1,25-Dihydroxyvitamin D3Inhibits the Differentiation
and Migration of TH17 Cells to Protect against
Experimental Autoimmune Encephalomyelitis
Jae-Hoon Chang1, Hye-Ran Cha1, Dong-Sup Lee2, Kyoung Yul Seo1,3, Mi-Na Kweon1*
1Mucosal Immunology Section, Laboratory Science Division, International Vaccine Institute, Seoul, Korea, 2Cancer Research Institute, Seoul National University College of
Medicine, Seoul, Korea, 3Department of Ophthalmology, Institute for Vision Research, Yonsei University College of Medicine, Seoul, Korea
Background: Vitamin D3, the most physiologically relevant form of vitamin D, is an essential organic compound that has
been shown to have a crucial effect on the immune responses. Vitamin D3ameliorates the onset of the experimental
autoimmune encephalomyelitis (EAE); however, the direct effect of vitamin D3on T cells is largely unknown.
Methodology/Principal Findings: In an in vitro system using cells from mice, the active form of vitamin D3 (1,25-
dihydroxyvitamin D3) suppresses both interleukin (IL)-17-producing T cells (TH17) and regulatory T cells (Treg) differentiation
via a vitamin D receptor signal. The ability of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) to reduce the amount of IL-2 regulates
the generation of Treg cells, but not TH17 cells. Under TH17-polarizing conditions, 1,25(OH)2D3helps to increase the
numbers of IL-10-producing T cells, but 1,25(OH)2D3’s negative regulation of TH17 development is still defined in the
IL-102/2T cells. Although the STAT1 signal reciprocally affects the secretion of IL-10 and IL-17, 1,25(OH)2D3inhibits IL-17
production in STAT12/2T cells. Most interestingly, 1,25(OH)2D3negatively regulates CCR6 expression which might be
essential for TH17 cells to enter the central nervous system and initiate EAE.
Conclusions/Significance: Our present results in an experimental murine model suggest that 1,25(OH)2D3can directly
regulate T cell differentiation and could be applied in preventive and therapeutic strategies for TH17-mediated autoimmune
Citation: Chang J-H, Cha H-R, Lee D-S, Seo KY, Kweon M-N (2010) 1,25-Dihydroxyvitamin D3Inhibits the Differentiation and Migration of TH17 Cells to Protect
against Experimental Autoimmune Encephalomyelitis. PLoS ONE 5(9): e12925. doi:10.1371/journal.pone.0012925
Editor: Derya Unutmaz, New York University, United States of America
Received June 15, 2010; Accepted August 29, 2010; Published September 23, 2010
Copyright: ? 2010 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Mid-Career Researcher Program through a NRF grant funded by the MEST (No. 2007-04213) http://www.nrf.go.kr/
html/kr/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Interleukin (IL)-17-producing T cells have been identified in the
mouse as a new lineage of CD4+T cells that can be differentiated
from naı ¨ve T cells by the polarizing cytokines TGF-b, IL-6, and
IL-23 [1–4]. TH17 cells can protect against bacterial pathogens by
recruiting neutrophils but have also been reported to develop into
an immunopathology in various models of autoimmunity [1–4].
Multiple sclerosis (MS) is a chronic autoimmune disease of the
central nervous system (CNS) characterized by inflammatory cell
infiltration and subsequent demyelination of axonal tracts in the
brain and spinal cord . Demyelination disturbs the conduction
of neuronal signals along axons, resulting in clinical symptoms
including pain, fatigue, muscle weakness, and visual disturbances
. Several studies report that TH17 cells are involved in the
initiation and maintenance of experimental autoimmune enceph-
alomyelitis (EAE), a murine model of MS [6,7]. In addition, recent
studies suggest that TH17 cells (i.e., IL-17+TH17 cells) have a high
inflammatory potential and may constitute a relevant inflamma-
tory subset in human MS [8,9]. Some of these TH17 cells secrete
IFN-c (i.e., IFN-c+TH17 cells), which preferentially migrates into
the CNS in human MS [10,11].
Although the exact cause of MS remains unclear, genetic
background and/or unknown environmental factors are believed
to contribute to the onset of the disease. Epidemiological studies
have shown that geographical location is associated with the
incidence of MS, which increases with latitude in both
hemispheres . One potential explanation is that susceptibility
to MS is related to exposure to sunlight and the subsequent
production of vitamin D . In one recent study, levels of
vitamin D were significantly lower in relapsing-remitting patients
than in healthy controls . In addition, the level of vitamin D
production in MS patients suffering a relapse was lower than in
patients during remission . Furthermore, vitamin D supple-
mentation and higher levels of vitamin D in circulation are
associated with a decreased incidence of MS [15,16].
Vitamin D is a well-known nutrient that acts as a modulator
of calcium homeostasis and the immune response , and
the vitamin D receptor (VDR) is expressed in several types of
immune cells, including monocytes, macrophages, dendritic cells
(DCs), and effector/memory T cells [18–20]. In in vitro studies,
1,25(OH)2D3 inhibits T cell proliferation, the production of
IL-2 and IFN-c and cytotoxicity [21–23]. 1,25(OH)2D3 nega-
tively regulates the differentiation, maturation, and immunosti-
PLoS ONE | www.plosone.org1 September 2010 | Volume 5 | Issue 9 | e12925
mulatory capacity of DCs by decreasing the expression of MHC
class II, CD40, CD80, and CD86 [24–26]. In addition,
1,25(OH)2D3decreases the synthesis of IL-6, IL-12, and IL-23
[27–29]. Hence it seems likely that 1,25(OH)2D3suppresses the
generation of TH1 and TH17 cells and probably induces the
development of forkhead box protein 3 (Foxp3)+Treg cells.
However, the direct effect of 1,25(OH)2D3on the function and
differentiation of T cells is largely unknown because VDR is not
expressed in naı ¨ve T cells . Thus, these inhibitory effects of
1,25(OH)2D3are most pronounced in the effector/memory T cells
which do express VDR or are mediated by 1,25(OH)2D3-treated
In this study, we addressed whether 1,25(OH)2D3 directly
down-regulates the development of both Treg and TH17 cells.
These inhibitory capabilities of 1,25(OH)2D3are dependent on
the VDR signal in activated CD4+
1,25(OH)2D3 regulates the migration of TH17 cells into the
CNS by suppressing CCR6 expression. Our findings establish
that oral treatment with systemic 1,25(OH)2D3 directly modu-
lates to T cells to prevent both the development of TH17 cells
and the expression of CCR6 in EAE-induced conditions.
Therefore, vitamin D3could be applicable in both preventive
and therapeutic strategies for TH17-mediated autoimmune
T cells. Importantly,
1,25(OH)2D3inhibits the onset of EAE and alters THcell
To develop an animal experimental model of EAE, B6 mice
were immunized subcutaneously with a peptide consisting of
myelin oligodendrocyte glycoprotein (MOG33–55) in complete
Freund’s adjuvant (CFA) and pertussis toxin as described
elsewhere [31–34]. The severity of the resulting paralysis was
determined as a disease score. Symptoms were shown at 9 days
after challenge and high severity of paralysis was shown at about
20 days (Figure 1A). To confirm whether vitamin D3inhibits EAE
initiation, mice were orally treated with 1,25(OH)2D3as described
elsewhere . Of note, most 1,25(OH)2D3-treated mice were
completely resistant to the development of EAE (Figure 1A). Since
previous studies demonstrated that autoreactive T cells, especially
TH1 and TH17, are essential to induce EAE, we further analyzed
THcells in EAE-induced mice. To this end, mononuclear cells in
the CNS (including the brain and spinal cord) were enriched by
density gradient and analyzed by flow cytometry. As depicted in
Figure 1B, significantly fewer infiltrated CD4+T cells were present
in the CNS of the 1,25(OH)2D3-treated EAE-induced mice than in
the CNS of PBS-treated EAE-induced mice. We further analyzed
the THdifferentiation in the spleen and CNS of EAE-induced
mice with and without oral 1,25(OH)2D3. As expected, IL-17-
secreting TH17 cells were predominant in the spleen of EAE-
induced mice when compared with the untreated wild-type B6
mice (Figure 1C). Of note, oral treatment with 1,25(OH)2D3
dramatically reduced the numbers of TH17 cells in the spleen of
EAE-inducedmice (Figure1C, p=0.00114). In addition,
increased numbers of TH17 cells were detected in the CNS of
EAE-induced mice (Figure 1C) whereas no TH17 cells were
detected in the CNS of 1,25(OH)2D3-treated mice (data not
shown). The number of Foxp3+cells in the spleen of 1,25(OH)2D3-
treated mice was slightly decreased, but the numbers of IL-10 and
IFN-c expressing cells in the spleen of all groups of mice were
identical. Taken together, these results suggest that vitamin D3
may regulate the differentiation and/or migration of CD4+T cells
in the EAE inductive phase.
1,25(OH)2D3inhibits in vitro differentiation of both Treg
and TH17 cells
We next examined the potential role of vitamin D3on TH
generation by using well-established in vitro conditions. An in vitro
treatment of 1,25(OH)2D3on MOG-specific CD4+T cells in the
presence of MOG peptide, antigen-presenting cells (APCs), and
TGF-b inhibited the expression of Foxp3 (Figure 2). Of note,
1,25(OH)2D3also inhibited the generation of IL-17-secreting cells
in the presence of TGF-b and IL-6 (Figure 2). In addition, since an
inhibitory role of vitamin D3on TH1 differentiation has been
reported , we investigated the effect of 1,25(OH)2D3under
TH1 polarizing-conditions. However, the effect of 1,25(OH)2D3on
the differentiation of IFN-c-secreting cells was not addressed in
our system (Figure 2). To make clear whether 1,25(OH)2D3can
directly inhibit TH17 T cell differentiation regardless of antigen
type, we used DO11.10 mice, which have OVA-specific CD4+
T cells. An in vitro culture of naı ¨ve KJ1-26+CD4+T cells with
1,25(OH)2D3 in the presence of OVA peptide and APCs
significantly inhibited the generation of both Foxp3 and IL-17-
secreting cells (Figure 3A). Similar to MOG-specific CD4+T cells,
1,25(OH)2D3did not affect the differentiation of IFN-c-secreting
cells (Figure 3A). The mRNA levels of Foxp3 and IL-17 also
declined in 1,25(OH)2D3-treated CD4+T cells (Figure 3B). We
also confirmed that 1,25(OH)2D3 inhibited Foxp3 and IL-17
expression in a dose-dependent manner (data not shown). Overall,
our results demonstrate that vitamin D3 has a significant
suppressive effect on Treg and TH17 generation but not on TH1
Inhibition of Treg and TH17 differentiation by
1,25(OH)2D3is dependent on the VDR on CD4+T cells
The biological actions of vitamin D3are mediated through the
VDR, a member of the nuclear receptor superfamily . To
investigate whether VDR is essential for vitamin D3to regulate TH
cell differentiation, we used VDR2/2
deficiency of the VDR did not influence Treg and TH17
differentiation (Figure 4A and B). Of note, CD4+T cells isolated
from VDR2/2mice were resistant to the inhibitory effect of
vitamin D3on the differentiation of Treg (Figure 4A) and TH17
(Figure 4B) under polarizing conditions. In contrast, the inhibitory
role of 1,25(OH)2D3on Treg and TH17 differentiation was still
shown when VDR2/2APCs were adopted (Figure 4A and B).
Therefore, the VDR signal on activated CD4+T cells was essential
to down-regulate the development of Treg and TH17 cells.
mice. As expected,
Down-regulation of Treg differentiation by 1,25(OH)2D3
is dependent on the low production of IL-2
Since vitamin D3inhibits the secretion of IL-2, which is essential
for the generation of Treg cells [37,38], we first measured IL-2
levels in the culture supernatant after stimulation with vitamin D3.
Interestingly, co-culture with vitamin D3decreased IL-2 produc-
tion by CD4+T cells in a dose-dependent manner (Figure 5A). To
investigate whether IL-2 recovers from the decrease of Treg
differentiation caused by 1,25(OH)2D3, we added recombinant
IL-2 (rIL-2) on the culture medium of CD4+T cells in the
presence of TGF-b and 1,25(OH)2D3. The addition of rIL-2
resulted in recovery of the Foxp3+Treg cells that had been
decreased by vitamin D3compared with the numbers of Treg cells
in the TGF-b-alone group (Figure 5B). However, in contrast to
recovery of Treg cells following the addition of rIL-2, addition of
rIL-2 did not reverse the inhibitory role of vitamin D3on the
generation of TH17 cells (Figure 5C). These results suggest that
vitamin D3’s ability to decrease the number of Treg cells may be
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the result of its inhibitory effect on the amount of IL-2 secreted by
Regulation of TH17 differentiation by vitamin D3is
independent of IL-10
Since a previous study showed that IL-10 plays a crucial role in
the vitamin D3-mediated inhibition of EAE , we further
assessed the role of IL-10 on the inhibition of IL-17 production by
1,25(OH)2D3in activated T cells upon stimulation with TGF-b
and IL-6. Treatment with 1,25(OH)2D3alone did not increase the
number of IL-10-producing T cells whereas co-treatment with
TGF-b and 1,25(OH)2D3led to a brisk increase in the number of
IL-10-producing CD4+T cells (Figure 6A and B), and co-
treatment with IL-6 synergistically helped to produce IL-10
1,25(OH)2D3on IL-10 secretion under TH17-polarizing condi-
tions. Treatment of CD4+T cells with 1,25(OH)2D3 in the
presence of TGF-b and IL-6 enhanced IL-10 production in a
dose-dependent manner (Figure 6D).
Previous studies reported that IL-27 was up-regulated in APCs
isolated from the CNS and lymph nodes of EAE-induced mice
. In addition, a combination of IL-27 and TGF-b has been
Figure 1. 1,25(OH)2D3inhibits the onset of EAE and modulates the composition of THcells. (A) Disease scores are shown for EAE in B6
mice at various time points after subcutaneous immunization with MOG35–55peptide in CFA and pertussis toxin. Results shown are mean 6 SD.
**p,0.01, ***p,0.001, compared with EAE-PBS group. (B) At 20 days after challenge, total mononuclear cells obtained from the brains of MOG35–55-
immunized wild-type mice and vitamin D3-treated mice and stained with anti-CD4 and anti-CD3 Abs. Data are representative of three independent
experiments with at least five mice per group. ***p,0.001, compared with EAE-PBS group. (C) Mononuclear cells from brains or splenocytes were
restimulated in vitro with PMA/ionomycin for 5 hr, then stained intracellularly for Foxp3, IL-17A, IL-10, and IFN-c. Data are representative of three
independent experiments with at least five mice per group. *p,0.05, compared with splenocytes of EAE-PBS group.
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shown to promote the differentiation of IL-10-producing Tr-1 cells
[34,40]. Therefore, it is possible that vitamin D3might cooperate
with IL-27 to suppress TH17 differentiation through IL-10.
Interestingly, under TH17-polarizing conditions, treatment with
a combination of IL-27 and 1,25(OH)2D3generated a significantly
higher number of IL-10-secreting cells when compared with
the number of IL-10-secreting cells produced following treatment
with IL-27 alone (Figure 6E). These data suggest that enhanced
IL-10 production following treatment with vitamin D3 may
regulate TH17 differentiation via an autocrine effect in the EAE
inductive phase. To clarify the exact role of IL-10 in the
suppression of TH17 differentiation by vitamin D3, we adopted
IL-102/2mice. Under TH17-polarizing conditions, treatment
with 1,25(OH)2D3decreased IL-17 expression in T cells isolated
from both IL-10+/+and IL-102/2mice (Figure 6F). These results
imply that IL-10 might not be directly involved in the suppressive
role that vitamin D3 has on TH17 differentiation. Moreover,
vitamin D3 may be a ‘‘helper’’ in the generation of IL-10-
producing cells in an inflammatory environment but the effect of
IL-10 is not essential for vitamin D3’s negative regulation of TH17
The mechanism of suppression of TH17 generation by
1,25(OH)2D3is independent on STAT1
Since the effect of vitamin D3is similar to that of IL-27, which
inhibits the development of TH17 cells through STAT1-dependent
mechanisms [41–43], we adopted STAT12/2mice to help us
address the role that STAT1 signaling has on vitamin D3’s
inhibitory effect on TH17 differentiation. As expected, IL-27 failed
to inhibit TH17 development in STAT12/2T cells under TH17-
polarizing conditions (Figure 7). However, under TH17-polarizing
conditions, 1,25(OH)2D3 suppressed IL-17 expression in both
STAT12/2and STAT1+/+CD4+T cells (Figure 7). These results
indicate that the negative regulation of TH17 by vitamin D3is
independent on STAT1.
1,25(OH)2D3negatively regulates the expression and
migration of CCR6+T cells
A recent study reported that the CCR6-CCL20 axis plays an
essential role in controlling the entry of TH17 cells into the CNS
and thus mediates the initiation of EAE . In our present study,
we found significantly reduced migration of CD4+T cells into the
Figure 2. 1,25(OH)2D3negatively regulates Treg and TH17 induction in neuro-antigen-specific CD4+ +T cells. CD4+T cells isolated from
MOG TCR-Tg mice (Va3.2 and Vb11 TCR, B6 background) were cultured with MOG35–55peptide (25 mg/ml) in the presence of CD3+T cell-depleted
splenocytes for 4 days under Treg-polarizing conditions (rTGF-b, 1 ng/ml; anti-IFN-c, 10 mg/ml; and anti-IL-4, 10 mg/ml) or TH17-polarizing conditions
(rTGF-b, 1 ng/ml; rIL-6, 20 ng/ml; anti-IFN-c, 10 mg/ml; and anti-IL-4, 10 mg/ml) or TH1-polarizing conditions (rIL-12, 10 ng/ml; and anti-IL-4, 10 mg/ml)
together with 1,25(OH)2D3(VitD, 100 nM). Cells were then stained intracellularly for Foxp3, IL-17, or IFN-c, respectively. The plots shown are gated on
CD4+Va3.2+cells with quadrants drawn based on isotype controls. Data are representative of two independent experiments with at least three mice
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CNS following oral feeding of 1,25(OH)2D3 (Figure 1B). To
investigate the direct effect of vitamin D3on the migration of
TH17 cells into the CNS, we analyzed the CCR6 expression of the
OVA-specific CD4+T cells under TH17-polarizing conditions.
Interestingly, 1,25(OH)2D3directly inhibited CCR6 expression in
the presence of TGF-b and IL-6 (Figure 8A). We further checked
the expression levels of CCR6 in an EAE-relevant T cell system.
Interestingly, 1,25(OH)2D3reduced the expression of CCR6 on
Figure 3. 1,25(OH)2D3negatively regulates Treg and TH17 induction in OVA-specific CD4+ +T cells. (A) Naı ¨ve CD4+T cells from Rag22/2
DO11.10 mice (BALB/c background) were cultured with 0.25 mM OVA323–339peptide in the presence of CD3+T cell-depleted splenocytes for 4 days
under polarizing conditions (Treg, TH17, or TH1) together with retinoic acid (RA, 100 nM) or 1,25(OH)2D3(VitD, 100 nM) as described for Figure 2. Then
cells were stained intracellularly for Foxp3, IL-17, or IFN-c, respectively. The plots shown are gated on CD4+KJ1-26+cells with quadrants drawn based
on isotype controls. The numbers in the quadrants indicate cell percentages (left). Means 6 SD of triplicate samples are plotted (right). Data are
representative of five independent experiments with at least three mice per group. **p,0.01 compared with each cytokine-alone group. (B)
Expression of Foxp3 and IL-17 genes was analyzed by quantitative PCR. Data are representative of five independent experiments with at least three
mice per group.
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Figure 4. Vitamin D receptor on CD4+ +T cells is required for regulation of Treg and TH17 differentiation by 1,25(OH)2D3. Purified naı ¨ve
CD4+T cells from wild-type (WT) or VDR2/2(KO) mice of B6 background were cultured with APCs from WT or VDR2/2mice in the presence of 1 mg/
ml anti-CD3 mAb for 4 days under Treg-polarizing conditions (rTGF-b, 1 ng/ml; anti-IFN-c, 10 mg/ml; and anti-IL-4, 10 mg/ml) or TH17-polarizing
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activated MOG-specific CD4+T cells (data not shown). To further
address the regulation of CCR6 expression by 1,25(OH)2D3, we
evaluated the migratory characteristics of TH17 cells generated in
vitro using the Transwell chemotaxis assay. Interestingly, we found
that TH17 cells elicited by TGF-b and IL-6 signals migrated
principally toward MIP-3a/CCL20 (Figure 8B). Of note,
1,25(OH)2D3, 1,25(OH)2D3-treated TH17 cells migrated, to a
much lesser degree, toward MIP-3a/CCL20 (Figure 8B). These
results suggest that vitamin D3inhibits the CCR6 expression on
TH17 cells, which may block TH17 cells from entering the CNS.
In this study, we found that oral administration with
1,25(OH)2D3significantly reduced the number of lymphocytes in
the CNS of EAE-induced mice. The active form of vitamin D3is a
direct inhibitor for TH17 differentiation via the VDR signal but
works independently of IL-2, IL-10, and STAT1 signals in vitro. In
addition, we studied whether vitamin D3negatively regulate the
expression of IL-6R to inhibit TH17 differentiation but we did not
see any significant differences in the IL-6R expression of CD4+T
cells after co-culture with IL-6, TGF-b and vitamin D3(data not
shown). Most importantly, 1,25(OH)2D3negatively regulates the
expression of CCR6on the TH17 cells. Recently the CCR6-CCL20
axis was reported to play an essential role in controlling the entry of
TH17 cells into the CNS, thus mediating the initiation of EAE .
Our data suggest the possibility that VDR activation modulates
CCR6 expression and leads to a functional hypo-responsiveness to
CCL20. Overall, our current results imply that oral administration
of vitamin D3could be an effective tool for the treatment of TH17-
mediated autoimmune diseases.
Several recent studies reported the immunomodulatory effects of
vitamin D3 on the differentiation and function of Treg cells,
specifically the ability of topically applied vitamin D3to increase the
suppressive activity of Treg cells and the in vivo expansion of antigen-
specific Treg cells following the topical application of calcipotriol, as
a vitamin D3analog [45,46]. In addition, vitamin D3-treated DCs
induce Treg cells via independence of an inhibitory receptor
immunoglobulin-like transcript 3 (ILT3) molecule, which is required
for induction of Treg . These studies suggest that topical
application of vitamin D3might alter DC function in the periphery
and affect the differentiation and functions of Treg cells. In contrast,
our present data show thatthe expressionofTGF-b mediatedFoxp3
was inhibited by 1,25(OH)2D3via the VDR signal on CD4+T cells
(Figure 4). In particular, in vitro treatment of 1,25(OH)2D3resulted in
decreased levels of IL-2 production by activated CD4+T cells in
concurrence with prior reports [48–50]. Thus, IL-2 might be crucial
for inhibiting Treg differentiation by vitamin D3.
Although IL-2 blocks the inhibitory role of 1,25(OH)2D3on
Treg generation, 1,25(OH)2D3and IL-2 synergistically constrain
IL-17 production in CD4+T cells (Figure 5). Thus, it seems likely
that the mechanisms by which 1,25(OH)2D3 inhibits the
generation of Treg and TH17 cells differ. The inhibitory effect
of vitamin D3seems to be similar to that of IL-27, which inhibits
the lineage commitment of TH17 cells [33, 41–43 51] and induces
IL-10 production, which, in turn, suppresses EAE initiation .
Since the ability of IL-27 to block the generation of TH17 cells is
dependent on the transcription factor STAT1 [41–43], we next
sought to determine whether STAT1 is involved in 1,25(OH)2D3-
mediated inhibitory effects on the development of TH17 cells.
However, unlike IL-27, 1,25(OH)2D3’s ability to inhibit the
development TH17 cells was independent on the STAT1.
A previous study demonstrated that Smad3, signal transducers
of the TGF-b superfamily, mediated cross-talk between TGF-b
and vitamin D3signaling pathways . The cooperative actions
of the Smad3-VDR complex can be synergistic or antagonistic in a
conditional manner . In addition, another study suggested that
Figure 5. Exogenous IL-2 recovers the decreased Treg but not TH17 generation by 1,25(OH)2D3. Naı ¨ve CD4+T cells from Rag22/2
DO11.10 mice (BALB/c background) were cultured with 0.25 mM OVA323–339peptide in the presence of CD3+T cell-depleted splenocytes for 4 days.
(A) Under Treg-polarizing conditions with 1,25(OH)2D3(0.1, 1, 10, and 100 nM), culture supernatants were analyzed for IL-2 production by ELISA. (B)
Under Treg-polarizing conditions, IL-2 cytokine was added in 1,25(OH)2D3-treated groups in a dose-dependent manner (IL-2: 0.1, 1, 10, and 20 ng/ml);
4 days later the CD4+T cells were stained intracellularly for Foxp3. (C) Under TH17-polarizing conditions, 1,25(OH)2D3(100 nM) and IL-2 (0.1, 1, 10, and
20 ng/ml) were added. The average frequency of IL-17A+T cells in gated CD4+KJ1-26+cells is shown. Means 6 SD of triplicate samples are plotted.
Data are representative of three independent experiments with at least three mice per group. *p,0.05, ***p,0.001 compared with cytokine-alone
conditions (rTGF-b, 1 ng/ml; rIL-6, 20 ng/ml; anti-IFN-c, 10 mg/ml; and anti-IL-4, 10 mg/ml). (A) Foxp3 expression in gated CD3+CD4+cells was
analyzed by flow cytometry. (B) For the IL-17A staining, CD4+T cells were restimulated with PMA/ionomycin for 5 hr. Numbers beside quadrants
indicate percentages of positive cells in each quadrant. Data are representative of three independent experiments with at least three mice per group.
***p,0.001 compared with cytokine-alone group.
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PLoS ONE | www.plosone.org 7September 2010 | Volume 5 | Issue 9 | e12925
the enhancement of TGF-b-driven Smad3 signaling by retinoic
acid increases the number of Foxp3-expressing T cells and inhibits
the development of TH17 cells . These several lines of study
lead us to speculate that Smad3 mediates vitamin D3’s ability to
inhibit the development of TH17 cells. However, as of yet we have
not been able to verify this hypothesis.
Another study reported that the combination of vitamin D3and
dexamethasone increased the frequency at which IL-10-producing
regulatory T cells are generated . Further, vitamin D3failed to
inhibit EAE in IL-102/2or IL-10R2/2B6 mice . However,
in our in vitro study, vitamin D3 alone failed to induce IL-10
production in activated T cells (Figure 6A and B). Thus, it requires
additional factors to protect against EAE through the IL-10 effect.
Vitamin D3helped TGF-b mediate IL-10 production and strongly
enhanced the generation of IL-27-mediated IL-10-producing
CD4+T cells in an in vitro system (Figure 6E). A recent study
Figure 6. 1,25(OH)2D3and TGF-b plus IL-6 partially induce IL-10 production, but IL-10 is not involved in the inhibitory mechanism of
vitamin D3. (A and B) Naı ¨ve CD4+T cells from Rag22/2DO11.10 mice (BALB/c background) were cultured with OVA323–339peptides in the presence of
the indicated cytokines with and without 1,25(OH)2D3(100 nM) for 4 days. The average frequency of IL-10-producing cells is shown. (C) The dose-
dependent effect of IL-6 on IL-10 production in CD4+T cells induced by TGF-b and 1,25(OH)2D3was determined by titrated doses of IL-6 (0.1, 1, 10, and
100 ng/ml). (D) IL-10 production by CD4+T cells cocultured with CD3-depleted splenocytes and OVA323–339peptide was determined by titrated doses of
1,25(OH)2D3(0.1, 1,10, and100 nM). (E) Average frequency ofIL-10+cells amongCD3+CD4+cells as determined byflow cytometryafter treatmentwithIL-
27 and/or other indicated cytokines with or without 1,25(OH)2D3. Plots show mean 6 SD of triplicate samples. *p,0.05, **p,0.01, ***p,0.001 compared
with medium alone. (F) To analyze the effect of autocrine IL-10 on TH17 differentiation, weused IL-102/2mice of C57BL/6 background. Naı ¨ve CD4+T cells
isolatedfrom IL-102/2orIL-10+/+micewere stimulatedwithanti-CD3mAb in the presence of the indicatedcondition for 4 days. IL-17 productionin CD4+
T cells was analyzed by flow cytometry. Data are representative of three independent experiments with at least three mice per group.
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PLoS ONE | www.plosone.org8 September 2010 | Volume 5 | Issue 9 | e12925
clearly showed that IL-27 plays a crucial role in the development
of IL-10-producing anti-inflammatory T cells . Others
reported that IL-27 and IL-27R are up-regulated in APCs from
the CNS and lymph nodes in EAE-induced mice . When
considered together, the facts that IL-27 is a good inducer of IL-
10-producing T cells and that 1,25(OH)2D3possesses synergistic
effects under TH17-polarizing conditions suggest that vitamin D3
requires the presence of TGF-b and IL-6 to increase the number
of IL-27-mediated IL-10-producing T cells. Thus, it is possible
that vitamin D3cooperates with IL-27 to protect against EAE
A recent study found that CCR6 plays an essential role in the
initiation of EAE and that CCL20, a CCR6 ligand, is
constitutively expressed in choroid plexus epithelial cells in mice
and humans . Further, TH17 cells predominantly express
CCR6 . In accordance, it has been suggested that the
recruitment of TH17 cells via the CCR6-CCL20 axis is necessary
for development of TH17 cell-mediated autoimmune disease. As
depicted in Figure 1B, CD4+T cells were highly infiltrated in
EAE-induced mice whereas 1,25(OH)2D3-treated mice had
extremely low numbers of CD4+T cells in their CNS. However,
although CCR6 are important for recruitment of TH17 cells into
Figure 7. The inhibitory mechanism of 1,25(OH)2D3is independent of the STAT1 signal. Naı ¨ve CD4+T cells from STAT1+/+and STAT12/2
mice (B6 background) were cultured with anti-CD3 Abs (1 mg/ml) in the presence of CD3-depleted splenocytes for 4 days under various cytokine
treatment conditions (IL-27, 10 ng/ml; TGF-b, 1 ng/ml; IL-6, 20 ng/ml; anti-IFN-c, 10 mg/ml; or anti-IL-4, 10 mg/ml) with 1,25(OH)2D3(100 nM) and
then stained intracellularly for IL-17A and IL-10. Data are representative of three independent experiments with at least three mice per group.
**p,0.01 compared with cytokine-alone group.
Figure 8. 1,25(OH)2D3inhibits the expression of the CCR6 molecule in activated T cells. (A) Flow cytometry analysis of CCR6 expression
on activated T cells under TH17-polarizing conditions (as described for Figure 2). Data are representative of three independent experiments with at
least three mice per group. *p,0.05 compared with cytokine-alone group. (B) MIP-3a/CCL20 was added to the lower chamber and in vitro-generated
TH17 cells were applied to the upper chamber well. Two hours later, cells in the lower chamber were counted. Plots are mean 6 SD of triplicate
samples. Data are representative of two independent experiments with at least three mice per group. **p,0.01.
D3Suppresses TH17 Cell
PLoS ONE | www.plosone.org9 September 2010 | Volume 5 | Issue 9 | e12925
the mouse CNS, this has not yet been shown in human MS.
Rather IL-17 and IL-22 receptors on blood-brain barrier
endothelial cells play a crucial role on ICAM-1-mediated
migration of TH17 in MS [8,11]. Further study is required to
elucidate differences between mouse and human receptors.
We raised two hypotheses to explain the absence of lymphocytes
in the CNS after vitamin D3treatment. First, we postulated that
vitamin D3causes lymphocyte death; however, vitamin D3did not
induce apoptosis and/or cell death of activated T cells under
TH17-polarizing conditions (data not shown). Our second
hypothesis was that regulation of TH17 cell recruitment occurs
via chemokine and chemokine receptors. As expected, we found
that 1,25(OH)2D3inhibited the expression of CCR6 on T cells
that had been activated by both TGF-b and IL-6 (Figure 8). Since
one recent study also showed that vitamin D3 induces the
expression of CCR10 on activated CD4+T cells in the presence of
IL-12 , we investigated the possibility that vitamin D3also
plays a role in the ability of TH17 cells to express CCR10 instead
of CCR6. Those investigations showed that 1,25(OH)2D3did not
induce CCR10 expression on the TH17 cells in the presence of
TGF-b and IL-6 (data not shown). Overall, we found that vitamin
D3 down-regulates CCR6 but not CCR10 expression in the
In summary, our study results suggest that vitamin D3 can
directly regulate T cell development and migratory function. The
VDR signal on the CD4+T cells inhibits the expression of IL-17,
IL-2, Foxp3, and CCR6 but enhances the expression of IL-10.
These characteristic features of vitamin D3could be applied to
preventive and therapeutic strategies for TH17-mediated autoim-
Materials and Methods
Female BALB/c and C57BL/6 mice (Charles River Laborato-
ries, Seoul, Korea) were used at ages 8–12 wks. Rag22/2
DO11.10 mice (BALB/c background), MOG-TCR (2D2) trans-
genic mice (B6 background), IL-102/2mice (B6 background), and
STAT12/2(B6 background) were purchased from Taconic
(Germantown, NY) and Jackson Laboratory (Bar Harbor, ME).
VDR2/2mice were kindly provided by Prof. S. Kato (University
of Tokyo, Tokyo, Japan). All mice were maintained under
pathogen-free conditions in the experimental facility at the
International Vaccine Institute (Seoul, Korea) where they received
sterilized food and water ad libitum and all experiments described in
this article were approved by Institutional Animal Care and Use
Committees (Approval No: PN 0901).
Vitamin D3treatment and induction of EAE
One mg/ml stock of 1,25(OH)2D3(Sigma-Aldrich, St. Louis,
MO) in DMSO was added to water (50 ng/day for females;
100 ng/day for males). Alternatively, 200 ng of 1,25(OH)2D3in
oil or oil only as a placebo was injected i.p. . To induce EAE,
myelin oligodendrocyte glycoprotein peptide (MOG33–55, MEVG-
WYRSPFSRVVHLY-RNGK) was resuspended in sterile PBS to a
concentration of 4 mg/ml and then emulsified with an equivalent
volume of complete Freund’s adjuvant (CFA) supplemented with
5 mg/ml Myocobacterium tuberculosis H37Ra (BD Diagnostic Sys-
tems, Sparks, MD). EAE was induced in 9- to 10-wk old female
C57BL/6 mice by s.c. injection of 100 ml of MOG35–55/CFA
homogenate delivering 200 mg of MOG35–55peptide. On days 1
and 3 after immunization, the mice were injected i.p. with 200 ng
of pertussis toxin (Sigma-Aldrich) diluted in PBS. The mice were
then scored daily for clinical signs of EAE using the following scale
for a ‘‘disease score’’: 0= no clinical disease, 1= loss of tail tone,
2= unsteady gait, 3= hind limb paralysis, 4= forelimb paralysis,
In vitro THgeneration
CD4+CD252naı ¨ve T cells (.95% purity). To purify naı ¨ve T
cells, erythrocyte-depleted splenocytes were first depleted of
CD25+cells via magnetic selection using anti-CD25 microbeads
(Miltenyi Biotec, Auburn, CA). In the remaining population,
CD4+cells were positively selected using anti-CD4 microbeads
(Miltenyi Biotec). Cells were cultured in complete RPMI 1640
supplemented with 10% FBS and 50 U/ml of penicillin and
streptomycin. For antigen-specific stimulation, purified CD4+T
cells from MOG TCR-Tg or Rag22/2DO11.10 mice were
incubated with MOG35–55(25 mg/ml) or OVA323–339(0.2 mM)
peptide presented by CD3-depleted splenocytes under Treg-
polarizing conditions (1 ng/ml rhTGF-b1, 10 mg/ml anti-IFN-c,
and 10 mg/ml anti-IL-4); under TH17-polarizing conditions (1 ng/
ml rhTGF-b1, 20 ng/ml rmIL-6, 10 mg/ml anti-IFN-c, and
10 mg/ml anti-IL-4); or under TH1-polarizing conditions (4 ng/
ml rmIL-12 and 10 mg/ml anti-IL-4). Death cells were confirmed
by propidium iodide (PI; BD Pharmingen, San Diego, CA)
staining and were excluded before analysis.
CD4+T cells were collected and stimulated with PMA (50 ng/
ml; Sigma-Aldrich) and ionomycin (750 ng/ml; Calbiochem, La
Jolla, CA) for 5 hr in the presence of Golgi Plug (BD Pharmingen).
Anti-mouse CD3e-PerCP (145-2C11; BioLegend, San Diego, CA),
anti-mouse CD4-FITC (RM4-5; BD Pharmingen), anti-mouse
DO-11.10 Clonotypic TCR (KJ1-26; BD Pharmingen), anti-
mouse TCR Va3.2-FITC (RR3-16; BD Pharmingen), anti-mouse
TCR Vb 11 PE (RR3-15; BD Pharmingen), anti-mouse IL-17A-
APC (eBio17B7; eBioscience, San Diego, CA), anti-mouse IFN-c-
APC (XMG1.2; BD Pharmingen), anti-mouse Foxp3-APC (FJK-
16s; eBioscience), and anti-mouse IL-10-PE Abs (JES5-16E3; BD
Pharmingen) were used according to manufacturers’ instructions.
Data were obtained using a FACSCalibur (BD Immunocytometry
Systems, San Jose, CA) with CellQuest software and the profiles
were analyzed using Flowjo flow cytometry software (TreeStar
Inc., Ashland, OR).
Real-time PCR and RT-PCR
To assess the expression of IL-17 and Foxp3, mRNA was
extracted using TRIzol (Invitrogen, Camarillo, CA) according to
the manufacturer’s instructions and then reverse transcribed into
cDNA. The primer sequences for amplification of each transcript
are as follows: IL-17, 59-GGTCAACCTCAAAGTCTTTAAC-
TC-39 and 59-TTAAAAAT GCAAGTAA GTTTGCTG-39;
Foxp3, 59-CAGCTGCCTACAGTGCCCCTAG-39 and 59-CA-
TTTGC CAGCAGTGGGTAG-39; b-actin, 59- ATCTGGCAC-
CACACCTTCTACAATGAGCT GCG-39 and 59-CGTCATA-
To evaluate the migration of TH17 cells, 5-mm Transwell inserts
(Corning, Cambridge, MA) containing 16105in vitro-generated
TH17 cells were placed in the 24-well plate so as to make contact
with 600 ml of the medium alone (basal) or with 100 nM MIP-3a/
CCL20 (R&D Systems, Minneapolis, MN). Two hours later, the
inserts were removed and the population that migrated to the well
bottoms was counted.
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PLoS ONE | www.plosone.org10 September 2010 | Volume 5 | Issue 9 | e12925
Data are expressed as the mean 6 SD. Statistical comparisons
between experimental groups were performed using the Student t-
Conceived and designed the experiments: JHC MNK. Performed the
experiments: JHC HRC. Analyzed the data: JHC HRC. Contributed
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