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The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

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Deletion of Nr4a2 attenuates iTreg induction.(a) Effects of Nr4a2-knockout on CD4+T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4+T cells from Nr4a2+/+Lck-Cre+ or Nr4a2fl/flLck-Cre+ mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (b) Quantification of the results in a of the iTreg condition at 0.2 and 0.5 ng ml−1 TGF-β. Top: ratio of the Foxp3+ fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. The value of Nr4a2+/+Lck-Cre+ cells at 0.2 ng ml−1 TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: Nr4a2+/+Lck-Cre+ cells; open bars: Nr4a2fl/flLck-Cre+ cells. (c–f) Effects of the ex vivo deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4+ T cells from Nr4a2+/+ERT2-Cre+ (c, d) or Nr4a2fl/flERT2-Cre+ (e, f) mice were treated (d, f) or untreated (c, e) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (g) Body weight of Nr4a2+/+Lck-Cre+ (open circles) or Nr4a2fl/flLck-Cre+ (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (h) Histological analysis of the experiments of (g) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (i) Analysis of colon and caecum lamina proprial CD4+ T cells from the experiments in g at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (j) Quantification of results in i. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: Nr4a2+/+Lck-Cre+ mice; open bars: Nr4a2fl/flLck-Cre+ mice. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. *P<0.05, **P<0.01 (two-tailed Student's t-test).
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f5: Deletion of Nr4a2 attenuates iTreg induction.(a) Effects of Nr4a2-knockout on CD4+T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4+T cells from Nr4a2+/+Lck-Cre+ or Nr4a2fl/flLck-Cre+ mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (b) Quantification of the results in a of the iTreg condition at 0.2 and 0.5 ng ml−1 TGF-β. Top: ratio of the Foxp3+ fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. The value of Nr4a2+/+Lck-Cre+ cells at 0.2 ng ml−1 TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: Nr4a2+/+Lck-Cre+ cells; open bars: Nr4a2fl/flLck-Cre+ cells. (c–f) Effects of the ex vivo deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4+ T cells from Nr4a2+/+ERT2-Cre+ (c, d) or Nr4a2fl/flERT2-Cre+ (e, f) mice were treated (d, f) or untreated (c, e) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (g) Body weight of Nr4a2+/+Lck-Cre+ (open circles) or Nr4a2fl/flLck-Cre+ (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (h) Histological analysis of the experiments of (g) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (i) Analysis of colon and caecum lamina proprial CD4+ T cells from the experiments in g at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (j) Quantification of results in i. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: Nr4a2+/+Lck-Cre+ mice; open bars: Nr4a2fl/flLck-Cre+ mice. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. *P<0.05, **P<0.01 (two-tailed Student's t-test).

Mentions: The findings above suggest the involvement of Nr4a2 in Foxp3 induction and Th1 suppression. To analyse the roles of Nr4a2 in T-cell development and differentiation, we crossed Nr4a2-floxed mice with Lck-Cre+ mice, which express Cre-recombinase specifically in T cells3536. Although more than half of the Nr4a2fl/flLck-Cre+ mice died neonatally with unknown reasons, surviving mice developed normally and were fertile, and had no symptoms of autoimmune diseases up to 4 months of age. However, the population of CD44highCD62Llow-activated CD4+ T cells, as well as that of IFN-γ+ cells were significantly increased in Nr4a2fl/flLck-Cre+ mice compared with Nr4a2+/+Lck-Cre+ littermates. On the other hand, the number of Tregs in the thymus and spleen was not altered in the Nr4a2fl/flLck-Cre+ mice (Supplementary Fig. S5a,b). We isolated naïve CD4+ T cells from Nr4a2fl/flLck-Cre+ and Nr4a2+/+Lck-Cre+ mice, and cultured them under neutral, Th1-, Th17- and iTreg-skewing conditions. We observed strong induction of Th1 under all culture conditions, and a decrease in iTreg and Th17 induction (Fig. 5a). Expression levels of Foxp3 protein were also lower in Nr4a2-deficient iTreg cells (Fig. 5b). These results are consistent with forced expression studies, which indicate the involvement of Nr4a2 in Th1 repression and Foxp3 induction.


The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.

Sekiya T, Kashiwagi I, Inoue N, Morita R, Hori S, Waldmann H, Rudensky AY, Ichinose H, Metzger D, Chambon P, Yoshimura A - Nat Commun (2011)

Deletion of Nr4a2 attenuates iTreg induction.(a) Effects of Nr4a2-knockout on CD4+T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4+T cells from Nr4a2+/+Lck-Cre+ or Nr4a2fl/flLck-Cre+ mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (b) Quantification of the results in a of the iTreg condition at 0.2 and 0.5 ng ml−1 TGF-β. Top: ratio of the Foxp3+ fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. The value of Nr4a2+/+Lck-Cre+ cells at 0.2 ng ml−1 TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: Nr4a2+/+Lck-Cre+ cells; open bars: Nr4a2fl/flLck-Cre+ cells. (c–f) Effects of the ex vivo deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4+ T cells from Nr4a2+/+ERT2-Cre+ (c, d) or Nr4a2fl/flERT2-Cre+ (e, f) mice were treated (d, f) or untreated (c, e) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (g) Body weight of Nr4a2+/+Lck-Cre+ (open circles) or Nr4a2fl/flLck-Cre+ (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (h) Histological analysis of the experiments of (g) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (i) Analysis of colon and caecum lamina proprial CD4+ T cells from the experiments in g at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (j) Quantification of results in i. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: Nr4a2+/+Lck-Cre+ mice; open bars: Nr4a2fl/flLck-Cre+ mice. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. *P<0.05, **P<0.01 (two-tailed Student's t-test).
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f5: Deletion of Nr4a2 attenuates iTreg induction.(a) Effects of Nr4a2-knockout on CD4+T cells differentiation. iTreg induction was carried out at the two different TGF-β concentrations indicated. Naïve CD4+T cells from Nr4a2+/+Lck-Cre+ or Nr4a2fl/flLck-Cre+ mice were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or IL-17 expressions were analysed. (b) Quantification of the results in a of the iTreg condition at 0.2 and 0.5 ng ml−1 TGF-β. Top: ratio of the Foxp3+ fractions. Bottom: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3+ fractions. The value of Nr4a2+/+Lck-Cre+ cells at 0.2 ng ml−1 TGF-β was set to 1. Data are representative of three independent experiments (mean±s.d. in triplicate). Filled bars: Nr4a2+/+Lck-Cre+ cells; open bars: Nr4a2fl/flLck-Cre+ cells. (c–f) Effects of the ex vivo deletion of Nr4a2 on Th and iTreg differentiation. Isolated naïve CD4+ T cells from Nr4a2+/+ERT2-Cre+ (c, d) or Nr4a2fl/flERT2-Cre+ (e, f) mice were treated (d, f) or untreated (c, e) with 4-hydroxy tamoxifen for 6 h in RPMI1640 10% FBS at 37 °C. Cells were cultured under the indicated conditions for 96 h. IFN-γ, Foxp3 or Il-17a expression were analysed. (g) Body weight of Nr4a2+/+Lck-Cre+ (open circles) or Nr4a2fl/flLck-Cre+ (filled squares) mice treated with 1.5% DSS. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). (h) Histological analysis of the experiments of (g) at day 11. Top: hematoxylin and eosin staining of colon sections. Scale bar, 100 μm. Bottom: histological score of colitis. Data are representative of two independent experiments, each with five mice per group (vertical bars: median of the colitis scores). (i) Analysis of colon and caecum lamina proprial CD4+ T cells from the experiments in g at day 11, examining IFN-γ and IL-17a expression (top) and Foxp3 expression (bottom). (j) Quantification of results in i. Data are representative of two independent experiments, each with five mice per group (mean±s.d. of five mice). Filled bars: Nr4a2+/+Lck-Cre+ mice; open bars: Nr4a2fl/flLck-Cre+ mice. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. *P<0.05, **P<0.01 (two-tailed Student's t-test).
Mentions: The findings above suggest the involvement of Nr4a2 in Foxp3 induction and Th1 suppression. To analyse the roles of Nr4a2 in T-cell development and differentiation, we crossed Nr4a2-floxed mice with Lck-Cre+ mice, which express Cre-recombinase specifically in T cells3536. Although more than half of the Nr4a2fl/flLck-Cre+ mice died neonatally with unknown reasons, surviving mice developed normally and were fertile, and had no symptoms of autoimmune diseases up to 4 months of age. However, the population of CD44highCD62Llow-activated CD4+ T cells, as well as that of IFN-γ+ cells were significantly increased in Nr4a2fl/flLck-Cre+ mice compared with Nr4a2+/+Lck-Cre+ littermates. On the other hand, the number of Tregs in the thymus and spleen was not altered in the Nr4a2fl/flLck-Cre+ mice (Supplementary Fig. S5a,b). We isolated naïve CD4+ T cells from Nr4a2fl/flLck-Cre+ and Nr4a2+/+Lck-Cre+ mice, and cultured them under neutral, Th1-, Th17- and iTreg-skewing conditions. We observed strong induction of Th1 under all culture conditions, and a decrease in iTreg and Th17 induction (Fig. 5a). Expression levels of Foxp3 protein were also lower in Nr4a2-deficient iTreg cells (Fig. 5b). These results are consistent with forced expression studies, which indicate the involvement of Nr4a2 in Th1 repression and Foxp3 induction.

Bottom Line: Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Show MeSH
Related in: MedlinePlus