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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

DUSP6 deficiency aggravates colitis in the IL-10 model(a) Paraffin-embedded colon samples were sectioned and stained with hematoxylin and eosin (H&E). Representative micrograph (100× magnification) of co-housed 12 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice maintained under specific pathogen-free conditions (n≥10 mice/group). (b) Quantitative measurement of crypt length and cellular infiltration in the different groups of mice at 12 weeks of age (n≥10 mice/group). (c) Cytokine levels in colonic explant supernatants after 24 hours of culture. Represented values are normalized to milligrams of cultured colonic tissue. (d) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from Il10−/− and Il10−/−/Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours. Data represent pooled results from two independent experiments with at least 6 mice per group (c–d). (e) Immunoblot analysis of phosphorylated levels of ERK12/ in splenic CD4+ T cells from both groups of mice stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments. Error bars represent standard deviation. n.s: not significant, *P<0.05, **P<0.01
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Figure 5: DUSP6 deficiency aggravates colitis in the IL-10 model(a) Paraffin-embedded colon samples were sectioned and stained with hematoxylin and eosin (H&E). Representative micrograph (100× magnification) of co-housed 12 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice maintained under specific pathogen-free conditions (n≥10 mice/group). (b) Quantitative measurement of crypt length and cellular infiltration in the different groups of mice at 12 weeks of age (n≥10 mice/group). (c) Cytokine levels in colonic explant supernatants after 24 hours of culture. Represented values are normalized to milligrams of cultured colonic tissue. (d) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from Il10−/− and Il10−/−/Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours. Data represent pooled results from two independent experiments with at least 6 mice per group (c–d). (e) Immunoblot analysis of phosphorylated levels of ERK12/ in splenic CD4+ T cells from both groups of mice stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments. Error bars represent standard deviation. n.s: not significant, *P<0.05, **P<0.01

Mentions: We have previously shown that TLR4 regulates the inflammatory profile of CD4+ T cells and exerts a tonic inhibition on certain TCR signaling events via the induction of DUSP610. Furthermore, the data shown herein suggests that DUSP6 exerts important regulatory functions in CD4+ T cells that may affect their pro- and anti-inflammatory potential. To delineate the role of this phosphatase in the development of colitis, we crossed Dusp6−/− mice onto Il10−/− animals to generate Il10−/−/Dusp6−/− double knock out mice. We then co-housed 4 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice to allow colonization of these two groups with the same microflora30, and followed them weekly for signs of inflammation for an additional period of 6 months. While Il10−/− mice did not show intestinal inflammation at the age of 7 months, Il10−/−/Dusp6−/− mice displayed macroscopic signs of intestinal inflammation between 10 to 14 weeks of age, such as diarrhea and thickening of the intestinal wall (Supplementary Figure S7). Histological evaluation (Figure 5a) and quantitative morphometric analysis (Figure 5b) of the colon revealed that Il10−/−/Dusp6−/− mice developed severe inflammation with a high degree of epithelial crypt hyperplasia, goblet cell depletion, and infiltration of mononuclear cells in the colonic lamina propria. To quantify the inflammatory mediators produced by the inflamed colons, we cultured ex vivo colonic explants from Il10−/− and Il10−/−/Dusp6−/− mice. Colonic explants from Il10−/−/Dusp6−/− mice released high amounts of IFN-γ and TNF-α, whereas the level of IL-17A was lower than in the supernatants harvested from Il10−/− colonic explants (Figure 5c). To further evaluate the inflammatory phenotype of these mice, effector CD4+ T cells were isolated from MLNs and spleen and stimulated with anti-CD3/28 antibodies. Similar to Dusp6−/− mice, MLN-derived CD4+ T cells from Il10−/−/Dusp6−/− mice produced higher levels of IFN-γ but lower levels of IL-17A when compared with CD4+ T cells from Il10−/− mice (Figure 5d). Similar results were obtained with splenic CD4+0er, these data demonstrate that the lack of DUSP6 expression accelerates intestinal inflammation in genetically susceptible hosts (i.e., Il10−/− mice), and impacts the inflammatory cytokine profile of IL-10-deficient CD4+ T cells.


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)

DUSP6 deficiency aggravates colitis in the IL-10 model(a) Paraffin-embedded colon samples were sectioned and stained with hematoxylin and eosin (H&E). Representative micrograph (100× magnification) of co-housed 12 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice maintained under specific pathogen-free conditions (n≥10 mice/group). (b) Quantitative measurement of crypt length and cellular infiltration in the different groups of mice at 12 weeks of age (n≥10 mice/group). (c) Cytokine levels in colonic explant supernatants after 24 hours of culture. Represented values are normalized to milligrams of cultured colonic tissue. (d) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from Il10−/− and Il10−/−/Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours. Data represent pooled results from two independent experiments with at least 6 mice per group (c–d). (e) Immunoblot analysis of phosphorylated levels of ERK12/ in splenic CD4+ T cells from both groups of mice stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments. Error bars represent standard deviation. n.s: not significant, *P<0.05, **P<0.01
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Related In: Results  -  Collection

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Figure 5: DUSP6 deficiency aggravates colitis in the IL-10 model(a) Paraffin-embedded colon samples were sectioned and stained with hematoxylin and eosin (H&E). Representative micrograph (100× magnification) of co-housed 12 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice maintained under specific pathogen-free conditions (n≥10 mice/group). (b) Quantitative measurement of crypt length and cellular infiltration in the different groups of mice at 12 weeks of age (n≥10 mice/group). (c) Cytokine levels in colonic explant supernatants after 24 hours of culture. Represented values are normalized to milligrams of cultured colonic tissue. (d) Cytokine levels in culture supernatants of MLN-derived CD4+ T cells isolated from Il10−/− and Il10−/−/Dusp6−/− mice and stimulated with anti-CD3/28 antibodies for 24 hours. Data represent pooled results from two independent experiments with at least 6 mice per group (c–d). (e) Immunoblot analysis of phosphorylated levels of ERK12/ in splenic CD4+ T cells from both groups of mice stimulated with anti-CD3/28 antibodies for the indicated time-points. Data are representative of three independent experiments. Error bars represent standard deviation. n.s: not significant, *P<0.05, **P<0.01
Mentions: We have previously shown that TLR4 regulates the inflammatory profile of CD4+ T cells and exerts a tonic inhibition on certain TCR signaling events via the induction of DUSP610. Furthermore, the data shown herein suggests that DUSP6 exerts important regulatory functions in CD4+ T cells that may affect their pro- and anti-inflammatory potential. To delineate the role of this phosphatase in the development of colitis, we crossed Dusp6−/− mice onto Il10−/− animals to generate Il10−/−/Dusp6−/− double knock out mice. We then co-housed 4 weeks-old Il10−/− and Il10−/−/Dusp6−/− mice to allow colonization of these two groups with the same microflora30, and followed them weekly for signs of inflammation for an additional period of 6 months. While Il10−/− mice did not show intestinal inflammation at the age of 7 months, Il10−/−/Dusp6−/− mice displayed macroscopic signs of intestinal inflammation between 10 to 14 weeks of age, such as diarrhea and thickening of the intestinal wall (Supplementary Figure S7). Histological evaluation (Figure 5a) and quantitative morphometric analysis (Figure 5b) of the colon revealed that Il10−/−/Dusp6−/− mice developed severe inflammation with a high degree of epithelial crypt hyperplasia, goblet cell depletion, and infiltration of mononuclear cells in the colonic lamina propria. To quantify the inflammatory mediators produced by the inflamed colons, we cultured ex vivo colonic explants from Il10−/− and Il10−/−/Dusp6−/− mice. Colonic explants from Il10−/−/Dusp6−/− mice released high amounts of IFN-γ and TNF-α, whereas the level of IL-17A was lower than in the supernatants harvested from Il10−/− colonic explants (Figure 5c). To further evaluate the inflammatory phenotype of these mice, effector CD4+ T cells were isolated from MLNs and spleen and stimulated with anti-CD3/28 antibodies. Similar to Dusp6−/− mice, MLN-derived CD4+ T cells from Il10−/−/Dusp6−/− mice produced higher levels of IFN-γ but lower levels of IL-17A when compared with CD4+ T cells from Il10−/− mice (Figure 5d). Similar results were obtained with splenic CD4+0er, these data demonstrate that the lack of DUSP6 expression accelerates intestinal inflammation in genetically susceptible hosts (i.e., Il10−/− mice), and impacts the inflammatory cytokine profile of IL-10-deficient CD4+ T cells.

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