1,25-Dihydroxyvitamin D3 Inhibits the Differentiation and Migration of TH17 Cells to Protect against Experimental Autoimmune Encephalomyelitis

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 D3 ameliorates the onset of the experimental autoimmune encephalomyelitis (EAE); however, the direct effect of vitamin D3 on 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)2D3 helps 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-10−/− T cells. Although the STAT1 signal reciprocally affects the secretion of IL-10 and IL-17, 1,25(OH)2D3 inhibits IL-17 production in STAT1−/− T cells. Most interestingly, 1,25(OH)2D3 negatively 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)2D3 can directly regulate T cell differentiation and could be applied in preventive and therapeutic strategies for TH17-mediated autoimmune diseases.


Introduction
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 naïve T cells by the polarizing cytokines TGF-b, IL-6, and IL-23 [1][2][3][4]. T H 17 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][2][3][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 [5]. Demyelination disturbs the conduction of neuronal signals along axons, resulting in clinical symptoms including pain, fatigue, muscle weakness, and visual disturbances [5]. Several studies report that T H 17 cells are involved in the initiation and maintenance of experimental autoimmune encephalomyelitis (EAE), a murine model of MS [6,7]. In addition, recent studies suggest that T H 17 cells (i.e., IL-17 + T H 17 cells) have a high inflammatory potential and may constitute a relevant inflammatory subset in human MS [8,9]. Some of these T H 17 cells secrete IFN-c (i.e., IFN-c + T H 17 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 [12]. One potential explanation is that susceptibility to MS is related to exposure to sunlight and the subsequent production of vitamin D [13]. In one recent study, levels of vitamin D were significantly lower in relapsing-remitting patients than in healthy controls [14]. In addition, the level of vitamin D production in MS patients suffering a relapse was lower than in patients during remission [14]. Furthermore, vitamin D supplementation 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 [17], 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][19][20]. In in vitro studies, 1,25(OH) 2 D 3 inhibits T cell proliferation, the production of IL-2 and IFN-c and cytotoxicity [21][22][23]. 1,25(OH) 2 D 3 negatively regulates the differentiation, maturation, and immunosti-mulatory capacity of DCs by decreasing the expression of MHC class II, CD40, CD80, and CD86 [24][25][26]. In addition, 1,25(OH) 2 D 3 decreases the synthesis of IL-6, IL-12, and IL-23 [27][28][29]. Hence it seems likely that 1,25(OH) 2 D 3 suppresses the generation of T H 1 and T H 17 cells and probably induces the development of forkhead box protein 3 (Foxp3) + Treg cells. However, the direct effect of 1,25(OH) 2 D 3 on the function and differentiation of T cells is largely unknown because VDR is not expressed in naïve T cells [30]. Thus, these inhibitory effects of 1,25(OH) 2 D 3 are most pronounced in the effector/memory T cells which do express VDR or are mediated by 1,25(OH) 2 D 3 -treated DCs.
In this study, we addressed whether 1,25(OH) 2 D 3 directly down-regulates the development of both Treg and T H 17 cells. These inhibitory capabilities of 1,25(OH) 2 D 3 are dependent on the VDR signal in activated CD4 + T cells. Importantly, 1,25(OH) 2 D 3 regulates the migration of T H 17 cells into the CNS by suppressing CCR6 expression. Our findings establish that oral treatment with systemic 1,25(OH) 2 D 3 directly modulates to T cells to prevent both the development of T H 17 cells and the expression of CCR6 in EAE-induced conditions. Therefore, vitamin D 3 could be applicable in both preventive and therapeutic strategies for T H 17-mediated autoimmune disease.

1,25(OH) 2 D 3 inhibits the onset of EAE and alters T H cell composition
To develop an animal experimental model of EAE, B6 mice were immunized subcutaneously with a peptide consisting of myelin oligodendrocyte glycoprotein (MOG  in complete Freund's adjuvant (CFA) and pertussis toxin as described elsewhere [31][32][33][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 D 3 inhibits EAE initiation, mice were orally treated with 1,25(OH) 2 D 3 as described elsewhere [32]. Of note, most 1,25(OH) 2 D 3 -treated mice were completely resistant to the development of EAE ( Figure 1A). Since previous studies demonstrated that autoreactive T cells, especially T H 1 and T H 17, are essential to induce EAE, we further analyzed T H cells 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) 2 D 3 -treated EAE-induced mice than in the CNS of PBS-treated EAE-induced mice. We further analyzed the T H differentiation in the spleen and CNS of EAE-induced mice with and without oral 1,25(OH) 2 D 3 . As expected, IL-17secreting T H 17 cells were predominant in the spleen of EAEinduced mice when compared with the untreated wild-type B6 mice ( Figure 1C). Of note, oral treatment with 1,25(OH) 2 D 3 dramatically reduced the numbers of T H 17 cells in the spleen of EAE-induced mice ( Figure 1C, p = 0.00114). In addition, increased numbers of T H 17 cells were detected in the CNS of EAE-induced mice ( Figure 1C) whereas no T H 17 cells were detected in the CNS of 1,25(OH) 2 D 3 -treated mice (data not shown). The number of Foxp3 + cells in the spleen of 1,25(OH) 2 D 3treated 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 D 3 may regulate the differentiation and/or migration of CD4 + T cells in the EAE inductive phase.

1,25(OH) 2 D 3 inhibits in vitro differentiation of both Treg and T H 17 cells
We next examined the potential role of vitamin D 3 on T H generation by using well-established in vitro conditions. An in vitro treatment of 1,25(OH) 2 D 3 on 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) 2 D 3 also 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 D 3 on T H 1 differentiation has been reported [35], we investigated the effect of 1,25(OH) 2 D 3 under T H 1 polarizing-conditions. However, the effect of 1,25(OH) 2 D 3 on the differentiation of IFN-c-secreting cells was not addressed in our system ( Figure 2). To make clear whether 1,25(OH) 2 D 3 can directly inhibit T H 17 T cell differentiation regardless of antigen type, we used DO11.10 mice, which have OVA-specific CD4 + T cells. An in vitro culture of naïve KJ1-26 + CD4 + T cells with 1,25(OH) 2 D 3 in the presence of OVA peptide and APCs significantly inhibited the generation of both Foxp3 and IL-17secreting cells ( Figure 3A). Similar to MOG-specific CD4 + T cells, 1,25(OH) 2 D 3 did 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) 2 D 3 -treated CD4 + T cells ( Figure 3B). We also confirmed that 1,25(OH) 2 D 3 inhibited Foxp3 and IL-17 expression in a dose-dependent manner (data not shown). Overall, our results demonstrate that vitamin D 3 has a significant suppressive effect on Treg and T H 17 generation but not on T H 1 differentiation.

Inhibition of Treg and T H 17 differentiation by 1,25(OH) 2 D 3 is dependent on the VDR on CD4 + T cells
The biological actions of vitamin D 3 are mediated through the VDR, a member of the nuclear receptor superfamily [36]. To investigate whether VDR is essential for vitamin D 3 to regulate T H cell differentiation, we used VDR 2/2 mice. As expected, deficiency of the VDR did not influence Treg and T H 17 differentiation ( Figure 4A and B). Of note, CD4 + T cells isolated from VDR 2/2 mice were resistant to the inhibitory effect of vitamin D 3 on the differentiation of Treg ( Figure 4A) and T H 17 ( Figure 4B) under polarizing conditions. In contrast, the inhibitory role of 1,25(OH) 2 D 3 on Treg and T H 17 differentiation was still shown when VDR 2/2 APCs 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 T H 17 cells.

Down-regulation of Treg differentiation by 1,25(OH) 2 D 3 is dependent on the low production of IL-2
Since vitamin D 3 inhibits 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 D 3 . Interestingly, co-culture with vitamin D 3 decreased IL-2 production 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) 2 D 3 , 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) 2 D 3 . The addition of rIL-2 resulted in recovery of the Foxp3 + Treg cells that had been decreased by vitamin D 3 compared 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 D 3 on the generation of T H 17 cells ( Figure 5C). These results suggest that vitamin D 3 's ability to decrease the number of Treg cells may be the result of its inhibitory effect on the amount of IL-2 secreted by CD4 + T cells.

Regulation of T H 17 differentiation by vitamin D 3 is independent of IL-10
Since a previous study showed that IL-10 plays a crucial role in the vitamin D 3 -mediated inhibition of EAE [32], we further assessed the role of IL-10 on the inhibition of IL-17 production by 1,25(OH) 2 D 3 in activated T cells upon stimulation with TGF-b and IL-6. Treatment with 1,25(OH) 2 D 3 alone did not increase the number of IL-10-producing T cells whereas co-treatment with TGF-b and 1,25(OH) 2 D 3 led to a brisk increase in the number of IL-10-producing CD4 + T cells ( Figure 6A and B), and cotreatment with IL-6 synergistically helped to produce IL-10 ( Figure 6C). We then explored the dose-dependency of 1,25(OH) 2 D 3 on IL-10 secretion under T H 17-polarizing conditions. Treatment of CD4 + T cells with 1,25(OH) 2 D 3 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 [39]. In addition, a combination of IL-27 and TGF-b has been shown to promote the differentiation of IL-10-producing Tr-1 cells [34,40]. Therefore, it is possible that vitamin D 3 might cooperate with IL-27 to suppress T H 17 differentiation through IL-10. Interestingly, under T H 17-polarizing conditions, treatment with a combination of IL-27 and 1,25(OH) 2 D 3 generated 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 D 3 may regulate T H 17 differentiation via an autocrine effect in the EAE inductive phase. To clarify the exact role of IL-10 in the suppression of T H 17 differentiation by vitamin D 3 , we adopted IL-10 2/2 mice. Under T H 17-polarizing conditions, treatment with 1,25(OH) 2 D 3 decreased IL-17 expression in T cells isolated from both IL-10 +/+ and IL-10 2/2 mice ( Figure 6F). These results imply that IL-10 might not be directly involved in the suppressive role that vitamin D 3 has on T H 17 differentiation. Moreover, vitamin D 3 may be a ''helper'' in the generation of IL-10producing cells in an inflammatory environment but the effect of IL-10 is not essential for vitamin D 3 's negative regulation of T H 17 generation.
The mechanism of suppression of T H 17 generation by 1,25(OH) 2

D 3 is independent on STAT1
Since the effect of vitamin D 3 is similar to that of IL-27, which inhibits the development of T H 17 cells through STAT1-dependent mechanisms [41][42][43], we adopted STAT1 2/2 mice to help us address the role that STAT1 signaling has on vitamin D3's inhibitory effect on T H 17 differentiation. As expected, IL-27 failed to inhibit T H 17 development in STAT1 2/2 T cells under T H 17polarizing conditions (Figure 7). However, under T H 17-polarizing conditions, 1,25(OH) 2 D 3 suppressed IL-17 expression in both STAT1 2/2 and STAT1 +/+ CD4 + T cells (Figure 7). These results indicate that the negative regulation of T H 17 by vitamin D 3 is independent on STAT1.
1,25(OH) 2 D 3 negatively 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 T H 17 cells into the CNS and thus mediates the initiation of EAE [44]. In our present study, we found significantly reduced migration of CD4 + T cells into the   activated MOG-specific CD4 + T cells (data not shown). To further address the regulation of CCR6 expression by 1,25(OH) 2 D 3 , we evaluated the migratory characteristics of T H 17 cells generated in vitro using the Transwell chemotaxis assay. Interestingly, we found that T H 17 cells elicited by TGF-b and IL-6 signals migrated principally toward MIP-3a/CCL20 ( Figure 8B). Of note, consistent with the suppression of CCR6 expression by 1,25(OH) 2 D 3 , 1,25(OH) 2 D 3 -treated T H 17 cells migrated, to a much lesser degree, toward MIP-3a/CCL20 ( Figure 8B). These results suggest that vitamin D 3 inhibits the CCR6 expression on T H 17 cells, which may block T H 17 cells from entering the CNS.

Discussion
In this study, we found that oral administration with 1,25(OH) 2 D 3 significantly reduced the number of lymphocytes in the CNS of EAE-induced mice. The active form of vitamin D 3 is a direct inhibitor for T H 17 differentiation via the VDR signal but works independently of IL-2, IL-10, and STAT1 signals in vitro. In addition, we studied whether vitamin D 3 negatively regulate the expression of IL-6R to inhibit T H 17 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 D 3 (data not shown). Most importantly, 1,25(OH) 2 D 3 negatively regulates the expression of CCR6 on the T H 17 cells. Recently the CCR6-CCL20 axis was reported to play an essential role in controlling the entry of T H 17 cells into the CNS, thus mediating the initiation of EAE [44]. 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 D 3 could be an effective tool for the treatment of T H 17mediated autoimmune diseases.
Several recent studies reported the immunomodulatory effects of vitamin D 3 on the differentiation and function of Treg cells, specifically the ability of topically applied vitamin D 3 to increase the suppressive activity of Treg cells and the in vivo expansion of antigenspecific Treg cells following the topical application of calcipotriol, as a vitamin D 3 analog [45,46]. In addition, vitamin D 3 -treated DCs induce Treg cells via independence of an inhibitory receptor immunoglobulin-like transcript 3 (ILT3) molecule, which is required for induction of Treg [47]. These studies suggest that topical application of vitamin D 3 might alter DC function in the periphery and affect the differentiation and functions of Treg cells. In contrast, our present data show that the expression of TGF-b mediated Foxp3 was inhibited by 1,25(OH) 2 D 3 via the VDR signal on CD4 + T cells (Figure 4). In particular, in vitro treatment of 1,25(OH) 2 D 3 resulted in decreased levels of IL-2 production by activated CD4 + T cells in concurrence with prior reports [48][49][50]. Thus, IL-2 might be crucial for inhibiting Treg differentiation by vitamin D 3 .
Although IL-2 blocks the inhibitory role of 1,25(OH) 2 D 3 on Treg generation, 1,25(OH) 2 D 3 and 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) 2 D 3 inhibits the generation of Treg and T H 17 cells differ. The inhibitory effect of vitamin D 3 seems to be similar to that of IL-27, which inhibits the lineage commitment of T H 17 cells [33, 41-43 51] and induces IL-10 production, which, in turn, suppresses EAE initiation [34]. Since the ability of IL-27 to block the generation of T H 17 cells is dependent on the transcription factor STAT1 [41][42][43], we next sought to determine whether STAT1 is involved in 1,25(OH) 2 D 3mediated inhibitory effects on the development of T H 17 cells. However, unlike IL-27, 1,25(OH) 2 D 3 's ability to inhibit the development T H 17 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 D 3 signaling pathways [52]. The cooperative actions of the Smad3-VDR complex can be synergistic or antagonistic in a conditional manner [53]. In addition, another study suggested that the enhancement of TGF-b-driven Smad3 signaling by retinoic acid increases the number of Foxp3-expressing T cells and inhibits the development of T H 17 cells [54]. These several lines of study lead us to speculate that Smad3 mediates vitamin D 3 's ability to inhibit the development of T H 17 cells. However, as of yet we have not been able to verify this hypothesis.
Another study reported that the combination of vitamin D 3 and dexamethasone increased the frequency at which IL-10-producing regulatory T cells are generated [55]. Further, vitamin D 3 failed to inhibit EAE in IL-10 2/2 or IL-10R 2/2 B6 mice [32]. However, in our in vitro study, vitamin D 3 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 D 3 helped 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 clearly showed that IL-27 plays a crucial role in the development of IL-10-producing anti-inflammatory T cells [40]. Others reported that IL-27 and IL-27R are up-regulated in APCs from the CNS and lymph nodes in EAE-induced mice [39]. When considered together, the facts that IL-27 is a good inducer of IL-10-producing T cells and that 1,25(OH) 2 D 3 possesses synergistic effects under T H 17-polarizing conditions suggest that vitamin D 3 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 D 3 cooperates with IL-27 to protect against EAE through IL-10.
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 [44]. Further, T H 17 cells predominantly express CCR6 [56]. In accordance, it has been suggested that the recruitment of T H 17 cells via the CCR6-CCL20 axis is necessary for development of T H 17 cell-mediated autoimmune disease. As depicted in Figure 1B, CD4 + T cells were highly infiltrated in EAE-induced mice whereas 1,25(OH) 2 D 3 -treated mice had extremely low numbers of CD4 + T cells in their CNS. However, although CCR6 are important for recruitment of T H 17 cells into  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 T H 17 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 D 3 treatment. First, we postulated that vitamin D 3 causes lymphocyte death; however, vitamin D 3 did not induce apoptosis and/or cell death of activated T cells under T H 17-polarizing conditions (data not shown). Our second hypothesis was that regulation of T H 17 cell recruitment occurs via chemokine and chemokine receptors. As expected, we found that 1,25(OH) 2 D 3 inhibited 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 D 3 induces the expression of CCR10 on activated CD4 + T cells in the presence of IL-12 [57], we investigated the possibility that vitamin D 3 also plays a role in the ability of T H 17 cells to express CCR10 instead of CCR6. Those investigations showed that 1,25(OH) 2 D 3 did not induce CCR10 expression on the T H 17 cells in the presence of TGF-b and IL-6 (data not shown). Overall, we found that vitamin D 3 down-regulates CCR6 but not CCR10 expression in the T H 17-conditioned circumstance.
In summary, our study results suggest that vitamin D 3 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 D 3 could be applied to preventive and therapeutic strategies for T H 17-mediated autoimmune diseases.

Mice
Female BALB/c and C57BL/6 mice (Charles River Laboratories, Seoul, Korea) were used at ages 8-12 wks. Rag2 2/2 DO11.10 mice (BALB/c background), MOG-TCR (2D2) transgenic mice (B6 background), IL-10 2/2 mice (B6 background), and STAT1 2/2 (B6 background) were purchased from Taconic (Germantown, NY) and Jackson Laboratory (Bar Harbor, ME). VDR 2/2 mice 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 D 3 treatment and induction of EAE
One mg/ml stock of 1,25(OH) 2 D 3 (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) 2 D 3 in oil or oil only as a placebo was injected i.p. [32]. To induce EAE, myelin oligodendrocyte glycoprotein peptide (MOG 33-55 , 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 Systems, Sparks, MD). EAE was induced in 9-to 10-wk old female C57BL/6 mice by s.c. injection of 100 ml of MOG 35-55 /CFA homogenate delivering 200 mg of MOG  peptide. 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, 5 = death.

Chemotaxis assay
To evaluate the migration of T H 17 cells, 5-mm Transwell inserts (Corning, Cambridge, MA) containing 1610 5 in vitro-generated T H 17 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.

Statistics
Data are expressed as the mean 6 SD. Statistical comparisons between experimental groups were performed using the Student ttest.