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 Citation[1]. Although genome-wide association studies have discovered numerous genetic variants predisposing to MS, the environment exerts a greater influence on susceptibility Citation[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 Citation[1,2]. Growing evidence lends strong support to vitamin D deficiency and Epstein–Barr virus (EBV) infection as being key environmental risk factors.
Evidence for vitamin D & Epstein–Barr virus in MS
The data supporting a role for vitamin D in the development of MS are overwhelming. The worldwide prevalence of MS positively correlates with latitude Citation[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 Citation[2,4]. Vitamin D intake significantly decreases the risk of MS, and vitamin D levels inversely correlate with risk of MS later in life Citation[5,6]. Furthermore, CYP27B1, the gene encoding 1α-hydroxylase (the enzyme that activates vitamin D) is associated with MS susceptibility Citation[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 Citation[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 Citation[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 Citation[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 Citation[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 Citation[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 Citation[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 Citation[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 Citation[15].
Evidence for vitamin D–EBV interaction
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 Citation[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 Citation[10,16]. Furthermore, serum levels of 25-hydroxyvitamin D correlate positively with the ability of Tregs to suppress T-cell proliferation Citation[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 Citation[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) Citation[19] and the finding that individuals with a high serum 25-hydroxyvitamin D were less likely to have viral (EBV) shedding in saliva Citation[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 Citation[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 Citation[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 Citation[23] and one of these mechanisms may be EBNA-3’s interaction with vitamin D, which has well-described antiviral effects Citation[24].
Vitamin D & EBV: causal cascade & disease prevention
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 Citation[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 Citation[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 Citation[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 Citation[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.
No writing assistance was utilized in the production of this manuscript.
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