Introduction

Vitamin D, in particular the biologically active metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), is a pleiotropic hormone exerting a variety of biological effects including the regulation of bone and mineral metabolism, as well as the modulation of immune responses. Vitamin D can either be ingested or is produced in the skin upon UV radiation (UVR). The active spectrum is within the UVB range (290–320 nm) and catalyzes the first step of the synthesis. The biologically active metabolite is then hydroxylated in the liver by one mitochondrial enzyme (CYP27A1) and five microsomal enzymes, of which one is CYP2D25, followed by further hydroxylation in the kidney via CYP27B1 (Zhu and Deluca, 2012). Whether the final hydroxylation can also take place in the skin is still a matter of debate (Bikle and Pillai, 1993; Vanhooke et al., 2006).

In the past years, the interest in vitamin D and its analogs as therapeutic agents has increased. This is because of the fact that vitamin D deficiency and low 25(OH)D₃ levels, respectively, have been suspected—although not undisputed—to correlate with a variety of pathologic conditions including multiple sclerosis, allergic asthma, diabetes, and even certain types of cancer (reviewed in Holick, 2007; Shahriari et al., 2010; Hart et al., 2011). Moreover, some studies indicated that vitamin D supplementation reduces the risk of developing multiple sclerosis and type-I diabetes (reviewed in Fletcher et al., 2012) or prevents asthma exacerbation triggered by acute respiratory infection (Majak et al., 2011). Again, these studies have been criticized for various reasons, and there is unanimous agreement that further studies are needed.

The fact that the vast majority of diseases that are supposedly influenced by vitamin D are immune-mediated is in accordance with the well-known immunosuppressive effects of vitamin D, which have been proven in numerous in vitro and in vivo studies (reviewed in Mora et al., 2008). 1,25(OH)₂D₃ exerts effects on the differentiation of macrophages and dendritic cells (DCs), modulates antigen presentation, enhances the differentiation and activity of regulatory T cells (Tregs), and reduces T helper 1 and T helper 17 differentiation, just to name a few (reviewed in Hart et al., 2011).

It is firmly established that UVR suppresses the immune system. One of the hallmark features of UVR-induced immunosuppression is the induction of Tregs. As UVR-Tregs act in an antigen-specific manner, they harbor therapeutic potential (Schwarz, 2008). This idea has been further fostered by the recent demonstration that UVR-Tregs can be induced in sensitized mice in vivo and reprogrammed in such a way that they inhibit the elicitation of contact hypersensitivity (CHS) and thus act in a therapeutic manner (Schwarz et al., 2011). However, DNA damage caused by UVR in Langerhans cells (LCs) appears to be essential for the induction of UVR-Tregs (Schwarz et al., 2005). As DNA damage carries the risk of potential malignant transformation, we are seeking for alternative methods to induce similar Tregs without the need for UVR. Topical application of 1,25(OH)₂D₃ and the vitamin D analog calcipotriol, respectively, has been described to induce Tregs in different models (Gorman et al., 2007; Ghoreishi et al., 2009). Thus, we were interested to study whether in the model of hapten-mediated CHS topical 1,25(OH)₂D₃ can induce Tregs and whether they differ from UVR-Tregs. In addition, we sought to clarify whether UVR-induced immunosuppression is mediated via vitamin D, which is still a matter of debate.

Results

1,25(OH)₂D₃-induced immunosuppression is mediated via Foxp3⁺ Tregs

Topical application of 1,25(OH)₂D₃ has been described to inhibit the induction of CHS and to induce Tregs, the phenotypic characterization of which, however, is still incomplete. To further characterize 1,25(OH)₂D₃-induced Tregs, we used the model of DNFB-mediated CHS. As described previously (Guo et al., 1992), topical application of 1,25(OH)₂D₃ onto naive mice inhibited the induction of CHS and induced lymphocytes with suppressor activity, as demonstrated by adoptive transfer experiments (data not shown). Foxp3 is a key transcription factor in the development of natural CD4⁺CD25⁺ Tregs. When overexpressed in T cells, Foxp3 promotes a suppressor phenotype (Fontenot et al., 2003; Hori et al., 2003). Thus, Foxp3 is currently regarded as the most specific marker for Tregs. Recently, it was shown that UVR-Tregs express Foxp3 (Schwarz et al., 2011).

To clarify whether the same applies for 1,25(OH)₂D₃-induced Tregs, we took advantage of DEREG (depletion of Tregs) mice. These mice express a diphtheria toxin (DT) receptor–enhanced GFP under control of the foxp3 gene locus (Lahl et al., 2007). Injection of DT results in the selective depletion of all Foxp3⁺ T cells. 1,25(OH)₂D₃ was topically applied on 4 consecutive days on the shaved backs of DEREG mice. At 24 hours after the last treatment, animals were sensitized by painting DNFB onto 1,25(OH)₂D₃-treated skin. After another 48 hours, DT was injected for 3 consecutive days. Five days after sensitization, lymph node cells and splenocytes were obtained and injected into naive wild-type (WT) mice. After 24 hours, the recipients were sensitized against DNFB and ear challenge was performed 5 days thereafter. Cells obtained from DEREG mice treated with 1,25(OH)₂D₃ significantly suppressed CHS in the recipients (Figure 1, no. 3). In contrast, sensitization was not reduced upon transfer of cells obtained from DEREG mice treated with 1,25(OH)₂D₃ and DT (Figure 1, no. 4). This indicates that similar to UVR 1,25(OH)₂D₃ induces Tregs that express Foxp3.

Figure 1.

Induction of immunosuppression by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated via Foxp3⁺ regulatory T cells (Tregs). DEREG (depletion of regulatory T cells) mice were painted on the backs with 1,25(OH)₂D₃ for 4 consecutive days. At 24 hours after the last treatment, mice were sensitized with DNFB painted onto 1,25(OH)₂D₃-treated skin. At 48 hours after sensitization, one group of animals received 1 μg diphtheria toxin (DT) on 3 consecutive days. Five days after sensitization, splenocytes and lymph node cells were obtained from 1,25(OH)₂D₃-treated DEREG mice and injected intravenously (i.v). into naive wild-type (WT) mice. Recipients were sensitized against DNFB 24 hours after transfer. Five days later, challenge with DNFB was performed. Positive control (Pos Co) mice were sensitized and challenged, whereas negative control (Neg Co) mice were only challenged. The ear swelling response is expressed as the difference (cm × 10⁻³, mean±SD) between the thickness of the challenged ear and the thickness of the vehicle-treated ear. ^*P<0.05 versus Pos Co.

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1,25(OH)₂D₃-induced Tregs suppress CHS in an antigen-specific way

One of the main features of UVR-Tregs is the antigen-specific mode of action (Schwarz, 2008). To address whether the same applies for 1,25(OH)₂D₃-induced Tregs, lymph node cells and splenocytes were obtained from mice painted with 1,25(OH)₂D₃ and sensitized with DNFB and injected intravenously into naive recipients, which were subsequently sensitized against either DNFB or the unrelated hapten oxazolone (Oxa). Five days later, recipients were challenged with DNFB or Oxa, respectively. As known from previous experiments, injection of cells obtained from sensitized mice does not alter the CHS response and does not induce Tregs in the recipients. Transferred cells obtained from 1,25(OH)₂D₃-treated and DNFB-sensitized donors suppressed in the recipients the CHS response only against DNFB, but not against Oxa (Figure 2). This indicates that 1,25(OH)₂D₃-induced Tregs act in an antigen-specific manner.

Figure 2.

1α,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃)–induced regulatory T cells (Tregs) act in an antigen-specific manner. (a) C57BL/6 mice were painted on the backs with 1,25(OH)₂D₃ for 4 consecutive days (VD₃). At 24 hours after the last treatment, mice were sensitized with DNFB painted onto 1,25(OH)₂D₃-treated skin. After 5 days, animals were challenged and ear swelling was measured 24 hours later. ^*P<0.005 versus Pos Co. (b) Immediately after measuring ear swelling, splenocytes and lymph node cells were obtained from 1,25(OH)₂D₃-treated and DNFB-sensitized donors depicted in Figure 2a (no. 3, VD) and injected into naive animals. Recipients were sensitized against DNFB or the irrelevant hapten oxazolone (Oxa) 24 hours after transfer. Five days later, challenge with the respective hapten was performed. Pos Co mice were sensitized and challenged, whereas Neg Co were only challenged. ^*P<0.005 versus Pos Co. Neg Co, negative control; Pos Co, positive control; VD, vitamin D.

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LCs are required for 1,25(OH)₂D₃-induced immunosuppression and induction of Tregs

It was recently shown that LCs are required for UVR-induced immunosuppression and the induction of UVR-Tregs (Schwarz et al., 2010). To elucidate the role of LCs in the development of 1,25(OH)₂D₃-induced Tregs, we used Langerin DT receptor (DTR) knock-in (Langerin-DTR) mice that harbor a targeted insertion of the human DTR into the Langerin locus (Bennett et al., 2005). Thus, injection of DT results in depletion of Langerin⁺ cells. Langerin-DTR mice were injected with DT and 72 hours later mice were painted with 1,25(OH)₂D₃ for 4 days. After 24 hours, animals were sensitized with DNFB through 1,25(OH)₂D₃-painted skin. Langerin-DTR mice that had not received DT behaved like WT animals. The positive controls responded with a significant ear swelling response to sensitization. As observed in WT mice, 1,25(OH)₂D₃ suppressed the CHS response in Langerin-DTR mice (Figure 3a, no DT). Animals in which Langerin⁺ cells were depleted by DT injection could be sensitized, although the sensitization response was weaker than in mice that did not receive DT, confirming previous observations (Bennett et al., 2005, 2007; Schwarz et al., 2010). In contrast, topically applied 1,25(OH)₂D₃ did not inhibit sensitization in mice depleted of Langerin-expressing cells (Figure 3a, DT).

Figure 3.

Langerhans cells are required for 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)–induced immunosuppression and induction of regulatory T cells (Tregs). (a) Langerin-diphtheria toxin receptor (DTR) mice were injected with diphtheria toxin (DT). After 72 hours, mice were painted on the backs with 1,25(OH)₂D₃ for 4 consecutive days (VD₃). At 24 hours after the last treatment, mice were sensitized with DNFB painted onto 1,25(OH)₂D₃-treated skin. After 5 days, animals were challenged and ear swelling was measured 24 hours later. Positive controls (Pos Co) comprised DT-treated Langerin-DTR mice that were sensitized and challenged, whereas negative controls (Neg Co) comprised those that were only challenged. As additional controls, Langerin-DTR mice were treated identically but not injected with DT (No DT). ^*P<0.005 versus Pos Co, No DT. (b) Immediately after measuring ear swelling, splenocytes and lymph node cells were obtained from 1,25(OH)₂D₃-treated Langerin-DTR mice depicted in Figure 3a (nos 3 and 6). Cells were injected intravenously into naive wild-type (WT) mice (Transfer). Recipients were sensitized against DNFB 24 hours after injection. Five days later, challenge with DNFB was performed. Pos Co mice were sensitized and challenged, whereas Neg Co were only challenged. ^*P<0.05 versus Pos Co. NS, nonsignificant; VD, vitamin D.

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To determine whether 1,25(OH)₂D₃ induced Tregs, lymph node cells and splenocytes were obtained from the 1,25(OH)₂D₃-painted donors (nos 3 and 6 of Figure 3a) and injected intravenously into naive WT mice. Recipients were sensitized against DNFB 24 hours later. After 5 days, mice were challenged and ear swelling was evaluated 24 hours later. Mice injected with cells obtained from 1,25(OH)₂D₃-painted Langerin-DTR donors that were not treated with DT were significantly suppressed in their CHS response, indicating that Tregs had developed in the donors (Figure 3b, no. 3). In contrast, recipients of cells from Langerin-DTR donors in which Langerin⁺ cells were depleted by injection of DT responded with a significant ear swelling reaction (Figure 3b, no. 4), suggesting that Tregs did not develop in donors depleted of Langerin⁺ cells upon treatment with 1,25(OH)₂D₃.

Langerin was initially described as a specific marker for LCs (Takahara et al., 2002). However, shortly thereafter it turned out that another DC subset located in the dermis also expresses Langerin (Merad et al., 2008) and is eliminated in Langerin-DTR mice upon injection of DT (Noordegraaf et al., 2010). As both LCs and dermal DCs contribute to inducing sensitization responses through the skin (Romani et al., 2010), we analyzed their role in 1,25(OH)₂D₃-induced immunosuppression and induction of Tregs. To discern the contribution of LCs and Langerin⁺ dermal DCs in this process, Langerin-DTR mice were treated with DT 10 days before 1,25(OH)₂D₃ application. At this time point, dermal DCs have repopulated the dermis, whereas LCs are still absent (Noordegraaf et al., 2010). Animals were sensitized after treatment with 1,25(OH)₂D₃ and 5 days later ear challenge was performed. The ear swelling response was reduced in mice treated with 1,25(OH)₂D₃ without injection of DT when compared with positive controls (Figure 4a, no. 3). This effect was not observed in animals injected with DT 10 days before the 1,25(OH)₂D₃ treatment, indicating that LCs but not dermal DCs are essential for 1,25(OH)₂D₃-mediated immunosuppression (Figure 4a, no. 6).

Figure 4.

Langerin⁺ dermal dendritic cells (DCs) are not involved in 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)–induced immunosuppression and induction of regulatory T cells (Tregs). (a) Langerin–diphtheria toxin receptor (DTR) mice were injected with diphtheria toxin (DT). After 10 days, mice were painted on the backs with 1,25(OH)₂D₃ for 4 consecutive days (VD₃). At 24 hours after the last treatment, mice were sensitized with DNFB painted onto VD₃-treated skin. After 5 days, animals were challenged and ear swelling was measured 24 hours later. Positive controls (Pos Co) comprised DT-treated Langerin-DTR mice that were sensitized and challenged, whereas negative controls (Neg Co) were only challenged. As additional controls, Langerin-DTR mice were treated identically but not injected with DT (No DT). ^*P<0.0005 versus Pos Co, No DT. (b) Immediately after measuring ear swelling, splenocytes and lymph node cells were obtained from 1,25(OH)₂D₃-treated Langerin-DTR mice depicted in Figure 4a (nos 3 and 6). Cells were injected intravenously (i.v.) into naive wild-type (WT) mice (Transfer). Recipients were sensitized against DNFB 24 hours after injection. Five days later, challenge with DNFB was performed. Pos Co mice were sensitized and challenged, whereas Neg Co mice were only challenged. ^*P<0.0005 versus Pos Co. VD, vitamin D.

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Adoptive transfer of lymph node cells and splenocytes obtained from 1,25(OH)₂D₃-treated but not DT-injected donors (no. 3 of Figure 4a) into naive WT mice resulted in a significant reduction of ear swelling, indicating that Tregs had developed in the donor mice (Figure 4b, no. 3). In contrast, animals that received cells from donors treated with DT 10 days before 1,25(OH)₂D₃ application were not suppressed in their CHS reaction (Figure 4b, no. 4). In these donor mice, dermal DCs were still present but LCs were absent, suggesting that LCs but not dermal DCs are required for induction of 1,25(OH)₂D₃-induced Tregs.

UVR suppresses the induction of CHS and induces Tregs in VDR-KO mice

According to the present findings, 1,25(OH)₂D₃-induced Tregs appear to be phenotypically very similar to UVR-Tregs. This nourishes the speculation that UVR-induced immunosuppression could be mediated via vitamin D. To clarify this, we used vitamin D receptor knockout (VDR-KO) mice that harbor a deletion of a fragment of genomic DNA encoding the second zinc finger of the receptor DNA-binding domain (Li et al., 1997). VDR-KO and WT controls were exposed to UVR (1.5 kJm⁻²) on 4 consecutive days. At 24 hours after the last exposure, animals were sensitized with DNFB through irradiated skin and challenged 5 days later. UVR-exposed WT mice responded to sensitization with a marginal ear swelling response, which was comparable to that of negative controls (Figure 5a, no. 3). Sensitized VDR-KO mice reacted with robust ear swelling upon DNFB challenge, indicating that these mice can be fully sensitized. Upon UVR exposure, the CHS reaction was significantly reduced comparable to that of UVR-exposed WT mice (Figure 5a, no. 6), indicating that inhibition of the induction of CHS by UVR is independent of a functional VDR and consequently of receptor-dependent action of vitamin D.

Figure 5.

UV radiation (UVR)-induced immunosuppression and development of regulatory T cells (Tregs) are not impaired in vitamin D receptor knockout (VDR-KO) mice. (a) C57BL/6 (wild type, WT) and VDR-KO mice were UVR exposed (1.5 kJm⁻²) for 4 consecutive days. At 24 hours after the last UVR exposure, mice were sensitized with DNFB painted onto UV-irradiated skin. After 5 days, animals were challenged and ear swelling was measured 24 hours later. Positive control (Pos Co) mice were sensitized and challenged, whereas negative controls (Neg Co) were only challenged. ^*P<0.0005 versus Pos Co WT; ^**P<0.0005 versus Pos Co VDR-KO. (b) Immediately after measurement, splenocytes and lymph node cells were obtained from UVR-exposed WT and VDR-KO mice (Figure 5a, nos 3 and 6) and injected intravenously (i.v.) into naive WT mice (Transfer). Recipients were sensitized against DNFB 24 hours after injection. Five days later, challenge was performed. ^*P<0.005 versus Pos Co; ^**P<0.0005 versus Pos Co.

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To determine whether UVR-Tregs developed, lymph nodes and spleens were obtained from UVR-exposed WT and VDR-KO mice (nos 3 and 6 of Figure 5a). Cells were injected intravenously into naive WT mice, which were sensitized against DNFB 24 hours later. Recipients injected with cells from UVR-exposed WT animals revealed a significantly attenuated CHS response, indicating that Tregs had developed in the donors (Figure 5b). A similar suppression was observed in recipients that received cells obtained from UV-irradiated VDR-KO mice, implying that development of UVR-Tregs is not dependent on vitamin D.

Discussion

Despite the ongoing controversy whether 1,25(OH)₂D₃ can be produced entirely in the skin (Bikle and Pillai, 1993; Vanhooke et al., 2006), we used 1,25(OH)₂D₃ as most of the similar studies were conducted with this compound. We also decided to apply 1,25(OH)₂D₃ on 4 consecutive days in analogy to the UVR regimen, as it was our primary intention not to imitate a physiological process but to identify an alternative route by which Tregs can be induced. Guo et al. (1992) were the first to describe the inhibition of CHS by topically applied 1,25(OH)₂D₃. On the basis of adoptive transfer experiments, which showed that suppression can be transferred at a cellular level into naive recipients, the authors concluded that suppressor lymphocytes were induced. It took several years until these cells were further characterized. Using a different model, Gorman et al. (2007) showed that 1,25(OH)₂D₃ induces CD4⁺Foxp3⁺ Tregs, which expanded in vivo in syngeneic recipients upon antigenic stimulation. Similarly, Ghoreishi et al. (2009) reported that transcutaneous immunization through calcipotriol-treated skin augments CD4⁺CD25⁺Foxp3⁺ Treg populations. As Foxp3 is expressed intracellularly, antibody-mediated depletion studies are not possible. Hence, the only strategy to prove the functional involvement of Foxp3-expressing Tregs in vivo is the utilization of DEREG mice in which Foxp3-expressing cells can be selectively depleted by the injection of DT (Lahl et al., 2007). DEREG mice not injected with DT behaved like WT mice both in terms of sensitization and adoptive transfer experiments. However, when 1,25(OH)₂D₃-treated DEREG mice were injected with DT after sensitization, suppression was lost upon cell transfer. This clearly indicates on a functional level that 1,25(OH)₂D₃-induced cells transferring suppression express Foxp3 and thus belong to the Foxp3⁺ subtype of Tregs. Comparable to UVR-Tregs, they act in an antigen-specific manner, as 1,25(OH)₂D₃-induced Tregs obtained from DNFB-sensitized donors inhibited the sensitization response only against DNFB but not against Oxa in the recipients.

Antigen-presenting cells appear to be one of the major cellular targets for 1,25(OH)₂D₃ to exert its immunosuppressive features. Guo et al. (1992) were the first to describe a reduction in the number of LCs upon topical application of 1,25(OH)₂D₃. The same applies for calcipotriol, as recently reported by Ghoreishi et al. (2009). As the dendrites of LCs decreased and the cells became oval or round after topical administration of 1,25(OH)₂D₃, Guo et al. (1992) already speculated that this may be associated with an inhibition of antigen presentation. According to recent data by Gorman et al. (2010), 1,25(OH)₂D₃ appears to induce the emigration of skin-derived DCs into the lymph nodes, where they alter the function of Tregs residing in the skin-draining lymph nodes. Indoleamine 2,3-dioxygenase may be involved in this process.

In their elegant work, Gorman et al. (2010) did not define the skin-derived DC population affected by topical 1,25(OH)₂D₃. To get more insight into this, we used Langerin-DTR mice in which Langerin-expressing cells can be depleted by DT (Bennett et al., 2007). Using this system, we previously showed that the primary target cell for UVR to exert its immunosuppressive features is the LC (Schwarz et al., 2010). Langerin-depleted mice turned out to be resistant to 1,25(OH)₂D₃-induced suppression of the induction of CHS and did not develop Tregs, as demonstrated by adoptive transfer experiments. Langerin, however, is not only expressed by LCs but also by dermal DCs (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007; Nagao et al., 2009). When 1,25(OH)₂D₃ was applied topically 10 days after DT injection, a time point at which LCs were selectively absent, CHS was not suppressed and Tregs did not develop. This indicates that LCs but not dermal DCs are the crucial target cell for 1,25(OH)₂D₃ to exert its immunosuppressive features. These data are in accordance with recent in vitro findings by van der Aar et al. (2011). In this report, 1,25(OH)₂D₃-primed human LCs induced CD25^hiFoxp3⁺Tregs, whereas 1,25(OH)₂D₃-primed dermal DCs induced Foxp3⁻ cells, which expressed IL-10 and thus appeared to belong to the Tr1 subtype.

The mechanism by which 1,25(OH)₂D₃ modifies LCs to induce Tregs still remains to be determined. On the basis of preliminary data, we know that DCs pretreated with 1,25(OH)₂D₃ before being hapten coupled lose their capacity to induce sensitization upon intracutaneous injection into naive recipients. Furthermore, the expression of major histocompatibility complex-II and B7-2 is suppressed by 1,25(OH)₂D₃. Hence, we speculate that 1,25(OH)₂D₃ similar to UVR induces Tregs by primarily affecting antigen-presenting cells.

The current characterization of 1,25(OH)₂D₃-induced Tregs, which is still ongoing, implies that they are similar, if not identical, to UVR-Tregs. Hence, it was obvious to speculate at this stage that UVR-induced immunosuppression might be mediated by vitamin D. This is an ongoing debate (Hart et al., 2011). The involvement of vitamin D was supported by the observation that topical treatment of human skin with the vitamin D analog calcipotriene inhibited immune responses to a similar extent as UVR (Hanneman et al., 2006). On the other hand, 1,25(OH)₂D₃ was reported to reduce UVR-induced DNA damage (Trémezaygues et al., 2009; Mason et al., 2010), which is supposed to be one of the major molecular triggers of UVR-induced immunosuppression. Accordingly, topical 1,25(OH)₂D₃ reduced systemic immunosuppression induced by UVR in mice (Dixon et al., 2007). Damian et al. (2010) added further to this discussion by showing that topical 1,25(OH)₂D₃, although protecting from UVR-induced DNA damage, suppresses cutaneous immunity in humans. Ghoreishi et al. (2009) proposed that UVR-induced tolerance is induced via a vitamin D receptor–dependent mechanism, as VDR-KO mice failed to increase Foxp3⁺ Tregs in the draining lymph nodes upon UVR exposure.

To clarify whether vitamin D is involved in the suppression of DNFB-mediated CHS by low-dose UVR and subsequent induction of Tregs, we used VDR-KO mice. In terms of suppression of CHS and induction of Tregs, they behaved similar to WT mice, suggesting that local UVR-induced immunosuppression is not mediated via vitamin D. There are reservations about the validity of mice in which the vitamin D metabolism or signaling has been genetically disrupted, as these animals on the long term suffer from developmental problems leading to skeletal, reproductive, and immune system dysfunction (Hart et al., 2011). For our purposes, we used 6–8-week-old mice. As the average experiment (induction of CHS and generation of Treg) lasted maximally for 10 days, the mice were off the experimental protocol at 10 weeks of age at the latest. At this age, the VDR-KO did not show immunologic abnormalities. In addition, the mice were kept under a rescue diet. As they responded equally well to sensitization as WT mice, obvious major alterations in the immune system can be excluded.

Taken together, the present data demonstrate that topically applied 1,25(OH)₂D₃ acts in a similar manner as UVR in terms of suppressing the induction of CHS and generating Tregs. On the other hand, vitamin D does not appear to be essentially involved in mediating local UVR-induced immunosuppression in mice. Independently of this, topical application of 1,25(OH)₂D₃ may represent a valid therapeutic strategy to evoke UVR-like immunosuppressive effects without the need of UVR exposure and its inherent risk of DNA damage.

Materials and Methods

Mice and reagents

Six- to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Langerin-DTR mice were previously described (Bennett et al., 2005). To deplete Langerin⁺ cells, mice were injected with DT (Merck, Darmstadt, Germany) intraperitoneally and subcutaneously (400 ng). DEREG mice were kindly provided by T Sparwasser (Lahl et al., 2007). VDR-KO mice (B6.129S4-Vdr^tm1Mbd/J) were purchased from Jackson Laboratory (Bar Harbor, ME) and raised from weaning on 2% calcium, 1.25% phosphorus, and 20% lactose rescue diets (Song et al., 2003). All mice were bred and kept in the central animal facilities of the University Clinics Schleswig-Holstein, Campus Kiel. Animal care was provided by expert personnel in compliance with the relevant laws and institutional guidelines.

1,25(OH)₂D₃-induced immunosuppression

1,25(OH)₂D₃ (Sigma-Aldrich, Taufkirchen, Germany) was diluted in dimethylsulfoxid (100 μgml⁻¹; Roth, Karlsruhe, Germany) and then in acetone/olive oil (4:1; 1 μgml⁻¹). A volume of 100 μl was applied on the shaved back for 4 days. Mice were sensitized with 50 μl of DNFB (Sigma-Aldrich) solution (0.5% in acetone/olive oil, 4:1) on the back 24 hours after the last application. Five days later, ears were challenged with 20 μl of 0.3% DNFB. Topical application of the vehicle alone did not affect sensitization and did not induce Tregs. Positive control mice were sensitized and challenged, whereas negative control mice were only challenged. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm × 10⁻³. Bars show mean±SD of increase in ear thickness.

UVR-induced immunosuppression

Mice were irradiated on their shaved backs with 1.5 kJm⁻² UVR on 4 consecutive days. UV irradiation was performed using fluorescent bulbs (TL12, Philips, Eindhoven, The Netherlands), which emit most of their energy within the UVB range (290–320 nm). At 24 hours after the last UVR exposure, mice were sensitized against DNFB.

Adoptive transfer

For adoptive transfer, cells were obtained from lymph nodes (axillary and inguinal) and spleens (ratio 1:5), as described previously (Maeda et al., 2008). Cells (5 × 10⁷) were injected intravenously into naive mice. Recipients were sensitized 24 hours after injection and ear challenge performed 5 days later.

Statistics

Data were analyzed by Student’s t-test. Differences were considered significant at P<0.05. Unless otherwise stated, one representative of three independently performed experiments is shown. Each group contained at least six mice.

Conflict of interest

The authors state no conflict of interest.

References

1. Bennett CL, Noordegraaf M, Martina CA et al. (2007) Langerhans cells are required for efficient presentation of topically applied hapten to T cells. J Immunol 179:6830–6835 | PubMed | ISI | CAS |
2. Bennett CL, van Rijn E, Jung S et al. (2005) Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity. J Cell Biol 169:569–576 | Article | PubMed | ISI | CAS |
3. Bikle DD, Pillai S (1993) Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14:3–19 | Article | PubMed | ISI | CAS |
4. Bursch LS, Wang L, Igyarto B et al. (2007) Identification of a novel population of Langerin+ dendritic cells. J Exp Med 204:3147–3156 | Article | PubMed | ISI | CAS |
5. Damian DL, Kim YJ, Dixon KM et al. (2010) Topical calcitriol protects from UV-induced genetic damage but suppresses cutaneous immunity in humans. Exp Dermatol 19:e23–e30 | Article | PubMed | ISI |
6. Dixon KM, Deo SS, Norman AW et al. (2007) In vivo relevance for photoprotection by the vitamin D rapid response pathway. J Steroid Biochem Mol Biol 103:451–456 | Article | PubMed | CAS |
7. Fletcher JM, Basdeo SA, Allen AC et al. (2012) Therapeutic use of vitamin D and its analogues in autoimmunity. Recent Pat Inflamm Allergy Drug Discov 6:22–34 | Article | PubMed |
8. Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336 | Article | PubMed | ISI | CAS |
9. Ghoreishi M, Bach P, Obst J et al. (2009) Expansion of antigen-specific regulatory T cells with the topical vitamin d analog calcipotriol. J Immunol 182:6071–6078 | Article | PubMed | ISI | CAS |
10. Ginhoux F, Collin MP, Bogunovic M et al. (2007) Blood-derived dermal Langerin+ dendritic cells survey the skin in the steady state. J Exp Med 204:3133–3146 | Article | PubMed | ISI | CAS |
11. Gorman S, Judge MA, Hart PH (2010) Topical 1,25-dihydroxyvitamin D3 subverts the priming ability of draining lymph node dendritic cells. Immunology 131:415–425 | Article | PubMed |
12. Gorman S, Kuritzky LA, Judge MA et al. (2007) Topically applied 1,25-dihydroxyvitamin D3 enhances the suppressive activity of CD4+CD25+ cells in the draining lymph nodes. J Immunol 179:6273–6283 | PubMed | ISI | CAS |
13. Guo Z, Okamoto H, Imamura S (1992) The effect of 1,25(OH)2-vitamin D3 on Langerhans cells and contact hypersensitivity in mice. Arch Dermatol Res 284:368–370 | Article | PubMed | CAS |
14. Hanneman KK, Scull HM, Cooper KD et al. (2006) Effect of topical vitamin D analogue on in vivo contact sensitization. Arch Dermatol 142:1332–1334 | Article | PubMed |
15. Hart PH, Gorman S, Finlay-Jones JJ (2011) Modulation of the immune system by UV radiation: more than just the effects of vitamin D? Nat Rev Immunol 11:584–596 | Article | PubMed | CAS |
16. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357:266–281 | Article | PubMed | ISI | CAS |
17. Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061 | Article | PubMed | ISI | CAS |
18. Lahl K, Loddenkemper C, Drouin C et al. (2007) Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 204:57–63 | Article | PubMed | ISI | CAS |
19. Li YC, Pirro AE, Amling M et al. (1997) Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 94:9831–9835 | Article | PubMed | CAS |
20. Maeda A, Beissert S, Schwarz T et al. (2008) Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells. J Immunol 180:3065–3071 | PubMed | ISI | CAS |
21. Majak P, Olszowiec-Chlebna M, Smejda K et al. (2011) Vitamin D supplementation in children may prevent asthma exacerbation triggered by acute respiratory infection. J Allergy Clin Immunol 127:1294–1296 | Article | PubMed |
22. Mason RS, Sequeira VB, Dixon KM et al. (2010) Photoprotection by 1alpha,25-dihydroxyvitamin D and analogs: further studies on mechanisms and implications for UV-damage. J Steroid Biochem Mol Biol 121:164–168 | Article | PubMed | ISI |
23. Merad M, Ginhoux F, Collin M (2008) Origin, homeostasis and function of Langerhans cells and other Langerin-expressing dendritic cells. Nat Rev Immunol 8:935–947 | Article | PubMed | ISI | CAS |
24. Mora JR, Iwata M, von Andrian UH (2008) Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol 8:685–698 | Article | PubMed | ISI | CAS |
25. Nagao K, Ginhoux F, Leitner WW et al. (2009) Murine epidermal Langerhans cells and Langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions. Proc Natl Acad Sci USA 2106:3312–3317 | Article |
26. Noordegraaf M, Flacher V, Stoitzner P et al. (2010) Functional redundancy of Langerhans cells and Langerin+ dermal dendritic cells in contact hypersensitivity. J Invest Dermatol 130:2752–2759 | Article | PubMed | ISI |
27. Poulin LF, Henri S, de Bovis B et al. (2007) The dermis contains Langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells. J Exp Med 204:3119–3131 | Article | PubMed | ISI | CAS |
28. Romani N, Clausen BE, Stoitzner P (2010) Langerhans cells and more: Langerin-expressing dendritic cell subsets in the skin. Immunol Rev 234:120–141 | Article | PubMed | ISI | CAS |
29. Schwarz A, Maeda A, Kernebeck K et al. (2005) Prevention of UV radiation-induced immunosuppression by IL-12 is dependent on DNA repair. J Exp Med 201:173–179 | Article | PubMed | ISI | CAS |
30. Schwarz A, Navid F, Sparwasser T et al. (2011) In vivo reprogramming of ultraviolet radiation-induced regulatory T cell migration to inhibit the elicitation of contact hypersensitivity. J Allergy Clin Immunol 128:826–833 | Article | PubMed |
31. Schwarz A, Noordegraaf M, Maeda A et al. (2010) Langerhans cells are required for UVR-induced immunosuppression. J Invest Dermatol 130:1419–1427 | Article | PubMed | ISI |
32. Schwarz T (2008) 25 years of UV-induced immunosuppression mediated by T cells-from disregarded T suppressor cells to highly respected regulatory T cells. Photochem Photobiol 84:10–18 | Article | PubMed | ISI | CAS |
33. Shahriari M, Kerr PE, Slade K et al. (2010) Vitamin D and the skin. Clin Dermatol 28:663–668 | Article | PubMed |
34. Song Y, Kato S, Fleet JC (2003) Vitamin D receptor (VDR) knockout mice reveal VDR-independent regulation of intestinal calcium absorption and ECaC2 and calbindin D9k mRNA. J Nutr 133:374–380 | PubMed |
35. Takahara K, Omatsu Y, Yashima Y et al. (2002) Identification and expression of mouse Langerin (CD207) in dendritic cells. Int Immunol 14:433–444 | Article | PubMed | ISI | CAS |
36. Trémezaygues L, Seifert M, Tilgen W et al. (2009) 1,25-dihydroxyvitamin D(3) protects human keratinocytes against UV-B-induced damage: in vitro analysis of cell viability/proliferation, DNA-damage and -repair. Dermatoendocrinol 1:239–245 | Article | PubMed |
37. van der Aar AM, Sibiryak DS, Bakdash G et al. (2011) Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells. J Allergy Clin Immunol 127:1532–1540 | Article | PubMed |
38. Vanhooke JL, Prahl JM, Kimmel-Jehan C et al. (2006) CYP27B1 null mice with LacZreporter gene display no 25-hydroxyvitamin D3-1alpha-hydroxylase promoter activity in the skin. Proc Natl Acad Sci USA 103:75–80 | Article | PubMed | CAS |
39. Zhu J, Deluca HF (2012) Vitamin D 25-hydroxylase—four decades of searching, are we there yet? Arch Biochem Biophys 523:30–36 | Article | PubMed |

Acknowledgments

We are grateful to Chris Opitz and Graziella Podda for excellent technical assistance and Arne Voss for help in preparing the graphs. This study was supported by grants from the German Research Foundation (SCHW625/4-1, SCHW625/6-1) and The Netherlands Organization for Scientific Research (NWO, VIDI 917-76-365 to BEC).