Contribution of vitamin D insufficiency to the pathogenesis of multiple sclerosis

Vitamin D metabolism

The main steps of vitamin D metabolism are well known and will not be detailed here [Lips, 2006; Holick et al. 2007; Norman and Bouillon, 2010] (Figure 1). After transformation of 7-dehydrocholesterol into cholecalciferol (vitamin D3) in the skin through the action of ultraviolet B radiation (UVB) or after direct oral intake of vitamin D3 (or D2), there is a first hydroxylation in the liver catalyzed by several vitamin D-25-hydroxylase enzymes, the most important being CYP2R1 [Prosser and Jones, 2004]: this results in 25-OH-D, which is the metabolite measured in the blood to evaluate the vitamin D status (see below). Then, a second hydroxylation takes place in the proximal tubule of the kidney, catalysed by the enzyme 1α-hydroxylase (CYP27B1) [Prosser and Jones, 2004], resulting in 1,25-OH-2D (calcitriol), which is the active metabolite of vitamin D. Low calcium intake and the parathyroid hormone (PTH) stimulate this renal hydroxylation and increase the calcitriol level in the blood, whereas the phosphaturic hormone fibroblast growth factor 23 (FGF23) and a high level of calcitriol have the opposite effect. Furthermore, the vitamin D 24-hydroxylase, another enzyme located in the renal tubule and encoded by the CYP24A1 gene, is also able to induce an inactivating pathway for vitamin D metabolites. This enzyme is tightly regulated by FGF23 and the level of calcitriol. The importance of this inactivating pathway has recently been highlighted in the literature with the demonstration that inactivating mutations of the CYP24A1 gene induced severe neonatal hypercalcaemia [Schlingman et al. 2011]. Vitamin D and its diverse metabolites, including calcitriol, are transported in the blood by the vitamin D-binding protein (DBP) (which is a serum globulin mainly produced in the liver) and to a lesser extent by albumin. The calcitriol dissociates from DBP when entering a target cell and first binds to a specific receptor of vitamin D (VDR) within the cytoplasm (Figure 1). Then, this complex enters the nucleus and forms a heterodimer by connecting to a nuclear receptor, that is, the retinoid X receptor (RXR). The heterodimer calcitriol–VDR–RXR finally binds to vitamin D-responsive elements (VDREs), which constitute a specific sequence of DNA within the promoter region of the target genes, the whole regulating (by activation or suppression) gene transcription and expression and finally protein synthesis (e.g. cytokines, etc.) in approximately 5–10% of the genome [Wang et al. 2005; Niino et al. 2008; Norman and Bouillon, 2010; Pike and Meyer, 2010] (Figure 1). Thus, calcitriol, secreted into the bloodstream by the kidney and exerting its actions in various other tissues by binding to a specific receptor, can be considered as a hormone.

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Figure 1.

Schematic representation of vitamin D metabolism.

Multiplicity of vitamin D actions

VDRs are widespread in almost all cells of the organism [Walters, 1992], including immunity cells: macrophages, monocytes, dentritic cells (DCs) and lymphocytes T and B [Bahlla et al. 1983; Provvedini et al, 1983, Morgan et al. 1996; Vedman et al. 2000; Chen et al. 2007]. VDRs are also present in all types of central nervous system (CNS) cells, that is, neurons, oligodendrocytes, astrocytes and glial cells [Walters, 1992; Baas et al. 2000; Garcion et al. 2002; Eyles et al. 2005]. These different immune and nervous cells express the CYP27B1 enzyme and are able to transform in situ the circulating 25-OH-D into calcitriol [Zehdner et al. 2001; van Etten et al. 2008] (Figure 1), resulting in intracrine and paracrine actions in these cells and neighbouring cells [Morris and Anderson, 2010]. Besides its role in calcium physiology and bone health, vitamin D also has numerous potential extra-bone actions: protective for the cardiovascular system, antiproliferative (in certain cancers), anti-infectious (innate immunity) and anti-inflammatory and immunomodulatory (adaptive immunity), an effect which could be involved in autoimmune diseases such as type 1 diabetes, Crohn’s disease, rheumatoid arthritis and MS [Holick, 2004, 2007; Vieth, 2007; Vieth et al. 2007; Borradale and Kimlin, 2009; Hewison, 2012]. The specific role of calcitriol within CNS cells remains to be clarified [Smolders et al. 2011b]: it may have potential actions in neuronal functioning, neuroprotection and myelination [Wergeland et al. 2011], but also in innate and adaptive immunity of the CNS, through the invading lymphocytes. Accordingly, the presence of VDRs and CYP27B1 in the different immune and nervous cells constitutes a first indication for potential actions of vitamin D in MS. The immunodulatory action of vitamin D through the general immune system, likely important for MS pathogenesis, is specifically reviewed in the following sections.

Immunodulatory effect of vitamin D in experimental autoimmune encephalomyelitis

Since experimental autoimmune encephalomyelitis (EAE) is the best animal model of MS, it is of interest to briefly review the effect of vitamin D in this disease, which has been studied for more than 20 years. Calcitriol has both a preventive and a curative effect in EAE [Lemire and Archer, 1991; Cantorna et al. 1996] but requires the presence of calcium [Cantorna et al. 1999] and VDRs [Mehan and DeLuca, 2002] for these actions. However, there are contradictory reports concerning the beneficial effect of vitamin D sufficiency [Fernandes de Abreu et al. 2011] or deficiency [DeLuca and Plum, 2011] on the severity and delay of onset of EAE. If vitamin D3 is used, the beneficial effect predominates in females, likely via a potentiation by oestrogens [Spach and Hayes, 2005; Nashold et al. 2009; Subramanian et al. 2012]. Various immunological mechanisms have been reported to explain the vitamin D and calcitriol effects: an anti-inflammatory effect [Spach et al., 2004], actions on macrophages [Nashold et al. 2000], on different types of cytokines [Cantorna et al. 1998; Spach et al. 2006; Pedersen et al. 2007], on regulatory T lymphocyte cells (Tregs), lymphocyte T helper 1 (Th1), Th17 and Th2 [Mattner et al. 2000; Muthian et al. 2006; Chang et al. 2010; Mayne et al. 2011] and invariant natural killer T-cells (iNKTs) [Cantorna et al. 2012]. Interestingly, with low VDR gene expression, EAE is facilitated, with an increase in Th1 and Th17, suggesting that, in a similar genetic situation in humans, the MS risk may be increased despite high ambient UV radiation (e.g. in Sardinia) [Spanier et al. 2012]. These diverse beneficial actions on T lymphocytes are favoured by some authors, who suggest that vitamin D positively influences Treg activity, restoring a better ratio between the Th2 (protective) and Th1 (aggressive) cells, the overall effect being a decrease in inflammation [Cantorna, 2006, 2008; Smolders et al. 2008a; Cantorna et al. 2012] (Figure 2). It should be noted that this mechanism is analogous to the mechanism of interferon β (IFNβ) [Axtell et al. 2010; Bushnell et al. 2012], used as an immunomodulatory treatment in MS, and that a potentiation exists between the beneficial effects of IFNβ and calcitriol analogue used together in EAE [Van Etten et al. 2007]. Despite all these findings observed with vitamin D or calcitriol in EAE, one research group recently reported results that led them to suggest that UVB plays the actual protective role in EAE instead of vitamin D [Becklund et al. 2010] and to question the role of VDR and vitamin D in EAE and MS [DeLuca and Plum, 2011; Wang et al. 2012]. However, even if it were the case that UVB exerts a specific immunosuppressive effect independent of vitamin D synthesis [Hart et al. 2011], such an effect has not yet been studied in humans and does not in fact rule out parallel, now well documented immunomodulatory mechanisms induced by vitamin D. Furthermore, whatever the current controversial points of view concerning the role of vitamin D in EAE, it should not be forgotten that this disease is not exactly comparable to human MS.

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Figure 2.

Schematic representation of one of the hypothetical immunomodulatory effects of vitamin D (through calcitriol).

General immunodulatory effect of vitamin D in humans

The general immunodulatory effect of vitamin D in humans (for a review, see Hewison [2012]), that is, outside the CNS, is currently the best known mechanism through which vitamin D appears to influence MS risk and course. Furthermore, vitamin D could enhance innate immunity via its actions on macrophages and monocytes and regulate adaptive immunity in multiple ways [Adorini and Penna, 2008]. The presence of VDRs in human T lymphocytes [Provvedini et al. 1983; Baeke et al. 2010], in greater number in CD8 than in CD4 lymphocytes [Vedman et al. 2000], as well as in B lymphocytes [Provvedini et al. 1983; Chen et al. 2007], and the expression of CYP27B1 in lymph nodes [Zehdner et al. 2001] and T lymphocytes [Sigmundsdottir et al. 2007] constitute important indications of a potential role of vitamin D in adaptive immunity. Furthermore, a number of mechanisms by which vitamin D and calcitriol could favourably influence immunity have been reported in the past 30 years: it has been shown that vitamin D (through calcitriol) reduces differentiation of monocytes to DCs and differentiation and proliferation of DCs, thus decreasing T-cell stimulation [Griffin et al. 2001]; controls T-cell activation [von Essen et al. 2010] and inhibits T-cell proliferation [Rigby et al. 1990; Lemire et al. 1984]; reduces the production of interleukin (IL)-2 (growth factor for T cells) [Müller et al. 1993]; suppresses in vitro and in vivo production of proinflammatory Th1 cell-derived IFNγ and tumour necrosis factor α [Reichel et al. 1987; Lemire et al. 1995; Baeke et al. 2010; Zhang et al. 2012]; reduces proinflammatory Th17 activity and IL-17 production [Tang et al. 2009; Ikeda et al. 2010; Bruce et al. 2011; Joshi et al. 2011; Allen et al. 2012]; enhances the production of the anti-inflammatory cytokine IL-10 [Heine et al. 2008; Baeke et al. 2010; Allen et al. 2012]; promotes in vitro and in vivo the development of Tregs expressing cytotoxic T lymphocyte antigen 4 and forkhead box P3, resulting in an anti-inflammatory effect [Jeffery et al. 2009; Prietl et al. 2010; Khoo et al. 2012; Urry et al. 2012]; enhances the transformation of CD4 T lymphocytes into a Th2 phenotype (with a protective role) [Boonstra et al. 2001; van Etten and Mathieu, 2005; Sloka et al. 2011b]; and furthermore, inhibits B-cell differentiation [Chen et al. 2007]. Accordingly, vitamin D has general immunomodulatory and anti-inflammatory effects not only by reducing DCs, Th1, Th17, B-cell proliferation and proinflammatory cytokines but also by promoting Th2 phenotype, Treg activity and anti-inflammatory cytokines. The vitamin D action on Tregs, as mentioned above in the context of EAE, could itself reduce Th1 activity and re-equilibrate the balance between Th1 and Th2 cells, resulting in a reduction of inflammation [Cantorna, 2006; Smolders et al. 2008a] (Figure 2). Such an action profile of vitamin D (through calcitriol) strongly suggests that an insufficiency of this vitamin could play a role in the pathophysiology of autoimmune diseases, including MS, and may constitute one of the risk factors involved in these diseases.

Immunomodulatory effect of vitamin D in patients with multiple sclerosis

The multiple immunological studies on patients with MS reported recently have shown that the general immunomodulatory actions of vitamin D on T and B cells already described in animals and normal humans likely also exist in this disease. One of the first studies dealing with the immunological action of vitamin D in patients with MS was a controlled trial in which it was observed that vitamin D supplementation (1000 IU/day for 6 months) significantly increased tumour growth factor β1, a cytokine inhibiting T cells and secreted by different types of cells, including Tregs [Mahon et al. 2003]. More recently, it has been shown in patients with MS that calcitriol inhibits in vitro T-cell proliferation, inhibits the development of IL-6- and IL-17-producing cells, enhances IL-10 production and the number of Tregs [Correale et al. 2009] and stimulates CD 46 and IL-10 [Kickler et al. 2012], all these mechanisms contributing to an anti-inflammatory action. Furthermore, a correlation was found between the vitamin D and calcitriol serum levels and the Treg number [Royal et al. 2009], or only between the vitamin D serum level and the inhibitory action of Tregs on Th1 cells, with a beneficial effect in IFNβ users [Smolders et al. 2009] and without correlation with calcitriol, PTH and calcium [Smolders et al. 2010a]. In a small controlled trial, MS-associated abnormal T reactivities were suppressed in vivo by vitamin D supplementation at serum 25-OH-D concentrations higher than 100 nmol/liter [Kimball et al. 2011b]. In another small study, in which patients with relapsing–remitting MS (RRMS) were supplemented with high doses of vitamin D (20,000 IU/day) for 3 months, Tregs were unchanged but the proportion of IL-10+ CD4+ T cells was increased [Smolders et al. 2010b]. Furthermore, in patients with MS, a low vitamin D serum level was associated with T-cell proliferation [Grau-Lopez et al. 2012], vitamin D inhibited in vitro the differentiation and maturation of DCs [Bartosik-Psujek et al. 2010] and enhanced in vivo anti-inflammatory cytokines [Moysayebi et al. 2011], and calcitriol reduced in vitro proinflammatory cytokines and enhanced anti-inflammatory cytokines [Lysandropoulos et al. 2011]. However, there was no substantial effect on phenotypic markers of B-cell differentiation in circulating B cells in a study using supplementation with high doses of vitamin D3 [Knippenberg et al. 2011]. Lastly, the immunomodulatory and anti-inflammatory effects of vitamin D appear to be more marked in women than in men in patients with MS as well as in healthy subjects, maybe due to synergic effects between calcitriol and 17-β estradiol [Correale et al. 2010]. Altogether, these different studies show that vitamin D has potentially beneficial immunomodulatory and anti-inflammatory effects in patients with MS, though their actual impact on the course of the disease remains to be accurately evaluated by randomized, controlled trials (RCTs) using vitamin D supplementation.

Optimal vitamin D serum level

25-OH-D is the vitamin D metabolite usually measured in the blood since it is representative of the vitamin D store in the organism [Heaney, 2000; Zerwekh, 2008]. The limits usually recommended are between 75 and 200 nmol/liter (i.e. 30 and 80 ng/mL) [Dawson-Hughes et al. 2005; Binkley and Krueger, 2008; Souberbielle et al. 2010]. The question of the lower cut-off (75 nmol/liter) is a key point to understand the whole vitamin D problem. This limit has not been determined from classical control groups of ‘normal’ adults (i.e. with the 2.5th or 5th percentile found in an apparently healthy population) since vitamin D insufficiency is widespread in general populations (see below), but it has not been empirically fixed either. Defining vitamin D insufficiency corresponds to determining the 25-OH-D serum level below which adverse outcomes may occur or above which beneficial effects of vitamin D may be observed. Ideally, this supposes that RCTs demonstrating positive effects of vitamin D compared with placebo on clinical (‘hard’) outcomes are available and that the 25-OH-D concentrations in the ‘vitamin D groups’ of theses RCTs have been evaluated. It must be emphasized that, with the exception of the effect on the risk of falls, the many lines of evidence concerning the various potential extra-skeletal effects of vitamin D are mostly based on observational and mechanistic studies. Although numerous prospective studies have shown that subjects in the highest quantile of 25-OH-D serum concentrations (usually >70–80 nmol/liter) have a lower relative risk for many diseases than those in the lowest quantile (usually <30–40 nmol/liter) [Munger et al. 2006; Bodnar et al. 2007; Leu and Giovannucci, 2011; Ma et al. 2011], the observational nature of these studies precludes any conclusion regarding a causal relationship between low vitamin D status and these diseases, and there are consequently no clear clinical cutoff(s) to optimize the potential vitamin D effects. It must be acknowledged that the 75 nmol/liter cutoff is only ‘reasonably’ evidence based (i.e. based on RCTs) for the musculoskeletal effects of vitamin D: in the RCTs that have shown positive effects of vitamin D on nonvertebral fractures [Bischoff-Ferrari et al. 2009b] and falls [Bischoff-Ferrari et al. 2009a], subjects in the ‘vitamin D groups’ generally had 25-OH-D levels of more than 75 nmol/liter, whereas those in the ‘placebo groups’ had levels mostly in the 30–60 nmol/liter range. Consistent with these RCTs, bone biopsy data showed that histomorphometric signs of defect in the mineralization of bone were not detected in subjects with a 25-OH-D serum level of more than 75 nmol/liter whereas they were present, as defined by the most conservative threshold of the osteoid volume/bone volume ratio of 2%, in approximately 20% of subjects with a 25-OH-D serum level between 50 and 75 nmol/liter [Bischoff-Ferrari et al. 2004; Priemel et al. 2010]. Furthermore, patients with a basal 25-OH-D serum level of up to 70 nmo/liter decreased their PTH serum concentration when they were given vitamin D (without calcium) [Okazaki et al. 2011], whereas it has been reported that the PTH serum concentration may increase when the 25-OH-D serum level is below 75–80 nmol/liter [Chapuy et al. 1996; Holick, 2007; Durazo-Arvizu et al. 2010]. It has also been shown that calcium absorption was improved in menopausal women when the 25-OH-D serum level increased to approximately 80 nmol/liter [Heaney et al. 2003b] and calcium excretion was no longer directly dependent on 25-OH-D serum concentrations below the level of 75 nmol/liter [Kimball et al. 2011a]. Lastly, recent data indicate that a 25-OH-D serum level of at least 82 nmol/liter is required to optimize the antifracture efficacy of bisphosphonates [Carmel et al. 2012].

Due to the convergence of the findings provided by all of these different approaches on the 75 nmol/liter level, most medical laboratories in the world have now adopted this level as the lower normal limit, even if this point is not yet consensual [Ross et al. 2011; Heaney and Holick, 2011; Holick et al. 2011]. Furthermore, it should be noted that the ‘physiological’ zone between the 75 and 200 nmol/liter 25-OH-D serum levels grossly corresponds to the serum levels observed in outdoor workers [Haddad and Chyu, 1971; Haddock et al., 1982; Barger-Lux and Heaney, 2002; Azizi et al. 2012], as well as in traditionally living populations in East Africa [Luxwolda et al. 2012]. This zone is far below the toxic zone, which appears to be located above the 400 nmol/liter serum level [Hathcock et al. 2007; Burton et al. 2010].

Vitamin D requirements

On the basis of these new metabolic and pathological findings, the daily requirement of vitamin D has recently been reassessed and is now thought to be far higher than the 200–400 IU/day dose that, until a few years ago, was generally estimated to be sufficient. The previously held belief regarding the optimal requirement was principally based on the results of experiments in the rat almost one century ago in the context of studies on rickets prevention. Nowadays, it is more readily accepted that humans are different from rats, as a species as well as in terms of weight for determining treatment doses, and that rickets prevention is not the only vitamin D action to be taken into account. The daily requirement does of course depend on what the optimal target 25-OH-D serum level is considered to be: for a 25-OH-D serum level of 50 nmol/liter, 800-1000 IU/day of vitamin D appears sufficient, but to bring most people above the 75 nmol/liter level, a dosage of between 1000 and 4000 IU/day (depending on the individual, but on average 2000 IU/day) is required [Heaney et al. 2003a, 2009; Grant and Holick, 2005; Hollis, 2005; Bischoff-Ferrari et al. 2006, 2009b, 2012; Vieth, 2006; Hall et al. 2010; Schwalfenberg et al. 2010; Whiting and Calvo, 2010; Cashman et al. 2011; Garrett-Mayer et al. 2012; Holick, 2011, 2012]. However, vitamin D intake via (unfortified) food is very marginal in normal Western diets, even in those considered well balanced, and generally provides less than 100–200 IU/day, rarely reaching little more than 400 IU/day with fortified food [Calvo et al. 2004; Moore et al. 2005; Välimäki et al. 2007; O’Donnell et al. 2008; Vatanparast et al. 2010; von Geldern and Mowry, 2012]. Sunshine therefore remains the principal natural source of vitamin D, providing 80–90% of the requirement in the absence of fortified food. However, in temperate and Nordic countries, vitamin D may be synthesized in the skin via UVB only a few months per year (around summer), that is, when the sun is seasonally sufficiently high in the sky for UVB to penetrate all the layers of the atmosphere, and vitamin D stocks disappear in a few weeks after exposure to the sun (or oral intake) if they are not regularly replenished [Holick, 2007]. It should also be noted that modern lifestyles have tended to reduce most people’s outdoor activities and exposure to the sun. Moreover, exposure to the sun is often avoided due to dermatological concerns and people with dark skins and older people synthesize vitamin D less easily than people with light skins and the young [Vieth, 1999; Armas et al. 2007; Binkley et al. 2007]. Lastly, in people who are overweight, (liposoluble) vitamin D is partly sequestered in adipocytes, which may contribute to a worsening of insufficiency [Earthman et al. 2012].

Widespread vitamin D insufficiency

The characteristics of vitamin D physiology, the effects of latitude and climate and multiple societal factors related to vitamin D synthesis result in an insufficiency in this vitamin in most people living beyond the 40th parallels, that is, in Europe, the northen half of the United States, Canada and the former Soviet Union for the northen hemisphere, and New Zealand and Tasmania for the southern hemisphere [Holick, 2007; Pierrot-Deseilligny and Souberbielle, 2011; van der Mei et al. 2012b]. In these countries (for a review, see Pierrot-Deseilligny and Souberbielle [Pierrot-Deseilligny and Souberbielle, 2010]), 25-OH-D serum levels in ‘normal’ adults are between 40 and 70 nmol/liter on average, with generally only slight differences depending upon the season and consequently, at least for a large part of the year, 75% of people are in a state of insufficiency with a 25-OH-D serum level cut-off of 75 nmol/liter and still almost half of the population is in a state of insufficiency if one considers that the cutoff should be 50 nmol/liter. In tropical or subtropical countries, vitamin D serum levels are generally higher, at least for people not systematically avoiding sun exposure, and a correlation exists between latitude and vitamin D serum levels in white people at the world scale [Hagenau et al. 2009]. Such a correlation has also been observed in France, a relatively small country [Chapuy et al. 1996].

Vitamin D insufficiency in patients with multiple sclerosis

In patients with MS living in temperate and Nordic countries, as in the general populations of these countries, vitamin D insufficiency is widespread, whatever the cutoff (50 or 75 nmol/liter) for the lower limit of the 25-OH-D serum level (Figure 3): indeed, as early as the earliest stages of the disease, that is, in patients with clinically isolated syndrome (CIS) or with RRMS, average serum levels are between 42 and 74 nmol/liter, depending on the studies and the seasons, with a general mean close to 60 nmol/liter [Soilu-Hänninen et al. 2005, 2012; Smolders et al. 2008b; Hiremath et al. 2009; Kragt et al. 2009; Mowry et al. 2010; Pierrot-Deseilligny and Souberbielle, 2010, 2012; Simpson et al. 2010; Banwell et al. 2011; Dabbaghmanesh and Yousefipour, 2011; Lonergan et al. 2011; Neau et al. 2011; Steffensen et al. 2011; Yildiz et al. 2011; Bäärnhielm et al. 2012; Kampman et al. 2012; Kirbas et al. 2012; Løken-Amsrud et al. 2012; Moen et al. 2012; Runia et al. 2012; Soilu- Hänninen et al. 2012; Šaltyte. Benth et al. 2012; Triantafyllou et al. 2012] (Table 1). In some of these studies, there was a control group in addition to the patient group and there was not always a significant difference in vitamin D serum levels between the two groups. However, this point may be considered secondary since we now know that most control ‘normal’ subjects are also in a state of more or less marked vitamin D insufficiency. Thus, the possible role of vitamin D status in any disease, including in MS, must always be interpreted in conjunction with the actions of multiple other environmental and genetic risk factors interacting with this status (see below). In this context, vitamin D insufficiency appears to be only one risk factor favouring the disease, interacting in concert with multiple other risk factors, which may explain a significant deleterious effect on MS risk of this widespread vitamin insufficiency at a population scale but does not account for the totality of individual situations of patients with MS, in whom the cumulative effects of several other risk factors may have at times a crucial role, independently of vitamin D status. In particular, in the relatively rare cases of patients with MS in whom normal spontaneous vitamin D serum levels are observed throughout the year, it may be hypothesized that, in these patients, either other environmental (infectious, toxic, etc.) and genetic risk factors play a determinant role or (genetic) errors in the metabolism and actions of vitamin D exist downstream to the 25-OH-D serum level determination. Conversely, the fact that the great majority of ‘normal’ subjects who are in a state of vitamin D insufficiency (on the same basis as patients with MS) do not eventually develop MS may be explained by the existence, in these subjects, of other protective environmental or genetic factors.

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Figure 3.

Schematic representation of the evolution of 25-OH-D serum level according to multiple sclerosis stage.

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Table 1.

25-OH-D serum levels in different cohorts of patients with multiple sclerosis, mainly at the earliest stages of the disease.

Reference	Cohort provenance: country (region or city)	Age (years) Mean ± SD (range)	Sample size, N	25-OH-D serum level (nmol/liter): mean ± SD (range)	MS form
Soilu-Hänninen et al. [2005]	Finland (Turku)	36 ± 1.4	40	41 ± 5 (W), 58 ± 3 (S)	RRMS
Smolders et al. [2008b]	The Netherlands (Maastricht)	NA	126	72 ± 31	RRMS
Hiremath et al. [2009]	USA (Baltimore)	42 ± 13	199	71 ± 39	CIS, RRMS, SPMS
Kragt et al. [2009]	The Netherlands (Amsterdam)	45 ± NA	103	59 ± 25 (W), 97 ± 34 (S)	RRMS, SPMS
Mowry et al. [2010]	USA (San Francisco and New York)	15 ± 3	110	55 ± 22	CIS, RRMS
Pierrot-Deseilligny and Souberbielle [2010]	France (Paris region)	36 ± 12	32	45 ± 16	CIS
Shaygannejad et al. [2010]	Iran (Isfahan)	36 (15–55)	50	48 ± NA	RRMS
Simpson et al. [2010]	Australia (Tasmania)	44 ± 10	145	42 ± NA (W), 74 ± NA (S)	RRMS
Banwell et al. [2011]	Canada (multiple centres)	9.5 ± 4.5	302	63 ± 28	CIS
Dabbaghmanesh and Yousefipour [2011]	Iran (Shiraz)	35 ± 8	82	55 ± 54	RRMS, SPMS
Lonergan et al. [2011]	Ireland (3 centres)	46 (21–80)	329	38 (13–161)	RRMS, SPMS
Neau et al. [2011]	France (Poitiers region)	46 ± 23	170	46 ± 23	RRMS, SPMS
Steffensen et al. [2011]	Norway (Tromsø)	39 (21–50)	35	55 ± 29	RRMS
Yildiz et al. [2011]	Switzerland (St Gallen)	38 ± 10	80	57 ± 29	RRMS
Bäärnhielm et al. [2012]	Sweden (Stockholm)	39 ± 10	1013	63 ± NA	RRMS
Kampmann et al. [2012]	Norway (Tromsø)	40 (21–50)	35	48 (20–120)	RRMS
Kirbas et al. [2012]	Turkey (Rize)	NA (18–40)	30	67 ± 35	RRMS
Løken-Amsrud et al. [2012]	Norway (multiple centres)	39 (19–58)	88	67 (26–121)	RRMS
Moen et al. [2012]	Norway (Oslo)	NA	99	68 ± 24	CIS, RRMS
Pierrot-Deseilligny et al. [2012]	France (Paris region)	39 ± 10	156	49 ± 22*	RRMS
Runia et al. [2012]	The Netherlands (Rotterdam)	39 (19–55)	73	69 ± NA	RRMS
Šaltyte. Benth et al. [2012]	Norway (multiple centres)	39 (19–48)	92	68 ± 26	RRMS
Soilu-Hänninen et al. [2012]	Finland (Turku)	39 (22–53)	66	54 (19–82)*	RRMS
Triantafyllou et al. [2012]	Greece (Athens)	39 ± 10	119	62 ± 25	RRMS

*

Before vitamin D supplementation.

CIS, clinically isolated syndrome; MS, multiple sclerosis; NA, not available; RRMS, relapsing–remitting multiple sclerosis; S, in summer; SD, standard deviation; SPMS, secondary progressive multiple sclerosis; W, in winter.

Furthermore, it should be noted that the vitamin D serum level usually tends to decrease throughout the course of MS because of the conjugate actions of three worsening factors for vitamin D insufficiency, successively intervening and accumulating during this course (Figure 3): as early as the beginning of MS, Uhtoff’s phenomenon (heat sensitivity) may lead some patients to spontaneously avoid sun exposure and the associated heat, an attitude that until recently was often encouraged by neurologists, leading to an accelerated decline in vitamin D synthesis; in the mid course of the disease, disability reduces outdoor activities and, consequently, sun exposure; in older patients, vitamin D synthesis is physiologically reduced by age. These different factors contributing to the deterioration of vitamin D status likely partly explain why vitamin D serum levels are lower in secondary progressive MS (SPMS) than at the earliest stages of the disease, with concentrations usually close to 40 nmol/liter [Nieves et al. 1994; Ozgocmen et al. 2005; Smolders et al. 2008b; Pierrot-Deseilligny and Souberbielle, 2010; Neau et al. 2011]. These associated factors might also contribute to ‘reverse causality’ (i.e. with the disease worsening the initial insufficiency in vitamin D), at least in mid and advanced stages of MS. However, it should not be ignored that a marked hypovitaminosis D is observed as early as the earliest stages of MS (i.e. before these associated factors can be exerted) and consequently may contribute to triggering the disease (see below). From a preventive point of view, it would also be of particular interest to study the vitamin D status of subjects with radiologically isolated syndromes and in siblings of patients with MS (Figure 3), since they all have an increased risk for MS.

Human leukocyte antigen system

The HLA system drives many immune responses. In the analysis performed by Sawcer and colleagues, the allele HLA-DRB1*1501, which is present in 14–30% of populations from countries with high risk for MS [Schmidt et al. 2007], has the strongest association with this disease, representing 11% of the whole heritability, but other HLA-DRB1 alleles are also involved to a lesser degree [Sawcer et al. 2011]. By contrast, HLA-A*0201 could be protective. The risk for MS is also increased in children with one or more HLA-DRB1*15 alleles [Banwell et al. 2011; Disanto et al. 2011a]. It should be noted that a VDRE exists within the promoter region of HLA-DRB1 [Ramagopalan et al. 2009b; Handunnetthi et al. 2010], that is, the main risk variant for MS: this VDRE was highly conserved (no mutations on over 600 chromosomes) in the major MS-associated DR2 haplotype bearing the HLA-DRB1*15 allele and not conserved generally among non-MS associated haplotypes. In addition, this VDRE influenced gene expression and conferred calcitriol sensitivity to HLA-DRB1*15, whereas the variant VDRE present on other, non-MS-associated HLA-DRB1 haplotypes was not responsive to calcitriol. The authors hypothesized that a lack of vitamin D in early childhood can affect the expression of HLA-DRB1 in the thymus and result in an increase in the risk of autoimmunity later in life [Handunnetthi et al. 2010]. It should also be noted that the frequency of the MS-associated HLA-DRB1*1501 allele is much higher in white individuals than in other racial types, which could explain why MS prevalence is relatively low in people with dark skins living in temperate countries, independently of their vitamin D status [Handunnetthi et al. 2010]. Furthermore, the frequency of HLA-DRB1*15 is higher in women than in men with MS [Hensiek et al. 2002; Chao et al. 2010; Irizar et al. 2012] and the penetrance of HLA-DRB1*15 has increased over time in women [Chao et al. 2009]. This could contribute to the well known recent increase in female predominance of the disease and also, through the calcitriol effect on HLA-DRB1*15 expression, to a lower incidence of MS in women with higher vitamin D serum levels [Kragt et al. 2009]. Furthermore, it may be that decreasing vitamin D serum levels in the general population [Yetley, 2008] due to diverse changes in lifestyle (fewer outdoor activities, more protection from sunshine and higher obesity levels) has increased the frequency of HLA-DRB1*15 in MS over time [Handunnetthi et al. 2010]. In a recent study, it was confirmed that the majority of HLA-DRB1 alleles (including HLA-DRB1*1501) express the VDRE, but an independent contribution of VDRE motif variation to an increased MS risk was not discernible [Nolan et al. 2012]; however, HLA-DRB1*04, *07 and *09 alleles, which express the ‘nonresponsive’ VDRE motif, were associated with a significantly reduced risk of MS. It has also been suggested that VDR variants could modulate the risk of MS conferred by HLA-DRB1*1501 [Agliardi et al. 2011; Huang and Xie, 2012] and, in a cohort of Sardinian patients with MS (i.e. a population with a specific high genetic risk for MS), VDREs do not seem to play a significant role in the promoter region of DRB1 in susceptibility to MS [Cocco et al. 2012]. Accordingly, even if there is no doubt that some HLA alleles play a crucial role in MS risk or protection, further studies are required to understand better the different HLA-VDRE mechanisms involved in this disease.

Other genes

The study by Sawcer and colleagues has shown the involvement in MS risk of two genes involved in vitamin D metabolism: CYP27B1, controlling 1-α-hydroxylase and therefore calcitriol synthesis, and CYP24A1, controlling calcitriol catabolism [Sawcer et al. 2011]. The genetic involvement of CYP27B1 in MS risk was also found in another GWAS considering a specific pathway in which eight genes within a module of 13 genes influenced by vitamin D were associated with MS [ANZgene and the Australia and New Zealand Multiple Sclerosis Genetics Consortium, 2009] and is now confirmed thanks to several other types of genetic research methods used to study patients with MS with vitamin D-dependent rickets [Torkildsen et al. 2008], with rare variants [Ramagopalan et al. 2011a] or with single nucleotide polymorphisms [Sundqvist et al. 2010; Simon et al. 2011]. However, it should be mentioned that no association was found between genes involved in vitamin D metabolism and MS risk in two other studies [Orton et al. 2011a; Smolders et al. 2011c], but these studies were performed with a much smaller sample than the one used by Sawcer and colleagues [Sawcer et al. 2011]. Furthermore, the genes encoding CYP27B1 and CYP24A1 could be epigenetically regulated [Kim et al. 2009; Novakovic et al. 2009], which may influence the vitamin D serum level and MS risk [Burrell et al. 2011]. Moreover, interactions between CYP27B1 and HLA-DRB1*15 may exist and influence the MS risk [Simon et al. 2011] (see below). Polymorphisms in the VDR gene such as the TaqI variant could be weakly linked to MS [Cox et al. 2012], but some other variants and single-nucleotide polymorphisms, located for example near DHCR7 [Alloza et al. 2012], may be involved in vitamin D insufficiency and the MS risk, a point that does, however, require confirmation. Recent findings have also provided new insights into how vitamin D influences the genetic regulation of B cells in MS (for a review, see Disanto and colleagues) [Disanto et al. 2012c]. Lastly, a genetic regulation of DBP may also influence the MS risk [Disanto et al. 2011c]. Accordingly, these different genetic links reported between vitamin D and MS, in particular those related to CYP27B1, strongly suggest that vitamin D insufficiency is involved in the pathogenesis of this disease. However, in a few of the aforementioned studies, some results are not concordant, which may simply result from methodological questions and require further studies. Furthermore, the exact molecular mechanisms as to how VDREs exert control over numerous genes and cells are not yet understood and are currently being studied [Berlanga-Taylor et al. 2011 Disanto et al. 2012e].

Epstein–Barr virus infection

Almost all, if not all, patients with MS have previously been infected by EBV [Wagner et al. 2000; Wandinger et al. 2000; Ascherio and Munger, 2007a; Pakpoor et al. 2012]. Following primary infection, EBV remains latent in the memory B-cell population for life and EBV antibody titres are increased with potentially deleterious immunologic effects, for example by favouring certain autoimmune diseases. EBV infection is ubiquitous and 95% of the general population has been infected by this virus, but MS is extremely rare in EBV-negative adult individuals [Ascherio and Munger, 2007a; Levin et al. 2010]. Like MS, but independently of this disease, EBV infection is also positively correlated with latitude [Disanto et al. 2012d]. Individuals with a history of late infectious mononucleosis (after childhood) have a twofold to threefold increased risk of developing MS [Thacker et al. 2006; Nielsen et al. 2007b; Ramagopalan et al. 2009c; Handel et al. 2010b]. Furthermore, plasma antibody titres against the EBV nuclear antigen 1 (EBNA1) increase several years before the clinical onset of MS [Sundström et al. 2004; Levin et al. 2005; Lünemann et al. 2010; Sundqvist et al. 2012a], EBNA1 levels are correlated with MS risk [Lucas et al. 2011a; Munger et al. 2011b; Simon et al. 2012b] and antibody response to EBV antigens is generally higher in MS [Lindsey et al. 2012]. It should particularly be noted that MS risk appears to be 30-fold higher in subjects with the highest anti-EBNA1 level (> 300) [Munger et al. 2011b; Ascherio et al. 2012a]. Therefore, there no longer appears to be any doubt that EBV infection contributes to the pathogenesis of MS, but the exact mechanisms that lead to the disease have yet to be determined [Tselis, 2012]. EBV was found in 90% of meningeal B-cell follicles and in perivascular cuffs of patients with MS in one study [Serafini et al. 2007] but was either not found or not frequently observed in a number of subsequent studies [Willis et al. 2009; Aloisi et al. 2010; Lassmann et al. 2010; Peferoen et al. 2010; Sargsyan et al. 2010; Torskilden et al. 2010; Owens and Bennett, 2012; Tracy et al. 2012]. Nevertheless, latent EBV infection, with residual viral particles possibly remaining chronically in B lymphocytes, may contribute to the inflammatory milieu in active MS lesions by activating an innate and adaptive immune response, including B-cell activation and proinflammatory IFNα production [Serafini et al. 2010; Tzartos et al. 2012; Ascherio et al. 2012a]. As an alternative, or in addition to this still uncertain direct effect of chronic or latent EBV infection, it may be that an altered long-lasting immune response following primary EBV infection contributes to trigger or perpetuate demyelinating disease [Niller et al. 2008; Mameli et al. 2012; Perron et al. 2012]. Links with MS risks appear to exist between EBV immunological response and HLA-DRB1*1501 [De Jager et al. 2008; Sundström et al. 2009; Lucas et al. 2011a; Sundqvist et al. 2012a], HLA-B*0702 [Jilek et al. 2012], vitamin D insufficiency [Hayes and Donald Acheson, 2008; Holmøy, 2008; Lossius et al. 2011] and smoking [Simon et al. 2010]. However, the latter link was not found in another study [Sundqvist et al. 2012b] and the exact deleterious mechanisms involved in these diverse cumulative risk factors remain currently unclear and require further studies. From a practical point of view, even if a vaccine against EBV is developed [Sokal et al. 2007], its efficacy in preventing autoimmune diseases (including MS) or even its total inocuity could be difficult to evaluate. By contrast, given the extreme rarity of MS in EBV-seronegative patients, the serological test may be useful in certain difficult diagnostic circumstances since a negative test result, at least in adults, or low anti-EBNA titres could constitute an argument against MS [Lünemann et al. 2010]. Accordingly, even if the simple encounter with EBV remains in the great majority of cases a banal infection without apparent deleterious long-term consequences, it could also, in some cases, be the first event triggering a long-lasting deleterious immunological cascade, worsened both by a late primo infection occurrence with symptomatic infectious mononucleosis and by the persistence of a high anti-EBNA1 level, eventually leading to the disease onset. These worsening infectious circumstances for the MS risk might also be cumulated with other risk factors (e.g. HLA-DRB1*1501, vitamin D insufficiency, smoking), which could increase even more the disease risk, in particular if the protective counterparts (e.g. HLA-A*0203 and normal vitamin D status) are absent (see below).

Vitamin D insufficiency

Although only a few epidemiological studies have so far directly implicated vitamin D status as an influencing factor in MS risk, multiple different environmental findings indirectly relating to vitamin D suggest that this vitamin plays an important role. It has long been known that latitude influences MS risk, the prevalence of the disease being minimal at the equator and increasing with either North or South latitude. This effect is observed on a world scale [Gale and Martyn, 1995; Alonso and Hernan, 2008; Simpson et al. 2011; Sloka et al. 2011a], at a continental level [Kurtzke, 1995; Puggliatti et al., 2006], in large countries, such as the United States [Acheson et al. 1960; Kurtzke et al. 1985, Kurtzke, 2008], the former Soviet Union [Boiko et al. 1995] and Australia [van der Mei et al. 2001; Taylor et al. 2010] and even in comparatively smaller countries, such as New Zealand [Taylor et al. 2008] and France, at least in farmers [Vukusic et al. 2007]. MS prevalence may change after migrations occurred during the second decade of life, with, for example, a beneficial effect for people who have migrated from a high-latitude region (with a high MS prevalence) to a sunnier, lower-latitude region (with a low MS prevalence) [Kurtzke et al. 1985; Gale and Martyn, 1995; Hammond et al. 2000; Ascherio and Munger, 2007a, 2007b; Handel et al. 2010a; McDowell et al. 2010; McLeod et al. 2011]. It should be noted that a reverse migration, that is, from a low-latitude region to a higher-latitude region, has less effect on MS prevalence, as though the environmental protection acquired in infancy and childhood, maybe related to the sunny climate of these regions (see below), is long lasting at adult age for these migrants. By contrast, no such protection seems to exist for their children, who have an MS risk similar to that of the natives of the high-latitude regions [Elian et al. 1990; Dean and Elian, 1997], which constitutes a further argument suggesting that this part of the risk is indeed environmental and not genetic.

However, MS prevalence does not depend upon latitude per se but upon more specific elements linked to latitude. Among these elements, climate and sun exposure likely play a crucial role. Indeed, there were very strong correlations between MS prevalence in the different states of the United States or in nine large-scale areas of North America and the corresponding mean annual amounts of UV in these areas [Beretich and Beretich, 2009]. In a meta-analysis performed on 52 studies from various countries around the world, a highly significant link (p < 10^-8) existed between MS prevalence and the annual amount of UVB in the different countries, this link being 20 times more significant than that existing between MS prevalence and simple latitude [Sloka et al. 2011a]. In France, sunshine maps show large climate areas analogous to those of the main zones of MS prevalence identified in farmers by Vukusic and colleagues [Vukusic et al. 2007; Ebers, 2008, 2009; Handel et al. 2010a]. Furthermore, in two successive independent studies, a strong correlation was observed between the regional MS prevalence in French farmers and the annual UV radiation of the 22 French regions [Pierrot-Deseilligny and Souberbielle, 2010; Orton et al. 2011b], with a much higher significance for the link between UV radiation and MS prevalence (p = 4 × 10^–6) than for that between MS prevalence and the latitude of the regions (p = 4 × 10^–4) [Pierrot-Deseilligny and Souberbielle, 2010], and more marked results in women than in men [Orton et al. 2011b]. Similar links have also been reported between MS prevalence and UV radiation in Canada [Acheson et al. 1960], the United States [Freedman et al. 2000] and in England and Wales [Ramagopalan et al. 2011b]. Furthermore, at identical latitudes, the risk of MS is lower in the sunniest regions [van Amerogen et al. 2004; van der Mei et al. 2007a, 2007b], in particular in high-altitude regions compared with lowland regions [Kurtzke, 1967]. Accordingly, there are no longer any doubts that UV radiation influences MS risk. However, it may be that a part of the protection provided by UV radiation results from its hypothetical direct immunosuppressive effect, that is, bypassing vitamin D synthesis by UVB and the immunomodulatory action of this vitamin [Hart et al. 2011], as mentioned above in the section on EAE. Nevertheless, it remains that vitamin D is also directly involved in MS, independently of UV radiation, as suggested by a number of experimental, epidemiological, genetic, immunological and clinical studies reviewed in the different chapters of this review. In fact, these two mechanisms involved in the MS risk, that is, a possible direct immunosuppressive action of UVB, not involving vitamin D, and the immunomodulatory effect of vitamin D, mainly synthesized thanks to UVB, may play parallel, independent roles, as suggested in a few recent studies [Lucas et al. 2011b; Bäärnhielm et al. 2012].

The timing for MS protection by UVB and vitamin D mechanisms is also a key point. Multiple types of studies suggest that protection/risk from these environmental factors may occur at different epochs during the first part of life until early adulthood (Figure 4). For the period of adulthood, we have already mentioned that living in a sunny country or migrating to such a country after the age of 15–20 years are favourable factors for avoiding MS, but other, nonclimatic factors may be involved in this protection. In some studies, oral intake of vitamin D in the form of diverse vitamin supplements [Munger et al. 2004] or oily fish [Kampmann et al. 2007; Kampmann and Brustad, 2008] was found to be linked with a lower MS risk, but other associated protective factors cannot be ruled out in these studies. Of greater significance, since it was based on the serum level of vitamin D itself, was a study performed in young American soldiers who had given at least two serum samples a few years before the onset of any neurological symptoms during their military service [Munger et al. 2006]. The group of white soldiers with levels of vitamin D in the highest quintile (i.e. between 99 and 152 nmol/liter) had a significantly lower risk of MS than those with the lowest levels of vitamin D (i.e. between 15 and 63 nmol/liter) (p < 0.01). Furthermore, in a recent Swedish nested case–control study, an association was found between relatively high 25-OH-D serum levels (≥75 nmol/liter) during the years preceding disease onset and a decreased risk of MS [Salzer et al. 2012a].

The protection afforded by UV radiation/vitamin D may also be acquired before adulthood, that is, during childhood and adolescence. The studies analysing these points are based on questionnaires assessing the amount of time spent outdoors during holidays and weekends during the first two decades of life in patients with MS and control subjects. The risk of MS was significantly lower in subjects who spent the most time outdoors during their youth [Acheson et al. 1960; van der Mei et al. 2003; Kampman et al. 2007; Dwyer et al. 2008; Sloka et al. 2008], including within pairs of monozygotic twins [Islam et al. 2007]. These results are also supported by studies of skin actinic activity, measured on the back of the hand and reflecting total accumulated exposure to sun; the subjects who had the highest level of actinic activity also had the lowest MS risk [van der Mei et al. 2003; Lucas et al. 2011b]. Furthermore, in patients with MS who resided in low-to-medium solar radiation areas, low sun exposure in the autumn/winter during the ages of 6–15 years was significantly associated with a 2.1 year earlier symptom onset [McDowell et al. 2011]. Therefore, relatively frequent outdoor activities in childhood and adolescence, resulting in significant sun exposure during these epochs and consequently the likelihood of more vitamin D synthesis than with a lifestyle consisting of almost exclusively indoor activities, could be protective in terms of MS risk. However, in a study evaluating vitamin D intake during adolescence, no association was found with MS risk [Munger et al. 2011a]. By contrast, childhood or adolescence obesity, which is a cause of vitamin D insufficiency, increases the MS risk [Munger et al. 2009; Hedström et al. 2012]. It may be that the relatively long-lasting protection from MS acquired before adolescence by sun exposure or vitamin D status corresponds to a critical step of maturation of the thymus and the immune system [Tulic et al. 2012], which could be favourably influenced by these environmental factors during the first part of life. To be more precise about this hypothetical mechanism, it has been suggested that vitamin D insufficiency in utero and during childhood may affect expression of HLA-DRB1 in the thymus, allowing autoreactive T cells to escape thymic deletion [Ramagopalan et al. 2009a].

The month of birth and the vitamin D status of the mother during pregnancy may also have an impact on the MS risk for offspring when they reach adulthood. The risk of MS is lower for people born in autumn (especially in November) and higher for those born in spring (especially in May) in the northern hemisphere [Templer et al. 1992; Willer et al. 2005; Sotgiu et al. 2006; Fernandes de Abreu et al. 2009; Ramagopalan et al. 2009a; Bayes et al. 2009; Disanto et al. 2012a], with a reverse pattern in the southern hemisphere [Staples et al. 2010]; see also a recent meta-analysis confirming these findings [Dobson et al. 2012]. This seasonal effect is correlated with the presence of a familial risk factor for MS [Sotgiu et al. 2006] or with the phenotype HLA-DRB1 in Canada [Ramagopalan et al. 2009a] but not in Finland [Saastoimanen et al. 2012]. These results may be related to the vitamin D status of pregnant women [Willer et al. 2005; Salzer et al. 2010], since 25-OH-D serum levels are at their highest in autumn and their lowest in spring in the general population as well as in pregnant women [Hypponen and Power, 2007; Holmes et al. 2009; Handel et al. 2010a; Lewis et al. 2010]. In line with this hypothesis, it may be consistent that in sunnier countries, that is, with less contrast between the seasons, no seasonal difference in the month of birth has been observed for MS risk [Givon et al. 2012; Fragoso et al. 2012]. Furthermore, in a cohort of 927 US army veterans with MS, those who were born in winter and whose birthplace was in low solar radiation areas had disease symptom onset on average 2.8 years earlier than those born in seasons other than winter and in medium- and high-solar radiation areas [McDowell et al. 2010]. The influence of month of birth could exist for different immune-related diseases, including MS [Disanto et al. 2012a]. Furthermore, the predicted 25-OH-D level in pregnant mothers was inversely associated with the risk of MS in their daughters [Mirzaei et al. 2011]. It has recently been suggested [Disanto et al. 2012b], based on experimental findings [Harvey et al. 2010; Yu and Cantorna, 2011], that the influence on MS risk of month of birth and vitamin D status during pregnancy may be related to a critical step of development of the immune system in utero requiring vitamin D, and that an insufficiency of vitamin D at this crucial time could result in a state that cannot be corrected later, after birth, by vitamin D intakes. Accordingly, besides the positive influences on the MS risk of outdoor activities in childhood and adolescence and the beneficial role of a normal vitamin D status in early adulthood, both the vitamin D status in the pregnant mother and the month of birth, which is likely related to this status or sun exposure of the mother during the last months of pregnancy, may also influence the MS risk for a long-lasting period.

Cigarette smoking

The role of smoking in MS has recently been reviewed [Wingerchuk, 2012] and this could be both a risk factor for MS and a deleterious element for the progression of the disease. In three large American and European cohort studies, it has been shown that cigarette smoking is a significant risk factor for MS, that this risk is dose dependent and perhaps due to cigarette smoking rather than nicotine [Hernan et al. 2001; Hedström et al. 2009; Carlens et al. 2010]. In smaller, less powered and, therefore, less important studies, a significant link also existed [Riise et al. 2003; Pekmezovic et al. 2006; Sundström et al. 2008] or was not found [Silva et al. 2009; Simon et al. 2010] between smoking and MS risk, but in a meta-analysis of 14 studies, smoking was indeed a significant risk for MS [Handel et al. 2011]. Passive smoking also constituted a risk factor for MS in a paediatric cohort for children exposed to parental smoking [Mikaeloff et al. 2007]. Furthermore, in a large Swedish cohort of adult patients with MS, the MS risk was increased among never smokers who had been exposed to passive smoking compared with never smokers who had never been exposed [Hedström et al. 2011a]. By contrast, no risk factor for MS was found for offspring of mothers who had smoked during their pregnancy [Montgomery et al. 2008]. As already emphasized in the preceding chapters, risk factors for MS are multiple and smoking may, therefore, interact with genetics and the main two other identified environmental risk factors. Smoking is associated with higher levels of EBNA1 [Nielsen et al. 2007a] and enhances the association between high EBNA1 titres and increased MS risk [Simon et al. 2010]. An interaction with HLA-DRB1*15 was also observed in smoker but not in nonsmoker patients with MS [Hedström et al. 2011b]. Smoking could also be a deleterious factor for the course of MS. Smoking significantly speeds conversion from CIS to confirmed MS [Di Pauli et al. 2008] and usually from RRMS to SPMS, also increasing the rate of accumulation of disability in established progressive forms of MS [Hernan et al. 2005; Sundström and Nyström, 2008; Healy et al. 2009; Pittas et al. 2009]. However, there was an exception to such worsening effects in one study [Koch et al. 2007] and only a trend for smoking to increase the risk of SPMS in a meta-analysis [Handel et al. 2011]. Accordingly, the findings already available strongly suggest that smoking increases the risk for MS and, in the course of the disease, is deleterious for disability, but further studies are required to confirm these points. A possible potentiation of MS risk by the association of smoking and vitamin D insufficiency also remains to be specifically studied.