Charles Pierrot-Deseilligny

Jean-Claude Souberbielle
email: jean-claude.souberbielle@aphp.fr

Vitamin D and multiple sclerosis: an update

Keywords: environment, genetics, multiple sclerosis, supplementation, vitamin D anti-EBNA1, antibodies against EBV antigen nuclear 1, CIS, clinically isolated syndrome, CNS, central nervous system, CYP27B1, enzyme 1α-hydroxylase, EAE, experimental autoimmune encephalopathy, EBV, Epstein-Barr virus, HLA, human leukocyte antigen, MS, multiple sclerosis, RCTs, randomized clinical trials, UVB, ultraviolet B rays, VDR, vitamin D receptor

come

Introduction

The key role of vitamin D in bone metabolism has been known for a long time, but our knowledge has developed considerably during the past 15 years about the physiological extra--skeletal actions of this vitamin, in particular its immunodulatory effects [START_REF] Hayes | Vitamin D actions on CD4(+) T cells in autoimmune disease[END_REF], which have implications in some inflammatory and autoimmune diseases such as multiple sclerosis (MS). In parallel, great advances have also been made in identifying a number of protective factors and risk factors for MS, including exposure to sun and vitamin D insufficiency, which complexly interact with one another [START_REF] Ascherio | Epidemiology of multiple sclerosis: from risk factors to prevention-An update[END_REF]. The main recent findings linking vitamin D to MS are reviewed in this update.

Figure 1. Unequal solar ultraviolet radiation in the world. UVB reach the ground and skin only when the sun is sufficiently high in the sky for UVB to penetrate all the layers of the atmosphere. This physical mechanism can only exist in the middle of the day and, beyond the 40th parallel in mid and high latitude regions, only for a few months of the year, essentially between the middle of spring and the end of summer. Thus, a vast portion of the northern hemisphere -comprising almost all of Europe and a large part of North America and of the former Soviet Union -as well as a few countries in the southern hemisphere -are involved. In all these countries, exposure to sun includes UVB only during these relatively reduced time slots in the day and year. These regions correspond to those where the highest multiple sclerosis prevalence is observed. By contrast, subtropical and tropical regions, where 80% of the world's population lives, have enough sunshine to provide the major source of natural vitamin D almost throughout the year. 40°, 40th parallel; UVB, ultraviolet B rays; kWh/m 2 kilowatt/hour per square meter. Adapted from Meteonorm values.

General and physiological considerations on sunshine and vitamin D

Vitamin D is principally produced by the action of ultraviolet B rays (UVB) in the skin, providing 80-90% of human requirements [START_REF] Holick | Vitamin D: evolutionary, physiological and health perspectives[END_REF][START_REF] Wacker | Sunlight and Vitamin D: A global perspective for health[END_REF], but this main natural solar source of vitamin is unequally distributed in the world (O'Neil et al., 2016), as shown in Figure 1. The metabolism of vitamin D is nowadays relatively well known and is summarized in Figure 2 [START_REF] Norman | Vitamin D nutritional policy needs a vision for the future[END_REF][START_REF] Pike | The vitamin D receptor: new paradigms for the regulation of gene expression by 1,25dihydroxyvitamin D3[END_REF]. 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 [START_REF] Souberbielle | Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: recommendations for clinical practice[END_REF]. The optimal levels for serum 25--OH--D concentration vary according to the target population [START_REF] Hoel | The risks and benefits of sun exposure 2016[END_REF][START_REF] Holick | Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline[END_REF][START_REF] Ross | The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know[END_REF].

For immune health and more generally for patients, the target 25--OH--D range usually accepted nowadays (30--60 ng/mL or 75--150 nmol/L) has been derived from multiple metabolic and bone clinical data [START_REF] Binkley | Evaluation and correction of low vitamin D status[END_REF][START_REF] Holick | Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline[END_REF][START_REF] Pierrot-Deseilligny | Contribution of vitamin D insufficiency to the pathogenesis of multiple sclerosis[END_REF]: these findings are very different in nature but, quite remarkably, they all indicate the critical feature of the lower limit (30 ng/mL), below which a pathological significance is reached. This is why those norms are nowadays often recommended [START_REF] Hoel | The risks and benefits of sun exposure 2016[END_REF], even though there is not yet a total consensus [START_REF] Holick | Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline[END_REF]Pludowski et al., 2013;[START_REF] Ross | The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know[END_REF][START_REF] Souberbielle | Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: recommendations for clinical practice[END_REF].

Oral%intake% (marginal%in% food)%

To reach a 25--OH--D serum level above 30 ng/mL and then permanently maintain it within the physiological zone (30--60 ng/mL), one must receive a daily vitamin D dose of between 2000 and 4000 IU (3000 IU on average) [START_REF] Bischoff-Ferrari | A pooled analysis of vitamin D requirements for fracture prevention[END_REF][START_REF] Hall | Vitamin D intake needed to maintain target serum 25-hydroxyvitamin D concentrations in participants with low sun exposure and dark skin pigmentation is substantially higher than current recommendations[END_REF][START_REF] Wacker | Sunlight and Vitamin D: A global perspective for health[END_REF], which is naturally observed only in outdoor workers during the summer [START_REF] Azizi | Serum levels of 25-hydroxy-vitamin D3 among sun-protected outdoor workers in Israel[END_REF]Bager--Lux and Heaney, 2002) or in tropical countries [START_REF] Luxwolda | Traditionally living populations in East Africa have a mean serum 25-hydroxyvitamin D concentration of 115 nmol/L[END_REF]. Indeed, food, even in a well-balanced diet, provides only about 100 IU/d and, if fortified, not more than 300--400 IU/d, which is in any case insufficient [START_REF] Holick | Vitamin D: extraskeletal health[END_REF]. Moreover, the organism does not durably store vitamin D, which progressively disappears within 6--8 weeks after a single solar or oral intake. The so--called 'toxic' area, potentially generating hypercalcemia, seems to be located largely above 150 ng/mL, corresponding to a daily intake far higher than 10,000 IU/d [START_REF] Hathcock | Risk assessment for vitamin D[END_REF].

These multiple considerations -physical, geographical, societal and physiological -explain why vitamin D insufficiency is widespread nowadays in the general population of temperate countries, and how this problem has progressively worsened during the last hundred years as a corollary of the development of the modern lifestyle which has progressively taken these populations away from direct exposure to sunshine (Pierrot--Deseilligny and Souberbielle, 2010).

Role of vitamin D in the risk for MS

Epidemiology of latitude and solar radiation effects on the MS risk

It has long been known that latitude influences MS risk, the prevalence of the disease being minimal at the equator and increasing with North or South latitude [START_REF] Simpson | Latitude is significantly associated with the prevalence of multiple sclerosis: a meta-analysis[END_REF]. Furthermore, MS prevalence may change after migrations that occurred during the second decade of life, with a beneficial effect for people who migrated from a high--latitude region (with a high MS prevalence) to a sunnier, lower--latitude region, with a low MS prevalence [START_REF] Handel | Environmental factors and their timing in adult-onset multiple sclerosis[END_REF][START_REF] Mcleod | Migration and multiple sclerosis in immigrants to Australia from United Kingdom and Ireland: a reassessment. I. Risk of MS by age at immigration[END_REF]. It has also been reported that the MS prevalence of a region is inversely correlated to the level of solar radiation of this region, always with a high level of significance, on a world scale [START_REF] Sloka | A quantitative analysis of suspected environmental causes of MS[END_REF], in the USA [START_REF] Beretich | Explaining multiple sclerosis prevalence by ultraviolet exposure: a geospatial analysis[END_REF], in France (Pierrot--Deseilligny and Souberbielle, 2010) and the United Kingdom [START_REF] Ramagopalan | Relationship of UV exposure to prevalence of multiple sclerosis in England[END_REF], thus explaining the well--known link between MS risk and latitude. It is also known that the time spent outdoors during childhood and adolescence has an impact on MS risk: people who practiced many outdoor activities during their youth, and thus received more UVB, have a significantly lower risk later for MS [START_REF] Bjørnevik | Sun exposure and multiple sclerosis risk in Norway and Italy: The EnvIMS study[END_REF]Laursen et al., 2016a;[START_REF] Van Der Mei | Past exposure to sun, skin phenotype, and risk of multiple sclerosis: case-control study[END_REF]. In spite of methodological debates (Fides et al., 2014), the month of birth does also appear to influence the MS risk: people born at the end of autumn, i.e. whose mothers could have had a relatively good exposure to sun in the major part of pregnancy, have a significantly reduced MS risk, whereas those born at the end of spring, whose mothers likely had much less exposure to sun in the major part of pregnancy, have an increased MS risk, these findings existing in both hemispheres, in which seasons are reversed [START_REF] Balbuena | Sunshine, sea, and season of birth: MS incidence in Wales[END_REF][START_REF] Dobson | The month of birth effect in multiple sclerosis: systematic review, meta-analysis and effect of latitude[END_REF]Rodriguez Cruz et al., 2016).

Specific UVB effects on the MS risk

From a general point of view, vitamin D is likely a major intermediary link between exposure to sun and the MS risk since UVB physiologically produce directly this vitamin in the organism. However, it may also be that UVB play a beneficial role in immunity through a specific immunomodulatory effect that could be independent of the vitamin D synthesis [START_REF] Breuer | Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity[END_REF]DelLuca and Plum, 2017;[START_REF] Hart | Exposure to UV wavelengths in sunlight suppresses immunity. To what extent is UV-induced vitamin D3 the mediator responsible?[END_REF] (see Figure 3). Although the respective immunological vitamin D and UVB effects from the skin and its different outlet pathways are not easy to differentiate and delineate in humans, the possibly specific UVB action may be exerted in parallel to the immunomodulatory effect of vitamin D [START_REF] Lucas | Ultraviolet radiation, vitamin D and multiple sclerosis[END_REF], which is currently far more widely documented (see below). In any case, these two mechanisms do not exclude each other and their possibly different specific actions have even been differentiated in a few epidemiological studies [START_REF] Bäärnhielm | Sunlight is associated with decreased MS risk: no interaction with HLA-DRB1*15[END_REF][START_REF] Lucas | Sun exposure and vitamin D are independent risk factors for CNS demyelination[END_REF]. , as yet much less well known, could be partly independent of the vitamin D synthesis: the UBV could also stimulate skin--derived tolerogenic dendritic cells, producing IL--10 (a favorable cytokine), resulting in stimulation of local Tregs and also, after moving through lymph draining, of Tregs located in the lymph nodes. Tregs finally join through the bloodstream the general immune system, in which they could also have a beneficial action. However, it should be noted that calcitriol may also be locally produced within the skin from 7--dehydrochlesterol or the circulating 25--OH--D in the keratinocytes and other skin cells, which have a complete enzymatic equipment to do this [START_REF] Lehmann | Role of the vitamin D3 pathway in healthy and diseased skin--facts, contradictions and hypotheses[END_REF]. Furthermore, calcitriol could be used in the skin [START_REF] Mccully | Skin metabolites define a new paradigm in the localization of skin tropic memory T cells[END_REF][START_REF] Schuessler | Skin is an autonomous organ in synthesis, two-step activation and degradation of vitamin D(3): CYP27 in epidermis completes the set of essential vitamin D(3)-hydroxylases[END_REF]) and captured, through lymph draining, by the lymph nodes [START_REF] Gorman | Topically applied 1,25dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes[END_REF], where it might also stimulate the local Tregs. Thus, this possible further pathway involving vitamin D within the skin itself complicates the study of a specific, independent role of UVB in immunity. 1,25--OH--2D, calcitriol; 25--OH--D, calcidiol; CNS, central nervous system; CYP27B1, enzyme 1α--hydroxylase; CYP2R1, enzyme 25--hydroxylase; Tregs, T regulator lymphocytes; UVB, ultraviolet B rays; V.3, vitamin D3.

Epidemiology of vitamin D effects on the MS risk

In fact, the direct epidemiological link between vitamin D and the MS risk has been suggested in studies in which the consumption of vitamin D supplements during youth [START_REF] Munger | Vitamin D intake and incidence of multiple sclerosis[END_REF], cod liver oil during adolescence [START_REF] Cortese | Timing of use of cod liver oil, a vitamin D source, and multiple sclerosis risk: The EnvIMS study[END_REF] or fatty fish (rich in vitamin D) [START_REF] Bäärnhielm | Fatty fish intake is associated with decreased occurrence of multiple sclerosis[END_REF] appeared to provide significant protective effects for the MS risk later in life. In another study, young American soldiers who had a spontaneous 25--OH--D serum level above 40 ng/mL had a 62% lower MS risk than those with less than 25 ng/mL [START_REF] Munger | Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis[END_REF]. More recently, analogous findings were observed in Swedish pregnant women in whom those with a 25--OH--D serum level above 30 ng/mL had a 61% lower MS risk than those with less than 30 ng/mL [START_REF] Salzer | Vitamin D as a protective factor in multiple sclerosis[END_REF]. Vitamin D status during

UVB$ 1,25%OH%2D) Skin) Lymph)) nodes) General)immunity) (including)CNS)) Cholecalciferol))))))))(V.D3)) 7%dehydro%) cholesterol) ?$ 25%OH%D) +) IL%10)producing) )dendriKc)cells) Tregs) Liver) Kidney) Lymph) draining) 1,25%OH%2D?) Skin)Tregs) Bloodstream) Bloodstream) Bloodstream) +) +) Tregs) CYP27B1) CYP2R1) KeraKnocytes)&)other)skin)cells) CYP27B1)))))))CYP2R1) 25%OH%D)))))))))))V.D3)
pregnancy also played a significant role in the MS risk of offspring in two other studies, in the USA [START_REF] Mirzaei | Gestational vitamin D and the risk of multiple sclerosis in offspring[END_REF] and in Finland [START_REF] Munger | Vitamin D status during pregnancy and risk of multiple sclerosis in offspring of women in the Finnish maternity cohort[END_REF]. Lastly, in a recent Danish study, the 25--OH--D serum level of newborns appeared to influence their MS risk 15--30 years later, with the observation that for each increase of 20 ng/mL in the serum level the MS risk was decreased by 60% [START_REF] Nielsen | Neonatal vitamin D status and risk of multiple sclerosis: A population-based case-control study[END_REF]. Accordingly, the results of these multiple studies strongly suggest that an insufficiency in exposure to sun and/or in vitamin D during the first part of life constitutes a major risk for MS, with as a corollary a consistent quantified reduction of about 60% of this risk resulting from a normal vitamin D serum level.

Contribution of genetic abnormalities of the vitamin D metabolism to the MS risk

Lastly, vitamin D insufficiency contributes to MS susceptibility as both an environmental and a genetic risk factor. Genetic studies in MS patients are numerous but we will focus here only on the major recent advances that have linked in a non--ambiguous way diverse specific genetic errors in the vitamin D metabolism to MS risk. Abnormalities of the gene of enzyme 1α--hydroxylase (CYP27B1) [START_REF] Sawcer | The International Multiple Sclerosis Genetics Consortium and Wellcome Trust Case Control Consortium 2 Genetic risk and primary role for cell-mediated immune mechanisms in multiple sclerosis[END_REF], which specifically controls calcitriol synthesis, significantly influenced the MS risk in studies testing the variants rs703842 [START_REF] Karaky | The multiple sclerosisassociated regulatory variant rs10877013 affects expression of CYP27B1 and VDR under inflammatory or vitamin D stimuli[END_REF][START_REF] Simon | Genetic predictors of 25hydroxyvitamin D levels and risk of multiple sclerosis[END_REF][START_REF] Sundqvist | Confirmation of association between multiple sclerosis and CYP27B1[END_REF] or rs10877013 [START_REF] Zhuang | Variants of CYP27B1 are associated with both multiple sclerosis and neuromyelitis optica patients in Han Chinese population[END_REF]. Genetic abnormalities of the CYP24A1 gene, which encodes the enzyme responsible for initiating calcitriol degradation, also influenced MS risk [START_REF] Ramasamy | Genetic evidence for a pathogenic role for the vitamin D3 metabolizing enzyme CYP24A1 in multiple sclerosis[END_REF]. Moreover, two vast studies recently aimed to link genetically low 25--OH--D serum levels to the MS risk: the first study, in Canada [START_REF] Mokry | Vitamin D and risk of multiple sclerosis: A mendelian randomization study[END_REF], and the second one, in both the USA and Sweden [START_REF] Rhead | Mendelian randomization shows a causal effect of low vitamin D on multiple sclerosis risk[END_REF], comprising further methodological criteria, involved thousands of MS patients and control subjects, who were tested using mendelian randomization techniques from several single nucleotide polymorphisms corresponding to different stages of vitamin D metabolism. In the results, very strong links existed between these vitamin D abnormalities and the risk for MS. Accordingly, such studies, in which there was methodologically very little room for reverse causality and confounding factors, state that a genetically induced hypovitaminosis D is a risk factor for MS. This finding has several important implications. First, even if such a relatively rare type of genetic hypovitaminosis D represents a very minor part of currently known forms of MS heritability, dominated by dysimmune abnormalities, it nevertheless provides evidence that hypovitaminosis D per se is a risk factor. Second, it may be inferred from such evidence that the most widespread vitamin D insufficiency, which is not genetically induced but results from unfavorable environmental conditions, is also an MS risk factor, as strongly suggested by the epidemiological studies reviewed above in which this environmental part of the risk seemed even to be a major component. Third, since these genetic studies specifically involve vitamin D metabolism, they also confirm that UVB do not play an exclusive beneficial role in immunity (see above).

Other risk factors for MS and interactions with vitamin D

Role of the human leukocyte antigen system

For MS there are in fact many other risk factors and protective factors, both genetic and environmental, which could cumulate or compensate one another. Among the other genetic risk factors for MS, the allele DRB1*1501 in the human leukocyte antigen (HLA) system accounts for 11% of heritability of the disease, whereas other alleles such as HLA--A*0201 could be protective [START_REF] Sawcer | The International Multiple Sclerosis Genetics Consortium and Wellcome Trust Case Control Consortium 2 Genetic risk and primary role for cell-mediated immune mechanisms in multiple sclerosis[END_REF]. There is a vitamin D responsive element (VDRE) zone in the promoter region of HLA DRB1*1501 gene which strongly suggests an involvement in mechanisms linked to vitamin D, even if these mechanisms are as yet little known [START_REF] Ramagopalan | Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D[END_REF]. Furthermore, in a recent pediatric study, the allele HLA DRB1*1501 was significantly associated with the 25--OH--D serum level and with the relapse rate [START_REF] Graves | Genetic predictors of relapse rate in pediatric MS[END_REF], whereas in another study this allele was related to low exposure to sun and to obesity [START_REF] Laursen | Genetic and environmental determinants of 25-hydroxyvitamin D levels in multiple sclerosis[END_REF]. Lastly HLA DRB1*1501 is over--represented in Caucasian populations and in women and it has been suggested that certain HLA alleles could have evolved during previous generations thanks to epigenetic mechanisms influenced by favorable or unfavorable environmental conditions, in which the vitamin D status could figure [START_REF] Handunnetthi | Multiple sclerosis, vitamin D, and HLA-DRB1*15[END_REF][START_REF] Koch | Epigenetic changes in patients with multiple sclerosis[END_REF] (Figure 4). P ( r ( o ( t ( e ( c ( t ( i ( o ( n ( ( ( ( ( W ( ( ( o ( ( ( r ( ( ( s ( ( ( e ( ( n ( ( ( i ( ( ( n ( ( ( g

Role of viruses and infections

Among the other environmental risk factors, Epstein--Barr virus (EBV) infection might play a major role [START_REF] Ascherio | Epidemiology of multiple sclerosis: from risk factors to prevention-An update[END_REF][START_REF] Olsson | Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis[END_REF][START_REF] Salzer | Epstein-Barr virus antibodies and vitamin D in prospective multiple sclerosis biobank samples[END_REF][START_REF] Waubant | Environmental and genetic factors in pediatric inflammatory demyelinating diseases[END_REF] (see more details in Figure 4). In MS patients, genetic arguments for interactions between EBV infection and vitamin D have been reported [START_REF] Ricigliano | EBNA2 binds to genomic intervals associated with multiple sclerosis and overlaps with vitamin D receptor occupancy[END_REF], with, furthermore, associations existing between HLA DRB1*1501, the 25--OH--D serum level and the serum level of antibodies against EBV antigen nuclear 1 (anti--EBNA1) [START_REF] Wergeland | Vitamin D, HLA-DRB1 and Epstein-Barr virus antibody levels in a prospective cohort of multiple sclerosis patients[END_REF], or between these last two factors [START_REF] Salzer | Epstein-Barr virus antibodies and vitamin D in prospective multiple sclerosis biobank samples[END_REF]. Moreover, vitamin D supplementation appears to decrease the anti--EBNA1 serum level [START_REF] Røsjø | Effect of high-dose vitamin D3 supplementation on antibody responses against Epstein-Barr virus in relapsing-remitting multiple sclerosis[END_REF], which might be a beneficial effect. Infections with human herpes virus of type 6 or human endogenous retrovirus could also be MS risk factors and/or factors worsening the disease [START_REF] Tao | Association between human herpesvirus & human endogenous retrovirus and MS onset & progression[END_REF]. By contrast, the accumulation of numerous common infections and/or infestations with parasites during childhood appears to be protective from the risk of autoimmune diseases occurring later, including MS (Correale and Gaetan, 2015;[START_REF] Gustavsen | Environmental exposures and the risk of multiple sclerosis investigated in a Norwegian case-control study[END_REF]: this constitutes the 'hygiene' hypothesis, in which a particular development of the protective Tregs could be favorable. Furthermore, links between the mechanisms involved in the hygiene hypothesis and vitamin D may exist, through the vitamin D receptor (VDR) and gut microbiome [START_REF] Clark | Role of vitamin D in the hygiene hypothesis: the interplay between vitamin D, vitamin D receptors, gut microbiota, and immune response[END_REF], the importance of which is beginning to be discovered for the pathogenesis of autoimmune diseases, including MS [START_REF] Budhram | Breaking down the gut microbiome composition in multiple sclerosis[END_REF]Mielcraz and Kasper, 2015;[START_REF] Tremlett | Associations between the gut microbiota and host immune markers in pediatric multiple sclerosis and controls[END_REF]. Serologic positivity to cytomegalovirus could also be protective [START_REF] Makhani | Viral exposures and MS outcome in a prospective cohort of children with acquired demyelination[END_REF][START_REF] Sundqvist | Cytomegalovirus seropositivity is negatively associated with multiple sclerosis[END_REF][START_REF] Waubant | Common viruses associated with lower pediatric multiple sclerosis risk[END_REF] due to complex immunological competition with EBV [START_REF] Chidrawar | Cytomegalovirus-seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals[END_REF], in which the 25--OH--D serum levels may also be associated [START_REF] Mowry | Vitamin D status and antibody levels to common viruses in pediatric-onset multiple sclerosis[END_REF].

Role of other risk and protective factors

Obesity has recently been identified as another risk factor for MS, in particular if it exists in childhood or adolescence [START_REF] Gianfrancesco | Evidence for a causal relationship between low vitamin D, high BMI and pediatric-onset MS[END_REF][START_REF] Mokry | Obesity and multiple sclerosis: a Mendelian randomization study[END_REF][START_REF] Munger | Childhood body mass index and multiple sclerosis risk: a long-term cohort study[END_REF][START_REF] Wesnes | Body size and the risk of multiple sclerosis in Norway and Italy: The EnvIMS study[END_REF].

Although obesity and vitamin D are partly linked since this vitamin is liposoluble and sequestered in fatty tissues and consequently less available in the blood [START_REF] Earthman | The link between obesity and low circulating 25hydroxyvitamin D concentrations: considerations and implications[END_REF], these two factors could also be partly independent in the MS risk [START_REF] Gianfrancesco | Evidence for a causal relationship between low vitamin D, high BMI and pediatric-onset MS[END_REF]. Sexual hormones, i.e. testosterone in men [START_REF] Bove | Low testosterone is associated with disability in men with multiple sclerosis[END_REF] and estrogens and progesterone in women [START_REF] Patas | Pregnancy and multiple sclerosis: feto-maternal immune cross talk and its implications for disease activity[END_REF], could also be protective, with possible links between vitamin D, estrogens and progesterone (see below). Lastly, there are other environmental risk factors for MS, such as cigarette smoking [START_REF] Ascherio | Epidemiology of multiple sclerosis: from risk factors to prevention-An update[END_REF][START_REF] Olsson | Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis[END_REF], even when intoxication is passive in children, whereas the α-linolenic acid [START_REF] Bjørnevik | Polyunsaturated fatty acids and the risk of multiple sclerosis[END_REF], nicotine (if not smoked), coffee (at high doses), alcohol (at moderate doses) [START_REF] Olsson | Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis[END_REF] could rather be protective. All these MS risk and protective factors, the list of which is likely not yet exhaustive, appear to be more or less independent of each other and could cumulate or compensate [START_REF] Ascherio | The initiation and prevention of multiple sclerosis[END_REF][START_REF] Ramien | Hypovitaminosis-D and EBV: no interdependence between two MS risk factors in a healthy young UK autumn cohort[END_REF].

General interactions between protective and risk factors in MS

Thus, vitamin D insufficiency is only one of many risk factors for MS. It is generally accepted that MS results from the association of several genetic and environmental risk factors and it may be assumed that multiple different deleterious combinations between these diverse potential risk factors could lead to its triggering. Likewise, vitamin D sufficiency appears to be one of many protective factors, including diverse genetic, infectious and pharmacological protective factors. Lastly, as emphasized above, all these MS risk and protective factors could interact at different times between conception and MS triggering, which generally occurs during adulthood. This leads to the relatively recent concept of a continuous modulation of the global MS risk taking place throughout the first part of life (Figure 4). However, it should be noted that determining events appear to happen more particularly between the mother's pregnancy and the end of adolescence. This period could correspond to that of the maturation of the immune system and thymus [START_REF] Tamblyn | Immunological role of vitamin D at the maternal-fetal interface[END_REF][START_REF] Tulic | Changes in thymic regulatory T-cell maturation from birth to puberty: differences in atopic children[END_REF]Vijayendra Chari et al., 2015), during which favorable or unfavorable immunological transformations could be durably constituted, which is likely related to the dysimmune nature of the disease and is reviewed in the next section.

Mechanisms of action of vitamin D in MS

Immunological mechanisms

There are potentially multiple mechanisms by which vitamin D influences MS, before or after the disease is triggered. The general immunological effects of this vitamin are currently the best known and are likely involved at the inflammatory period of the disease, but other, central neurological mechanisms could also exert an effect in parallel. MS is a general dysimmune disease, which exclusively involves the central nervous system (CNS). Numerous dysimmune mechanisms have been discussed in MS [START_REF] Yadav | Advances in the immunopathogenesis of multiple sclerosis[END_REF] and it should be noted that a majority of the genes involved in the disease are related to such mechanisms [START_REF] Sawcer | The International Multiple Sclerosis Genetics Consortium and Wellcome Trust Case Control Consortium 2 Genetic risk and primary role for cell-mediated immune mechanisms in multiple sclerosis[END_REF]. The general immunomodulatory role of vitamin D has been abundantly documented in recent years, involving the different categories of T and B lymphocytes and several cytokines [START_REF] Allen | A pilot study of immunological effects of high-dose vitamin D in healthy volunteers[END_REF][START_REF] Cantorna | Vitamin D and 1,25(OH)2D regulation of T cells[END_REF][START_REF] Danner | Vitamin D3 suppresses class II invariant chain peptide expression on activated B-lymphocytes: A plausible mechanism for downregulation of acute inflammatory conditions[END_REF][START_REF] Fawaz | Comparative effect of 25(OH)D3 and 1,25(OH)2D3 on Th17 cell differentiation[END_REF][START_REF] Rolf | Vitamin D effects on B cell function in autoimmunity[END_REF][START_REF] Sommer | Vitamin D regulates cytokine patterns secreted by dendritic cells to promote differentiation of IL-22-producing T cells[END_REF][START_REF] Von Essen | Vitamin D controls T cell antigen receptor signaling and activation of human T cells[END_REF]. It is likely through these diverse immunomodulatory mechanisms that vitamin D intervenes in MS, as recently confirmed in numerous immunological studies performed in MS patients [START_REF] Correale | Immunomodulatory aspects of vitamin D in multiple sclerosis[END_REF][START_REF] Grau-Lopez | Regulatory role of vitamin D in T-cell reactivity against myelin peptides in relapsing-remitting multiple sclerosis patients[END_REF][START_REF] Lysandropoulos | Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and in healthy controls[END_REF][START_REF] Smolders | Vitamin D status is positively correlated with regulatory T cell function in patients with multiple sclerosis[END_REF][START_REF] Smolders | Regulatory T cell function correlates with serum 25-hydroxyvitamin D, but not with 1,25 dihydroxyvitamin D, parathyroid hormone and calcium levels in patients with relapsing remitting multiple sclerosis[END_REF] that cannot be detailed here but in some of which vitamin D supplementation resulted in multiple beneficial immunological effects: in particular, with stimulation of Tregs (Murris et al., 2016c) and of the favorable IL--10 cytokine [START_REF] Ashtari | Short-term effect of high-dose vitamin D on the level of interleukin 10 in patients with multiple sclerosis: a randomized, double-blind, placebo-controlled clinical trial[END_REF][START_REF] Mosayebi | Therapeutic effect of vitamin D3 in multiple sclerosis patients[END_REF], diminution of the pro--inflammatory Th17 lymphocytes [START_REF] Da Costa | Vitamin D modulates different IL-17-secreting T cell subsets in multiple sclerosis patients[END_REF] and the deleterious cytokine IL--17 [START_REF] Sotirchos | Safety and immunologic effects of high-vs low-dose cholecalciferol in multiple sclerosis[END_REF], and attenuation of B--cell immunoreactivity [START_REF] Haas | Hypovitaminosis D upscales B-cell immunoreactivity in multiple sclerosis[END_REF]. Although these studies usually comprised small numbers of patients, with a follow--up of less than 6 months and without analysis of clinical parameters, they were nevertheless usually controlled and thus have allowed it to be stated unequivocally that vitamin D has positive immunological effects in MS.

The same immunomodulatory mechanisms also appear to be involved in experimental autoimmune encephalopathy (EAE) [START_REF] Cantorna | Vitamin D and 1,25(OH)2D regulation of T cells[END_REF], which is the best experimental model of MS. The numerous studies on vitamin D in EAE published during the past 25 years cannot be detailed here but they have generally shown a preventive and curative action of this vitamin, more particularly in female mice [START_REF] Cantorna | Vitamin D and 1,25(OH)2D regulation of T cells[END_REF]. Moreover, it was experimentally reported that vitamin D and estrogens act synergistically in stimulating different favorable immunomodulatory effects [START_REF] Spanier | Vitamin D and estrogen synergy in Vdrexpressing CD4(+) T cells is essential to induce Helios(+)FoxP3(+) T cells and prevent autoimmune demyelinating disease[END_REF]. An analogous synergy between progesterone and vitamin D could also stimulate immunomodulation [START_REF] Thangamani | Cutting edge: progesterone directly upregulates vitamin d receptor gene expression for efficient regulation of T cells by calcitriol[END_REF]. In humans, it is known that estrogens (Nadkami and McArthur, 2013) and progesterone [START_REF] Tan | Hormonal modulation of the immune system -A spotlight on the role of progestogens[END_REF] have specific immunomodulatory actions and that pregnancy has rather a protective effect in terms of risk for MS development, most likely due to the physiologically elevated hormonal levels reinforcing general immunomodulation [START_REF] Nair | Immune-endocrine crosstalk during pregnancy[END_REF]. In the context of the widespread hypovitaminosis D in countries with little sunshine, a phenomenon that has increased over the past tens of years due to changing lifestyles [START_REF] Looker | Serum 25-hydroxyvitamin D status of the US population: 1988-1994 compared with 2000-2004[END_REF], a decrease in immunomodulatory synergies between vitamin D and female hormones, without a similar potentiation existing between vitamin D and testosterone [START_REF] Bove | Low testosterone is associated with disability in men with multiple sclerosis[END_REF][START_REF] Zhao | Serum vitamin D and sex hormones levels in men and women: The Multi-Ethnic Study of Atherosclerosis (MESA)[END_REF], could have had a more deleterious impact in women than in men and thus might have contributed to the well--known and increasing female predominance in MS [START_REF] Spanier | Vitamin D and estrogen synergy in Vdrexpressing CD4(+) T cells is essential to induce Helios(+)FoxP3(+) T cells and prevent autoimmune demyelinating disease[END_REF]. Furthermore, such immunomodulatory synergies could account for a more beneficial effect of vitamin D supplementation observed in women [START_REF] Kragt | Higher levels of 25hydroxyvitamin D are associated with a lower incidence of multiple sclerosis only in women[END_REF].

Other possible mechanisms

Vitamin D, among its diverse extra--skeletal effects, likely has other non--immunomodulatory actions. In particular, this vitamin enters the different types of cells in the CNS -neurons, astrocytes, microglia and oligodendrocytes, which all have VDR - and it could exert at the central neurological level other modes of action, e.g. neuroprotector, neurotrophic, remyelinating, etc. [START_REF] Smolders | Vitamin D in the healthy and inflamed central nervous system: access and function[END_REF]. Some experimental results have already been reported in this field (de la [START_REF] De La Fuente | Vitamin D receptor-retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation[END_REF][START_REF] Shirazi | 1,25-Dihydroxyvitamin D3 enhances neural stem cell proliferation and oligodendrocyte differentiation[END_REF], and a link between axonal degeneration and vitamin D seems to exist in MS patients [START_REF] Sandberg | Vitamin D and axonal injury in multiple sclerosis[END_REF]. However, these other possible central mechanisms remain largely to be explored in human physiology and pathology, i.e. in MS as in other central neurological degenerative diseases.

Clinical data on vitamin D in MS

Practical difficulties of clinical studies using vitamin D supplementation in MS patients

Although there is currently very little doubt that vitamin D insufficiency constitutes one of the risk factors for MS before the triggering of the disease, important questions and doubts remain regarding its action once the disease has started. Many studies using vitamin D supplementation have been performed but they were insufficiently powered, most often without a long--lasting follow--up and/or with diverse methodological bias, to be able to produce really conclusive results. A few other, longer, phase 2 or 3 randomized clinical trials (RCTs) are still ongoing but they may also prove to be under--powered due to their already known baseline characteristics and because of the diverse, quasi insurmountable practical difficulties that exist nowadays with conducting and/or completing studies of this type using vitamin D. Indeed, since patients being enrolled in such RCTs testing vitamin D supplementation will necessarily be aware before they sign their initial consent form that they have an insufficiency of a 'vitamin', they often prefer not to participate in the study and, if they do participate, they quit it more or less rapidly or even change their own behavior in regard to vitamin D intake -going out in the sun more frequently and/or using more fortified food and diverse vitamin D supplements than previously -ultimately downsizing the studies or introducing major methodological bias. Only the already published and most reliable clinical results are discussed here.

Vitamin D serum levels in MS patients

Low 25--OH--D serum levels (around 20 ng/mL) are usually observed in MS patients as early as the beginning of the disease, i.e. at the stage of the clinically isolated syndrome (CIS) or of the first relapses [START_REF] Ascherio | Vitamin D as an early predictor of multiple sclerosis activity and progression[END_REF][START_REF] Behrens | Low 25-hydroxyvitamin D, but not the bioavailable fraction of 25-hydroxyvitamin D, is a risk factor for multiple sclerosis[END_REF][START_REF] Kuhle | Conversion from clinically isolated syndrome to multiple sclerosis: A large multicentre study[END_REF][START_REF] Martinelli | Vitamin D levels and risk of multiple sclerosis in patients with clinically isolated syndromes[END_REF][START_REF] Pierrot-Deseilligny | Is hypovitaminosis D one of the environmental risk factors for multiple sclerosis?[END_REF]. However, spontaneously normal serum levels (above 30 ng/mL) may also be observed in rare MS patients since the disease is multifactorial. The vitamin D insufficiency in MS patients, compared to matched control subjects, could be partly explained by abnormalities in the vitamin metabolism existing in patients [START_REF] Bhargava | Multiple sclerosis patients have a diminished serologic response to vitamin D supplementation compared to healthy controls[END_REF]. Later in the disease, vitamin D serum levels have a clear tendency to decrease even more, but two associated factors may then favor such a decrease (Pierrot--Deseilligny and Souberbielle, 2013): first, the Uthoff phenomenon, where patients tend to avoid direct sunlight because heat aggravates their symptoms, then disability, with fewer outdoor activities and, consequently, less exposure to UVB. If these two factors can indeed contribute to a reverse causality, in which the disease itself widens the vitamin D deficit, it should not be forgotten that a marked vitamin D insufficiency often already exists as early as the beginning of the disease in young and non--disabled patients in whom such factors cannot yet have played any significant role. 1. Association studies between relapses and 25--OH--D serum levels in MS patients. These five studies were comparable in terms of their methodologies, the statistic models used (linear regression, with multivariate analyses), cohort size and the duration of follow--ups. Note that the first study was pediatric and that the third and fifth studies were association--intervention studies with vitamin D supplementation. In these five studies, statistical models predicted a reduction of 50% to 70% of relapses, depending on the study, for an increase of 20 ng/mg in the 25--OH--D serum level (right column). For more details, see text. CIS, clinically isolated syndrome; IMT, immunomodulatory treatment; MS, multiple sclerosis; RR, relapsing--remitting form of MS. 6.

Association studies comparing vitamin D serum levels to other variables in MS patients

A number of association studies linking vitamin D insufficiency to diverse parameters of the disease have been reported. An exhaustive review of such studies cannot be done here. Some association studies using MRI and testing 25--OH--D serum levels in MS patients have shown that relatively high serum levels were associated with a decrease in the number of new T2 and gadolinium--enhancing lesions [START_REF] Ascherio | Vitamin D as an early predictor of multiple sclerosis activity and progression[END_REF][START_REF] Mowry | Vitamin D status predicts new brain magnetic resonance imaging activity in multiple sclerosis[END_REF], thus suggesting a beneficial action of this vitamin in the inflammatory component of the disease. Furthermore, five association or association--intervention studies reported between 2010 and 2016 [START_REF] Ascherio | Vitamin D as an early predictor of multiple sclerosis activity and progression[END_REF][START_REF] Mowry | Vitamin D status is associated with relapse rate in pediatric-onset MS[END_REF]Laursen et al., 2016b;[START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF][START_REF] Simpson | Higher 25-hydroxyvitamin D is associated with lower relapse risk in MS[END_REF] and analyzing both 25--OH--D serum levels and clinical relapses in MS patients seem to be of particular interest: their main characteristics are given in Table 1. These five studies were methodologically comparable, and their analogous robust statistical models (comprising multivariate analyses) predicted a marked reduction of relapses of 50%--70%, according to the study, for each increase of 20 ng/mL in the 25--OH--D serum level. It may already be considered remarkable that such close figures were observed in five different studies, made on three continents, under diverse climates, in adolescents or adults, with a relapsing--remitting (RR) form of MS or with a CIS, but all at the inflammatory stage of the disease, with or without an associated MS preventive treatment, which could be a first--line or second--line medication, and lastly with or without associated vitamin D supplementation. The fact that so many various contexts led to quasi--similar figures suggests that such quantification may finally represent a good reflection of the specific 'vitamin D effect'. One can be legitimately astonished by the very extent of this effect, which also necessarily has an upper limit. However, if one considers the curves produced by the statistical model in one of the association--intervention studies of this series [START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF], it is first clear that the marked vitamin D effect on relapses was mainly between 26 and 44 ng/mL, i.e. when the initial insufficiency in vitamin D of this cohort was corrected by the supplementation to reach a serum level located within the range generally recommended nowadays (Figure 5). Then, it may be observed that the statistical model generated a clear, unexpected inflexion of the curves resulting in a 'plateau' effect beyond 44 ng/mg, without a further decrease in relapses at higher vitamin D serum levels. For the moment, such a plateau effect has only been observed in this association-intervention study [START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF] since this is the sole study to have used relatively substantial doses of vitamin D (3000 IU/d) resulting in sufficiently high serum levels to reach this plateau and then to confirm it (with serum tests performed every 6 months) throughout a long--lasting follow--up of several years. Furthermore, this study suggests that it is pointless to prescribe very high doses of vitamin D resulting in supra--physiological serum levels (above 60 ng/mL), but that it appears nevertheless useful to reach at least the 40--50 ng/mL zone of serum levels (and not simply 30 ng/mL) to make full use of the vitamin D effect. It should also be noted that most patients of these association--intervention studies likely benefited from this marked effect. Finally, in the durable and maybe definitive absence of major RCTs testing vitamin D supplementation (see above), these different association studies have already given interesting information to decide to supplement MS patients with vitamin D and to choose a supplementation dose at a relatively moderate level (between 2000 and 4000 IU/d).

Figure 5. Example of the effect of a continuous moderate supplementation of vitamin D on MS relapses.

In this study [START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF], 156 consecutive patients with a relapsing--remitting form of MS (with mean EDSS = 2.01) were given 100 000 IU of vitamin D monthly (i.e. with a mean of 3000 IU/d) in addition to an injectable first--line IMT and were followed during 2.5 years on average. By contrast, results have been somewhat contradictory for the degenerative, progressive stage of MS, with, in particular, non--significant results in most recent, relatively well--powered studies (Muris et al., 2016b;[START_REF] University Of California | Long-term evolution of multiple sclerosis disability in the treatment era[END_REF]. Even if an association between vitamin D serum level and disability may at times be found [START_REF] Hempel | A systematic review of modifiable risk factors in the progression of multiple sclerosis[END_REF], this particular symptom, often resulting in fewer outdoor activities in disabled patients, could contribute to reverse causality (see above), which makes the interpretation of results more difficult. Furthermore, the prevailing impression in observational clinical practice is that vitamin D is not much more efficient than the other immunomodulatory treatments when the progression of disability is already clinically obvious, i.e. in the secondary or primary progressive forms of the disease. However, it cannot be ruled out that relatively normal vitamin D serum levels existing at an early stage of the disease may decrease the progression of brain atrophy (Mowry et al., 2016) or delay the occurrence of the secondary progressive phase (Murris et al., 2016a), as previously observed with other immunomodulators.

Concluding remarks on clinical studies analyzing the vitamin D effect in MS patients

In conclusion, from all these studies currently available in MS patients, it appears highly likely that vitamin D markedly decreases the inflammatory component of the disease. Indeed, it can no longer be reasonably argued nowadays that all the positive effects of vitamin D observed on this inflammatory component in the numerous and diverse (clinical, radiological, immunological) reported MS studies, often comprising multivariate analyses, were systematically due to reverse causality and/or to confounding factors, as has at times been suggested [START_REF] Autier | Vitamin D status and ill health: a systematic review[END_REF]. Even if the inflammation mechanism supposed to 'consume' vitamin D in certain systemic diseases [START_REF] Ghashut | The effect of the systemic inflammatory response on plasma vitamin 25 (OH) D concentrations adjusted for albumin[END_REF]) may be theoretically extended to MS inflammation and then be unlikely incriminated as a factor of reverse causality for all MS association studies (whatever their diverse nature), such a mechanism could not in any case account: 1) for the results of genetic studies specifically linking vitamin D metabolism to MS risk (see above); 2) for the findings of numerous epidemiological studies related to latitude or performed throughout the first part of life, i.e. often a long time before the onset of the disease and prior to any inflammatory stage; 3) for the beneficial immunodulatory effects of vitamin D supplementation observed in immunological controlled studies performed in MS patients. Consequently, the main question is no longer to evidence an anti--inflammatory vitamin D effect in MS but rather to calibrate it with greater precision, with however already an approximate answer provided by the association studies reviewed above. Although major RCTs are still required for scientific validation, they are practically more and more difficult to conduct on a topic such as vitamin D and that is why a systematic vitamin D supplementation of MS patients has already been recommended in clinical practice for several years by different specialized teams around the world [START_REF] Ascherio | Vitamin D as an early predictor of multiple sclerosis activity and progression[END_REF][START_REF] Holick | Vitamin D: extraskeletal health[END_REF][START_REF] Holmøy | Vitamin D in multiple sclerosis: implications for assessment and treatment[END_REF][START_REF] Jahromi | Iranian consensus on use of vitamin D in patients with multiple sclerosis[END_REF][START_REF] Jelinek | Latitude, sun exposure and vitamin D supplementation: associations with quality of life and disease outcomes in a large international cohort of people with multiple sclerosis[END_REF][START_REF] Pierrot-Deseilligny | Clinical implications of a possible role of vitamin D in multiple sclerosis[END_REF][START_REF] Salzer | Vitamin D and multiple sclerosis: where do we go from here? Expert[END_REF].

Clinical practice of vitamin D supplementation in MS

From a pragmatic and practical point of view, while waiting for new scientific information, a vitamin D3 supplementation using moderate oral doses (between 2000 and 4000 IU/d) can already be undertaken in all types of MS patients, including pregnant women [START_REF] Wagner | Vitamin D administration during pregnancy as prevention for pregnancy, neonatal and postnatal complications[END_REF]. Such a supplementation dose has several advantages: 1) it corrects the vitamin D insufficiency existing in the great majority of MS patients, with 25--OH--D serum levels thus increasing up to the currently recommended zone (30--60 ng/mL) in most of them [START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF]; 2) furthermore, in patients in marked, often deep vitamin D insufficiency, a supplementation should be considered obligatory from a general medical point of view, to prevent osteoporosis, attenuate infections, etc. (Pierrot--Deseilligny and [START_REF] Pierrot-Deseilligny | Contribution of vitamin D insufficiency to the pathogenesis of multiple sclerosis[END_REF]; 3) a moderate supplementation dose is harmless, in particular without the risk of hypercalcemia or any other serious significant side effect [START_REF] Hathcock | Risk assessment for vitamin D[END_REF][START_REF] Pierrot-Deseilligny | Relationship between 25-OH-D serum level and relapse rate in multiple sclerosis patients before and after vitamin D supplementation[END_REF]; 4) this supplementation might also have beneficial effects on the inflammatory component of the disease. In temperate countries, the supplementation should never be stopped since there is no durable storage of this vitamin in the organism. A blood test is useful before supplementation to check the initial calcemia and 25--OH--D serum level, which may be extremely low (below 10 ng/mL) as early as the beginning of the disease, but this is often in particular contexts: people systematically avoiding sunshine and/or with dark skin (absorbing less UVB) and/or with skin--covering clothes and/or with obesity. In these cases, a loading dose may be useful at the beginning of supplementation to reach more rapidly an optimal vitamin D status. A control test, around six months after the beginning of supplementation, should be used to check that the patient has been compliant, the optimal 25--OH--D serum zone (40--60 ng/mL) has been reached and there is no hypercalcemia. Then, even if the first control has been satisfactory, tests should be repeated yearly to check that compliance has been maintained. Lastly, it also appears useful to encourage the avoidance of other risk factors, by recommending that cigarette smokers stop smoking and obese patients lose weight, since risk factors for MS are generally also worsening factors after the start of the disease. These various recommendations may be extended to MS patients' relatives [START_REF] Sundström | Vitamin D and multiple sclerosis-from epidemiology to prevention[END_REF] since they have a genetically increased risk for the disease.

  almost)all)of)Europe)and)a)large)part) )of)North)America)and)the)former)Soviet)Union))

Figure 2 .

 2 Figure 2. Schematic representation of vitamin D metabolism.Vitamin D3 is mainly synthesized at the skin level thanks to UVB and, after a double hydroxylation in the liver and the kidney, is transformed into calcitriol, i.e. the active metabolite. Calcitriol can also be directly synthesized in the cell cytoplasm from the circulating 25--OH--D and links at this level to a specific receptor (VDR), which is present in numerous cells of the organism, including immune and nervous cells. Then, this complex penetrates into the nucleus, forms a heterodimer with the receptor RXR and this heterodimer finally binds to VDRE, which constitutes a specific sequence of DNA within the promoter region of the target genes. The whole regulates (by activation or suppression) gene transcription and expression and, ultimately, protein synthesis in approximately 5-10% of the genome. Thus, for immune cells, which are particularly involved in MS pathogenesis, the synthesis of the diverse favorable and unfavorable cytokines related to inflammatory mechanisms could be either increased or decreased depending on the genes and the vitamin D status at the time (see below in the text). Note that the allele HLA DRB1*1501, which is particularly deleterious in terms of MS risk and taken here as example, possesses a VDRE area. C, calcitriol; CYP27B, enzyme 1α--hydroxylase; CYP2R, enzyme 25-hydroxylase; HLA, human leukocyte antigen; PTH, parathyroid hormone; RXR, retinoid X receptor; UVB, ultraviolet B rays; VDR, vitamin D receptor; VDRE, vitamin D responsive element.

Figure 3 .

 3 Figure 3. The two main pathways through which UVB may influence immunity. The first, classical pathway (right) starts in the skin under the influence of UVB with the transformation of 7-dehydrocholesterol into cholecalciferol (vitamin D3), which then passes through the bloodstream into the liver and the kidneys and results in calcitriol (the active metabolite) stimulating Tregs, having a beneficial immunomodulatory effect on the general immunity (see below in the text). The second pathway(left), as yet much less well known, could be partly independent of the vitamin D synthesis: the UBV could also stimulate skin--derived tolerogenic dendritic cells, producing IL--10 (a favorable cytokine), resulting in stimulation of local Tregs and also, after moving through lymph draining, of Tregs located in the lymph nodes. Tregs finally join through the bloodstream the general immune system, in which they could also have a beneficial action. However, it should be noted that calcitriol may also be locally produced within the skin from 7--dehydrochlesterol or the circulating 25--OH--D in the keratinocytes and other skin cells, which have a complete enzymatic equipment to do this[START_REF] Lehmann | Role of the vitamin D3 pathway in healthy and diseased skin--facts, contradictions and hypotheses[END_REF]. Furthermore, calcitriol could be used in the skin[START_REF] Mccully | Skin metabolites define a new paradigm in the localization of skin tropic memory T cells[END_REF][START_REF] Schuessler | Skin is an autonomous organ in synthesis, two-step activation and degradation of vitamin D(3): CYP27 in epidermis completes the set of essential vitamin D(3)-hydroxylases[END_REF]) and captured, through lymph draining, by the lymph nodes[START_REF] Gorman | Topically applied 1,25dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes[END_REF], where it might also stimulate the local Tregs. Thus, this possible further pathway involving vitamin D within the skin itself complicates the study of a specific, independent role of UVB in immunity. 1,25--OH--2D, calcitriol; 25--OH--D, calcidiol; CNS, central nervous system; CYP27B1, enzyme 1α--hydroxylase; CYP2R1, enzyme 25--hydroxylase; Tregs, T regulator lymphocytes;UVB, ultraviolet B rays; V.3, vitamin D3.

Figure 4 .

 4 Figure 4. Schematic continuous modulation of protective and risk factors in MS. For more details see text. A schematic curve of the global MS risk, fluctuating with time, is shown in the middle. Hypothetical risk factors for MS (bottom) and protective factors for the disease (top) are both genetic and environmental.The numerous interactions between these different factors may occur (from right to left) in adulthood, in adolescence, in childhood, during the mother's pregnancy and even during previous generations, through epigenetic mechanisms that could shape protective or deleterious alleles in the HLA system depending on favorable or unfavorable environmental conditions. The evolution of the MS risk (left to right) starts at conception, where protective and deleterious genetic factors are transmitted, in particular HLA DRB1*1501 and genes involved in abnormalities of vitamin D metabolism (bottom). Then, a birthday in spring, a high vitamin D serum level during pregnancy, in the newborn or later during the first part of life, but also outside activities during youth (with UVB and/or vitamin D) and migration to a sunny country in adolescence would all be protective elements, whereas the reverse conditions could increase the MS risk. Infection with EBV, responsible for infectious mononucleosis, appears to be a major and possibly founding risk factor for MS, in particular if primo--infection occurs late, after childhood, and/or if it leaves a high level of anti--EBNA1, whereas a negative serologic status to EBV could be protective (5% of population). The viruses HHV6 and HERVs could also have deleterious effects in terms of MS risk, maybe more particularly during the disease course. By contrast, numerous common infections or parasitic infestations during childhood could be protective, according to the hygiene hypothesis, as would also be positive serology for CMV. Sexual hormones, i.e. testosterone in men and estrogens and progesterone in women, could be protective. Obesity, in particular in adolescence, and cigarette smoking are other risk factors for MS, whereas coffee (at high dose), ALA and other substances could be protective. All these protective and risk factors interact between conception and the triggering of MS, usually occurring in young adulthood, leading to the concept of a continuous modulation of the global MS risk throughout the first part of life. However, many of these interactions intervene between pregnancy and the end of adolescence, in a period corresponding to that of the maturation of the immune system and thymus, which could be related to the dysimmune nature of the disease. Certain combinations of deleterious risk factors could finally trigger the disease, whereas diverse protective effects could prevent it durably. If the disease starts (right), some risk factors could continue to have deleterious effects by worsening its course and favoring relapses, whereas certain protective factors could contribute to remissions. ALA, α--linoleic acid; anti--EBNA1, antibodies against EBV antigen nuclear 1; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HERV, human endogenous retrovirus; HHV6, human herpes virus of type 6; HLA, human leukocyte antigen; M, men;MS, multiple sclerosis; Nl, normal; UVB, ultraviolet B rays; V.D, vitamin D; W, women.

  #started#vitamin#D# 4.2#±#2.7#years#(at#least#6# months)#aAer#IMT#(n#=#80)# Pa&ents#who#started# vitamin#D#at#the#same# #&me#as#IMT#(n#=#76

Reference Cohort MS form Number of Age Follow--up Associated Associated Decrease in relapses location patients (years) (mean, years) treatment vitamin D for each increase of 20 ng/mL in 25--OH--D Mowry et al.,

		USA	RR--CIS	n = 110	15 ± 3	1.7	No	No	-68%
	2010
	Simpson et al.,	Australia	RR	n = 145	44 ± 10	3	IMT	No	-50%
	2010
	Pierrot--Deseilligny Paris, France	RR	n = 156	39 ± 10	2.5	IMT	Yes	-68%
	et al., 2012
	Ascherio et al.,	Multicentric	CIS	n = 336	35 ± 9	2	IMT	No	-57%
	2014
	Laursen et al.,	Denmark	RR	n = 134	41 ± 9	1	Natalizumab	Yes	-70%
	2016b

Table

Relapse'IRR' 10'' ng/mL' 60'' ng/mL' 44'' ng/mL' 26' 'ng/mL' 20' 'ng/mL' 11#

	Most%pa(ents%before% vitamin%D%supplementa(on%	Most%pa(ents%under%vitamin%D% supplementa(on%(3000%IU/day)%