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Keywords:

  • Blimp-1;
  • Egr-2;
  • IL-10;
  • IL-27;
  • inducible regulatory T (Treg) cells;
  • Prdm1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Interleukin-27 (IL-27) suppresses immune responses through inhibition of the development of IL-17 producing Th17 cells and induction of IL-10 production. We previously showed that forced expression of early growth response gene 2 (Egr-2), a transcription factor required for T-cell anergy induction, induces IL-10 and lymphocyte activation gene 3 expression and confers regulatory activity on CD4+ T cells in vivo. Here, we evaluated the role of Egr-2 in IL-27-induced IL-10 production. Among various IL-10-inducing factors, only IL-27 induced high levels of Egr-2 and lymphocyte activation gene 3 expression. Intriguingly, IL-27 failed to induce IL-10 in Egr-2-deficient T cells. IL-27-mediated induction of Prdm1 that codes B lymphocyte induced maturation protein-1, a transcriptional regulator important for IL-10 production in CD4+ T cells, was also impaired in the absence of Egr-2. Although IL-27-mediated IL-10 induction was dependent on both STAT1 and STAT3, only STAT3 was required for IL-27-mediated Egr-2 induction. These results suggest that IL-27 signal transduction through Egr-2 and B lymphocyte induced maturation protein-1 plays an important role in IL-10 production. Furthermore, Egr-2-deficient CD4+ T cells showed dysregulated production of IFN-γ and IL-17 in response to IL-27 stimulation. Therefore, Egr-2 may play key roles in controlling the balance between regulatory and effector cytokines.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Naïve CD4+ T cells play central roles in immune regulation by differentiating into effector as well as Treg-cell subsets. Recently, a number of Treg-cell subsets, which are important for suppressing effector T cells, tissue inflammation, and autoimmunity, have also been identified. On one hand, CD4+CD25+ Treg cells, which express the transcription factor Foxp3, have a dominant function in immune suppression and the maintenance of immune homeostasis [1, 2]. On the other hand, other Treg cells, which arise in the periphery, such as Treg type I (Tr1) cells and Th3 cells produce the suppressive cytokines IL-10 and TGF-β1, and contribute to the suppression of immune responses in a Foxp3-independent manner [3, 4]. IL-10 is an anti-inflammatory cytokine which was initially described as a cytokine associated with Th2 cells that inhibits the production of IFN-γ by Th1 cells [5, 6]. A number of reports have revealed that IL-10 suppresses cytokine production and proliferation of T cells [7, 8] and inhibits the T-cell-stimulating capacity of APCs [9]. IL-10-deficient mice die with spontaneously developed inflammatory bowel disease [10].

Interleukin-27 (IL-27), a member of the IL-12/IL-23 hetero-dimeric family of cytokines produced by APCs, is composed of two chains, p28 and EBV-induced gene 3 [11]. IL-27 induces the expansion of Th1 cells by activating the STAT1-mediated T-bet pathway [12], but IL-27Rα-deficient mice developed severe EAE with enhanced Th17-cell responses [13]. The immuno-suppressive effects of IL-27 depend on inhibition of the development of Th17 cells and induction of IL-10 production [14]. Recently, IL-27 has been identified as a differentiation factor for IL-10-producing Tr1 cells [15-17]. On the other hand, B lymphocyte induced maturation protein-1 (Blimp-1) (coded by Prdm1 gene), a zinc finger-containing transcriptional regulator that is well known to be a regulator of plasma cell differentiation, is also important for IL-10 production in naïve CD4+ T cells. Martins et al. [18, 19] reported that Blimp-1-deficient CD4+ T cells proliferated more and produced excess IL-2 and IFN-γ, but reduced IL-10 after TCR stimulation.

Early growth response gene 2 (Egr-2) and Egr-3 have been reported to be transcription factors for TCR-induced negative regulatory program controlling Cbl-b expression [20]. We previously identified a Treg population expressing lymphocyte activation gene 3 (LAG-3) in a fraction of CD4+CD25CD45RBlow T cells and showed that forced expression of Egr-2 induces IL-10, LAG-3, and Blimp-1 expressions and confers regulatory activity in vivo on CD4+ T cells [21]. We here describe that IL-27 induces Egr-2 and LAG-3 as well as IL-10 in CD4+ T cells. Moreover, Egr-2-deficient CD4+ T cells exhibited reduced expression of IL-10 and Blimp-1 and reciprocally enhanced secretion of IFN-γ and IL-17 in response to IL-27. Results from a LUC assay and ChIP assay show that Egr-2 binds to the promoter lesion of Prdm1 to activate its transcription. These results indicate that IL-27 signal transduction through Egr-2 and Blimp-1 is required for IL-10 production in CD4+ T cells and controls the balance between regulatory and inflammatory cytokines.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

IL-27 induces Egr-2, IL-10, and LAG-3 expression in naïve CD4+ T cells

We previously reported that the forced expression of Egr-2 induces IL-10 production in CD4+ T cells and confers the phenotype of CD4+CD25LAG3+ Treg cells [21]. First, we confirmed the moderate induction of intracellular Egr-2 in TCR-stiumulated CD4+ T cells and observed that IL-10 production was restricted to cells expressing intracellular Egr-2 (Fig. 1A). Then, we explored the factor inducing Egr-2, which confers the pheno-type of CD4+CD25LAG3+ Treg cells. Various IL-10-inducible cytokines, such as IL-27, TGF-β [22], IL-21 [23], and IL-10, were added to a co-culture of splenic CD4+ T cells from TEα TCR transgenic mice expressing I-Eα-specific TCR [24] and B cells from B6 WT mice in the presence of Eα52–68 peptides. In addition, the effect of the IL-10-inducible chemical substance zymosan was examined because it induces DCs to secrete abundant IL-10 in a TLR-2- and dectin-1-mediated activation of ERK/MAPK-dependent manner [25]. Notably, IL-27 predominantly induced both Egr-2 and LAG-3 mRNA expressions relative to the other cytokines and zymosan. IL-27 did not induce Foxp3 mRNA expression (Fig. 1B), which is compatible with previous reports [15] and the fact that CD4+CD25LAG3+ Treg cells hardly expressed Foxp3 protein [21]. When we added IL-27 to naïve CD4+ T cells stimulated with plate-coated anti-CD3ε and anti-CD28 mAbs, Egr-2 protein was clearly detected by intracellular staining. This induction was abolished in Egr-2-deficient CD4+ T cells cultured with IL-27 and also in IL-27Rα (WSX-1)-deficient CD4+ T cells (Fig. 1C). Interestingly, LAG-3 was predominantly induced in B6 WT CD4+ T cells expressing Egr-2, and IL-27 alone did not induce Egr-2 in the absence of TCR stimulation. IL-27 more efficiently induced Egr2+LAG3+ cells than the other IL-12 family cytokines, IL-12 and IL-23 (Fig. 1D). Although IL-2 is required for IL-27-induced IL-10 expression through Blimp-1 in CD8+ T cells [26], IL-2 by itself could not induce Egr2+LAG3+ cells and showed no additive effect on IL-27-induced Egr-2 and LAG-3 expressions (Fig. 1D). No significant association was seen between the extent of cell division and the amount of Egr-2 expression, while Egr-2 induction was limited to proliferating cells (Fig. 1E).

image

Figure 1. IL-27 induces the simultaneous expression of Egr-2 and LAG-3 in CD4+ T cells. (A) Naïve CD4+ T cells from C57BL/6 WT mice were cultured with anti-CD3/CD28 mAb. On day 5, Egr-2 and IL-10 expression were analyzed by intracellular staining. Data shown are representative of three experiments performed. (B) Naïve CD4+ T cells from TEα TCR transgenic mice were cultured with B cells from WT mice with Eα52−68 peptide in the presence of indicated factors. Expression of Egr-2, LAG-3, and Foxp3 was measured by quantitative RT-PCR. Data are presented as mean + SD (n = 3; replicate wells). Experiments were performed three times. (C) The induction of Egr-2 in CD4+ T cells stimulated with IL-27. Naïve CD4+ T cells from C57BL/6 WT, Egr-2 CKO, Blimp-1 CKO, and WSX-1 KO mice were cultured with anti-CD3/CD28 mAb in the presence or absence of IL-27. Egr-2 expression was detected by intracellular staining on day 5. Data shown are representative of five experiments performed. (D) CD4+Egr-2+LAG3+ T-cell induction was analyzed. Naïve CD4+ T cells from C57BL/6 WT mice were cultured with or without anti-CD3/CD28 mAb under the indicated conditions. Data shown are representative of two or three experiments performed. (E) Carboxyfluorescein diacetate succinimidyl diester labeled naïve CD4+ T cells from C57BL/6 WT mice were stimulated as in (C). On day 5, T-cell proliferation and Egr-2 expression were determined by flow cytometry. Data shown are representative of five experiments performed. *p < 0.05; Student's t-test.

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IL-27-mediated induction of IL-10 and Blimp-1 is impaired in Egr-2 conditional KO (CKO) mice

Multiple observations support the idea that Blimp-1 regulates T-cell responsiveness by attenuating IL-2 production. IL-2 production in Blimp-1-deficient CD4+ T cells is elevated by stimulation via TCR [18]. As IL-2 signaling induces Blimp-1 transcription, Blimp-1 makes a negative feedback loop for Il2 transcription in T cells [19]. Recently, it was shown that Blimp-1 positively regulates IL-10 production in CD4+ T cells [18, 27]. Blimp-1 is required for IL-10 production and high ICOS expression in CD4+CD25+Foxp3+ Treg cells [28]. Therefore, the role of Egr-2 and Blimp-1 in IL-27-induced IL-10 production was examined using naïve CD4+ T cells from Egr-2 CKO (Egr2fl/fl-CD4-Cre+) and Blimp-1 CKO (Prdm1fl/fl-CD4Cre+) mice. Consistent with our previous observation that the forced expression of Egr-2 induced the high mRNA expression levels of Blimp-1 in CD4+ T cells [21], Egr-2-induction by IL-27 was not affected in the absence of Blimp-1 (Fig. 1C). In CD4+ T cells both from Egr-2 CKO mice and Blimp-1 CKO mice, the induction of Il10 transcription and IL-10 protein expression by IL-27 was impaired (Fig. 2A and B), and these inductions were not observed in CD4+ T cells from WSX-1 KO mice (Fig. 2A and B). Moreover, Blimp-1 mRNA induction by IL-27 was also impaired in Egr-2-deficient CD4+ T cells (Fig. 2A). This result suggested that Egr-2 is essential for IL-10 production via Blimp-1 expression in IL-27-stimulated CD4+ T cells. When we analyzed the induction of IL-10 and Blimp-1 mRNA expressions by other IL-12 family cytokines, IL-12 showed only marginal induction of IL-10 and Blimp-1 mRNA expressions and IL-23 induced no up-regulation of IL-10 and Blimp-1 mRNA expressions (Fig. 2C). We also found that IL-2 had no additive effect on IL-27-induced IL-10 and Blimp-1 mRNA expressions in CD4+ T cells (Fig. 2C).

image

Figure 2. IL-27-mediated induction of IL-10 and Blimp-1. (A) Expression of IL-10 (left) and Blimp-1 (right) mRNA was measured by quantitative RT-PCR. Naïve CD4+ T cells were isolated from C57BL/6 WT, Egr-2 CKO, Blimp-1 CKO, or WSX-1 KO mice and cultured with anti-CD3/CD28 mAb in the presence or absence of IL-27. Data are shown as mean + SD (n = 3; replicate wells). Experiments were performed four times. (B) IL-10 concentrations in culture supernatants of stimulated CD4+ T cells from C57BL/6 WT, Egr-2 CKO, Blimp-1 CKO, or WSX-1 KO mice were measured by ELISA. Data are shown as mean + SD (n = 3; replicate wells). Experiments were performed three times. (C) Expression of IL-10 and Blimp-1 mRNA was measured by quantitative RT-PCR. Naïve CD4+ T cells were isolated from C57BL/6 WT and cultured with anti-CD3/CD28 mAb under the indicated condition. Data are shown as mean + SD (n = 3; replicate wells). Experiments were performed two times. *p < 0.05; Student's t-test.

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Egr-2 directly binds to the promoter region of Prdm1 and enhances its activity

Next, we investigated whether Egr-2 regulates Blimp-1 transcription. To address this possibility, we performed a LUC reporter assay. A pGL3-LUC vector subcloned with the promoter region from –1500 bp to the Prdm1 transcription start site [29] was co-transfected with a pMIG-Egr-2 vector to 293T cells. As shown in Figure 3A, Egr-2 significantly enhanced the activity of the Prdm1 promoter. Next, a ChIP assay was performed with antibodies against Egr-2 to investigate whether Egr-2 directly binds to the promoter region of Blimp-1 in CD4+ T cells. Among four promoter regions examined (−3000 bp, −2000 bp, −1000 bp, and +1000 bp from its transcription site) of Blimp-1, only one region (−1000 bp) showed significant enrichment compared with control, indicating that Egr-2 specially binds to the Blimp-1 promoter, but not to Lag3 and Il10 promoters (Fig. 3B and Supporting Information Fig. 2A). Cretney et al. reported that Blimp-1 binds to intron 1 of the Il10 locus and, together with IFN regulatory factor-4, directly regulates IL-10 expression in CD4+CD25+ Treg cells by the remodeling of active chromatin at the Il10 locus [28]. Our observation suggested that IL-10 regulation with Blimp-1 was controlled by Egr-2.

image

Figure 3. Blimp-1 promoter activity is directly regulated by Egr-2. (A) Egr-2 enhanced LUC activity regulated by the Blimp-1 promoter. 293T cells were co-transfected with pGL-3-(-1500 Blimp-1)-LUC vector or pGL-3-Basic-LUC control vector and a pMIG-Egr-2 vector or a pMIG mock vector. LUC activity was measured 24 h after transfection. Data are shown as mean + SD of duplicates, from one experiment representative of at least three performed. (B) ChIP-coupled quantitative PCR analysis of Egr-2 binding to promoter regions in mouse CD4+ T cells. Normal IgG and anti-Egr-2 antibodies were used for IP assays. The guanosine monophosphate reductase locus was used as a negative control. The enrichment of Egr-2 binding to each promoter was determined. Mean + SD of triplicates done in one experiment representative of at least three performed are shown.

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IL-27-induced Egr-2 expression is dependent on STAT3

STAT1 and STAT3 have been shown to be crucial for IL-10 production from IL-27-stimulated naïve CD4+ T cells [17]. We investigated the effect of STAT1 and STAT3 deficiencies on IL-27-induced Egr-2 expression. As shown in Figure 4A and B, Egr-2 induction by IL-27 in CD4+ T cells was impaired by a STAT3 deficiency, but not by a STAT1 deficiency. When we analyzed the induction of Il10 transcription and IL-10 protein expression by IL-27 in STAT1- and STAT3-deficient CD4+ T cells, IL-10 protein induction by IL-27 was abolished both in STAT1 KO and in STAT3 CKO CD4+ T cells, although IL-10 mRNA expression levels were slightly up-regulated by IL-27 in STAT1 KO CD4+ T cells (Fig. 4C and D). These results suggest that IL-27-induced Egr-2 expression in CD4+ T cells is mostly dependent on STAT3, although both STAT1 and STAT3 are important for IL-10 production by IL-27. Next, we investigated the effect of other STAT1 or STAT3 activating cytokines for Egr-2 induction. IL-6 and IFN-γ were selected as the representatives of cytokines activating STAT3- and STAT1-mediated pathways, respectively. As shown in Figure 4E, IL-6 induced Egr-2 expression as effectively as IL-27 in CD4+ T cells, but IFN-γ did not. Interestingly, both IL-10 and Blimp-1 mRNA expressions were also elevated by IL-6, but expression levels seemed to be lower than those by IL-27 (Fig. 4F). IL-6 is a type I cytokine that shares structural homology and a receptor subunit, gp130, with IL-27 and has already been shown to induce IL-10 in CD4+ T cells [17]. These results suggest that Egr-2 is important for IL-10 production mediated both by IL-27 and by IL-6 through the STAT3-dependent pathway.

image

Figure 4. IL-27-induced Egr-2 expression in STAT1 and STAT3 deficiencies. (A) Naïve CD4+ T cells from STAT1 KO, STAT3 CKO, and control mice were cultured with or without IL-27 in the presence of anti-CD3/CD28 mAb. Egr-2 expression was analyzed by flow cytometry on day 5. Plots are gated-on CD4+ cells. Data shown are representative of three experiments performed. (B) Naïve CD4+ T cells were cultured as shown in (A). The percentage of Egr-2-expressing cells in CD4+ T cells was analyzed by flow cytometry on day 5. Data are shown as mean ± SD (n = 3; replicate experiments). (C) The expression of IL-10 mRNA was measured by quantitative RT-PCR. Naïve CD4+ T cells were isolated from STAT1 KO, STAT3 CKO, or control mice and cultured with anti-CD3/CD28 mAb in the presence or absence of IL-27. Data are shown as mean ± SD (n = 3; replicate wells). Experiments were performed two times. (D) IL-10 concentrations in culture supernatants of stimulated naïve CD4+ T cells as in (C) from STAT1 KO, STAT3 CKO, or each control mice were measured by ELISA. Data are shown as mean ± SD (n = 3; replicate wells). Experiments were performed three times. (E) Naïve CD4+ T cells were cultured as shown in (A) under the indicated condition. Egr-2 expression was analyzed by intracellular staining. Data shown are representative of three experiments performed. (F) Expression of IL-10 and Blimp-1 mRNA were measured by quantitative RT-PCR. Naïve CD4+ T cells were cultured as shown in (A) under the indicated condition. Data are shown as mean ± SD (n = 3; replicate wells). Experiments were performed two times. *p < 0.05; Student's t-test.

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Enhanced production of IFN-γ and IL-17 by IL-27 stimulation in Egr-2 CKO mice

To examine the role of Egr-2 in inflammatory cytokine production, we investigated the production of IFN-γ and IL-17 in response to IL-27 stimulation. It has already been shown that Egr-2-deficient CD4+ T cells produce high amounts of IFN-γ and IL-17 after TCR stimulation [30]. As shown in Fig. 5, IFN-γ and IL-17 production from IL-27-stiumulated CD4+ T cells was enhanced by an Egr-2 deficiency, which suggests that Egr-2 may also play an important role in controlling effector cytokine production.

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Figure 5. IL-10, IFN-γ, and IL-17 production in response to IL-27 stimulation. Naïve CD4+ T cells from C57BL/6 WT and Egr-2 CKO mice were cultured with anti-CD3/CD28 mAb in the presence of IL-27. On day 5, IL-10, IFN-γ, and IL-17 concentrations in culture supernatants were measured by ELISA. Data are shown mean + SD (n = 3; replicate wells). Experiments were performed two times. *p < 0.05; Student's t-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Recently, Tr1 cells, characterized by their high secretion of IL-10 and lack of Foxp3 expression, were induced by IL-27 [15-17, 31]. STAT1 and STAT3 have been shown to play an important role in the molecular mechanism of IL-10 production by IL-27 in CD4+ T cells [17]. Although it is clear that STAT1-driven IL-10 production is independent of T-bet, the precise mechanism still remains unclear [17]. The underlying mechanism of IL-10 production through the activation of STAT3 is that the activation of STAT3 leads to the induction of transcription factor c-Maf [32], which is essential for IL-10 production induced by IL-27 [33]. Motomura et al. [34] have reported that transcription factor E4 promoter-binding protein 4 is important for IL-10 production from IL-27-stimulated CD4+ T cells cultured under a Th1 skewing condition. E4 promoter-binding protein 4-deficient Th1 cells failed to produce IL-10 by IL-27 stimulation. It seems that IL-10 production from T cells is controlled by a complex pathway, depending on each subset or surrounding cytokine condition. In this study, we found that another transcription factor Egr-2 mediates IL-10 expression in IL-27-stimulated CD4+ T cells via direct binding to the Blimp-1 promoter. Furthermore, we have shown that IL-27-induced Egr-2 expression in CD4+ T cells is dependent on STAT3, but not on STAT1. Although Egr-2 may be less involved in STAT1- and T-bet-mediated pathways, which are required for IL-10 production, Egr-2 is associated with STAT3-mediated IL-10 production.

IL-27-induced IL-10 production has been considered to be important for gut immunity. In IL-27 receptor (WSX-1)-deficient mice, higher steady-state levels of Th17 cells were observed in the lamina propria and these mice were susceptible to high-dose dextran sulfate, a model of acute intestinal inflammation-induced colitis [35]. Similar to IL-10-deficient mice [36], WSX-1-deficient mice infected with Toxoplasma gondii develop a lethal CD4+ T-cell-mediated response characterized by excessive production of proinflammatory cytokines and massive lymphocytic infiltrates in multiple organs [37]. WSX-1-deficient CD4+ T cells have been shown to be impaired in IL-10 production in CD4+ T cells [17]. Although the Foxp3+ Treg cell is one of the IL-10 producers, it has been shown that there are IL-10-producing T cells other than Foxp3+ Treg cells in the intestine [38]. Moreover, CD4-specific IL-10-deficient mice have been shown to develop more severe colitis than Foxp3+ Treg-specific IL-10-deficient mice [39], suggesting that Foxp3-negative, IL-10-producing T cells may be important for the maintenance of homeostasis in gut immunity. Egr-2-expressing CD4+CD25LAG3+ Treg cells are Foxp3-negative, IL-10-producing T cells and are enriched in Peyer's patch [21]. Our observation that IL-27 induces CD4+Egr2+LAG3+ T cells may be associated with IL-27-mediated control of gut homeostasis; however, a more detailed investigation is required to elucidate the role of IL-27 in keeping intestinal homeostasis.

It has been well documented that stimulation of T cells through TCR in the absence of co-stimulation can result in long-term hyporesponsiveness to subsequent stimulation, which is termed anergy. It has been also reported that Egr-2 is required for the full induction of T-cell anergy [20, 40]. Egr-2 expression is rapidly induced within 6 h after TCR stimulation [41] and our results indicated that although IL-27-mediated Egr-2 induction was dependent on TCR stimulation, the TCR signal was not sufficient to support sustained Egr-2 expression. In addition to IL-27, another STAT3 activating cytokine, IL-6, also induced expressions of Egr-2, Blimp-1, and IL-10. This result was consistent with a previous report in which IL-6 induced STAT3-mediated production of IL-10 in CD4+ T cells [17] and suggested that not only STAT1-STAT3 heterodimers in response to IL-27 stimulation but also STAT3 homodimers in response to IL-6 stimulation could induce Egr-2 expression. However, IL-27 induces Blimp-1 and IL-10 more efficiently than IL-6 and the involvement of STAT1 should be addressed further.

It is well known that IL-2 has paradoxical functions in T-cell homeostasis, acting as a T-cell growth factor and having a crucial function in the maintenance of self-tolerance. Sun et al. [26] reported that the effective induction of IL-10-producing CD8+ CTLs by IL-27 requires the presence of IL-2, and that the IL-2-IL-27-mediated induction of IL-10 as well as the IL-27-mediated induction of IL-10 was Blimp-1 dependent. However, we observed that the addition of IL-2 did not up-regulate IL-10 and Blimp-1 mRNA induction levels by IL-27 in CD4+ T cells. In addition, IL-2 showed no synergistic effect on IL-27-induced Egr-2 and LAG-3 expressions in our experiments. This result is consistent with the fact that increased Egr-2 level by Ag activation was not affected by the addition of IL-2 in peptide treatment-induced CD4+ Treg cells [42]. These observations suggest that Blimp-1 is important for IL-27-induced IL-10 production both in CD4+ and CD8+ T cells, but the pathway leading to the activation of Blimp-1 is differently regulated between these cells.

Egr-2-expressing CD4+CD25LAG3+ Treg cells are anergic and have regulatory activities at least in part via IL-10 production. Because our results showed that Egr-2 is indispensable for the full production of IL-10 in CD4+ T cells after IL-27 stimulation, Egr-2 could be one of the molecular links between anergy and IL-10 production in CD4+ T cells. Further studies will be required to elucidate the relationship between the Egr-2-Blimp-1 pathway and other pathways, which have already been reported to contribute to IL-10 production by IL-27 stimulation.

We also found enhanced production of IFN-γ and IL-17 in Egr-2 CKO mice after IL-27 stimulation. Egr-2 CKO mice develop autoimmune disease characterized by the accumulation of IFN-γ and IL-17-producing CD4+ T cells, and massive infiltration of T cells into multiple organs. The expressions of T-bet, a Th1 transcription factor, IL-6, IL-21, and IL-23, which can induce Th17 differentiation in CD4+ T cells, were not altered in aged Egr-2 CKO mice [30]. Blimp-1 CKO mice develop severe colitis with age and Blimp-1-deficient CD4+ T cells have been shown to produce more IFN-γ than WT after stimulation with PMA plus ionomycin or with TCR plus IL-2 [18]. Recently, Lin et al. [43] reported that NOD-background Blimp-1-deficient CD4+ T cells exhibit significantly enhanced IL-17 production in a steady-state as well as in a Th17-polarizing condition. These observations indicate that increased IFN-γ and IL-17 production in IL-27-stimulated Egr-2-deficient CD4+ T cells may be a direct consequence of reduced Egr-2-Blimp-1 signaling. Although Egr-2 CKO mice did not exhibit colitis, a single-nucleotide polymorphism in a locus at chromosome 10q21, which was identified by genome-wide analysis to have a strong relationship with Crohn's disease susceptibility, exists in a strong linkage disequilibrium region of Egr-2 [44, 45].

In summary, we have shown that Egr-2 mediates IL-27-induced IL-10 production through Blimp-1 transcription in CD4+ T cells. Additionally, IFN-γ and IL-17 production by IL-27 was reciprocally regulated by Egr-2. Egr-2 may play a crucial role in maintaining the balance between regulatory and inflammatory cytokines. Our observation could contribute to the elucidation of the molecular regulation of IL-10 production in CD4+ T cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

Mice

C57BL/6 mice and Prdm1-floxed mice were purchased from Japan SLC and The Jackson Laboratory, respectively. Blimp-1 CKO mice were generated by crossing Prdm1-floxed mice with CD4-Cre transgenic mice in which Cre-induced recombination was detected only in CD4+ T cells. Egr-2 CKO mice were generated by crossing Egr-2-floxed mice [46] with CD4-Cre transgenic mice. TEα TCR transgenic mice were purchased from The Jackson Laboratory. WSX-1 deficient (WSX-1 KO) mice were prepared as described previously [47]. STAT1 KO mice were purchased from Taconic. STAT3 CKO mice (STAT3fl/fl-CD4-Cre+) were generated by crossing STAT3-floxed mice with CD4-Cre transgenic mice. CD4-Cre transgenic mice (line 4196), originally generated by Wilson and colleagues [48], were purchased from Taconic. All mice were used at 7–10 weeks of age. All animal experiments were conducted in accordance with Institutional and National Guidelines.

Reagents, antibodies, and media

The following reagents were purchased from BD Pharmingen: purified mAbs for CD3ε (145–2C11) and CD28 (37.51), Fc block (anti-CD16/32), FITC anti-CD45RB (16A), phycoerythrin (PE) anti-LAG-3 (C9B7W), PE anti-IgG2a (R35–95), PE anti-CD62L (MEL-14), allophycyanin-anti-CD25 (PC61), allophycyanin-Cy7 anti-CD25 (PC61), allophycyanin anti-CD4 (RM4–5), allophycyanin-Cy7 anti-CD4 (RM4–5), allophycyanin anti-LAG-3 (C9B7W), allophycyanin anti-IL-10 (JES5–16E3), allophycyanin anti-IgG2b (MPC-11), biotinylated mAb for CD8α (53–6.7), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD25 (PC61), CD62L (MEL-14), Ter119 (TER119), and streptavidin (SA)- allophycyanin, SA-allophycyanin Cy7, SA-FITC. Qdot605 anti-CD4 (RM4–5) and SA-Qd605 were obtained from Invitrogen. Alexa Fluor 488 anti-LAG-3 (C9B7W) was obtained from AbD Serotec. PE anti-Egr-2 (erongr2) was obtained from e-Bioscience. Streptavidin-conjugated microbeads were purchased from Miltenyi Biotec. Recombinant murine IL-2, IL-10, IL-12, IL-21, and IL-27 were obtained from R&D Systems. Recombinant human TGF-β1 was purchased from R&D Systems. Recombinant murine IL-23 was obtained from Biolegend. Zymosan was obtained from Sigma. Eα52−68 peptide was purchased from Takara (Otsu, Japan). T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 μg/mL L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol (all purchased from Sigma).

In vitro T-cell differentiation

Naïve CD4+ T cells (CD4+CD45RBhiCD62LhiCD25) from C57BL/6 WT, Egr-2 CKO, or Blimp-1 CKO mice, WSX-1 KO mice, and STAT1 KO, or STAT3 CKO mice were isolated from their splenocytes. Briefly, single cell suspensions were first purified by negative selection with MACS (Miltenyi Biotec) using anti-CD8α mAb, anti-CD11b mAb, anti-CD11c mAb, anti-CD19 mAb, anti-CD25mAb, and anti-Ter119 mAb, and were then purified by positive selection with anti-CD62L microbeads. The purity of MACS sorted cells was >90%. Purified cells were cultured in flat-bottomed 24-well plates coated with anti-CD3ε (2 μg/mL) and anti-CD28 (2 μg/mL). Mouse IL-27 (25 ng/mL) was added at the start of culturing. To assess T-cell proliferation, purified naïve CD4+ T cells were labeled with 1 μM carboxyfluorescein diacetate succinimidyl diester (Invitrogen) by incubation for 5 min at 37°C in the dark at a density of 2 × 106 cells/mL in RPMI medium. Other cytokines used were as follows: IL-2; 20 ng/mL, IL-6; 10 ng/mL, IL-12; 20 ng/mL, IL-23; 20 ng/mL and IFN-γ; 10 ng/mL.

In vitro response of TCR transgenic CD4+ T cells to peptide

A total of 1 × 106 cells of CD4+ T cells from Eα52−68/I-Ab-specific transgenic mice were purified by positive selection with anti-CD4 microbeads and cultured with 5 × 105 cells of B cells from C57BL/6 WT mice in the presence of Eα52−68 peptide (3 μM) in flat-bottomed 24-well plates. IL-27 (20 ng/mL), TGF-β1 (20 ng/mL), IL-21 (50 ng/mL), IL-10 (50 ng/mL), and zymosan (25 μg/mL) were added, respectively.

RNA isolation, cDNA synthesis, and quantitative real-time PCR

CD4+ T-cell RNA was prepared using an RNeasy Micro Kit (Qiagen). RNA was reverse-transcribed to cDNA with random primers (Invitrogen) and Superscript III (Invitrogen) in accordance with the manufacturer's protocol (Invitrogen). The cellular expression level of each gene was determined by quantitative real-time PCR analysis using an iCycler (Bio-Rad). The PCR mixture consisted of 25 μL of SYBR Green Master Mix (Qiagen), 15 pmol of forward and reverse primers, and cDNA samples in a total volume of 50 μL. Expression was normalized to the expression of β-actin. Specific primers for each indicated promoter were listed in Supporting Information Table 1.

Flow cytometry and cell sorting

Cultured T cells were harvested and stained using predetermined optimal concentrations of the respective antibodies. After Fc blocking (antimouse CD16/CD32 mAb), prepared cells were stained with the indicated mAbs: Qdot605 anti-CD4, allophycocyanin anti-LAG-3, and SA-allophycocyanin Cy7. For intracellular anti-Egr-2 staining, cells were stained using the Foxp3 staining buffer set (e-Bioscience). For co-staining of Egr-2 and IL-10, cells were re-stimulated for 4 h at 37°C with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma), and for final 2 h with GolgiStop (1 μL/mL; BD Biosciences), followed by surface staining. Cells were then fixed with 2% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% saponin (Sigma) containing anti-Egr-2 and anti-IL-10 antibodies for 30 min at room temperature in the dark. Analysis and cell sorting of CD4+ T cells were performed using FACSVantage with CellQuest (Becton Dickinson). Data were processed with FlowJo software. A full gating strategy was shown in Supporting Information Fig. 1.

ELISA

Cytokines in culture supernatants of CD4+ T cells were analyzed using ELISA kits according to the manufacturer's instructions (Thermo Scientific and Biolegend).

LUC reporter assay

The Dual-Luciferase Reporter Assay System was used (Promega). 293T cells were cultured in 96-well plates and transfected with pGL-3-(-1500 Blimp-1) LUC reporter plasmids and phRL-(thymidine kinase) LUC control plasmids with either a pMIG vector or pMIG vector containing Egr-2 using Fugene6 (Roche). Cells were harvested 48 h later and LUC activity was assessed using MicroLumat Plus LB96V Luminometer (Berthold).

ChIP assay

Splenocytes from C57BL/6 mice were cultured for 24 h with anti-CD3 Ab (10 μg/mL) and CD4+ T cells were then purified using the MACS system. The ChIP assay was carried out using a Simple ChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Briefly, CD4+ T cells were fixed with formaldehyde and quenched with glycine. Crude nuclei were isolated and digested enzymatically using Micrococcal Nuclease and then sonicated to reduce chromatin DNA length to approximately 500 bp. Chromatin solutions was diluted in IP dilution buffer containing protease inhibitor and incubated with anti-Egr-2 Ab (Covance) or normal rabbit IgG. Cross-links were reversed by incubation overnight at 65°C, and immunoprecipitated chromatin (DNA) was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR analysis was performed using primers; corresponding sequences −3000 bp (assay position; −2399 bp), −2000 bp (assay position; −1294 bp), −1000 bp (assay position; −388 bp), and +1000 bp (assay position; +601 bp) from the transcription start site of Blimp-1, −3000 bp (assay position; −2386 bp), −2000 bp (assay position; −1388 bp), −1000 bp (assay position; −398 bp), and +1000 bp (assay position; +602 bp) from the transcription start site of LAG-3, and −1000 bp (assay position; −399 bp) and +1000 bp (assay position; +605 bp) from the transcription start site of IL10. Each primer was obtained from SA Bioscience. The promoter sequence of guanosine monophosphate reductase was used as a control. PCR products were subjected to gel electrophoresis to check the amplicon size (Supporting Information Fig. 2B).

Statistical analysis

Statistical analysis was performed using the Student's t-test. A p-value of <0.05 was considered to indicate a significant difference.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

We thank Dr. Kathryn L. Calame for kindly providing us with pGL-3-(-1500 Blimp-1) LUC reporter plasmids. We also thank the following people for their technological expertise and support: Ms. K. Sakashita, Ms. K. Watada, and Mr. M. Anraku. This work was supported by grants from the Japan Society for the Promotion of Science, Ministry of Health, Labor and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (in part by Global COE Program Chemical Biology of the Diseases, by MEXT), Japan.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information
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Abbreviations
Blimp-1

B lymphocyte induced maturation protein-1

CKO

conditional KO

Egr-2

early growth response gene 2

LAG-3

lymphocyte activation gene 3

Tr1

type I Treg

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
  10. Supporting Information

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eji2563-sup-0001-FigureS1.pdf1366K

Figure 1. The full gating strategy used in our experiments.

Cells were gated based on side scatter and forward scatter to exclude debris. Cells were then gated for CD4 and CD4+ cells and were divided using Egr-2 and LAG-3 expressions. To assessing proliferation, we labeled cells with CFSE at the start of the culture and the relationship between Egr-2 expression and the CFSE dilution level was examined in CD4+ cells.

Figure 2.

(A) TheChIP assay result shown in Figure 2B was re-calculated. The result was presented as % input. (B) A gel picture of quantitative real-time PCR products. PCR products from Input DNA and immunoprecipitated DNA with anti-Egr-2 IgG or anti-control IgG amplified with the designed primers detecting Blimp-1 promoter sequences (# GPM1042845(-)01A; SA Biosciences) were subjected to gel electrophoresis. The amplicon size was 112 bp.

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