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Clin Exp Immunol. 2011 May; 164(2): 236–247.
PMCID: PMC3087916

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis

Abstract

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.

Keywords: collagen-induced arthritis, glycolipid ligand, natural killer T cells, Th1, Th17

Introduction

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation of the synovial tissues and subsequent destruction of multiple joints [1]. Although the pathogenesis of RA remains unclear, proinflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, play a central role in this process [2]. There is general agreement that interferon (IFN)-γ-producing T helper type 1 (Th1) cells play a pathogenic role in the development of RA. However, several recent studies on animal models of autoimmune diseases suggested that IL-17-producing Th17 cells, but not IFN-γ-producing Th1 cells, play a crucial role in the development of RA. For example, mice deficient in Th1 cytokines, such as IFN-γ- and IL-12-deficient mice, exhibited severe symptoms in collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) [35], whereas those deficient in Th17 cytokines, such as IL-17- and IL-23-deficient mice, were resistant to these diseases [69]. Harrington et al. [10] suggested that IFN-γ suppresses the differentiation of naive CD4 T cells to Th17 cells. Furthermore, Chu et al. [11] showed that IFN-γ also suppressed IL-17 production by differentiated Th17 cells. In addition, IFN-γ plays a suppressor role by inducing myeloid suppressor cells that induce apoptosis of activated T cells in the chronic immune response, such as the late phase of mycobacterial infection or the autoimmune response [12,13]. It has also been reported that IFN-γ is necessary for the conversion of CD4+ CD25- T cells to CD4+ regulatory T cells (Tregs) during EAE [14]. Thus, IFN-γ is thought to be a suppressive cytokine in several animal models of autoimmune diseases.

Natural killer T (NK T) cells are a subset of T lymphocytes that express NK cell markers, such as NK1·1, in mice. In mice, the majority of NK T cells express an invariant T cell receptor (TCR) encoded by Vα14Jα18, which is associated with highly skewed sets of Vβs, mainly Vβ8·2. The receptor recognizes glycolipid antigen presented by CD1d, a non-classical antigen-presenting molecule [15,16]. Stimulation of TCR induces NK T cells to rapidly secrete large amounts of proinflammatory and anti-inflammatory cytokines, such as IL-4 and IFN-γ[16]. Because of this property, NK T cells are known as immune regulators. Functional defects within NK T cells and reduced numbers of these cells are associated with various human autoimmune diseases [1720]. In animal models, NK T cells suppress the development and progression of diabetes mellitus [21], EAE [22] and systemic lupus erythematosus (SLE) [23]. However, NK T cells also act as effector cells in some murine models of RA by promoting Th17 responses, producing IL-17 and suppressing the production of transforming growth factor (TGF)-β[2428]. These evidences suggest a dual function for NK T cells in autoimmunity.

Alpha-galactosylceramide (α-GalCer) is a potent NK T cell ligand. The synthetic ligand induces NK T cell activation and secretion of various cytokines, such as IFN-γ, IL-4, and IL-17 [29]. To control NK T cell activation and cytokine secretion, several analogues of α-GalCer have been synthesized. OCH, which is an α-GalCer analogue with a shorter sphingosine chain, stimulates IL-4 production selectively by NK T cells [30]. The α-GalCer analogue suppressed the EAE-inducing antigen-specific Th2 response. Conversely, α-C-GalCer, a C-glycoside (carbon glycoside) analogue of α-GalCer, activated iNK T cells at very low concentrations and promoted Th1 responses in vivo[31]. More recently, Tashiro et al. [32] synthesized α-carba-GalCer, which strongly induced NK T cell-mediated Th1 cytokines in a fashion similar to α-C-GalCer [32].

In the present study, we found that α-carba-GalCer inhibited the development of CIA. This suppressive effect was dependent on IFN-γ induced by NK T cells. The results also showed that α-carba-GalCer suppressed the production of both anti-type II collagen (CII) antibodies in serum and IL-17 in draining lymph nodes (DLNs) in response to CII. This lower pathogenic Th17 response resulted from enhancement of the Th1 response via α-carba-GalCer-dependent IFN-γ. Thus, α-carba-GalCer could be a potentially useful therapeutic agent for Th17-mediated autoimmune diseases.

Materials and methods

Mice

Male DBA/1J mice were purchased from Charles River Japan (Tokyo, Japan). The animals were kept under specific pathogen-free conditions and studied at 6–9 weeks of age. The Institutional Animal Care and Use Committee of the University of Tsukuba approved all experimental plans.

Reagents

α-GalCer was purchased from Funakoshi (Tokyo, Japan) and α-carba-GalCer (RCAI-56) was kindly provided by Dr Masaru Taniguchi (Riken Research Center for Allergy and Immunology, Yokohama, Japan). The structures of these two reagents are shown in Fig. 1a. The stock solutions of these glycolipids were dissolved originally in 100% dimethyl sulphoxide (DMSO) at 1 mg/ml and diluted in phosphate-buffered saline (PBS) just before injection into the mice. The following monoclonal antibodies (mAbs) were used for flow cytometric analysis: allophycocyanin (APC)-conjugated anti-mouse CD4 (clone: GK1·5; eBioscience, San Diego, CA, USA), peridinin chlorophyll (PerCP)-conjugated anti-CD3, fluorescein isothiocyanate (FITC)- and PerCP-conjugated anti-CD19, FITC- or APC-conjugated anti-IFN-γ (clone XMG1·2; BioLegend, San Diego, CA, USA); and FITC- or phycoerythin (PE)-conjugated anti-IL-17 (clone TC11-18H10·1; BD Pharmingen, Franklin Lakes, NJ, USA), and PE-conjugated CD1d-tetramer (MBL International, Woburn, MA, USA). The following mAbs were used for anti-CII specific IgGs enzyme-linked immunosorbent assay (ELISA): polyclonal rabbit anti-mouse immunoglobulins/HRP (Dako, Glostrup, Denmark), rabbit anti-mouse IgG1-horseradish peroxidase (HRP) (Zymed Laboratories, San Francisco, CA, USA) and rat anti-mouse IgG2a-HRP (Zymed). Bovine type II collagen was purchased from Collagen Research Center (Tokyo, Japan) and dissolved under constant stirring overnight at 4°C in 0·05 m acetic acid in phosphate-buffered saline (PBS) to be used for immunization, or in 0·05 mm Tris-HCl, 0·2 m NaCl, pH 7·4 for ELISA.

Fig. 1
Natural killer (NK) T cell response to α-carba-GalCer in CIA-susceptible mice. (a) Structure of α-galactosylceramide (α-GalCer) and α-carba-GalCer. (b) DBA/1 mice were injected intravenously (i.v.) with 2 µg of ...

Cell preparation

Lymphocytes were isolated from the liver, spleen or DLN, as described previously [33].

Induction of CIA and glycolipid administration

Mice were immunized subcutaneously (s.c.) at the base of their tails with 100 µg of bovine CII emulsified with complete Freund's adjuvant (CFA) (Difco, Detroit, MI, USA). An emulsion was formed by 2 mg/ml of CII with an equal volume of CFA. Two micrograms of either α-GalCer or α-carba-GalCer was added to and emulsified with CII/CFA. The emulsion was injected s.c. into the tail base. A booster dose of 100 µg of CII solution was injected intraperitoneally (i.p.) on day 21. For intracellular cytokine staining, 50 µg was injected into each footpad of the hind paw. Joint swelling was monitored and scored as follows: 0, no swelling or redness; 1, swelling or redness in one joint; 2, involvement of > 2 joints; and 3, severe arthritis affecting all paws and joints. The score for each animal represented the sum of the score for all four paws. The clinical score was calculated using the results of all mice in the group.

Antibody treatment

Systemic IFN-γ neutralization was carried out by treatment with anti-IFN-γ mAb, at 150 µg/mouse injected i.p. on day 0.

Enzyme-linked immunosorbent assay

To determine the CII-specific IgG subtype, bovine CII (10 µg/ml) was coated onto ELISA plates and incubated at 4°C overnight. After two washes with washing buffer (0·05% Tween 20 in PBS), the blocking solution [2% bovine serum albumin (BSA) in PBS] was applied for 1 h at room temperature. After two washes, serially diluted serum samples were added to the CII-coated wells for 1 h. After three washes, HRP-conjugated anti-mouse IgG, IgG1, or IgG2a was added at a final dilution of 1:4000 and incubated for 1 h. After three washes, colour was developed using peroxidase substrate (KPL). The plates were incubated for 15 min at room temperature, and the optical density was read at 450 nm using a microplate reader.

The concentrations of IL-2, IL-4, IL-10, IL-12, IL-17 and IFN-γ in the serum and in the culture supernatants were measured using an ELISA kit (Duoset; R&D Systems, Abingdon, UK), according to the protocols supplied by the manufacturer.

NK T cell response to glycolipid ligand

For the in vivo assay, naive DBA/1 mice (n = 3) were injected intravenously (i.v.) with 2 µg/mouse of glycolipid ligands and serum was collected at various time points. The concentration of cytokines in serum was determined by ELISA.

CII-reactive T cell response

Twelve days after CII/glycolipid injection, the DLN (inguinal) cells were collected and restimulated with 100 µg/ml of denatured bovine CII (60°C, 10 min) for 72 h. The cells were cultured in complete RPMI-1640 medium containing antibiotics and 5% fetal calf serum (FCS) and incubated at 37°C. The concentration of cytokines in the culture supernatants was determined by ELISA.

Flow cytometry

Cells were stained at 4°C in PBS containing 2% heat-inactivated FCS, incubated for 5 min with anti-CD16/32 to block Fcr receptors, and then incubated for 30 min with various mAbs at appropriate dilutions. A mouse Treg cell staining kit (eBioscience) was used to stain Treg cells following the protocol provided by the manufacturer. Apoptosis was examined by the annexin V/propidium iodide (PI) assay (eBioscience) using the protocol supplied by the manufacturer. Intracellular cytokines were stained using an intracellular staining kit (BD Pharmingen). Lymphocytes from CII-immunized mice were stimulated with phorbol myristate acetate (PMA) (50 µg/ml) and ionomycin (1 g/ml) in the presence of GolgiStop solution (BD Pharmingen) for 4 to 6 h. Flow cytometry was performed on a four-colour fluorescence activated cell sorter (FACS)Calibur. Dead cells were excluded based on the forward- and side-scatter characteristics. The results were analysed using Mac CellQuest software (BD Biosciences, San Jose, CA, USA).

Quantification of cytokine transcripts

Total RNA was extracted with an RNA extraction kit (Isogen; Nippon Gene, Tokyo, Japan) in accordance with the instructions provided by the manufacturer. cDNA was obtained by reverse transcription with a commercially available kit (Fermentas, Glen Burnie, MD, USA). We used a TaqMan assay-on-demand gene expression product (Applied Biosystems, Foster City, CA, USA). The expression levels of IL-6, IL-23p19, TGF-β and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (assay ID IL-6: Mm00446191; IL-23p19: D1160011; TGF- β: 01178819; GAPDH: 99999915, respectively; Applied Biosystems) were normalized relative to the expression of GAPDH. Analysis was performed with an ABI Prism 7500 apparatus (Applied Biosystems).

Statistical analysis

Values were expressed as the mean ± standard error of the mean (s.e.m.). Differences between groups were examined for statistical significance using the t-test. Probability values less than 0·05 were considered significant.

Results

NK T cell response to α-carba-GalCer in CIA-susceptible DBA/1 mice

First, we examined whether α-carba-GalCer causes a differential simulation of Th1 cytokine production in DBA/1 mice (known as the CIA-susceptible strain). Mice were injected with 2 µg of either α-carba-GalCer or α-GalCer and their blood cytokine levels were then measured at various time-points by ELISA. α-GalCer, but not α-carba-GalCer, increased IL-4 concentrations at 3 h after injection (Fig. 1b). Conversely, IFN-γ and IL-12 production was induced by both glycolipids, but the levels in α-carba-GalCer-treated mice were higher than those in mice treated with α-GalCer (Fig. 1b). IL-2-production was observed in both α-GalCer- and α-carba-GalCer-treated mice and the concentration of IL-2 in α-carba-GalCer-treated mice was comparable to that in α-GalCer-treated mice (Fig. 1b).

These data support the findings of a previous study [32], and suggest that α-carba-GalCer is a potent ligand for NK T cells and can selectively induce a Th1-type response.

α-carba-GalCer suppresses CIA in an IFN-γ-dependent manner

To examine the effects of α-carba-GalCer on the onset and severity of CIA, male DBA/1 mice with type II collagen (CII) were injected s.c. with α-carba-GalCer, α-GalCer or vehicle on day 0. As shown in Fig. 2a, α-carba-GalCer treatment tended to reduce the incidence of CIA compared with the vehicle treatment, although the difference was not significant. In contrast, α-GalCer-treatment did not affect the incidence of the disease. The clinical score of arthritis of the α-carba-GalCer-treated group was significantly lower than that of the vehicle-treated groups (P < 0·05, Fig. 2b). To determine whether this therapeutic effect was dependent on IFN-γ, IFN-γ was neutralized in the α-carba-GalCer-treated mice. IFN-γ neutralization at the time of CII immunization abolished the beneficial effect of α-carba-GalCer on CIA, but had no effect in the vehicle-treated mice (Fig. 2c). These data indicate that α-carba-GalCer ameliorates CIA and that this action is mediated through IFN-γ.

Fig. 2
Effects of α-carba-GalCer on CIA. DBA/1 mice were immunized with CII in CFA and 2 µg of α-galactosylceramide (α-GalCer) (n = 5), α-carba-GalCer (n = 5) or vehicle (n = 5). (a) Incidence and (b) clinical score of ...

α-carba-GalCer suppresses anti-CII antibodies and CII-reactive IL-17 production

In general terms, CIA is thought to be an autoreactive T and B cell-dependent arthritis [34]. Therefore, we determined the anti-CII antibody titre in α-carba-GalCer-treated mice. As shown in Fig. 3a, the anti-CII IgG titre was significantly lower in the α-carba-GalCer-treated mice than in the control mice. Specifically, the anti-CII IgG2a titre was lower in α-carba-GalCer-treated mice, but there were no differences in the anti-CII IgG1 subclass titres among the groups (Fig. 3b,c). Twelve days after injection of CII/α-carba-GalCer, cells were collected from the DLN and restimulated with CII in vitro. IL-17 production by DLN cells was lower in the α-carba-GalCer-treated mice than in the control mice (Fig. 4). In contrast, IFN-γ production in α-carba-GalCer-treated mice was comparable to that in α-GalCer- and vehicle-treated mice. None of the cultures showed production of IL-4 and IL-10 (data not shown). These results suggest that α-carba-GalCer treatment suppresses antigen-specific Th17 cell and B cell responses in the development of CIA.

Fig. 3
Production of anti-type II collagen (CII) antibodies in α-carba-GalCer-treated mice. DBA/1 mice were immunized with CII in complete Freund's adjuvant (CFA) and 2 µg of α-galactosylceramide (α-GalCer) (n = 5), α-carba-GalCer ...
Fig. 4
CII-reactive T cell response in α-carba-GalCer-treated mice. DBA/1 mice were immunized with type II collagen (CII) in complete Freund's adjuvant (CFA) and 2 µg of α-galactosylceramide (α-GalCer) (n = 3), α-carba-GalCer ...

α-carba-GalCer does not alter the number of forkhead box P3 (FoxP3+) Tregs or apoptotic T cells

IFN-γ is reported to play an important role in the induction of apoptosis and Tregs in autoimmune disease [13,14]. Therefore, we examined whether the beneficial effects of α-carba-GalCer were mediated by induction of apoptosis of T cells or T regs. As shown in Fig. 5a, treatment with α-carba-GalCer did not increase apoptosis, as assessed by the annexin/PI assay. Further analysis indicated that the proportion of FoxP3+ Tregs was not significantly different between the α-carba-GalCer-treated mice and the control mice (Fig. 5b). These results suggest that the beneficial effects of α-carba-GalCer on CIA are unlikely to be mediated by induction of apoptosis or Tregs.

Fig. 5
α-carba-GalCer does not alter the number of forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) or apoptotic T cells. DBA/1 mice were immunized with type II collagen (CII) in complete Freund's adjuvant (CFA) and 2 µg of α-carba-GalCer ...

Alternation of the Th1/Th17 cytokine balance in α-carba-GalCer-treated mice

Because recent studies have shown that IFN-γ suppresses IL-17 production in CIA [10,11], we examined the hypothesis that the beneficial effect of α-carba-GalCer was due to the suppression of IL-17 production by IFN-γ. For this purpose, we determined the proportion of IFN-γ- and IL-17-producing T cells in α-carba-GalCer-treated mice. The proportion of IL-17-producing T cells in α-carba-GalCer-treated mice was significantly smaller than in vehicle-treated mice when analysed immediately ex vivo 10 days after α-carba-GalCer immunization with CII (Fig. 6a,b, upper panel). The smaller proportion of IL-17-producing cells was observed in both the CD4+ and CD4- (DN, CD8+) T cell population (Fig. 6a,b, middle and lower panels). In contrast, the proportion of IFN-γ-producing T cells was larger in α-carba-GalCer-treated mice (Fig. 6a,b, upper panel). The larger proportion of IFN-γ-producing T cells was observed only in the CD4- (DN, CD8+) T cell population (Fig. 6a,b, lower panel). The proportion of IL-17-producing T cells was also lower in α-GalCer-treated mice, but the effect was smaller than that in α-carba-GalCer-treated mice. In addition, the ratio of IFN-γ-/IL-17-producing T cells was significantly higher in α-carba-GalCer-treated mice (Fig. 6c). The Th1 polarization was evident in both the CD4+ T cell and CD4- T cell populations (Fig. 6d,e). These findings suggest that treatment with α-carba-GalCer polarizes the systemic T cell response towards Th1 and suppresses Th17 cell differentiation or activation.

Fig. 6
α-carba-GalCer treatment polarizes T cell response towards T helper type 1 (Th1) and suppresses Th17 cell activation or differentiation. Ten days after type II collagen (CII) and vehicle, α-galactosylceramide (α-GalCer) or α-carba-GalCer ...

α-carba-GC-treatment attenuates IL-6 and IL-23 gene expression in the initial phase of CIA

It has been suggested that TGF-β and IL-6 induce Th17 differentiation and that IL-23 is required for expansion and maintenance of Th17 cells. Thus, we determined whether this Th17-related gene expression occurs in CII/α-carba-GalCer-immunized mice. RNA was purified from DLN cells 3 days and 10 days after immunization and the expression of Th17-related cytokine transcripts was determined by quantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis. At day 3, the expression of IL-6 and IL-23p19 transcripts was significantly more reduced in mice treated with α-carba-GalCer than in mice treated with vehicle, while the expression of TGF-β transcripts in mice treated with α-carba-GalCer was comparable to that in control mice (Fig. 7a). α-GalCer treatment had no effect on these gene expressions at day 3 (Fig. 7a). In addition, all the Th17-related gene expressions were not significantly different among vehicle-, α-GalCer- and α-carba-GalCer-treated mice at day 10 (Fig. 7b). These results suggested that α-carba-GalCer suppresses IL-6 and IL-23p19 expression in the initial phase of CIA.

Fig. 7
α-carba-GalCer-treatment attenuates T helper type 17 (Th17)-related gene expression in the initial phase of collagen-induced arthritis (CIA). RNA was purified from draining lymph node (DLN) cells of each mouse (a) 3 days and (b) 10 days after ...

α-carba-GalCer treatment enhanced NK T cell activation and IFN-γ production in the initial phase of CIA

To analyse the activation state of NK T cells in the initial phase of CIA, we determined the frequency and cytokine production of NK T cells in the liver, spleen and DLN 3 days after CII/glycolipid immunization. The frequency of NK T cells in α-carba-GalCer-treated mice was comparable to that in the vehicle-treated mice (Fig. 8a). It has been reported that TCR down-modulation of NK T cells was observed when NK T cells were activated with glycolipid [35]. Notably, in the current study, a lower expression of CD1d-tetramers that bind to the invariant TCR of NK T cells was observed in α-carba-GalCer-treated mice, suggesting that NK T cells were activated at this time (Fig. 8a). The TCR down-modulation was also observed in α-GalCer-treated mice, but only partially (Fig. 8a). Moreover, intracellular cytokine staining showed that IFN-γ production by liver, splenic and DLN NK T cells in α-carba-GalCer-treated mice was higher than that in vehicle-treated mice (Fig. 8b,c). The α-GalCer teatment also induced higher IFN-γ production by NK T cells, but was lower than α-carba-GalCer treatment (Fig. 8b,c). Although we observed abundant IL-17-producing NK T cells in the peripheral lymph nodes but not in the liver and spleen, as reported previously [36], α-carba-GalCer treatment had no effect on IL-17 production by NK T cells in the DLN (Fig. 8b). These findings suggest that α-carba-GalCer treatment enhanced the activation and IFN-γ production of NK T cells in the initial phase of CIA. Thus, α-carba-GalCer treatment could regulate Th17-mediated autoimmune diseases negatively through NK T cell-derived IFN-γ in the initial phase of CIA.

Fig. 8
α-carba-GalCer treatment enhanced natural killer (NK) T cell activation and interferon (IFN)-γ production in the initial phase of collagen-induced arthritis (CIA). Three days after type II collagen (CII) and vehicle, α-galactosylceramide ...

Discussion

NK T cells are unconventional T cells that recognize glycolipid antigens and secrete several types of proinflammatory and anti-inflammatory cytokines [15,16,29]. Although endogenous ligands for NK T cells have not yet been identified, several synthetic ligands have been used in immunotherapy for cancer and autoimmune disease models [16]. The present study demonstrated that treatment with α-carba-GalCer, a novel synthetic NK T cell ligand, suppressed the development of CIA and that this effect was mediated by IFN-γ.

In the present study, we first showed the biological function of α-carba-GalCer in CIA-susceptible DBA/1 mice, because several reports suggested that there were some differences in NK T cell response among mouse strains [22]. We demonstrated that i.v. injection of α-carba-GalCer selectively induced serum Th1 cytokines in DBA/1 mice, similar to a previous report on C57BL6 mice, although the concentration of cytokines in DBA/1 mice was lower than that in C57BL/6 mice (data not shown). Therefore, we concluded that α-carba-GalCer could be used as a Th1-type glycolipid ligand for therapy of CIA in DBA/1 mice.

Interestingly, s.c. injection, unlike i.v. injection, of α-GalCer or α-carba-GalCer with CFA did not lead to detectable IFN-γ in serum (data not shown), although intracellular staining for IFN-γ showed clearly that α-carba-GalCer induced strong IFN-γ production by NK T cells. We believe that the activation profile of NK T cell in vivo is dependent on the route of administration of NK T cell ligand. It is well known that i.v. (or i.p.) injection of α-GalCer or its analogues rapidly induce several cytokines in serum and TCR down-modulation on NK T cells within 24 h after stimulation. TCR expression levels recover gradually and NK T cells then proliferate rapidly and the expansion of NK T cells peaks at about 72 h after stimulation. In contrast, our data of s.c. injection of NK T cell ligand show that NK T cell expansion is not so strong compared with that in i.v. injection reported previously, and TCR down-modulation is still observed at 72 h after stimulation in addition to the loss of induction of serum IFN-γ. Based on these observations, we speculate that the effect of s.c. injection of glycolipid might be weaker, but it sustained longer than that of i.v. injection. It might also be possible that emulsifying with adjuvant (CFA) made glycolipid remain longer in the injected site.

IFN-γ is an important proinflammatory cytokine in infection and tumour rejection. Conversely, IFN-γ also exhibits anti-inflammatory properties in autoimmune diseases, acting as an inducer of apoptosis and Tregs[1214]. In our study, neither apoptotic T cells nor FoxP3+ Tregs were identified in α-carba-GalCer-treated mice. These results indicate that apoptosis and FoxP3+ Tregs did not mediate the suppressive effect of α-carba-GalCer on CIA.

The results also showed an increased proportion of IFN-γ-producing T cells and a decreased proportion of IL-17-producing T cells in α-carba-GalCer-treated mice, suggesting that this NK T ligand polarizes the Th1/Th17 cytokine balance to Th1. Chu et al. [11] reported that IFN-γ suppressed Th17 cell differentiation and IL-17 production in CIA. IFN-γ maintains IFN-γ-producing Th1 cells by themselves and induces the production of IL-12, another cytokine important for Th1 differentiation from dendritic cells [37,38]. Much evidence suggests that TGF-β and IL-6 induce Th17 differentiation and that IL-23 is required for expansion and maintenance of Th17 cells. In this study, we showed that treatment with α-carba-GalCer attenuated IL-6 and IL-23 expression in the initial phase of CIA. Recently, Chu et al. [11] observed that IFN-γ deficiency leads to increased IL-6 production in CIA, indicating that IFN-γ regulates IL-6 production negatively. In addition, Sheikh et al. [39] suggested that IFN-γ is a negative regulator of IL-23 in murine macrophages. Thus, we speculate that IFN-γ induced by the treatment with α-carba-GalCer suppressed IL-6 and IL-23 production in the initial phase of CIA and that the reduction of these Th17-related cytokines leads to the amelioration of Th17 cell activation and expansion.

Our results showed an increased population of IFN-γ-producing CD4- T cells (non-Th cells) in α-carba-GalCer-treated mice, indicating that CD4- NK T cells, CD8+T cells or other CD4- T cells are associated with this Th1 polarization. These data support the results of a previous study showing that NK T cells can induce bystander T cell activation [40]. Thus, we believe that α-carba-GalCer treatment polarizes the systemic T cell response, including CII-reactive and CII non-reactive T cells, to the Th1-type response. We confirmed that IL-17-producing CII-reactive T cells were reduced in α-carba-GalCer-treated mice, although IFN-γ production was not significantly different between the α-carba-GalCer-treated mice and the control mice. These results also suggest that α-carba-GalCer can alter the Th1/Th17 balance to Th1 in the CII-reactive T cell response.

It has been reported that anti-CII antibodies are required for the development of CIA [41,42], and that Th1 and Th2 cells are involved in class-switching to IgG2a and IgG1. In fact, IL-4 directs murine IgE and IgG1 production, whereas IFN-γ stimulates selectively the production of IgG2a as well as that of IgG3 under certain circumstances [43,44]. Other studies have reported the association of IL-17 with IgG production in animals with autoimmune disease and the presence of low levels of anti-CII IgG2a antibodies in IL-17-deficient mice [8,45]. In our study, anti-CII IgG2a antibodies but not IgG1 were reduced significantly in α-carba-GalCer-treated mice, implying that the reduction in anti-CII IgG2a antibody in these mice could be due to the decreased number of CII-reactive Th17 cells.

It has been reported that mice deficient in IFN-γ exhibit severe CIA symptoms, suggesting that IFN- γ plays a role as a suppressor cytokine [11]. In the current study, as expected, IFN-γ neutralization abolished the beneficial effect of α-carba-GalCer in CIA. Thus, we concluded that α-carba-GalCer suppressed CIA in an IFN-γ-dependent manner. However, in the α-carba-GalCer-untreated condition, IFN-γ neutralization seemed to ameliorate the symptoms of CIA compared to isotype Ig treatment (Fig. 2c). Although it is difficult to explain the discrepancy between these results and those of the previous study of IFN-γ deficient mice [11], we speculate an alternative and complementary possibility that IFN-γ has a dual function in CIA. Jacob et al. showed that in vivo administration of IFN-γ 24 h before CFA immunization caused an exacerbation of arthritis, whereas administration of IFN-γ 24–48 h after CFA immunization suppressed the disease in an adjuvant arthritis model [46]. These observations indicate that IFN-γ plays a proinflammatory role at steady state or early stage of inflammation (0–24 h after CFA immunization) and subsequently plays an anti-inflammatory role. In fact, several reports have suggested that IFN-γ activates innate immune cells, such as enhancing phagocytosis, proinflammatory cytokine production in response to bacterial component and antigen presentation, although it is also a fact that IFN-γ regulates adaptive Th17 cells negatively [47]. Thus, in our study, anti-IFN-γ treatment at the time of CII/CFA immunization might inhibit the proinflammatory effect of IFN-γ in the early stage of inflammation (0–24 h after immunization) and it might lead to a decreased baseline severity of disease. Meanwhile, although we could not know when the effect of α-carba-GalCer were started after s.c. injection and how long the effect was sustained, it is indisputable that NK T cells in α-carba-GaCer-treated mice were activating and had a higher capacity of IFN-γ-production at 72 h after CII/CFA immunization, which is the time when IFN-γ could play an anti-inflammatory effect in adjuvant arthritis [46]. Thus, we think that α-carba-GalCer-mediated IFN-γ played an anti-inflammatory effect at 72 h after CII/CFA immunization, and anti-IFN-γ mAb could have blocked the IFN-γ and abolished the beneficial effect of α-carba-GalCer. Considering these complex effects of IFN-γ in the development of CIA, IFN-γ should be neutralized for a longer period of time by repeated injections of anti-IFN-γ mAb or by using IFN-γ-deficient mice to evaluate more clearly the IFN-γ dependency of the α-carba-GalCer effect.

Our results are somewhat different from those of previous studies describing the effects of NK T cells on CIA. For example, Miellot et al. [48] demonstrated that treatment with α-GalCer induced IL-10-producing T cells and suppressed CIA. However, in our study, the same treatment had no effect on the development of CIA and we found no IL-10-producing cells in the DLN (data not shown). Coppieters et al. [49] suggested that the protective effect of α-C-GalCer on CIA is IFN-γ-independent. In contrast to their results, the beneficial effect of α-carba-GalCer in our study was IFN-γ-dependent. Although the reason for this discrepancy is not known, we speculate that the route and timing of administration of α-carba-GalCer might have influenced the results. Hermans et al. [50] reported that enhanced CD4+ and CD8+ T cell responses were observed when α-GalCer was administered at the same time as T cell antigen, and they suggested that the enhancement of T cell responses requires the presentation of T cell antigen and α-GalCer by the same dendritic cell. In our study, CII and glycolipid were administered at the same time and by the same route. Thus, it is possible that CII and glycolipids were captured and presented by the same dendritic cell, which was effective in influencing the T cell response through NK T cell activation. Conversely, Coppieters et al. [49] administered glycolipids 5 days after CII immunization, and the route of administration of glycolipids (i.p.) was different from that of CII (intradermally). Further studies are required to examine the effects of several glycolipids on CIA under the same conditions such as dose, route and timing of glycolipid immunization.

In human autoimmune diseases such as RA, Sjögren's syndrome, systemic sclerosis and SLE, the number of NK T cells is decreased and NK T cells function as regulatory cells that inhibit autoimmunity [1720]. It is anticipated that increasing the population of IFN-γ-producing NK T cells by administration of α-carba-GalCer could be therapeutically useful in autoimmune diseases such as RA, owing to the similarities in the pathogenic processes of RA and CIA.

In conclusion, the present study demonstrated that NK T cells are multi-potent cells and act as regulatory cells by the induction of α-carba-GalCer in CIA. Based on these properties, we believe that further studies are warranted to explore the potential therapeutic benefits of α-carba-GalCer in Th17-mediated autoimmune diseases.

Disclosure

None.

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