**4. Epidemiological evidence that VitD is relevant in MS**

There are few studies concerning the impact of VitD on B cells. *In vitro* assays indicated that the active form of VitD inhibited the production of immunoglobulin E and increased IL‐10 production by B cells [46,47]. Similarly to the effect over DCs, active VitD also downregulated the expression of co‐stimulatory molecules at the surface of human B cells. Drozdenko et al. [48] demonstrated that the antigen‐presenting function of B cells was compromised by *in vitro* addition of active VitD to B and T cell co‐cultures. The authors detected a reduced expression of the co‐stimulatory molecule CD86 in B cells along with diminished T‐cell expansion and lower cytokine production by these cells. A general scheme indicating some of the most

**Figure 1.** VitD action on the immune and the central nervous systems. (A) Effect of active VitD on the innate and the

The immunomodulatory potential of VitD has been widely explored in the field of autoim‐ mune diseases. Epidemiological studies demonstrated that low VitD is correlated with a higher incidence of autoimmune diseases. Besides, genetic factors as VDR polymorphisms are also linked to autoimmune disorder susceptibility. The association between VitD and systemic and organ‐specific autoimmune diseases, including multiple sclerosis (MS), was carefully re‐

adaptive immunity cells and (B) direct and indirect effects of active VitD on the central nervous system.

viewed by Agmon‐Levin et al. [49].

210 A Critical Evaluation of Vitamin D - Clinical Overview

relevant effects of VitD on innate and adaptive immunity is displayed in **Figure 1**.

MS is an autoimmune disease characterized by the activation of self‐reactive T cells specific for CNS antigens. This immune response triggers an initial inflammation in brain and spinal cord that is then followed by demyelination, axonal damage, and scar formation [50]. The pathogenic immune response observed in MS is mainly mediated by Th1 and Th17 [51]. About 85% of MS patients present with a biphasic disease characterized by alternating epi‐ sodes of neurological disability and recovery, which is entitled as relapsing remitting MS (RRMS). Within 20–25 years, 60–70% of these patients progress to a secondary‐progressive disease that is characterized by progressive neurological deterioration. Approximately 10% of the patients display a disease course classified as primary progressive MS, which is char‐ acterized by a continuous decline in neurological performance without any recovery epi‐ sode [52]. Magnetic resonance imaging (MRI) is playing a prominent role in the diagnosis and also in the analysis of MS therapy efficacy [53]. As mentioned before, autoimmune dis‐ eases result from the interactions of environmental and genetic risk factors. Environmental risk factors considered essential for MS development include infections and non‐infectious factors that comprise differences in diet and other behaviors, such as cigarette smoking and sunlight exposure [54,55]. The development of MS has been strongly associated with viral and bacterial infections [54,56]. More recently, a possible relationship between MS and Can‐ dida species was proposed [57–59]. Our research team recently demonstrated that previous infection with *Candida albicans*, a commensal and opportunistic human pathogen, aggravates the clinical signs of EAE [60].

Epidemiological data on MS incidence and prevalence drew attention to a possible link between the geographical distribution of the disease and exposure to the sun, UV radiation/ intensity, and VitD levels. This sunshine hypothesis also known as latitude‐gradient effect was initially proposed by Limburg [61] that suggested a correlation between higher MS occurrence and increasing distance from the equator. According to the World Health Organization [62], the highest prevalence of MS occurs in Europe (80 per 100,000 people) and the lowest preva‐ lence in Africa (0.3 per 100,000). More recently it was reported that, until 2013, the number of MS was higher in northern hemisphere and lower in southern hemisphere, with the exception of Australia and New Zealand [63]. A latitudinal variation was also identified in the continents. For example, geospatial analysis carried out in North American regions showed an inverse correlation between MS and UV radiation, that is, higher MS rates have been associated with lower UV radiation due to a south‐north latitudinal gradient [64]. Interestingly, a series of lifestyle changes that include sun evasion associated with skin protection and extra time indoors, or increased charter tourism to warmer countries during the winter, seems to abolish latitude effects on UV radiation exposure [65]. According to these authors, this association between sun exposure and MS can be determined by distinct effects: by the VitD generated by sun exposure, by direct sun effects, or by a combination of both. These possibilities are reinforced by data from experimental animals and also from dietary studies in human populations. Dermal application of VitD ointments and UV radiation in VDR knockout mice were both able to induce Treg cells [66]. Further study indicated that these UV‐induced Treg

cells were able to migrate to the CNS of mice with EAE where they downregulated the inflammatory activity [67].

A lower prevalence of MS in some northern countries, which in a general way are expected to have a higher number of patients with the disease, could be explained by VitD‐related dietary factors. For example, VitD sufficiency could be achieved through a traditional diet that includes fatty fish and cod liver oil. This possibility has been suggested to explain the reduced risk of MS in Norway that is located at the north of the Arctic Circle [68]. The relevant role of dietary VitD intake in MS was examined in two large cohorts of women: the Nurses' Health Study (NHS; 92,253 women followed between 1980 and 2000) and the Nurses' Health Study II (NHS II; 95,310 women followed between 1991 and 2001). The authors concluded that intake of VitD from supplements had a protective effect on the risk of developing MS [69]. A recent study with 953 MS patients indicated an inverse association between MS risk and the dose of cod liver oil during adolescence, suggesting that this stage of life is an important susceptible period for adult‐onset MS, reinforcing the importance of dietary VitD as a risk factor for MS [70]. Altogether these data supported the possibility that MS patients could have lower levels of VitD. Regarding this, the largest study to date compared VitD levels present in Iranian MS patients (*n* = 700) to the ones found in healthy individuals (*n* = 1000) and demonstrated that VitD levels were significantly lower in patients with MS [71]. Strong evidences also support the likelihood that low VitD levels can be related to disability and progression of this disease. In a study with 267 patients, lower serum VitD levels were also associated with higher rates of both relapse and disability [72]. Other authors showed an association between a low VitD status at the start of RRMS and the early conversion to secondary progressive MS [73]. The possible effect of VitD levels in the therapeutic efficacy of interferon beta 1b(IFN‐β‐1b), fingolimod (FTY), and glatiramer acetate (GA) was also investigated. Among patients treated with IFN‐β‐1b, higher VitD levels were associated with a reduced risk of relapse [74], whereas lower VitD levels early in the disease course correlated with a strong risk factor for long‐term MS activity and progression [75]. In a similar way, in FTY‐treated patients, higher VitD levels were associated with an approximately 50% reduction in new inflammatory events and in relapses [76]. By contrast, there was no significant benefit of higher VitD levels with respect to inflammatory events, relapses, or disability progression in GA‐treated patients [76]. The strong correlation between low VitD levels and higher MS susceptibility reinforces the hypothesis that VitD deficiency leads to MS and/or disease progression and stimulates new researches focused on supplementation of these patients with VitD.

## **5. Supplementation of MS patients with VitD**

The recent identification of VitD as a risk factor for MS susceptibility, and more recently as a potential modifier of disease course, inspired several clinical trials in relapsing MS [77]. It has been proposed that VitD supplementation is a low‐cost and a low‐risk intervention that may potentiate the efficacy of certain treatments against MS, without the risk of provoking serious adverse events as occurs with other combination therapies [76]. In effect, many patients are being already supplemented with VitD. However, it is not known whether supplementation has a significant impact on MS progression. A clinical trial (NCTO1339676) employing oral supplementation with active VitD (20,000 IU/week, cholecalciferol, Dekristol) administered once a week during 12 months together with IFN‐β‐1b resulted in reduction of MRI lesions in the brain of MS patients [78]. In another clinical trial (NCT 00785473), this same dose (20,000 IU/ week, cholecalciferol, Dekristol) was administered during 24 months in RRMS patients under treatment with IFN‐β‐1b, GA, or natalizumab. Even though the patients presented a significant increase in serum VitD levels, the markers of systemic inflammation were not modified. The authors suggested that the anti‐inflammatory effects of VitD supplementation are limited to RRMS patients with VitD insufficiency or to earlier stages of the disease [79]. A higher dose of VitD3 (50,000 IU/week) administered by oral route during a short period (2 months) reduced disability in RRMS patients and surprisingly upregulated IL‐6 and IL‐17 gene expression in the peripheral blood mononuclear cells of these patients [80]. Similarly, the same VitD dose (50,000 IU) administered by oral route every five days for 3 months in 94 RRMS patients under treatment with IFN‐β‐1b reduced disability of these patients but also increased IL‐17 serum levels in comparison to a placebo group [81]. Investigations in this area suggested that changes in IL‐17 levels could be related to the adopted VitD doses. For example, Golan et al. [82] demonstrated that IL‐17 serum levels were significantly increased in a lower dose group (800 IU/per day), whereas patients that were taking higher doses (4370 IU/per day) presented heterogeneous IL‐17 responses: 40% of them had decreased serum IL‐17 levels, whereas 45% had increased IL‐17 levels after three months of supplementation. These authors suggested that IL‐17 data must be interpreted with caution as serum IL‐17 is not an established biomarker of MS disease activity. Furthermore, IL‐17 serum levels before treatment with IFN‐β could not be correlated to disease activity parameters [83]; IL‐17 also showed a trend toward higher levels in MS patients with inactive disease compared to those with active disease [84]. More recently, 40 patients with RRMS were randomized to receive 10,400 IU or 800 IU of cholecalciferol daily for 6 months. Mean increase of VitD levels from baseline to the ones detected at final visit was larger in the high‐dose group than in the low‐dose one and adverse events were minor and did not differ between the two groups. Interestingly, in the high‐dose group, but not in the low‐dose one, there was a reduction in the proportion of IL‐17+CD4+ T cells. The authors concluded that daily cholecalciferol supplementation with 10,400 IU is safe and well tolerated in patients with MS and determines *in vivo* pleiotropic immunomodulatory effects [85]. Considering that IL‐17 is an important cytokine involved in MS pathogenesis, further studies are needed to clarify the role of VitD on these unexpected elevated IL‐17 levels. Therefore, until nowadays it is not possible to consider IL‐17 as a biological marker for VitD levels in human body.

cells were able to migrate to the CNS of mice with EAE where they downregulated the

A lower prevalence of MS in some northern countries, which in a general way are expected to have a higher number of patients with the disease, could be explained by VitD‐related dietary factors. For example, VitD sufficiency could be achieved through a traditional diet that includes fatty fish and cod liver oil. This possibility has been suggested to explain the reduced risk of MS in Norway that is located at the north of the Arctic Circle [68]. The relevant role of dietary VitD intake in MS was examined in two large cohorts of women: the Nurses' Health Study (NHS; 92,253 women followed between 1980 and 2000) and the Nurses' Health Study II (NHS II; 95,310 women followed between 1991 and 2001). The authors concluded that intake of VitD from supplements had a protective effect on the risk of developing MS [69]. A recent study with 953 MS patients indicated an inverse association between MS risk and the dose of cod liver oil during adolescence, suggesting that this stage of life is an important susceptible period for adult‐onset MS, reinforcing the importance of dietary VitD as a risk factor for MS [70]. Altogether these data supported the possibility that MS patients could have lower levels of VitD. Regarding this, the largest study to date compared VitD levels present in Iranian MS patients (*n* = 700) to the ones found in healthy individuals (*n* = 1000) and demonstrated that VitD levels were significantly lower in patients with MS [71]. Strong evidences also support the likelihood that low VitD levels can be related to disability and progression of this disease. In a study with 267 patients, lower serum VitD levels were also associated with higher rates of both relapse and disability [72]. Other authors showed an association between a low VitD status at the start of RRMS and the early conversion to secondary progressive MS [73]. The possible effect of VitD levels in the therapeutic efficacy of interferon beta 1b(IFN‐β‐1b), fingolimod (FTY), and glatiramer acetate (GA) was also investigated. Among patients treated with IFN‐β‐1b, higher VitD levels were associated with a reduced risk of relapse [74], whereas lower VitD levels early in the disease course correlated with a strong risk factor for long‐term MS activity and progression [75]. In a similar way, in FTY‐treated patients, higher VitD levels were associated with an approximately 50% reduction in new inflammatory events and in relapses [76]. By contrast, there was no significant benefit of higher VitD levels with respect to inflammatory events, relapses, or disability progression in GA‐treated patients [76]. The strong correlation between low VitD levels and higher MS susceptibility reinforces the hypothesis that VitD deficiency leads to MS and/or disease progression and stimulates new researches

inflammatory activity [67].

212 A Critical Evaluation of Vitamin D - Clinical Overview

focused on supplementation of these patients with VitD.

**5. Supplementation of MS patients with VitD**

The recent identification of VitD as a risk factor for MS susceptibility, and more recently as a potential modifier of disease course, inspired several clinical trials in relapsing MS [77]. It has been proposed that VitD supplementation is a low‐cost and a low‐risk intervention that may potentiate the efficacy of certain treatments against MS, without the risk of provoking serious adverse events as occurs with other combination therapies [76]. In effect, many patients are being already supplemented with VitD. However, it is not known whether supplementation has a significant impact on MS progression. A clinical trial (NCTO1339676) employing oral

The researches done so far strongly suggest that VitD supplementation could be useful in MS treatment. However, the exact doses to be prescribed to patients presenting different clinical symptoms are still waiting to be determined [86]. Regarding the side effects of VitD that include hypercalcemia [87] and the imbalance in serum concentration of parathyroid hormone [88], monitoring serum VitD would also be extremely important. In spite of the findings that VitD directly regulates the nervous system development and function [89], there is no scientific evidence to support its use as a monotherapy for MS in clinical practice [90]. Recent human trials concerning VitD supplementation in MS patients suggest that higher VitD doses are more efficient to control the symptoms and disease inflammatory markers. Nonetheless, to fix the ideal dose, it is essential to measure VitD serum levels before supplementation and to follow up the patients by constantly monitoring side effects. It is important, however, to highlight that the ideal dose could vary from one patient to another. The possible use of VitD analogs devoid of side effects must be also evaluated. World Health Organization (WHO) and Multiple Sclerosis International Federation (MSIF) published in 2008 the first Atlas of MS [62], corre‐ lating the epidemiology, diagnosis, and therapy. To the best of our knowledge, WHO did not define a specific VitD dose to treat MS.

#### **6. Therapeutic effect of VitD in EAE**

Experimental autoimmune encephalomyelitis (EAE) is an animal model universally employed to investigate mechanisms of inflammation in the CNS in the context of MS. EAE is mainly induced in rodents either by active immunization with CNS antigens associated with adjuvant or by passive transfer of CNS‐specific T cells. Most of the therapeutic procedures adopted nowadays were initially tested in murine EAE [91]. In 1991, it was demonstrated that VitD administration every other day for 15 days, starting 3 days before EAE induction, significantly prevented disease development and prolonged the survival of SJL/J mice [92]. This was the first report concerning the therapeutic potential of VitD on EAE. To avoid undesirable hypercalcemia *in vivo*, the immunomodulatory activity of VitD analogs were confirmed and they were equally efficient to suppress EAE development [93,94]. Since then, EAE has been widely employed to understand the mechanisms involved in VitD efficacy against MS. In this regard, one of the first studies was done with the Lewis rat model. The authors observed that VitD administered after the beginning of clinical signs determined significant clinical im‐ provement. This therapeutic effect was associated with a striking decrease in the number of CD4+ cells, macrophages, and activated microglia in the CNS [95]. VDR is also essential for the beneficial effects of VitD on EAE since VitD treatment was not able to prevent disease manifestations in VDR‐knockout mice [96]. The efficacy of VitD over EAE has also been attributed to effects on cells from the innate immunity. It decreases macrophage accumulation [97], inhibits chemokine synthesis and inducible NOS, and also suppresses CD11b+ monocyte recruitment into the CNS [98]. NKT cells also contribute to the protective effect of VitD on murine EAE. All mice lacking NKT cells [CD1d(−/−)] presented EAE symptomatology upon VitD administration, whereas the same treatment completely avoided EAE development in wild‐type mice [99]. More recent data revealed that VitD administration induces tolerogenic DCs in the lymph nodes, which leads to suppression of encephalitogenic T cells, resulting in less inflammatory response in the CNS [100].

Critical effects of VitD on CD4+ T cells have been reported, whereas it is not evident if this vitamin affects CD8+ T cells, which express the highest concentrations of VDR. The effect of VitD on CD8+ T cells in EAE was evaluated in one report. The authors demonstrated that VitD inhibits EAE development even in mice lacking functional CD8+ cells, suggesting that they were not essential for VitD‐suppressive effect in murine EAE [101]. The conception that the CD4+ T‐cell subset was the main VitD target during EAE therapy was then established. VitD treatment triggered a reduction in the total number of lymphocytes, while the amount of IL‐4 and TGF‐β‐1 transcripts increased in the CNS of EAE mice [102]. Still regarding anti‐inflam‐ matory cytokines, VitD therapy was reported to be much less effective in preventing EAE symptoms in IL‐4‐deficient mice [103] and also failed to inhibit EAE in mice with a disrupted IL‐10 or IL‐10R gene [104]. A more recently described profile of CD4+ T cells termed Th17 plays a critical role in numerous inflammatory conditions and autoimmune diseases. In this context, researchers showed that VitD can inhibit the differentiation and migration of Th17 cells to the CNS, ameliorating EAE symptoms [41,105].

ideal dose, it is essential to measure VitD serum levels before supplementation and to follow up the patients by constantly monitoring side effects. It is important, however, to highlight that the ideal dose could vary from one patient to another. The possible use of VitD analogs devoid of side effects must be also evaluated. World Health Organization (WHO) and Multiple Sclerosis International Federation (MSIF) published in 2008 the first Atlas of MS [62], corre‐ lating the epidemiology, diagnosis, and therapy. To the best of our knowledge, WHO did not

Experimental autoimmune encephalomyelitis (EAE) is an animal model universally employed to investigate mechanisms of inflammation in the CNS in the context of MS. EAE is mainly induced in rodents either by active immunization with CNS antigens associated with adjuvant or by passive transfer of CNS‐specific T cells. Most of the therapeutic procedures adopted nowadays were initially tested in murine EAE [91]. In 1991, it was demonstrated that VitD administration every other day for 15 days, starting 3 days before EAE induction, significantly prevented disease development and prolonged the survival of SJL/J mice [92]. This was the first report concerning the therapeutic potential of VitD on EAE. To avoid undesirable hypercalcemia *in vivo*, the immunomodulatory activity of VitD analogs were confirmed and they were equally efficient to suppress EAE development [93,94]. Since then, EAE has been widely employed to understand the mechanisms involved in VitD efficacy against MS. In this regard, one of the first studies was done with the Lewis rat model. The authors observed that VitD administered after the beginning of clinical signs determined significant clinical im‐ provement. This therapeutic effect was associated with a striking decrease in the number of CD4+ cells, macrophages, and activated microglia in the CNS [95]. VDR is also essential for the beneficial effects of VitD on EAE since VitD treatment was not able to prevent disease manifestations in VDR‐knockout mice [96]. The efficacy of VitD over EAE has also been attributed to effects on cells from the innate immunity. It decreases macrophage accumulation [97], inhibits chemokine synthesis and inducible NOS, and also suppresses CD11b+ monocyte recruitment into the CNS [98]. NKT cells also contribute to the protective effect of VitD on murine EAE. All mice lacking NKT cells [CD1d(−/−)] presented EAE symptomatology upon VitD administration, whereas the same treatment completely avoided EAE development in wild‐type mice [99]. More recent data revealed that VitD administration induces tolerogenic DCs in the lymph nodes, which leads to suppression of encephalitogenic T cells, resulting in

Critical effects of VitD on CD4+ T cells have been reported, whereas it is not evident if this vitamin affects CD8+ T cells, which express the highest concentrations of VDR. The effect of VitD on CD8+ T cells in EAE was evaluated in one report. The authors demonstrated that VitD inhibits EAE development even in mice lacking functional CD8+ cells, suggesting that they were not essential for VitD‐suppressive effect in murine EAE [101]. The conception that the CD4+ T‐cell subset was the main VitD target during EAE therapy was then established. VitD treatment triggered a reduction in the total number of lymphocytes, while the amount of IL‐4

define a specific VitD dose to treat MS.

214 A Critical Evaluation of Vitamin D - Clinical Overview

**6. Therapeutic effect of VitD in EAE**

less inflammatory response in the CNS [100].

After the first demonstration that VitD leads to induction of CD4+CD25+Foxp3+ cells with suppressive activity *in vitro* [106] and that these regulatory cells are directly involved in the natural resolution of EAE [107], many studies validated the correlation between VitD treatment and the increment of a Foxp3+ regulatory profile in EAE [99,103,104] (**Figure 1B**). The potential for reversing inflammatory and demyelinating processes in the CNS has been attributed to an augmented generation of Foxp3+ Treg cells in the periphery and their further migration to the CNS [100,108]. New therapeutic approaches have also been tested to improve VitD efficacy in EAE. A synergistic effect was found by association of VitD with estrogen, which determined more CD4+Helios+Foxp3+ Treg cells and fewer CD4+ T cells among CNS mononuclear cells, preventing EAE development [109]. In addition to the large contribution of VitD immunomo‐ dulatory activity in EAE, this treatment can also directly act on neural cells promoting CNS remyelination and other neuroprotective effects (**Figure 1B**). *In vitro* assays indicated that this vitamin significantly enhanced proliferation of neural stem cells and their differentiation into neurons and oligodendrocytes [110]. In addition, VitD treatment modulated autophagic activity and neuroapoptosis in EAE mice. As autophagy is an evolutionarily conserved cellular catabolic process that recycles damaged organelles and its inhibition causes neurodegenera‐ tion in mature neurons, this process plays an essential role in maintaining neuronal homeo‐ stasis [111]. In summary, VitD controls EAE symptoms through reduction of inflammatory immune response and elicitation of a regulatory profile. As EAE reproduces specific features of the histopathology and neurobiology of MS [112], highlighting these mechanisms in rodent models is essential to translate VitD supplementation to MS patients.

Emphasis has been given to specific therapies, that is, to procedures that target CNS anti‐ gen and that would be, therefore, more efficient and devoid of side effects. In this context, MOG administration by different routes as intravenous [113], oral [114] or nasal [115], was able to suppress EAE symptoms. Various formulations containing myelin antigens were tested to control EAE. MOG conjugated with nanoparticles [116], mannan, [117] or inserted into a plasmid DNA [118] reduced EAE symptoms through induction of Foxp3+ Treg cells and dowmodulation of Th17 and Th1 cells. Our research group has been working in this context. Considering that an antigen from the CNS can provide the required specificity and that VitD is endowed with a strong downmodulatory potential, we anticipated that VitD could work as a tolerogenic adjuvant. Differently from the conventional immunogenic ad‐ juvants that reinforce the immune response, the denominated tolerogenic adjuvants have the ability to downmodulate or modify the specific immune response when associated with specific antigens. Confirming this hypothesis, we recently demonstrated that a combined therapy with MOG + VitD blocked EAE development. This elevated efficacy was correlated with reduced production of IL‐6 and IL‐17 by spleen and CNS cell cultures stimulated with MOG, reduced splenic DC maturation, and also a striking decline in CNS inflammation [119] (**Figure 2**).

**Figure 2.** MOG + active vitamin D3 association strategy for EAE prophylaxis and treatment. C57BL/6 mice were vacci‐ nated or treated with this association and the effect on EAE was evaluated in the acute EAE phase. Both strategies de‐ creased production of inflammatory cytokines by CNS mononuclear cells, frequency of CD4+CD25+Foxp3+ Treg cells, and inflammation in the CNS.
