Augmentation of Antigen-Presenting and Th1-Promoting Functions of Dendritic Cells by WSX-1(IL-27R) Deficiency

Sen Wang, Yoshiyuki Miyazaki, Yukari Shinozaki and Hiroki Yoshida
J Immunol November 15, 2007, 179 (10) 6421-6428; DOI: https://doi.org/10.4049/jimmunol.179.10.6421

Abstract

WSX-1 is the α subunit of the IL-27R complex expressed by T, B, NK/NKT cells, as well as macrophages and dendritic cells (DCs). Although it has been shown that IL-27 has both stimulatory and inhibitory effects on T cells, little is known on the role of IL-27/WSX-1 on DCs. LPS stimulation of splenic DCs in vivo resulted in prolonged CD80/CD86 expression on WSX-1-deficient DCs over wild-type DCs. Upon LPS stimulation in vitro, WSX-1-deficient DCs expressed Th1-promoting molecules higher than wild-type DCs. In an allogeneic MLR assay, WSX-1-deficient DCs were more potent than wild-type DCs in the induction of proliferation of and IFN-γ production by responder cell proliferation. When cocultured with purified NK cells, WSX-1-deficient DCs induced higher IFN-γ production and killing activity of NK cells than wild-type DCs. As such, Ag-pulsed WSX-1-deficient DCs induced Th1-biased strong immune responses over wild-type DCs when transferred in vivo. WSX-1-deficient DCs were hyperreactive to LPS stimulation as compared with wild-type DCs by cytokine production. IL-27 suppressed LPS-induced CD80/86 expression and cytokine production by DCs in vitro. Thus, our study demonstrated that IL-27/WSX-1 signaling potently down-regulates APC function and Th1-promoting function of DCs to modulate overall immune responses.

Dendritic cells (DCs)3 are the most potent APCs that initiate both innate and acquired immunity upon recognition and/or capture of pathogenic Ags (1). Once activated by inflammatory stimuli or infectious agents, immature DC precursors undergo a maturation process, migrate into lymphoid organs, and acquire the capacity to activate naive T lymphocyte for activation and differentiation. In addition to presenting Ags on MHC along with costimulatory molecules, such as CD80 and CD86, DCs produce various cytokines for augmentation of innate immunity and activation of acquired immunity. Among the cytokine produced, IL-12 is the most potent Th1-inducing cytokine. Recently, IL-27 and IL-23, produced mainly by DCs, have been identified as cytokines structurally and functionally similar to IL-12 (2). Analyses of mice deficient for WSX-1 (IL-27Rα) revealed that IL-27/WSX-1 is critical for initial commitment of Th1 differentiation through Stat1-mediated T-bet activation (3, 4). Along with other reports, these IL-12 cytokine family members have been reported to differentially regulate Th1 responses in a chronological way: IL-27 for commitment of naive CD4+ T cells for Th1 differentiation, IL-12 for consolidation of effector Th1 cells, and IL-23 for proliferation of memory Th1 cells (see Ref 5 for review).

Despite its critical role in Th1 differentiation, however, recent bodies of evidence have shown that IL-27 also has a suppressive effect on production of various cytokines, including IFN-γ. Mice deficient for WSX-1 developed lethal hepatic injury due to hyperproduction of various inflammatory cytokines upon protozoan infection (6, 7). The suppressive effects of WSX-1 have been also shown in Con A-induced hepatitis models or Ag-induced airway hypersensitivity models (8, 9). More recently, IL-27 has also been shown to suppress encephalomyelitis of either infection or autoimmune origin by inhibiting differentiation of IL-17-producing “Th17” cells (10, 11). Thus, although the precise mechanism of the suppression has yet to be elucidated, IL-27 diminishes cytokine-mediated immunopathology by down-regulating cytokine production. Taken together, IL-27/WSX-1 has two-sided roles: one as an initiator of immune responses and the other as an attenuator of immune/inflammatory responses (5).

Besides its highest expression in T cells, WSX-1 is also expressed in other types of cells such as B cells, NK/NKT cells, and macrophages (3). Although IL-27 is produced mainly by DCs (12), little is known about the function of IL-27 on DCs themselves. In the current study, we examined the roles of WSX-1 on DC functions and demonstrated that IL-27WSX-1 also has suppressive effects on APC function of DCs. WSX-1-deficient DCs expressed higher levels of costimulatory molecules and induced stronger Th1-biased immune responses in allogeneic MLR assays or in mice transferred with Ag-pulsed DCs. Our analyses indicated a new mechanism of regulation of DC functions by IL-27/WSX-1 and a possibility of augmenting DC functions by intervening IL-27/WSX-1 signals.

Materials and Methods

Animals

WSX-1-deficient (WSX-1−/−) mice were generated as described previously and were backcrossed more than nine times to C57BL/6 mice (continual backcrossing). Mice were housed in microisolator cages and were used between 8 and 14 wk of age. Age- and sex-matched wild-type (WT) C57BL/6 mice (Seac Yoshitomi) were used as controls. All experiments were approved by the institutional animal research committee of Saga University and conformed to the animal care guidelines of the American Physiologic Society.

Reagens

LPS was purchased from Sigma-Aldrich. Anti-mouse IL-6 Ab and anti-mouse IL-10 Ab were purchased from R&D Systems and were used at 1 μg/ml for in vitro blocking of each cytokine. rIL-27 was purchased from R&D Systems or generated in our laboratory as described previously (4). Anti-STAT3, STAT1, and anti-IκB Abs were purchased from Santa Cruz Biotechnology and Abs against p38 and anti-phosphorylated proteins were purchased from Cell Signaling.

Cell preparation

Bone marrow (BM)-derived DCs were prepared from BM suspensions from femurs and tibias of mice as described elsewhere (13). Briefly, bone marrow cells were cultured with 10 ng/ml murine GM-CSF (R&D Systems) and 10 ng/ml murine IL-4 (R&D Systems) for 10 days and used as DCs. For preparation of splenic DCs, spleen cells were stained with FITC-conjugated anti-CD11c Ab (BD Pharmingen) followed by anti-FITC magnetic beads (MACS; Miltenyi Biotec). NK cells and CD8+ cells also were purified using PE-anti-NK1.1 Ab (BD Pharmingen) and PE-anti-CD8 Ab, respectively, followed by anti-PE magnetic beads (Miltenyi Biotec). Splenic DCs cultured without any simulation for 24 h were regarded as mature (14).

LPS stimulation of DCs in vivo and in vitro

WT or WSX-1-deficient mice were injected i.p. with LPS (200 μg/mouse). After LPS injection, CD11c+ cells in the spleen were analyzed for CD80 or CD86 expressions. For in vitro stimulation of DCs, splenic DCs were prepared and were stimulated with LPS (100 ng/ml) for 48 h. Cells were examined for surface expressions of CD80 or CD86 as above.

Quantitative real-time PCR and RT-PCR analyses

Total RNAs were extracted from cells using TRIzol solution (Invitrogen Life Technologies) and reverse-transcribed with a Revertra-plus-kit (Toyobo). Expression levels of WSX-1, IL-27 EBI-3, IL-27p28, IL-12 p40, Jagged-1, and Delta-4 in DCs were determined relative to that of β-actin using TaqMan-PCR (Qiagen) and an Applied Biosystems PRISM 7700 sequence detection system according to the manufacturer’s instructions. Oligonucleotide primers and probes were designed using a Primer Express program (Applied Biosystems). The relative expression of each mRNA was determined and normalized to the expression of β-actin. For the expression of perforin and granzyme B in NK cells, RNAs were prepared and reverse-transcribed as above and the same amounts of cDNAs normalized to β-actin were amplified for the gene expression by PCR. Similarly, suppressor of cytokine signaling (SOCS) 3 expression was examined in LPS-stimulated DCs. Primer and probe sequences used are described elsewhere (15, 16).

Flow cytometry

For flow cytometric analyses of surface molecules on DCs, cells were treated with Fc Block (BD Biosciences) followed by staining with FITC-conjugated anti-CD11c Ab plus biotin-anti-CD80 Ab or biotin-anti-CD86 Ab (both from BD Pharmingen) and streptavidin-PE staining and analyzed with a FACSCalibur (BD Biosciences) and CellQuest software (BD Biosciences). As negative staining controls, cells were treated likewise except isotype-matched Ig was used instead of anti-CD80/86 Abs.

Allogeneic MLR assay

Allogeneic MLR experiments were performed by culturing CD4+ T cells from BALB/c (H-2d) mice as responder with splenic or BM-derived DCs from WT or WSX-1-deficient mice (H-2b) as stimulator. Briefly, 2 × 105 of CD4+ T cells were cocultured with DCs at 300–30,000/200 μl/well for 96 h in either the presence or absence of LPS (100 ng/ml; Sigma-Aldrich). During the last 18 h of culture, cell proliferation was measured by uptake of [3H]thymidine. IFN-γ production in the supernatants was measured by an ELISA development kit (R&D Systems).

In vivo transfer of Ag-pulsed DCs

Splenic DCs were prepared and incubated at 2 × 106/ml with 50 μg/ml keyhole limpet hemocyanin (KLH; Sigma-Aldrich) for 18 h. DCs were washed three times, resuspended in PBS, and then administered into the hind footpad of WT mice (1 × 106 cell/40 μl of PBS per mouse). The draining lymph nodes (LN) were removed 5 days after transfer and lymphocytes were cultured at 1 × 105 cells/200 μl per well with or without KLH (20 μg/ml) for 96 h. Ag-specific proliferation and IFN-γ production were measured as above. KLH-specific IgGa2b and IgG1 in the sera of mice on day 5 after transfer were detected as described elsewhere (8).

Parasite infection and vaccination with DC

BM-derived DCs (5 × 105/ml) were cocultured with Leishmania major (MHOM/SU/73-5-ASKH) promastigotes lysates (2.5 × 107/ml) at ratio of 10:1 for 18 h, washed with PBS three times, and injected i.v. (5 × 105 DC/40 μl PBS per mouse). Control mice were treated with PBS. One week later, mice were infected intradermally with 1 × 107 promastigotes of L. major at the footpad. The course of infection was monitored daily by measuring the increase in thickness of the infected footpad over uninfected footpad as described elsewhere (3). Draining LN cells were removed (day 2 after infection for WSX-1-deficient DC-transferred mice or 4 wk after infection for control and WT DC-transferred mice) and examined for proliferation and IFN-γ production as described elsewhere (3). Parasite numbers in the footpads were measured as follows. Footpads were removed from mice, minced, and incubated in RPMI 1640 medium at 25°C. After 3 days of incubation, parasite number in the culture medium was counted microscopically.

NK cells assay

Purified NK1.1+ NK cells (5 × 104/200 μl per well) were cocultured with WT or WSX-1-deficient CD11c+ splenic DCs in the presence of LPS (100 ng/ml) at indicated mixing ratios for 24 h. Cytokine production in the supernatants was measured by ELISA. For cytotoxicity activity assay, NK cells were cocultured with WT or WSX-1-deficient DCs at 10:1 ratio for 24 h and were measured for killing activity against 51Cr-labeled YAC-1 cells as target. The percentage of specific lysis was calculated as follows: percent lysis = [(cpm experimental well − cpm spontaneous release)/(cpm maximum release − cpm spontaneous release)] × 100. Expression of perforin and granzyme B was also examined by real-time PCR analyses.

Western blotting

Splenic DCs (1 × 106/ml) were prepared and stimulated with LPS (100 ng/ml) in either the presence or absence of rIL-27 (10 ng/ml; R&D Systems). Immunoblotting to detect phosphorylated STAT3, STAAT1, p38, and I-κB was performed as described previously (17).

Determination of cytokine production by DCs

Splenic DCs were prepared and stimulated with LPS (100 ng/ml) in the presence of IL-27 (10 ng/ml), anti-IL-6, or anti-IL-10 Ab for 24 h. Culture supernatants were examined for IL-12p40, TNF-α, or IL-6 by ELISA (Quantikine HS ELISA Kit; R&D Systems) according to the manufacturer’s direction.

Results

Augmented expression of WSX-1 in DCs after activation

WSX-1 is expressed in most immune cells, such as T cells, B cells, NK cells, and macrophages. Although IL-27 is mainly produced by DCs, the precise activation/maturation status of IL-27-producing DCs was not known. To examine the expression of WSX-1/IL-27 in DCs, we first assessed the expression of both subunits of IL-27, p28, and EBI-3 in WT DCs after activation with LPS using the real-time PCR method. Although the expression of p28 and EBI-3 was very low in DCs before LPS stimulation, splenic and BM-derived DCs showed substantial augmentation of both p28 and EBI-3 after activation of DCs with LPS (Fig. 1⇓A), consistent with a pervious report (12). The expression of WSX-1 was also low in immature DCs but increased after LPS stimulation (Fig. 1⇓B). Because the DCs did not proliferate in this experimental condition (data not show), these data demonstrated that activated DCs expressed WSX-1 as well as both subunits of IL-27 in an activation-dependent manner.

FIGURE 1.
FIGURE 1.

Augmented expression of WSX-1 and IL-27 in LPS-stimulated DCs. A, DCs were prepared from spleens or BM of WT mice and stimulated in vitro with LPS as described in Materials and Methods. Total RNAs were prepared from immature (im.), mature (m.) DCs, or DCs stimulated with LPS for the indicated hours and reverse-transcribed. Relative expressions of p28 (□) and EBI-3 (▪) were examined by real-time PCR methods. B, Relative expression of WSX-1 was examined as in A. Experiments were repeated three times with similar results.

Prolonged expression of CD80 and CD86 on WSX-1-deficient DCs after in vivo stimulation with LPS

To examine the effect of IL-27/WSX-1 on DC function, we took advantage of WSX-1 knockout (KO) mice (3) and analyzed the expression of some cell surface proteins. The expression of CD80 and CD86, both costimulatory molecules, was slightly lower in WSX-1-deficient DCs than in WT DCs (Fig. 2⇓A). The expression levels of MHC class I molecules (H-2Kd) and the percentages of CD4+ or CD8+ cells within DCs, however, were comparable between the two groups of mice (data not shown), suggesting that there were no differences in the cellular composition between WSX-1-deficient DCs and wide-type DCs. Upon i.p. injection of LPS into mice, the expression of CD80 and CD86 in DCs of both groups began to augment at 6 h after stimulation (data not shown) and at 24 h, ∼60% of DCs were positive for CD80 and CD86 expression. At this time point, there was no difference in the percentages of CD80- and CD86-positive cells between the two groups of mice (Fig. 2⇓A). Also, there were no significant differences in the mean fluorescence intensity (MFI) for the respective molecules between the two groups of mice (Fig. 2⇓B). Although the percentages of CD80- and CD86-positive DCs gradually decreased thereafter by 72 h after stimulation, WSX-1-deficient DCs still expressed high levels of CD80 and CD86 at 48 h after stimulation (Fig. 2⇓A). The expression levels were still higher in WSX-1-deficient DCs than in WT DCs at 72 h, albeit showing a decrease at 48 h. MFI for both CD80 and CD86 was also higher in WSX-1-deficient DCs than in WT DCs at 48 h and later (Fig. 2⇓B). The prolonged expression of CD80 and CD86 on WSX-1-deficient DCs indicated that DCs without IL-27/WSX-1 stimulation remained in activated status longer than WT DCs after stimulation. Percentages of viable DCs after in vitro stimulation with LPS were almost equivalent between WT and WSX-1-deficient DCs (data not shown).

FIGURE 2.
FIGURE 2.

Prolonged expression of costimulatory molecules on LPS-stimulated WSX-1-deficient DCs. A, WT or WSX-1-deficient (KO) mice were injected i.p. with LPS (200 μg/mouse). Before or 24, 48, and 72 h after LPS administration, CD80+ or CD86+CD11c+ cells in the spleen were analyzed. Numbers in each panel show percentage of CD80+ or CD86+ cells in the CD11c+ population. Gray histograms show control staining with isotype-matched Ig plus streptavidin-PE. Experiments were repeated two times with similar results. B, MFI for both CD80 and CD86 was measured in the same samples as in A. MFI was measured for CD80- or CD86-positive cell population. □, WT mice; ▪, WSX-1-deficient mice. ∗∗, p < 0.05 by paired Student’s t test.

In vitro suppression of CD80/86 expression by IL-27

The prolonged expression of CD80/86 by WSX-1-defucuebt DCs observed above could be secondary but not primary events induced by cytokines produced by in vivo LPS stimulation. To address the direct effect of WSX-1-deficiency on DCs and, more practically, to address the direct suppressive effect of IL-27 on DCs, spleen-derived DCs from WT or WSX-1-deficient mice were stimulated with LPS in vitro. As shown in Fig. 3⇓, WSX-1-deficient spleen-derived DCs showed activated phenotypes even before LPS stimulation in terms of CD80 expression. Although less evident than in vivo observation, there was a little but reproducible increase in the percentage of WSX-1-deficient CD80-positive cells as compared with that of WT cells. Presumably, in vitro stimulation of DCs with LPS (1–100 ng/ml used in our experiments) was strong enough to induce full induction of CD80 expression even in WT cells. As expected, however, addition of rIL-27 (10 ng/ml) significantly reduced CD80 expression by WT but not WSX-1-deficient DCs. IL-27 also suppressed the expression of CD86 by WT but not WSX-1-deficient DCs. Addition of anti-IL-6 Ab to the stimulation culture did not affect the CD80/86 expression by both WT and WSX-1-deficient DCs. Thus, it was unlikely that the higher expression of CD80/86 by WSX-1-deficient DCs is secondary to IL-6 production by stimulated DCs. Addition of IL-10 Ab did not affect the CD80/86 expression either (data not shown). Taken together, IL-27 had an inhibitory effect on LPS-stimulated DC function and, in the absence of its receptor, WSX-1, DCs showed higher sensitivity to LPS stimulation by CD80/86 expression both in vivo and in vitro, although the effect observed in vivo may be partially due to secondary cytokines produced after LPS stimulation.

FIGURE 3.
FIGURE 3.

Direct suppression of costimulatory molecule expression by IL-27 in vitro. Spleen-derived DCs from WT or WSX-1-deficient mice were stimulated with LPS (100 ng/ml) in vitro for 24 h. rIL-27 (10 ng/ml) or anti-IL-6 Ab was added to the culture as indicated. Cells were stained for CD80 or CD86 as described in the legend to Fig. 2⇑. Solid lines in control samples show negative control staining with isotype-matched Ig plus streptavidin-PE. Experiments were repeated two times with similar results.

Th1-inducing properties of WSX-1-deficient DCs

To further examine the impact of WSX-1 deficiency on DC function, we examined the expression of cytokines and some Notch ligands. Although the production of IL-12p70 was very low in WT and WSX-1-deficient DCs, WSX-1-deficient DCs produced significantly higher IL-12p70 before and after in vitro LPS stimulation (Fig. 4⇓A). Production of p40 subunit of IL-12 was also higher by WSX-1-deficient DCs than by WT DCs irrespective of LPS stimulation. Quantitative RT-PCR analyses also confirmed higher expression of IL-12p40 in WSX-1-deficient DCs than in WT DCs (Fig. 4⇓B). The expression of EBI-3, a subunit of IL-27, was also higher in WSX-1-deficient DCs than in WT DCs. There was no significant difference in the expression of p28 of IL-27 between WT and WSX-1-defieicnt DCs (data not shown). Interaction of Notch with its ligands, such as Jagged and Delta, affects the differentiation of CD4+ T cells (16). The expression of Delta-4, a Th1-inducing Notch ligand, was drastically induced and was much higher in LPS-stimulated WSX-1-deficient DCs than in WT DCs. Interestingly, the expression of Jagged-1, a Th2-inducing Notch ligand, was also induced by LPS stimulation in WT DCs and was higher in WT DCs than in WSX-1-deficient DCs. Expression of TLR4, the receptor for LPS, was not affected by WSX-1 deficiency (data not shown). These data indicated that WSX-1-deficient DCs were more potent in the induction of Th1 responses than WT DCs.

FIGURE 4.
FIGURE 4.

Expression of cytokines and Notch ligands. A, Splenic DCs were either untreated or stimulated with LPS (100 ng/ml, 24 h). Cytokine production in the culture supernatants were measured for production of IL-12. Shown are mean + SD of triplicate samples. B, Total RNAs were prepared from DCs prepared as in A. Expression of each gene was analyzed by the quantitative real-time PCR method. Relative expression of genes normalized to β-actin expression is shown. ∗, p < 0.05 and ∗∗, p < 0.01 by paired Student’s t test.

To substantiate the possible augmentation of Ag-presenting function as well as the possible augmentation of Th1-inducing function of WSX-1-defieicnt DCs, an allogeneic MLR assay was performed where responder CD4+ T cells from BALB/c mice (H-2d) were mixed with either WT DCs or WSX-1-deficient DCs (H-2b) as stimulator and cytokine production and responder proliferation was measured. When splenic DCs were used as stimulator, WSX-1-deficient DCs induced higher proliferation of responder cells than WT DCs in the presence or absence of LPS (Fig. 5⇓A). IFN-γ production was also higher in responder cells plus WSX-1-deficient DCs than in responder plus WT DCs. BM-derived DCs also showed similar results, albeit less in degree in that both IFN-γ production and responder cell proliferation were higher when WSX-1-deficient DCs were used as stimulator (Fig. 5⇓B). There was no significant difference in IL-2 production between cultures with WT DCs and WSX-1-deficient DCs (data not shown). Production of IL-4 was below detectable levels in all culture conditions (data not show). These results demonstrated that WSX-1-deficient DCs were more potent as Th1-inducing APCs than WT DCs.

FIGURE 5.
FIGURE 5.

Augmented APC functions of WSX-1-deficient DCs in MLR assay. Splenic (A) or BM (B)-derived DCs were prepared from WT (□, ○) or WSX-1-deficient (▪, •). Purified CD4+ T cells from BALB/c mice (2 × 105/200 μl/well) were mixed with the indicated numbers of DCs in either the presence (upper panels) or absence (lower panels) of LPS for 96 h. Cell proliferation was measured by uptake of [3H]thymidine. IFN-γ production in the supernatants was also measured. Data shown are mean ± SD of triplicate samples. ∗, p < 0.05; ∗∗, p < 0.01. Experiments were repeated three times with similar results.

Augmented APC functions of WSX-1-deficient DCs in vivo

Next, we analyzed the APC function of WSX-1-deficient DCs in vivo by adoptive transfer of Ag-pulsed DCs into WT mice. BM-derived DCs were prepared either from WT or WSX-1-deficient mice, pulsed with KLH, and then transferred into footpads of the syngeneic C57BL/6 mice. First, there was no difference in the number of lymphocytes in the draining LN between the two groups of mice after cell transfer (data not shown). Although CD4+ T cells from WSX-1-defieicnt DC-transferred mice proliferated reproducibly (but not significantly) higher than the cells from WT DC-transferred mice in response to KLH stimulation, these CD4+ T cells from WSX-1-deficient DC-transferred mice produced more IFN-γ than cells from WT DC-transferred mice (Fig. 6⇓A). IL-4 production was below detectable levels in this experimental condition. These data demonstrated that WSX-1-defieicnt DCs were more potent as Th1-inducing APCs in vivo than WT DCs. Consistent with this, mice transferred with WSX-1-deficient DCs contained higher levels of KLH-specific IgG2a, a subtype dependent on Th1-type immune responses, than WT DC-transferred mice (Fig. 6⇓B). The levels of KLH-specific IgG1, a subtype dependent mainly on Th2-type responses, were similar between the two groups of mice. All of these data strongly suggested that WSX-1-deficient DCs initiate higher levels of Th1-biased immune responses than WT DCs in vivo.

FIGURE 6.
FIGURE 6.

Induction of Th1 responses in vivo by Ag-pulsed DC transfer. A, Splenic DCs were prepared and incubated with KLH. DCs were washed and administered into the hind footpad of WT mice. The draining LN were removed 5 days after transfer and lymphocytes were cultured with or without KLH. Ag-specific proliferation and IFN-γ production were measured. □, No Ag; ▪, with KLH Ag; #, not significant; ∗∗, p < 0.05. B, Sera were taken from the DC-transferred mice as in A and analyzed for KLH-specific Ab titers. ▴, WSX-1-deficient DC transfer; ▪, WT DC transfer; □, no DC transfer. Numbers below the panels show log10 dilution. C, WT mice were transferred with L. major lysate-pulsed DCs and infected with L. major in their hind footpad as described in Materials and Methods. Footpad swelling was then measured. ○, Untreated mice; □, WT DC transferred; ▪, WSX-1-deficient (KO) DC transferred; ∗∗; p < 0.01 and ∗, p < 0.05 as compared with KO DC-transferred mice. D, On day 14 after infection, parasite numbers in the footpads from control (□), WT DC- transferred (▦), or WSX-1-deficient DC-transferred (KO DC, ▪) mice. Data shown are mean ± SD (n = 5 per group). ∗∗, p < 0.01. E, On day 2 after infection, CD4+ T cells from the draining LN were stimulated with L. major Ag and were examined for IFN-γ and IL-4 production. □, No Ag; ▪, with L. major Ag; ∗∗, p < 0.01. Experiments were repeated three times with similar results.

Protection against L. major infection bestowed by WSX-1-deficient DC transfer

L. major is intracellular protozoa and that the clearance of the parasites exclusively dependent on proper Th1 responses and IFN-γ production has been reported (18). Given the augmented APC function of WSX-1-deficient DCs with Th1-inducing features, we then examined the effect of the adoptive transfer of DCs pulsed with L. major lysates. Since the untreated WT mice with an H-2b haplotype were a resistant strain, a slight but significant swelling of the footpad, up to 1 mm, was observed on day 13 after infection (Fig. 6⇑C). In mice transferred with WT DCs pulsed with L. major, lysates showed footpad swelling on day 15 after infection, whose peak was lower than in untreated mice. Mice transferred with WSX-1-deficient DCs pulsed with L. major lysates showed no peak swelling of the footpad, demonstrating successful protection against L. major infection by WSX-1-deficient DC transfer. Consistent with these results, parasite numbers in the footpads were significantly lower in WSX-1-deficient DC-transferred mice than in control or WT DC-transferred mice (Fig. 6⇑D). In consistence with this observation, the draining LN cells produced significantly higher levels of IFN-γ in response to L. major lysates than cells from WT DC-transferred mice or untreated control mice (Fig. 6⇑E). This protective effect of WSX-1-deficient DC transfer is detectable fairly early after cell transfer (9 days after transfer; 2 days after infection). T cells from both untreated control and WT DC-transferred mice produced substantial amounts of IFN-γ, similar to those by cells from WSX-1-deficient DC-transferred mice, at 4 wk after infection, later than in WSX-1-deficient DC-transferred mice (data not shown). These data demonstrated that WSX-1-deficient DCs elicited stronger Th1-biased immune responses in vivo after transfer than did WT DCs.

Augmented NK cell IFN-γ production by WSX-1-deficient DCs

Recent lines of evidence show that DCs also augment cytotoxic functions and IFN-γ production of NK cells (19). To address whether WSX-1-deficient DCs are also potent in augmenting NK functions, purified WT NK cells were cocultured with either WT or WSX-1-deficient DCs and production of cytokines, expression of cytotoxic molecules, and killing activity were measured. NK cells primed with WSX-1-deficient DCs secreted more IFN-γ than did NK cells with WT DCs, especially at high DC:NK ratios (Fig. 7⇓A). Although not significant, NK cells with WSX-1-deficient DCs produced slightly more TNF-α than did NK cells with WT DCs. In RT-PCR analyses, expression of perforin was augmented in NK cells with WSX-1-deficient DCs than with WT DCs, while there was no apparent difference in the expression of granzyme B (Fig. 7⇓B). There was only a small increase in the killing activity against YAC-1 cells by NK cells primed with WSX-1-deficient DCs over those primed with WT DCs (Fig. 7⇓C). Thus, WSX-1 deficiency also resulted in up-regulation of DC function for NK cell priming, especially for IFN-γ production and perforin expression.

FIGURE 7.
FIGURE 7.

Augmented NK cell IFN-γ production by WSX-1-deficient DCs. A, Purified NK1.1+ NK cells were cocultured with WT or WSX-1-deficient CD11c+ splenic DCs. Cytokine production in the supernatants was measured by ELISA. □, With WT DCs; ▪, with WSX-1-deficient DCs; ∗∗, p < 0.01. B, NK cells cultured as in A (at 5:1) were analyzed for expression of perforin and granzyme B (Gr B) by RT-PCR analyses and also by real-time PCR analyses. □, NK cells without DCs; ▦, NK cells with WT DCs; ▪, NK cells with WSX-1-deficient DCs. C, Cytotoxicity activity was measured using 51Cr-labeled YAC-1 cells as target. ∗, p < 0.05. Experiments were repeated three times with similar results.

Enhanced responses of WSX-1-deficient DCs to LPS stimulation

Finally, we examined the functions of WSX-1-deficient DCs (Fig. 8⇓). TNF-α production by LPS-stimulated WSX-1-deficient DCs was significantly higher than that by WT DCs (Fig. 8⇓A). Although IL-12p40 production was also slightly augmented in WSX-1-deficient DCs, there was no difference in IL-6 production between WT and KO DCs (data not shown). Addition of rIL-27 suppressed both TNF-α and IL-12 production by WT DCs but not WSX-1-deficient DCs, demonstrating the suppressive effect of IL-27 on cytokine production by DCs (Fig. 8⇓A). Then we examined the signal pathways in LPS-stimulated DCs, including phosphorylation of STAT1/3, IκB, and p38 (20) (Fig. 8⇓, B and C). Phosphorylation of STAT3 was augmented in WSX-1-deficient DCs over that of WT DCs upon LPS stimulation (Fig. 8⇓B). Similarly, phosphorylation of p38 and IκB, both phosphorylated downstream of LPS signaling through TNFR-associated factor 6, was augmented in WSX-1-deficient DCs over that of WT DCs. Although simultaneous IL-27 stimulation augmented the STAT3 phosphorylation in WT DCs, it had no effect in WSX-1-deficient DCs. STAT1 phosphorylation was faintly detected only in WT DCs, which was augmented by IL-27 stimulation. Because the faint STAT1 activation was not detected in WSX-1-deficient DCs, this was presumably induced by endogenously produced IL-27. Time course experiments also detected augmented STAT3 phosphorylation in WSX-1-deficient DCs. By 60 min after LPS stimulation, STAT3 phosphorylation was detectable in WSX-1-deficient DCs when it was not detectable in WT DCs (Fig. 8⇓C). STAT3 is known to be activated downstream of IL-6R and IL-10R. Addition of anti-IL-6 Ab showed no effects on TNF-α and IL-12 production both by WT and KO DCs (Fig. 8⇓A). Addition of anti-IL-10 Ab slightly augmented TNF-α production by WT DCs, up to almost similar levels to those by KO DCs. However, there was no significant difference in IL-10 produced in the supernatants, 1785.5 ± 72.9 and 1963.3 ± 70.3 ng/ml by WT and WSX-1-deficient DCs, respectively. Anti-IL-10 Ab addition showed no apparent effect on IL-12 production. The expression of suppressor of cytokine signaling (SOCS) 3, induced downstream of STAT3 activation, was drastically augmented in WSX-1-deficient DCs over that of WT DCs upon LPS stimulation (Fig. 8⇓D). These data in toto demonstrated that IL-27 showed direct suppressive effects and also that WSX-1-deficient DCs were hyperreactive to LPS stimulation by cytokine production, including TNF-α and IL-12, and also by downstream signaling, including STAT3, p38, and IκB phosphorylation. Thus, it was indicated that IL-27/WSX-1 plays an inhibitory role in the LPS activation of DCs and the lack of this inhibition resulted in the augmentation of overactivation and cytokine production of DCs.

FIGURE 8.
FIGURE 8.

Hyperreactivity of WSX-1-deficient DCs to LPS stimulation. A, Splenic DCs were prepared and stimulated with LPS for 24 h. In some cultures, rIL-27, anti-IL-6 Ab, or anti-IL-10 Ab was added as indicated. Culture supernatants were analyzed for cytokine production by ELISA. □, WT DCs; ▪, WSX-1-deficient DCs. Shown are representative data (mean + SD) of three independent experiments. ∗, p < 0.05. B, Similarly, DCs were stimulated with LPS in either the presence or absence of rIL-27 for 12 h. Cells were lysed and analyzed for the presence of respective molecules. P-STAT3, P-STAT1, P-p38, and P-IκBα stand for phosphorylated forms of respective protein. C, Splenic DCs were similarly stimulated with LPS for the indicated minutes and were analyzed for STAT1 and STAT3 phosphorylation. D, DCs stimulated as in B were examined for SOCS3 expression using real-time PCR. Experiments were repeated three times with similar results.

Discussion

Our results illustrated the augmented functions of WSX-1 (IL-27Rα)-deficient DCs as APCs. Since the immunosuppressive roles of IL-27/WSX-1 have been proved mainly in T cells and macrophages (10, 11, 21, 22, 23), this is the first demonstration that IL-27R signaling also suppresses the function of DCs. The data also show a possibility that WSX-1-deficient DCs with augmented Th1-inducing function be used as an adjuvant for vaccination against infection and cancer.

As shown in Fig. 1⇑, although the expression of IL-27 (p28 plus EBI-3) was observed even in mature DCs before activation, the expression of WSX-1 in DCs was only observed in LPS-stimulated DCs but not in immature or mature DCs. Therefore, while being required for initial Th1 development, IL-27/WSX-1 has a suppressive role on activated DCs as a negative feedback system to attenuate the excess of immune responses, just like IL-27/WSX-1 is suppressive on activated (but not naive) T cells (24). As has been reported previously (6, 7), IL-27/WSX-1 suppresses possibly lethal immunopathology induced by cytokine secretion and, without the suppressive effect, the mice died from lethal inflammation. Similarly, in Mycobacterium tuberculosis infection, inflammatory cytokines, including TNF-α, were overproduced in WSX-1-deficient mice (21). Although these inflammatory cytokines such as TNF-α and IFN-γ are beneficial for bacterial clearance, WSX-1-deficient mice died from cachexia induced by the cytokines (21). Because lack of EBI-3 (a subunit of IL-27) resulted in enhanced neutrophil migration and oxidative burst during experimental peritonitis concomitant with high bacterial clearance (25), the high oxidative burst in the absence of IL-27/WSX-1 signaling may induce tissue inflammation while being beneficial for bacterial clearance.

Recently, two groups independently reported the inhibition of IL-2 production by IL-27 (22, 23). For the distinct functions of IL-27, Villarino et al. (26) proposed an intriguing model in their report. In their model, because naive T cells express barely detectable levels of WSX-1 (26), they received little inhibitory signal of IL-27 through WSX-1 and the naive T cells with their IL-2 production began to proliferate at activation phase. During the polarizing phase, activated and committed T cells now expressed WSX-1 and subsequently IL-12Rβ2 for IFN-γ production (with relatively little IL-2 production, presumably to avoid excess proliferation). Our current findings are in line with this model; at the initial T cell activation phase, expression of WSX-1 (and IL-27) by DCs was relatively low and the DCs were capable of delivering stimulatory signals through CD80/86 to T cells. At later phases, DCs expressed substantial levels of WSX-1 and, by receiving an inhibitory signal through IL-27/WSX-1, DCs were less stimulatory to attenuate excessive T cell proliferation. IL-27/WSX-1 thus seems to warrant effective (but not excessive) proliferation and properly controlled differentiation of Th1 cells by regulating cytokine production by T cells and also APC function of DCs.

Although Th1-promoting mechanisms through IL-27/WSX-1 have been clearly shown (4, 27), the suppressive mechanisms have not been fully understood. Although IL-2 suppression by IL-27 has been reported (22, 23), we have no evidence that IL-2 suppression explains the suppression of APC function in DCs in the current study. Because both STAT1 and STAT3 are activated downstream of the WSX-1-gp130 receptor complex upon binding of IL-27 (24, 28), one or both of these transcription factors may be responsible of the suppression. Actually, STAT1 is responsible for the inhibition of Th17 development by IL-27 (11, 29). Thus, it is possible that the lack of STAT1 activation in response to LPS stimulation in WSX-1-deficient DCs (Fig. 8⇑, B and C) led to insufficient SOCS1 induction and subsequent failure of LPS signaling inhibition, as demonstrated in SOCS1-deficient cells (20, 30). In contrast, we reported that STAT3 was partially involved in IL-27-mediated suppression of cytokine production including IL-17 by T cells (24). Holscher et al. (21) reported similar overactivation of WSX-1-deficient macrophages and direct suppression of macrophage activation by IL-27. Because IL-27 stimulation induced STAT3 phosphorylation in macrophages, the STAT3 activation was assumed responsible for IL-27-mediated suppression, as observed in IL-10 stimulation. However, as shown in Fig. 8⇑, B and C, WSX-1-deficient DCs showed overphosphorylation of STAT3 in response to LPS stimulation. Roles of STAT3 in IL-27/WSX-1-mediated suppression may be distinct in LPS activation of DCs.

Addition of anti-IL-10 Ab increased the production of TNF-α and IL-12 by LPS-stimulated WT DCs (Fig. 8⇑A). Thus, IL-10 produced by stimulated DCs appears to be responsible for IL-27-mediated suppression at least partially. However, since there was no difference in IL-10 production by WT and WSX-1-deficient DCs, other suppressive mechanisms, in addition to IL-10 production, should also be responsible for the IL-27-mediated DC suppression. Ruckerl et al. (31) reported that IL-10, IL-4, and IL-27 modulate macrophage activation in a distinct manner, respectively. Interestingly, these cytokines collaborate in attenuation of macrophage activation by successive up-regulation of the IL-4Rα and then WSX-1 on macrophages. It is reasonable to assume that similar factors exert their suppressive effects in a synergistic manner on DCs.

In addition to their augmented APC function, WSX-1-deficient DCs are characterized by their high Th1-inducing function. The WSX-1-deficient DCs with augmented APC function and Th1-inducing potential may have great advantage in eventual therapeutic usage. As shown in an L. major infection assay (Fig. 6⇑, C and D), WSX-1-deficient DCs pulsed with L. major lysates bestowed Th1-biased protective immunity. Because WSX-1-deficient mice were in the resistant C57BL/6 background, the footpad swelling in the control C57BL/6 mice was small. However, the footpad swelling in WSX-1-deficient DC-transferred mice was reproducibly less than control and WT DC-transferred mice (Fig. 6⇑). Of note, IFN-γ production by draining LN cells from WSX-1-deficient DC-transferred mice was remarkably higher than that from control and WT DC-transferred mice early after infection (day 2 after infection). In susceptible backgrounds such as BALB/c, the same effect of WSX-1 deficiency could induce more drastic curative effects over control mice, which should show exacerbated footpad swelling with far less IFN- γ production upon infection. So far, various approaches have been taken into consideration to augment antipathogen or antitumor vaccination, such as expression of cytokines or costimulatory molecules by DCs. WSX-1-deficient DCs, or WSX-1-knockdown DCs, may be a promising adjuvant to augment Th1-skewed immunity. In addition to their Th1-promoting function, WSX-1-deficient DCs also effectively activated NK cells through increasing perforin expression and especially through increasing IFN-γ production (Fig. 7⇑). Although NK cells activated with WSX-1-deficient DCs showed only a marginal increase in the killing activity against YAC-1 cells over NK cells activated with WT DCs, the increase in perforin and IFN-γ expression may cause augmented NK activity against other target cells. Thus, WSX-1-deficient DCs may be of potential importance in NK cell-mediated defense against viral infection and tumors. Possible augmentation of APC function of WSX-1-deficient DCs toward CD8+ T cells is currently under investigation.

Acknowledgments

We thank members of “Project W” and Dr. Hiromitsu Hara for helpful discussion, Yukiko Baba for animal husbandry, and Shizuko Furukawa for secretarial work.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture (to H.Y.), Japan Society for the Promotion of Science (to H.Y.), Japan Research Foundation for Clinical Pharmacology (to H.Y.), Sumitomo Foundation, Basic Science Research Projects (to H.Y.), Naito Foundation (to H.Y.), and Takeda Science Foundation. This work was also supported by the president’s research project expenditure of Saga University (to H.Y.). W.S. is a recipient of the Japan Society for the Promotion of Science Postdoctoral Fellowship for foreign researchers.

  • ↵2 Address correspondence and reprint requests to Dr. Hiroki Yoshida, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima, Saga, Japan. E-mail address: yoshidah{at}med.saga-u.ac.jp

  • ↵3 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; WT, wild type; KLH, keyhole limpet hemocyanin; LN, lymph node; MFI, mean fluorescence intensity; SOCS, suppressor of cytokine signaling; BM, bone marrow; KO, knockout.

  • Received January 10, 2007.
  • Accepted August 27, 2007.

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