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Dual-specificity phosphatase 6 regulates CD4+ T-cell functions and restrains spontaneous colitis in IL-10-deficient mice.

Bertin S, Lozano-Ruiz B, Bachiller V, García-Martínez I, Herdman S, Zapater P, Francés R, Such J, Lee J, Raz E, González-Navajas JM - Mucosal Immunol (2014)

Bottom Line: DUSP6 preferentially dephosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1/2) rendering them inactive.In summary, DUSP6 regulates CD4(+) T-cell activation and differentiation by inhibiting the TCR-dependent ERK1/2 activation.DUSP6 might therefore be a potential intervention target for limiting aberrant T-cell responses in T-cell-mediated diseases, such as inflammatory bowel disease.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, University of California San Diego, La Jolla, California, USA.

ABSTRACT
Mitogen-activated protein kinase (MAPK) phosphatases are dual-specificity phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues within MAPKs. DUSP6 preferentially dephosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1/2) rendering them inactive. Here, we study the role of DUSP6 in CD4(+) T-cell function, differentiation, and inflammatory profile in the colon. Upon T-cell receptor (TCR) stimulation, DUSP6 knockout (Dusp6(-/-)) CD4(+) T cells showed increased ERK1/2 activation, proliferation, T helper 1 differentiation, and interferon-γ production, as well as a marked decrease in survival, interleukin- 17A (IL-17A) secretion, and regulatory T-cell function. To analyze the role of DUSP6 in vivo, we employed the Il10(-/-) model of colitis and generated Il10(-/-)/Dusp6(-/-) double-knockout mice. Il10(-/-)/Dusp6(-/-) mice suffered from accelerated and exacerbated spontaneous colitis, which was prevented by ERK1/2 inhibition. ERK1/2 inhibition also augmented regulatory T-cell differentiation in vitro and in vivo in both C57Bl/6 and Dusp6(-/-) mice. In summary, DUSP6 regulates CD4(+) T-cell activation and differentiation by inhibiting the TCR-dependent ERK1/2 activation. DUSP6 might therefore be a potential intervention target for limiting aberrant T-cell responses in T-cell-mediated diseases, such as inflammatory bowel disease.

No MeSH data available.


Related in: MedlinePlus

Increased ERK1/2 activation and IFN-γ production in Dusp6−/−CD4+ T cells(a) Immunoblot analysis of DUSP6 expression in CD4+ T cells from WT mice upon activation of either TLR4 (100 µg LPS) or T cell receptor (αCD3/28 antibodies). (b) Immunoblot analysis of phosphorylated levels of MAPKs in CD4+ T cells stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments (a,b). (c) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours (n≥9 mice per group). (d) qPCR analysis of the expression of different transcription factors in CD4+ T cells from WT and Dusp6−/− mice stimulated with anti-CD3/28 antibodies (n≥5 mice per group). The mRNA fold induction in Dusp6−/− CD4+ T cells was normalized according to the mRNA expression in WT T cells. GAPDH was used as internal control. Error bars represent standard deviation Data represent results from either three (a–c) or two (d) independent experiments. *P<0.05, **P<0.01.
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Figure 1: Increased ERK1/2 activation and IFN-γ production in Dusp6−/−CD4+ T cells(a) Immunoblot analysis of DUSP6 expression in CD4+ T cells from WT mice upon activation of either TLR4 (100 µg LPS) or T cell receptor (αCD3/28 antibodies). (b) Immunoblot analysis of phosphorylated levels of MAPKs in CD4+ T cells stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments (a,b). (c) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours (n≥9 mice per group). (d) qPCR analysis of the expression of different transcription factors in CD4+ T cells from WT and Dusp6−/− mice stimulated with anti-CD3/28 antibodies (n≥5 mice per group). The mRNA fold induction in Dusp6−/− CD4+ T cells was normalized according to the mRNA expression in WT T cells. GAPDH was used as internal control. Error bars represent standard deviation Data represent results from either three (a–c) or two (d) independent experiments. *P<0.05, **P<0.01.

Mentions: Although the expression of DUSP6 in naïve CD4+ T cells has been reported in mice and humans10,23,24, the role of DUSP6 in CD4+ T cell activation and function has not been examined. We observed a time-dependent increase in DUSP6 protein expression after TCR or TLR4 stimulation of splenic CD4+ T cells from WT mice with anti-CD3/28 antibodies or LPS, respectively (Figure 1a). Next, we isolated CD4+ T cells from WT and Dusp6−/− mice to investigate the role of this phosphatase in CD4+ T cell functions. TCR stimulation of Dusp6−/− CD4+ T cells resulted in higher phosphorylation levels of ERK1/2, but not of p38 or JNK1/2 MAPKs compared to WT cells (Figure 1b). We then analyzed the cytokine production by CD4+ T cells harvested from spleen and mesenteric lymph nodes (MLNs) of WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies. While there was no significant difference in the production of IL-2, 4, 6 and 10, we found that Dusp6−/− CD4+ T cells, from both MLNs (Figure 1c) and spleen (Supplementary Figure S1), produced higher amounts of IFN-γ and lower amounts of IL-17A when compared to WT CD4+ T cells. In accordance with the cytokine data, TCR stimulation of splenic Dusp6−/− CD4+ T cells resulted in a transcriptional increase in the Th1-related transcription factor T-box expressed in T cells (T-bet), as well as in a decrease in the Th17-related RAR-related orphan receptor (ROR)γt, whereas the expression of GATA-binding protein 3 (GATA3) and forkhead box P3 (FOXP3), which function as master regulators of Th2 and regulatory T (Treg) cells respectively, remained unaltered when compared to WT CD4+ T cells (Figure 1d).


Dual-specificity phosphatase 6 regulates CD4+ T-cell functions and restrains spontaneous colitis in IL-10-deficient mice.

Bertin S, Lozano-Ruiz B, Bachiller V, García-Martínez I, Herdman S, Zapater P, Francés R, Such J, Lee J, Raz E, González-Navajas JM - Mucosal Immunol (2014)

Increased ERK1/2 activation and IFN-γ production in Dusp6−/−CD4+ T cells(a) Immunoblot analysis of DUSP6 expression in CD4+ T cells from WT mice upon activation of either TLR4 (100 µg LPS) or T cell receptor (αCD3/28 antibodies). (b) Immunoblot analysis of phosphorylated levels of MAPKs in CD4+ T cells stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments (a,b). (c) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours (n≥9 mice per group). (d) qPCR analysis of the expression of different transcription factors in CD4+ T cells from WT and Dusp6−/− mice stimulated with anti-CD3/28 antibodies (n≥5 mice per group). The mRNA fold induction in Dusp6−/− CD4+ T cells was normalized according to the mRNA expression in WT T cells. GAPDH was used as internal control. Error bars represent standard deviation Data represent results from either three (a–c) or two (d) independent experiments. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363301&req=5

Figure 1: Increased ERK1/2 activation and IFN-γ production in Dusp6−/−CD4+ T cells(a) Immunoblot analysis of DUSP6 expression in CD4+ T cells from WT mice upon activation of either TLR4 (100 µg LPS) or T cell receptor (αCD3/28 antibodies). (b) Immunoblot analysis of phosphorylated levels of MAPKs in CD4+ T cells stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments (a,b). (c) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours (n≥9 mice per group). (d) qPCR analysis of the expression of different transcription factors in CD4+ T cells from WT and Dusp6−/− mice stimulated with anti-CD3/28 antibodies (n≥5 mice per group). The mRNA fold induction in Dusp6−/− CD4+ T cells was normalized according to the mRNA expression in WT T cells. GAPDH was used as internal control. Error bars represent standard deviation Data represent results from either three (a–c) or two (d) independent experiments. *P<0.05, **P<0.01.
Mentions: Although the expression of DUSP6 in naïve CD4+ T cells has been reported in mice and humans10,23,24, the role of DUSP6 in CD4+ T cell activation and function has not been examined. We observed a time-dependent increase in DUSP6 protein expression after TCR or TLR4 stimulation of splenic CD4+ T cells from WT mice with anti-CD3/28 antibodies or LPS, respectively (Figure 1a). Next, we isolated CD4+ T cells from WT and Dusp6−/− mice to investigate the role of this phosphatase in CD4+ T cell functions. TCR stimulation of Dusp6−/− CD4+ T cells resulted in higher phosphorylation levels of ERK1/2, but not of p38 or JNK1/2 MAPKs compared to WT cells (Figure 1b). We then analyzed the cytokine production by CD4+ T cells harvested from spleen and mesenteric lymph nodes (MLNs) of WT and Dusp6−/− mice and stimulated with anti-CD3/28 antibodies. While there was no significant difference in the production of IL-2, 4, 6 and 10, we found that Dusp6−/− CD4+ T cells, from both MLNs (Figure 1c) and spleen (Supplementary Figure S1), produced higher amounts of IFN-γ and lower amounts of IL-17A when compared to WT CD4+ T cells. In accordance with the cytokine data, TCR stimulation of splenic Dusp6−/− CD4+ T cells resulted in a transcriptional increase in the Th1-related transcription factor T-box expressed in T cells (T-bet), as well as in a decrease in the Th17-related RAR-related orphan receptor (ROR)γt, whereas the expression of GATA-binding protein 3 (GATA3) and forkhead box P3 (FOXP3), which function as master regulators of Th2 and regulatory T (Treg) cells respectively, remained unaltered when compared to WT CD4+ T cells (Figure 1d).

Bottom Line: DUSP6 preferentially dephosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1/2) rendering them inactive.In summary, DUSP6 regulates CD4(+) T-cell activation and differentiation by inhibiting the TCR-dependent ERK1/2 activation.DUSP6 might therefore be a potential intervention target for limiting aberrant T-cell responses in T-cell-mediated diseases, such as inflammatory bowel disease.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, University of California San Diego, La Jolla, California, USA.

ABSTRACT
Mitogen-activated protein kinase (MAPK) phosphatases are dual-specificity phosphatases (DUSPs) that dephosphorylate phosphothreonine and phosphotyrosine residues within MAPKs. DUSP6 preferentially dephosphorylates extracellular signal-regulated kinases 1 and 2 (ERK1/2) rendering them inactive. Here, we study the role of DUSP6 in CD4(+) T-cell function, differentiation, and inflammatory profile in the colon. Upon T-cell receptor (TCR) stimulation, DUSP6 knockout (Dusp6(-/-)) CD4(+) T cells showed increased ERK1/2 activation, proliferation, T helper 1 differentiation, and interferon-γ production, as well as a marked decrease in survival, interleukin- 17A (IL-17A) secretion, and regulatory T-cell function. To analyze the role of DUSP6 in vivo, we employed the Il10(-/-) model of colitis and generated Il10(-/-)/Dusp6(-/-) double-knockout mice. Il10(-/-)/Dusp6(-/-) mice suffered from accelerated and exacerbated spontaneous colitis, which was prevented by ERK1/2 inhibition. ERK1/2 inhibition also augmented regulatory T-cell differentiation in vitro and in vivo in both C57Bl/6 and Dusp6(-/-) mice. In summary, DUSP6 regulates CD4(+) T-cell activation and differentiation by inhibiting the TCR-dependent ERK1/2 activation. DUSP6 might therefore be a potential intervention target for limiting aberrant T-cell responses in T-cell-mediated diseases, such as inflammatory bowel disease.

No MeSH data available.


Related in: MedlinePlus