**3. Immunomodulatory properties of VitD**

First evidences of VitD role in the immune system regulation date from the 80s. Haq [21] demonstrated that active VitD, but not its non‐active form, blocked the production of IL (interleukin)‐2 and consequently inhibited T‐cell proliferation. Based on this downmodulatory effect, the potential of VitD to increase organ survival in experimental allograft transplantation was also evaluated. First studies in this field were based on the *in vitro* immunosuppressive effects of VitD and its analogs. One of the most evident toxic effects of high VitD doses, which are usually required to avoid transplant rejection, is hypercalcemia. To avoid this and other toxic effects such as bone resorption, many efforts were done to develop synthetic structural analogs of active VitD that still preserved its immunomodulatory properties [22]. When tested *in vivo*, a 20‐epi‐vitamin D3 analog did not prolong renal allograft survival in Lewis rats and also led to the development of hypercalcemia [23]. These authors emphasized the importance of more experimental studies to evaluate the potential of VitD and its analogs to prevent graft rejection. Later, Hullett et al. [24] successfully demonstrated that Lewis rats orally receiving active VitD presented prolonged survival heart allografts without hypercalcemia. Over the years, a much broader role of VitD in the immune system was disclosed and the mechanisms underlying its immunomodulatory effects were progressively elucidated. Currently, calcitriol is largely known to modulate both innate and adaptive immunity through its binding to VDR, which is present in a multitude of immune cells. Although VitD can bind to both genomic and non‐genomic targets, the most important immunomodulatory properties are elicited by genomic mechanisms [25].

It is well known that VitD stimulates the innate immune system by enhancing the antimicrobial ability of monocytes and macrophages. This effect is mainly associated with TLRs activation and increased release of cathelicidin and IL‐1β by these cells [26]. Clinical evidences suggested a strong correlation between a poor VitD status and an increased susceptibility to infections. VitD has also been linked to more severe infectious diseases [27–29]. Moreover, Nouari et al. [30] recently demonstrated that active VitD can enhance the microbicidal activity of human monocyte‐derived macrophages against *Pseudomonas aeruginosa*.

Conversely, VitD has an inhibitory effect on the adaptive immune system. It directly targets APCs, which are a very important link between the innate and adaptive immunity. In this sense, conventional APCs as DCs are profoundly affected by VitD. The mechanisms underly‐ ing the effects of VitD on DC function were recently reviewed by Barragan et al. [31]. *In vitro* treatment with active VitD or its analogs inhibits both differentiation and maturation of human and murine DCs leading to changes in its phenotype and function [32]. The immature or semi‐ mature state induced by VitD is generally characterized by a decreased expression of co‐ stimulatory molecules such as CD40, CD80, and CD86. This state determines a tolerogenic DC phenotype associated with reduced IL‐12 and increased IL‐10 production. The addition of VDR agonists or active VitD during differentiation of DCs *in vitro* determines a reduction in subsequent T‐cell proliferation and also in interferon‐gamma (IFN‐γ) production [33]. Tolerogenic DCs are also able to induce the development of Treg cells that are mainly charac‐ terized by the expression of CD4 and CD25 molecules and production of anti‐inflammatory cytokines such as IL‐10 and transforming growth factor‐β (TGF‐β) [34]. As mentioned before, Treg cells play a major role in controlling inflammatory immune responses. The main mech‐ anisms underlying their suppressive activity include the induction of inhibitory molecules such as cytotoxic T‐lymphocyte antigen 4, the production of inhibitory cytokines that leads to impaired T‐cell expansion and the release of granzymes and perforin that trigger T‐cell death [35]. Chambers et al. [36] demonstrated that addition of active VitD on human CD4+ T lymphocytes significantly increased the expression of forkhead box protein P3 (Foxp3) that characterizes Treg cells.

**3. Immunomodulatory properties of VitD**

208 A Critical Evaluation of Vitamin D - Clinical Overview

genomic mechanisms [25].

First evidences of VitD role in the immune system regulation date from the 80s. Haq [21] demonstrated that active VitD, but not its non‐active form, blocked the production of IL (interleukin)‐2 and consequently inhibited T‐cell proliferation. Based on this downmodulatory effect, the potential of VitD to increase organ survival in experimental allograft transplantation was also evaluated. First studies in this field were based on the *in vitro* immunosuppressive effects of VitD and its analogs. One of the most evident toxic effects of high VitD doses, which are usually required to avoid transplant rejection, is hypercalcemia. To avoid this and other toxic effects such as bone resorption, many efforts were done to develop synthetic structural analogs of active VitD that still preserved its immunomodulatory properties [22]. When tested *in vivo*, a 20‐epi‐vitamin D3 analog did not prolong renal allograft survival in Lewis rats and also led to the development of hypercalcemia [23]. These authors emphasized the importance of more experimental studies to evaluate the potential of VitD and its analogs to prevent graft rejection. Later, Hullett et al. [24] successfully demonstrated that Lewis rats orally receiving active VitD presented prolonged survival heart allografts without hypercalcemia. Over the years, a much broader role of VitD in the immune system was disclosed and the mechanisms underlying its immunomodulatory effects were progressively elucidated. Currently, calcitriol is largely known to modulate both innate and adaptive immunity through its binding to VDR, which is present in a multitude of immune cells. Although VitD can bind to both genomic and non‐genomic targets, the most important immunomodulatory properties are elicited by

It is well known that VitD stimulates the innate immune system by enhancing the antimicrobial ability of monocytes and macrophages. This effect is mainly associated with TLRs activation and increased release of cathelicidin and IL‐1β by these cells [26]. Clinical evidences suggested a strong correlation between a poor VitD status and an increased susceptibility to infections. VitD has also been linked to more severe infectious diseases [27–29]. Moreover, Nouari et al. [30] recently demonstrated that active VitD can enhance the microbicidal activity of human

Conversely, VitD has an inhibitory effect on the adaptive immune system. It directly targets APCs, which are a very important link between the innate and adaptive immunity. In this sense, conventional APCs as DCs are profoundly affected by VitD. The mechanisms underly‐ ing the effects of VitD on DC function were recently reviewed by Barragan et al. [31]. *In vitro* treatment with active VitD or its analogs inhibits both differentiation and maturation of human and murine DCs leading to changes in its phenotype and function [32]. The immature or semi‐ mature state induced by VitD is generally characterized by a decreased expression of co‐ stimulatory molecules such as CD40, CD80, and CD86. This state determines a tolerogenic DC phenotype associated with reduced IL‐12 and increased IL‐10 production. The addition of VDR agonists or active VitD during differentiation of DCs *in vitro* determines a reduction in subsequent T‐cell proliferation and also in interferon‐gamma (IFN‐γ) production [33]. Tolerogenic DCs are also able to induce the development of Treg cells that are mainly charac‐ terized by the expression of CD4 and CD25 molecules and production of anti‐inflammatory

monocyte‐derived macrophages against *Pseudomonas aeruginosa*.

The direct effect of VitD on T cells was the first evidence of the immunomodulatory activity of this hormone. Active VitD suppresses Th1 inflammatory immune response through inhibition of IL‐2 and IFN‐γ production, which are the main cytokines produced by this Th cell subset. This subject was revised by Lemire et al. [37]. These authors described that VitD preferentially inhibited Th1 functions having little effects over Th2 cells. At that time, they already suggested that this vitamin could have a potential therapeutic application in Th1‐ mediated diseases as is the case of some autoimmune pathologies.

Many inflammatory responses are also related to the development of Th17 cells and its signature cytokine named IL‐17. It is largely known that this T‐cell subpopulation is involved in the pathogenesis of a variety of inflammatory and autoimmune disorders [38]. In this context, Th17 cell pathogenicity is frequently related to a Th17‐Th1 functional plasticity that is regulated by the cytokine milieu [39]. The immunomodulatory effects of VitD on Th17 cells are not clear and depend upon the disease. Most of what is known concerning VitD effect on these cells is based on experimental studies. For example, oral treatment with active VitD prevented and partly reversed experimental autoimmune uveitis in mice. This effect was related to both decreased IL‐17 production and impaired development of Th17 cells [40]. Moreover, Chang et al. [41] demonstrated that active VitD treatment protected mice from experimental autoimmune encephalomyelitis (EAE) by inhibiting the differentiation and further migration of Th17 cells to the central nervous system (CNS). Even though the effect of VitD on animal models is evident, human data are controversial and there is not a consensus in the literature yet.

Data on the effects of VitD on the development of Th2 cells are also conflicting. This T‐cell subset is able to suppress Th1 inflammatory immune response through the production of anti‐ inflammatory cytokines such as IL‐4 and IL‐5. A direct effect of active VitD on Th2 cells was demonstrated by Boonstra et al. [42]. Even in the absence of APCs, these authors observed an increased frequency of IL‐4‐, IL‐5‐, and IL‐10‐producing murine CD4+ T cells after *in vitro* stimulation with VitD. In addition, there was a decrease in the frequency of IFN‐γ‐producing cells. However, Staeva‐Vieira and Freedman [43] demonstrated that active VitD inhibited the *in vitro* production of both, IFN‐γ and IL‐4 by murine CD4+ T cells.

Other T‐cell subsets such as CD8+ T cells and natural‐killer T cells (NKT) are also targets of VitD. Chen et al. [44] demonstrated that active VitD signaling through VDR is essential to control pathogenic CD8+ T cells in inflammatory bowel diseases. The importance of VDR was also highlighted by Yu et al. [45] who demonstrated a critical role of VDR expression in the development of induced NKT cells from mice fed with synthetic diets containing active VitD. 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 relevant effects of VitD on innate and adaptive immunity is displayed in **Figure 1**.

**Figure 1.** VitD action on the immune and the central nervous systems. (A) Effect of active VitD on the innate and the adaptive immunity cells and (B) direct and indirect effects of active VitD on the central nervous system.

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‐ viewed by Agmon‐Levin et al. [49].
