamin D increases the production of the anti-microbial peptide cathelicidin, which may protect against EBV (10,16). Furthermore, serum levels of 25-hydroxyvitamin D correlate positively with the ability of Tregs to suppress T-cell proliferation (17), and it has been suggested that Tregs are important in controlling primary EBV infection to a subclinical level in most cases and that IM represents a failure of this protective mechanism (18). This idea is somewhat supported by the epidemiological evidence indicating that early vitamin D deficiency influences MS risk prior to EBV infection (i.e., place and month of birth precedes IM) (19) and the finding that individuals with a high serum 25-hydroxyvitamin D were less likely to have viral (EBV) shedding in saliva (20). However, as vitamin D deficiency affects all ages there needs to be an explanation as to why the peak age of IM is in the early teens/late 20s. Furthermore, given the variable nature of vitamin D status, it is not clear how this interaction could predispose to MS over the long term if EBV is suppressed when vitamin D levels are high. It is possible that this could be related to an ‘immunological imprint’ that is left as a result of vitamin D deficiency during a critical time period of development or after infection with EBV.

Another theory comes from the demonstration that the EBV protein EBNA-3 is able to stop the expression of the vitamin D-regulated genes C-FOS, CYCLIN C, CYP24A1, GADD45A and P21 by blocking VDR activity (21). A recent study identified the expression profiles of lymphoblastoid cell lines obtained by infecting primary B cells with either wild-type EBV or an EBV mutant strain lacking the EBNA-3 gene (22). To explore to what extent EBNA-3 may influence the expression of vitamin D responsive genes we calculated how many of the putative EBNA-3 regulated genes are characterized by the presence of a VDRE using data from our VDR ChIP-seq map. Interestingly, of the 296 genes that were differentially expressed between EBNA-3 positive and EBNA-3 negative lymphoblastoid cell lines, 82 (27.7%) were characterized by the presence of a VDRE, which was much greater than expected by chance (p = 0.003). A gene ontology analysis demonstrated that the genes with both an EBNA-3 and VDR influence were involved in cell proliferation, apoptosis and immune response processes. This analysis suggests that a substantial proportion of the genes regulated by the viral protein EBNA-3 are likely to be regulated by vitamin D, so it may be that EBV potentiates vitamin D deficiency by blocking the effect of vitamin D. The extent to which EBNA-3 is expressed may be dependent on the host immune response, which may include MS susceptibility genes. From an evolutionary perspective, EBV has evolved several mechanisms to evade immunological attack (23) and one of these mechanisms may be EBNA-3’s interaction with vitamin D, which has well-described antiviral effects (24).

Understanding how risk factors for MS integrate to lead to MS development is critical for disease prevention. The proposition that low vitamin D levels may be influencing the immune response against EBV is not surprising since vitamin D is a potent modulator of the adaptive immune system and low levels are known to increase the risk of other viral infections, such as influenza (25,26). However, further work is needed to address this question. In particular, future prospective studies should investigate whether the risk of IM and EBV antibody titers are influenced by vitamin D status at the time of infection.

If confirmed, this notion would open up the question as to whether the association between EBV and MS is merely a consequence of vitamin D deficiency predisposing to both MS and EBV infection. Although this possibility cannot currently be ruled out, it appears highly unlikely given the amount of evidence (mentioned above) supporting EBV as being specifically involved in MS causation. It is particularly intriguing to suppose that vitamin D-deficient individuals may be more likely to develop IM after EBV infection, and MS risk is increased in some of these individuals who have higher expression of EBNA-3, which blocks vitamin D action in B cells, which are gaining increasing recognition as being key players in the pathogenesis of MS (27,28). As EBV has coevolved with us over millions of years it may be of interest to study EBV–vitamin D interactions and responses between ancestral populations from Africa and Caucasians.

Great strides are being made in cataloguing the pleiotropic effects of vitamin D in the immune and nervous systems (16). A major facet to understanding better the interaction between vitamin D and EBV in MS is to better understand how EBV predisposes to MS – that is, is it through molecular mimicry, bystander activation or another mechanism (29)? More basic research is needed to fully understand the environmental risk factors in MS, as this will provide new strategies for both disease prevention and treatment. Longitudinal studies of at-risk populations will be extremely useful.
Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.


Papers of special note have been highlighted as: • of interest
1 Ramagopalan SV, Dobson R, Meier UC, Giovannoni G. Multiple sclerosis: risk factors, prodromes, and potential causal pathways. Lancet Neurol.9(7),727–739 (2010). (CrossRef)
2 Ebers GC. Environmental factors and multiple sclerosis. Lancet Neurol.7(3),268–277 (2008). (CrossRef) (Medline)
3 Simpson S Jr, Blizzard L, Otahal P, Van Der Mei I, Taylor B. Latitude is significantly associated with the prevalence of multiple sclerosis: a meta-analysis. J. Neurol. Neurosurg. Psychiatry DOI: 10.1136/jnnp.2011.240432 (2011) (Epub ahead of print).
4 Ramagopalan SV, Handel AE, Giovannoni G, Rutherford Siegel S, Ebers GC, Chaplin G. Relationship of UV exposure to prevalence of multiple sclerosis in England. Neurology76(16),1410–1414 (2011).
• By using data on ultraviolet B radiation, multiple sclerosis (MS) and infectious mononucleosis (IM) prevalence, the authors demonstrate that UVB exposure and IM together can explain a substantial proportion of the variance of MS prevalence in England. (CrossRef) (CAS)
5 Munger Kl, Zhang SM, O’Reilly E et al. Vitamin D intake and incidence of multiple sclerosis. Neurology62(1),60–65 (2004). (Medline) (CAS)
6 Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA296(23),2832–2838 (2006).
• In a prospective, nested case–control study among more than 7 million US military personnel, 25-hydroxyvitamin D levels were shown to inversely correlate with the risk of MS later in life. (CrossRef) (Medline) (CAS)
7 Anzgene. Genome-wide association study identifies new multiple sclerosis susceptibility loci on chromosomes 12 and 20. Nat. Genet.41(7),824–828 (2009). (CrossRef) (Medline)
8 Simpson S Jr, Taylor B, Blizzard L et al. Higher 25-hydroxyvitamin D is associated with lower relapse risk in multiple sclerosis. Ann. Neurol.68(2),193–203 (2010). (Medline) (CAS)
9 Walker VP, Modlin RL. The vitamin D connection to pediatric infections and immune function. Pediatr. Res.65(5 Pt 2),106R–113R (2009). (CrossRef)
10 Kamen DL, Tangpricha V. Vitamin D and molecular actions on the immune system: modulation of innate and autoimmunity. J. Mol. Med.88(5),441–450 (2010). (CrossRef) (Medline) (CAS)
11 Ramagopalan SV, Heger A, Berlanga AJ et al. A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution. Genome Res.20(10),1352–1360 (2010). (CrossRef) (CAS)
12 Ascherio A, Munger Kl. Epstein–Barr virus infection and multiple sclerosis: a review. J. Neuroimmune Pharmacol.5(3),271–277 (2010). (CrossRef)
13 Ascherio A, Munger Kl. Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann. Neurol.61(4),288–299 (2007).
• In this review, Ascherio et al. provide a comprehensive overview of the epidemiological evidence supporting a role for Epstein–Barr virus in MS. (CrossRef) (Medline)
14 Ramagopalan SV, Meier UC, Conacher M et al. Role of the HLA system in the association between multiple sclerosis and infectious mononucleosis. Arch. Neurol.68(4),469–472 (2011). (CrossRef)
15 Banwell B, Bar-Or A, Arnold Dl et al. Clinical, environmental, and genetic determinants of multiple sclerosis in children with acute demyelination: a prospective national cohort study. Lancet Neurol.10(5),436–445 (2011). (CrossRef) (CAS)
16 Holick MF. Vitamin D deficiency. N. Engl. J. Med.357(3),266–281 (2007). (CrossRef) (Medline) (CAS)
17 Smolders J, Thewissen M, Peelen E et al. Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis. PLoS One4(8),e6635 (2009).
• In this study, the authors demonstrate a direct correlation between 25-hydroxyvitamin D levels and functionality of regulatory T cells in MS patients. (CrossRef) (Medline)
18 Wingate PJ, McAulay KA, Anthony IC, Crawford DH. Regulatory T cell activity in primary and persistent Epstein–Barr virus infection. J. Med. Virol.81(5),870–877 (2009).
• This work provides evidence for a potential role played by regulatory T cells in controlling primary Epstein–Barr virus infection and IM onset.
19 Handel AE, Giovannoni G, Ebers GC, Ramagopalan SV. Environmental factors and their timing in adult-onset multiple sclerosis. Nat. Rev. Neurol.6(3),156–166 (2010). (CrossRef) (Medline)
20 Zwart SR, Mehta SK, Ploutz-Snyder R et al. Response to vitamin D supplementation during Antarctic winter is related to BMI, and supplementation can mitigate Epstein–Barr virus reactivation. J. Nutr.141(4),692–697 (2011). (CrossRef) (CAS)
21 Yenamandra SP, Hellman U, Kempkes B et al. Epstein–Barr virus encoded EBNA-3 binds to vitamin D receptor and blocks activation of its target genes. Cell Mol. Life Sci.67(24),4249–4256 (2010).
• The Epstein–Barr virus protein EBNA3 is able to bind the vitamin D receptor and block the activation of vitamin D-regulated genes against vitamin D-induced growth arrest and/or apoptosis. (CrossRef) (CAS)
22 Hertle ML, Popp C, Petermann S et al. Differential gene expression patterns of EBV infected EBNA-3A positive and negative human B lymphocytes. PLoS Pathog.5(7),e1000506 (2009). (CrossRef)
23 Means RE, Lang SM, Jung JU. Human ?-herpesvirus immune evasion strategies. In: Human Herpesviruses: Biology, Therapy and Immunoprophylaxis. Arvin A, Campadelli-Fiume G, Mocarski E et al. (Eds). Cambridge University Press, Cambridge, UK (2007).
24 Beard JA, Bearden A, Striker R. Vitamin D and the anti-viral state. J. Clin. Virol.50(3),194–200 (2011). (CrossRef) (CAS)
25 Cannell JJ, Vieth R, Umhau JC et al. Epidemic influenza and vitamin D. Epidemiol. Infect.134(6),1129–1140 (2006). (CrossRef) (Medline) (CAS)
26 Aloia JF, Li-Ng M. Re: epidemic influenza and vitamin D. Epidemiol. Infect.135(7),1095–1096; author reply 1097–1098 (2007). (CrossRef) (Medline)
27 Bar-Or A, Fawaz L, Fan B et al. Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS? Ann. Neurol.67(4),452–461 (2010). (CrossRef) (CAS)
28 Khademi M, Kockum I, Andersson ML et al. Cerebrospinal fluid CXCL13 in multiple sclerosis: a suggestive prognostic marker for the disease course. Mult. cler.17(3),335–343 (2011). (CrossRef) (CAS)
29 Kakalacheva K, Lunemann JD. Environmental triggers of multiple sclerosis. FEBS Lett. DOI: 10.1016/j.febslet.2011.04.006 (2011) (Epub ahead of print).

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