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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Jun 11;105(24):8381–8386. doi: 10.1073/pnas.0803454105

Orphan nuclear receptor NR4A2 expressed in T cells from multiple sclerosis mediates production of inflammatory cytokines

Yoshimitsu Doi *, Shinji Oki *,†, Tomoko Ozawa *, Hirohiko Hohjoh ‡, Sachiko Miyake *, Takashi Yamamura *,†
PMCID: PMC2426110  PMID: 18550828

Abstract

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and Th1 cells. DNA microarray analysis previously showed that NR4A2, an orphan nuclear receptor, is strongly up-regulated in the peripheral blood T cells of MS. Here, we report that NR4A2 plays a pivotal role for mediating cytokine production from pathogenic T cells. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, NR4A2, was selectively up-regulated in the T cells isolated from the CNS. Strikingly, a forced expression of NR4A2 augmented promoter activities of IL-17 and IFN-γ genes, leading to an excessive production of these cytokines. Conversely, treatment with siRNA for NR4A2, resulted in a significant reduction in the production of IL-17 and IFN-γ. Furthermore, treatment with NR4A2 siRNA reduced the ability of encephalitogenic T cells to transfer EAE in recipient mice. Thus, NR4A2 is an essential transcription factor for triggering the inflammatory cascade of MS/EAE and may serve as a therapeutic target.

Keywords: IL-17, interferon-γ, EAE, Th17, siRNA

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS), accompanying multiple foci of inflammatory lesions. MS is thought to have an autoimmune pathogenesis, involving autoimmune T cells reactive to myelin antigens (1). Development of the CNS inflammation is triggered by proinflammatory cytokines produced by the autoimmune T cells, which penetrate into the CNS parenchyma after being activated in the periphery (2, 3). Although the precise mechanism for the peripheral T cell activation remains obscure, studies indicated possible roles for cross-reactive peptides, cytokines, or superantigen (4).

Experimental autoimmune encephalomyelitis (EAE) is a prototype autoimmune disease model (5) that can be induced in laboratory animals by active immunization with myelin antigens (mAg) or by passive transfer of mAg-specific T cells. Because Th1 cell clones reactive to mAg are capable of inducing clinical and pathological manifestations of EAE in naive mice, it has long been believed that Th1 cells producing IFN-γ play a central role in the pathogenesis of EAE and MS. This postulate is also supported by the past experience that clinical application of IFN-γ treatment for MS turned out to worsen the disease (6). Furthermore, treatment with a peptide analogue of myelin basic protein (MBP) resulted in disease exacerbation along with an expansion of MBP-reactive Th1 cells (7). These results have been repeatedly mentioned to support the Th1-mediated pathogenesis of MS. However, this dogma has recently been challenged. Namely, despite an obvious reduction of Th1 cells, mice deficient for IFN-γ or IFN-γ receptor (8) or for IL-12 signaling were susceptible to EAE (9, 10). Subsequent studies have clarified that IL-23 rather than IL-12 is essential for EAE induction. Lately, the IL-23-dependent pathogenic T cells were identified as Th17 cells, a novel helper T cells producing IL-17 (11, 12). Currently, it is widely appreciated that Th17 cells are crucial in the development of autoimmune diseases either independently or collaboratively with Th1 cells (13).

DNA microarray analysis revealed an up-regulation of IL-17 in the brain lesions of MS (14). More recently, a pathological study has demonstrated that IL-17 secreting T cells are present in active lesions of MS (15). Gene expression profiling provided a number of potential candidate molecules that might be appropriate as a therapeutic target (14, 16). We recently characterized gene signature of peripheral blood T cells from Japanese MS patients and found that a nuclear orphan receptor NR4A2 is most significantly overexpressed in MS (17). NR4A2 mutations are reported to cause familial Parkinson's disease, reflecting its essential role in the development and survival of substantia nigra neurons (18). In contrast, much less attention has been paid onto its role in T cells. NR4A family members (NR4A1 and -3) were shown to mediate apoptotic processes of mature (19, 20) and immature T cells (21, 22). However, these studies do not give insights into an overexpressed NR4A2 in MS. Here, we report that NR4A2 is a transcription factor regulating the expression of key cytokines in the pathogenesis of MS, including IL-17. Furthermore, we revealed that silencing NR4A2 expression by specific siRNA effectively prevents the production of the cytokines, thereby inhibiting their pathogenic potentials to mediate EAE.

Results

Up-Regulation of NR4A2 in Peripheral Blood T Cells of MS.

We analyzed gene expression profiles of peripheral blood T cells from MS and control subjects (17, 23). Comparison of the patients and healthy donors has revealed that 286 of 1,263 genes are differentially expressed between MS and controls. Among genes up-regulated in MS, NR4A2 was most significantly overexpressed in MS in statistical P values and an increase ratio (3.6-fold). To consolidate the overexpression of NR4A2 in MS, we performed quantitative RT-PCR for NR4A2 expression, using the same samples previously analyzed. Expression of NR4A2 in T cells from MS increased ≈5-fold on average compared with healthy donors (Fig. 1; P < 0.01).

Fig. 1.

Fig. 1.

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Quantitative analysis of NR4A2 transcription between MS and controls. CD3+ T cells were isolated from PBMC of 57 MS patients and of 19 healthy donors, and total RNA was extracted. cDNA was synthesized and the expression levels of NR4A2 transcript were analyzed by quantitative RT-PCR. Each sample was normalized to GAPDH to adjust for variations. Open circles, MS patients; filled circles, healthy controls. Bars indicate mean values of each group. The statistical difference was determined by two-sided Student t test (∗∗, P < 0.01).

T Cell Expression of NR4A2 in EAE.

NR4A2 is a transcription factor of steroid/thyroid receptor family implicated in various cellular responses such as steroidogenesis, neuronal development, atherogenesis, and cell cycle regulation (24). However, its role in T cell-mediated autoimmune diseases is unknown. Therefore, we explored the functional involvement of NR4A2 in EAE induced in C57BL/6 (B6) mice by immunization with MOG35–55. CD3+ T cells were isolated from SPL, dLN, and PBMC after EAE induction and the expression levels of NR4A2 gene were measured by quantitative RT-PCR (Fig. 2a Right). NR4A2 expression was detectable in PBMC-T cells on days 14, 21, and 28, showing a maximum value on day 21, which was well correlated with the clinical severity of EAE (Fig. 2a Left). NR4A2 expression in SPL-T cells and dLN-T cells was also correlated with the severity of EAE, but only marginally.

Fig. 2.

Fig. 2.

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Kinetic analysis of NR4A2 expression in the disease course of EAE. (a) (Left) EAE was induced in B6 mice by immunization with MOG35-55 in CFA. Mice were killed on days 7, 14, 21 and 28 after immunization, and T cells were isolated from dLN, SPL, or PBMC, using anti-CD3 magnetic beads. (Right) Total RNAs were isolated from the T cell populations, and the expression levels of NR4A2 were determined by quantitative RT-PCR. One representative data from three independent experiments is shown, and data are expressed as mean ± SEM (n = 5 for each). (b) EAE induced in B6 mice with MOG35-55. Clinical scores were expressed as mean ± SEM (n = 4). Here, we determined NR4A2 expression in CD3+ T cells isolated by using EPICS ALTRA cell sorter. The lymphoid cells (SPL, dLN, and CNS) were pooled from four mice on days 0, 9, 15, and 21 and used for cell sorting and RT-PCR analysis. The purity of the CNS-derived CD3+ T cells was >93%.

In the course of EAE, mAg-primed T cells would accumulate into the CNS and produce inflammatory cytokines, leading to the formation of inflammatory lesions (25). We next examined a kinetic change of NR4A2 in the T cells infiltrating into the CNS. As assessed by quantitative RT-PCR, remarkable expression of NR4A2 was observed in the CNS-T cells on day 9, when an early EAE sign became evident (Fig. 2b). The expression level decreased gradually thereafter, but was still significant until day 21. These results suggest that the CNS-T cells also express NR4A2, but the expression kinetics significantly differed from that of PBMC-T cells.

Accumulation of IL-17- and IFN-γ-Producing T Cells in the CNS of EAE.

Th1 cells specific for mAg have long been thought to induce EAE through their production of IFN-γ. However, recent studies indicate that Th17 rather than Th1 cells may play a central role (13). To make this point clear in our experimental setting, we examined the ability of the CNS-T cells to produce IFN-γ and IL-17. Mononuclear cells were recovered from the CNS and SPL on day 17, and stimulated with PMA and ionomycin (P/I). After immunostaining, expression of IL-17 or IFN-γ in the CD4+ T cells was analyzed by flow cytometry. Major proportions of the CNS-T cells were found to produce IL-17 (21.7% of the cells) or IFN-γ (28.1%) after stimulation (Fig. 3). In contrast, spleen cells contained a lower number of cells producing these cytokines.

Fig. 3.

Fig. 3.

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Accumulation of IL-17 or IFN-γ-producing inflammatory T cells in the CNS. Mononuclear cells were isolated from spleen or CNS on day 17 after immunization and stimulated with PMA (20 ng/ml) and ionomycin (1 μg/ml) in the presence of 2 mM monensin for 4 h. Production of IL-17 and IFN-γ was analyzed for the gated CD4+ T cell population by intracellular cytokine staining. Black line represents samples stained with either anti-IL-17 or anti-IFN-γ Ab, and the filled histogram represents samples stained with isotype control. Given values show the percentage of cytokine producing-T cells present in each panel.

Transcriptional Up-Regulation of IL-17 and IFN-γ After Introduction of NR4A2.

The concomitant expression of inflammatory cytokines and NR4A2 has guided us to investigate whether NR4A2 directly affects cytokine gene expression as a transcription factor, using luciferase reporter plasmids containing the promoter fragment of IL-17, IFN-γ, or IL-2. NR4A2 gene transduction would result in a twofold augmentation of IL-17 promoter activity and, for IFN-γ, an even higher (5-fold) induction (Fig. 4a). A significant induction of IL-2 promoter activity was also noted. Intriguingly, an introduction of NR4A2 plasmid without P/I stimulation also augmented basal promoter activity of IL-17 genes in a dose dependent manner (Fig. 4b). Similarly, basal promoter activity of IFN-γ was promoted (data not shown).

Fig. 4.

Fig. 4.

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Promoter activities of cytokine genes in the presence of NR4A2. (a) The effect of NR4A2 expression on IL-17, IFN-γ, and IL-2 promoter activity. A reporter plasmid containing promoter of cytokine gene (10 μg) and Renilla luciferase plasmid (100 ng) were introduced into EL4 cells by electroporation, together with pcDNA4-NR4A2 or pcDNA4-LacZ (10 μg). Cells were stimulated for 18 h with P/I. Luciferase activity was determined for each cell lysate after normalization to the Renilla luciferase activity. One representative data from three independent experiments is shown. Data are expressed as mean ± SD. (b) The effect of NR4A2 expression on basal promoter activity of IL-17 gene. EL4 cells transfected with pcDNA4-NR4A2 or pcDNA4-LacZ together with IL-17 reporter plasmid and Renilla luciferase plasmid as desribed in a were cultured for 18 h without stimulation. One representative data from three independent experiments is shown. Data are expressed as mean ± SD.

Retroviral Transduction of NR4A2 Gene Enhances Expression of Inflammatory Cytokine in Primary T Cells.

The results obtained in EL4 lymphoma cells need to be verified in more physiological settings. Next, we examined whether forced expression of NR4A2 may affect the expression of cytokines in primary rodent T cells. Bicistronic retroviral vector containing NR4A2 gene fragment (pMIG-NR4A2) or empty vector (pMIG) were used for production of retroviruses (Fig. 5a). We infected the B6 T cells with either of the retroviruses as described in ref. 26 and compared the cytokine production between GFP-positive (infected) and GFP-negative (uninfected) CD4+ T cells by intracellular cytokine staining (Fig. 5b Top). CD4+ T cells infected with pMIG-NR4A2-introduced retrovirus showed a twofold enhancement of IL-17 expression (8.4%) compared with those infected with control retrovirus (4.1%) after stimulation with P/I. In contrast, IL-17 production by uninfected T cells in either panel was almost equivalent (Fig. 5b Middle). Furthermore, one-third of the CD4+ T cells infected with pMIG-NR4A2-introduced retrovirus showed a massive IFN-γ expression (35.1%) compared with control retrovirus (14.1%) (Fig. 5b Bottom).

Fig. 5.

Fig. 5.

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The effect of retrovirally transduced NR4A2 on cytokine production by primary murine CD4+ T cells. (a) DNA fragments encoding wild-type NR4A2 were cloned into the pMIG(W) bicistronic retroviral vector. LTR, long terminal repeat; IRES, internal ribosome entry site; eGFP, enhanced green fluorescence protein b. (b) Splenic CD4+ T cells were infected with retrovirus encoding NR4A2 or control retrovirus, and CD4+ GFP− T cells and CD4+ GFP+ T cells were gated as R1 and R2, respectively. Forced expression of NR4A2 increased the number of CD4+ T cells producing IL-17 or IFN-γ. The histogram shows intracellular cytokine staining on the gated cells (R1 or R2). Black line represents cells in R2 gate (GFP+) stained with either anti-IL-17 or anti-IFN-γ Ab, and the filled histogram represents cells in R1 gate (GFP−) stained with isotype control. Given values show the percentage of cytokine producing-T cells present.

Silencing of NR4A2 Gene Expression Results in a Reduced Production of IL-17 and IFN-γ.

Reporter gene analysis and retroviral transduction experiments demonstrated that T cell production of IL-17 and IFN-γ is controlled by NR4A2 (Figs. 4 and 5). We further explored whether silencing of NR4A2 gene may affect the production of inflammatory cytokines by CD4+ T cells. An NR4A2-specific siRNA was selected from three siRNAs based on the inhibitory efficacy. The targeting sequence of the NR4A2 siRNA is completely conserved between mice and human. Therefore, we could apply it to human T cells and study whether NR4A2 could be a therapeutic target in human MS. In a preparatory experiment, using FITC-labeled siRNA, the transfection efficiency was found to be 95%. We purified CD4+ T cells from human PBMC and transfected them with the NR4A2 siRNA or control RNA, using nucleofector II. The cells were stimulated with immobilized anti-CD3 Ab. As shown in Fig. 6a, silencing NR4A2 gene expression resulted in a 50% reduction of IL-17 and IFN-γ production. However, production of TNF-α, IL-4, or IL-5 was not changed significantly after siRNA treatment (Fig. 6b). Intriguingly, the siRNA treatment also induced a modest reduction of IL-10 production. The molecular mechanism of this inhibition is not clarified yet. Because silencing of NR4A2 expression rather selectively inhibited the expression of inflammatory cytokines, it is arguable that NR4A2 may be a good target for therapeutic intervention of MS. In this line, we next examined whether the NR4A2 siRNA is effective for inhibiting a production of inflammatory cytokines in MS. For this aim, CD4+ T cells were isolated from pairs of an MS patient and an age- and sex-matched healthy donor and were stimulated with anti-CD3 Ab after being transfected with the NR4A2 siRNA or control RNA. We found that the siRNA treatment significantly reduced the production of IL-17 and IFN-γ by T cells from MS or healthy donors [supporting information (SI) Fig. S1]. Again we observed some reduction of IL-10 after siRNA treatment. However, the siRNA showed little effect on production of TNF-α, IL-5, and IL-4 from T cells used for assays (Table S1).

Fig. 6.

Fig. 6.

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The effect of NR4A2 gene silencing on T cell cytokine production. (a) Specific inhibition of T cell production of IL-17 and IFN-γ by siRNA treatment. Human CD4+ T cells derived from PBMC were transfected with siRNA or control RNA and stimulated by immobilized anti-CD3 Ab for 48 h. Cytokine levels in the culture supernatant were determined by ELISA or a CBA human Th1/2 cytokine kit. Proliferation rate was measured by 3H-TdR uptake. (b) Effect of siRNA treatment for T cell production of TNF-α, IL-10, IL-5, and IL-4 after stimulation with immobilized anti-CD3 Ab. The data are expressed as mean ± SD (∗, P < 0.05; ∗∗, P < 0.01; Mann–Whitney U test).

Amelioration of EAE by Silencing of NR4A2.

Finally, we investigated the therapeutic implication of the siRNA experiments in a model of passively induced EAE, induced by adoptive transfer of mAg-activated LN cells. We prepared lymphoid cells from dLN of SJL/J mice 10 days after immunization with PLP139–151. The dLN cells were transfected with the NR4A2 siRNA or control RNA and stimulated with PLP139–151 in vitro. Three days later, the cultured cells enriched in lymphoblasts were transferred to irradiated naïve SJL/J mice. In addition to evaluating clinical manifestations, histology was assessed by hematoxylin-eosin (HE) and luxol fast blue (LFB) staining of paraffin-embedded spinal cord sections. Notably, severity of clinical (Fig. 7a) and histological EAE on day 31 (Fig. 7b) was significantly prevented in siRNA-treated group compared with control RNA-treated group (Fig. 7b). These results suggest that modulation of NR4A2 expression by specific siRNAs or other chemical compounds might be a promising treatment for active MS that are harboring potent encephalitogenic T cells.

Fig. 7.

Fig. 7.

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The effect of T cell silencing of NR4A2 expression on passive EAE. (a) Inguinal and popliteal LNs cells were collected from female SJL/J mice 10 days after immunization with PLP139-151, and were transfected with siRNA for NR4A2 or control RNA, using HVJ-E vector kit. The cells were cultured in complete media for 8 h. Then the media were replaced with fresh complete media containing 35 μg/ml PLP139-151, and the cells were stimulated for another 3 days. After expansion, cells were harvested and transferred i.p. (5 × 106 cells per mouse) into 3Gy-irradiated naïve SJL/J mice (n = 10) followed by i.p. injection of PT. Mean ± SEM clinical scores were indicated. (∗, P < 0.05 by Mann–Whitney U test.) (b) Histological analysis of spinal cords removed on day 31 after adoptive transfer of PLP139-151-reactive T cells. Sections obtained from cervical cord regions were stained with HE or LFB. Infiltration of mononuclear cells and demyelination of the cervical cord regions were analyzed for mice injected with PLP139-151-reactive T cells pretreated with control RNA or siRNA for NR4A2.

Discussion

Although mAg-specific T cell clones isolated from the peripheral blood has been widely used to gain insights into the pathogenesis of MS (27), analysis of polyclonal T cells has been undervalued for a long time. However, it was recently demonstrated that peripheral T cells from MS and healthy subjects significantly differ in surface phenotype or gene expression profiling (17, 23, 28). Using cDNA microarray, we have identified NR4A2 as a gene most significantly up-regulated in the peripheral T cells of MS (17). We conducted the present study to clarify the implication of this interesting observation. Inspired by the recent discovery that retinoid-related orphan receptor γt (RORγt) is essential for Th17 cell differentiation (29) and that retinoic acids play a regulatory role in Th17 cell differentiation (30), we have focused our efforts to explore the possible role of NR4A2 in cytokine regulation. Reporter gene analysis and retroviral transduction of NR4A2 clearly demonstrated that T cell production of inflammatory cytokines, including IL-17 and IFN-γ, is regulated by NR4A2, whereas silencing of NR4A2 by a specific siRNA prevents expression of these cytokines. Furthermore, treatment with the siRNA reduced the ability of pathogenic T cells to adoptively transfer EAE. These results have identified a previously uncharacterized role for NR4A2 in the regulation of T cell production of inflammatory cytokines.

NR4A2 is a member of the orphan nuclear NR4A subfamily that consists of NR4A1 (also referred to as Nur77), NR4A2 (Nurr1), and NR4A3 (NOR-1) (24). The NR4A members share a highly conserved zing finger DNA binding domain and a less conserved putative ligand-binding domain. All these members bind to the DNA sequence NBRE (AAAGGTCA) or NurRE to activate target gene expression. NR4A1 and NR4A2 can also heterodimerize with retinoic X receptor (RXR) and activate gene expression through DR5 (24). They exert pleiotropic functions and are classified as immediate early genes induced by physiological and physical stimuli. Studies of gene-targeted mice have shown that NR4A1 and NR4A3 play a critical role in T cell apoptosis during the thymocyte development (20–22, 31). In contrast, developing thymocytes in NR4A2 deficient mice appear to be normal (21, 32), which distinguishes NR4A2 from other NR4A members.

Involvement of orphan nuclear receptor in T cell differentiation has recently attracted broad attention, because RORγt, a splice variant of RORγ, was found to play an essential role in the development of Th17 cells (29). RORγ/RORγt were reported to play an essential function in survival of CD4+CD8+ thymocytes (33, 34) and in the generation of fetal lymphoid tissue inducer (LTi) cells (35). It is particularly intriguing that the consensus binding sequence for RORγ [(A/T)5AGGTCA] overlaps with that for NR4A (NBRE; AAAGGTCA), which has encouraged us to explore the functional role of NR4A2 in the production of IL-17 and IFN-γ. Although the molecular mechanism of cytokine production through the induced expression of NR4A2 is not clear yet, NR4A2 and RORγt may have an overlapping role in regulating the development and effector functions of Th17 cells.

NR4A2 expression in the CNS-infiltrating T cells showed a peak value at a very early phase of EAE (day 9–12) (Fig. 2b). We speculate that this probably coincides with the entry of encephalitogenic cells into the CNS (2, 3). Consistently, a similar kinetic change was found in expression of T-bet and RORγt in the CNS-T cells (data not shown). In contrast, up-regulation of NR4A2 in peripheral blood T cells was significantly delayed. This is likely to result from a late activation of peripheral T cells after peripheral recruitment of antigen presenting cells engulfing myelin and/or peripheral dispersion of myelin protein or its fragments.

By applying a specific siRNA, we showed that blocking NR4A2 expression is effective for inhibiting production of IL-17 and IFN-γ from T cells from healthy donors and MS patients. Therapeutic implication was further demonstrated by using an adoptive transfer EAE model. Because Th17 cells were identified as a major player in autoimmunity (12, 15), it is sometimes argued that Th17 cells would be a sole potent inducer of autoimmune inflammation. However, T-bet-deficient mice and Stat4-deficient mice that obviously lack Th1 cells would resist against induction of EAE, although they maintain a large number of Th17 cells (36, 37). This suggests that both Th1 and Th17 cells are required for induction of full-blown EAE (38). In this context, the ability of the NR4A2 siRNA to inhibit production of both IL-17 and IFN-γ suggests the advantage of NR4A2 targeting therapy in controlling autoimmune inflammation.

Materials and Methods

EAE Induction.

Active EAE was induced with myelin oligodendrocyte glycoprotein (MOG) amino acids 35–55 (MOG35-55; MEVGWYRSPFSRVVHLYRNGK) as described in ref. 39. Female B6 mice were immunized s.c. with 100 μg of MOG35–55 mixed with 1 mg of heat-killed Mycobacterium tuberculosis H37RA emulsified in Freund's adjuvant (CFA). Pertussis toxin (PT) (200 ng) was injected i.p. on days 0 and 2 after immunization. Clinical signs were scored daily as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, flaccid tail; 3, partial hind limb paralysis; 4, total hind limb paralysis; and 5, fore and hind limb paralysis.

Quantitative RT-PCR.

DNase-treated total RNAs were processed for cDNA synthesis, using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen). cDNAs were amplified by PCR on Light Cycler ST300 (Roche Diagnostics) by using a Light Cycler-FastStart DNA Master SYBR Green I kit (Roche). Values for each gene were normalized to those of a housekeeping gene GAPDH to adjust for variations between different samples. Forward primer for amplifying human NR4A2 gene was 5′-CGACATTTCTGCCTTCTCC-3′ and reverse primer 5′-GGTAAAGTGTCCAGGAAAAG-3′. Mouse NR4A2 forward primer was designed as 5′-GCATACAGGTCCAACCCAGT-3′ and reverse primer 5′-AATGCAGGAGAAGGCAGAAA-3′. To evaluate silencing efficacy of NR4A2-specific siRNAs, expression of NR4A2 gene was quantified by RT-PCR, using the primers to flank the siRNA target sequence (forward, 5′-TGCCACCACTTCTCTCCCCA-3′; reverse, 5′-GCGGCATCATCTCCTCAGAC-3′).

Luciferase Assays.

Ten million of EL4 thymoma cells suspended in 500 μl of cold PBS and transfected with 4–20 μg of pcDNA4-NR4A2 or pcDNA4-LacZ in the presence of 10 μg of reporter plasmid, 100 ng of Renilla luciferase plasmid, and 5 μg of DEAE-DEXTRAN (Sigma) by electroporation (250 V, 975 μF, time constant = 30–34 ms) with a GenePulser electroporator II (Bio-Rad). Six hours later, cells were stimulated with 20 ng/ml PMA and 1 μg/ml ionomycin for 24 h, followed by analysis for luciferase activity. The data were normalized for internal controls of Renilla luciferase activity.

Retroviral Infection.

Mouse CD4+ T cells purified by AutoMACS using mouse CD4 T isolation kit (Miltenyi Biotec) were stimulated with immobilized anti-CD3 Ab and soluble anti-CD28 Ab in complete medium supplemented with IL-2 (100 units/ml) for 24–48 h before infection. The primed CD4+ T cells were infected twice with retroviruses produced by 293T cells cotransfected with pMIG retroviral vector and pCL-Eco packaging vector. The T cells were cultured in the presence of 30 units/ml of IL-2 for 3 days and were then subjected to intracellular cytokine staining.

Silencing Effects of NR4A2 siRNA on Passive EAE.

To evaluate an effect of NR4A2 siRNA, an adoptive transfer EAE model in SJL/J mice was applied, because consistent disease could be induced relatively easily. Female SJL/J mice (8–12 weeks old) (Charles River Laboratories) were immunized s.c. with 100 μg of proteolipid protein (PLP) amino acids 139–151 (PLP139-151; HSLGKWLGHPDKF) and 1 mg of heat-killed M. tuberculosis H37RA in CFA. Inguinal and popliteal LNs harvested 10 days after immunization were transfected with siRNAs, using hemaggultinating Virus of Japan envelope (HVJ-E) vector kit (GENOMEONE; Ishihara Sangyo). Eight hours later, the cells were stimulated with PLP139-151 peptide (35 μg/ml). After 3 days, collected cells were injected i.p. (5 × 106 cells per body) into irradiated mice (3 Gy/body) with intrapelitoneal injection of PT. For conventional histological analysis of EAE, paraffin-embedded spinal cords were stained with either HE or LFB.

Statistics.

For statistical analysis, a nonparametric Mann–Whitney U test or Student t test was used. P < 0.05 was considered statistically significant.

Supporting Information.

For further details, see SI Materials and Methods.

Supplementary Material

Supporting Information
0803454105_index.html (757B, html)

Acknowledgments.

We thank Mayumi Fujita for EAE induction, Miho Mizuno, Chiharu Tomi, and Yuki Kikai for excellent technical assistance. This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0803454105/DCSupplemental.

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