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Counter-regulation of T cell effector function by differentially activated p38.

Alam MS, Gaida MM, Ogawa Y, Kolios AG, Lasitschka F, Ashwell JD - J. Exp. Med. (2014)

Bottom Line: Unlike the MAP kinase (MAPK) cascade that phosphorylates p38 on the activation loop, T cell receptor (TCR) signaling results in phosphorylation on Tyr-323 (pY323, alternative pathway).Notably, UVB treatment of human psoriatic lesions reduced skin-infiltrating p38 pY323(+) T cell IRF4 and IL-17 production.Thus, distinct mechanisms of p38 activation converge on NFATc1 with opposing effects on T cell immunity, which may underlie the beneficial effect of phototherapy on psoriasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immune Cell Biology, Center for Cancer Research; Dermatology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Unlike the MAP kinase (MAPK) cascade that phosphorylates p38 on the activation loop, T cell receptor (TCR) signaling results in phosphorylation on Tyr-323 (pY323, alternative pathway). Using mice expressing p38α and p38β with Y323F substitutions, we show that alternatively but not MAPK cascade-activated p38 up-regulates the transcription factors NFATc1 and IRF4, which are required for proliferation and cytokine production. Conversely, activation of p38 with UV or osmotic shock mitigated TCR-mediated activation by phosphorylation and cytoplasmic retention of NFATc1. Notably, UVB treatment of human psoriatic lesions reduced skin-infiltrating p38 pY323(+) T cell IRF4 and IL-17 production. Thus, distinct mechanisms of p38 activation converge on NFATc1 with opposing effects on T cell immunity, which may underlie the beneficial effect of phototherapy on psoriasis.

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p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4+ T cells. (A) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4+ T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4+ T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).
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fig1: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4+ T cells. (A) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4+ T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4+ T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).

Mentions: The finding that up-regulation of several NFAT-dependent genes was decreased in TCR-signaled DKI T cells prompted us to first ask if expression of NFATc1, the only family member that is induced at the transcriptional level, and IRF4, also upstream of cytokine expression, are regulated by p38 in T cells. Anti-CD3 induced NFATc1 and IRF4 up-regulation in CD4+ T cells was prevented by SB203580, a p38α and p38β catalytic inhibitor (Fig. 1 A). The effect was specific in that up-regulation of another inducible IRF family member, IRF8, was not prevented by inhibiting p38. To determine if p38-dependent up-regulation of IRF4 is independent, or downstream, of NFAT, CD4+ T cells were stimulated via the TCR in the presence of the cell-permeable, NFAT-specific inhibitor 11R-VIVIT. Induction of IRF4 mRNA (Fig. 1 B) and protein (Fig. 1 C) was prevented by 11R-VIVIT but not the inactive peptide 11R-VEET. The contribution of alternatively activated as opposed to MAPK cascade-activated p38 was addressed with CD4+ T cells from DKI mice. Induction of NFATc1 and IRF4 was markedly impaired in DKI CD4+ T cells at both the mRNA (Fig. 1 D) and protein (Fig. 1 E) levels. In contrast, expression of other NFAT (Nfatc2, Nfatc3, and Nfatc5) and IRF (Irf8) family members did not differ between TCR-stimulated WT and DKI mice (unpublished data). Remarkably, stimulation with PMA and ionomycin, which bypasses the TCR and activates p38 via the MAPK cascade, induced much less NFATc1 and IRF4 compared with stimulation via the TCR (Fig. 1 E). Blockade of NFAT pathway by CsA impairs IFN-γ and IL-17 expression and Th17 development (Granelli-Piperno et al., 1984; Tsuda et al., 2012). Moreover, CsA inhibits the expression of the transcription factor IRF4, and Th17 development is blocked in IRF4 knockout mice (Cristillo and Bierer, 2002; Brüstle et al., 2007; Tsuda et al., 2012). Therefore, the ability of stimulation via the TCR versus PMA plus ionomycin to skew CD4+ T cells was investigated. As shown in Fig. 1 F, naive CD4+ T cells cultured under neutral (Th0) or Th17-skewing (Th17) conditions expressed more IFN-γ and IL-17A (both in terms of numbers of positive cells and the amount of cytokine produced), respectively, when skewing was performed in the presence of anti-CD3/CD28 compared with PMA and ionomycin at a concentration that results in robust T cell activation (see also Fig. 4 C, right). Together, these results indicate that activation of p38 via the TCR, but not the MAPK cascade, is required for induction of NFATc1 and IRF4 and efficient cytokine production.


Counter-regulation of T cell effector function by differentially activated p38.

Alam MS, Gaida MM, Ogawa Y, Kolios AG, Lasitschka F, Ashwell JD - J. Exp. Med. (2014)

p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4+ T cells. (A) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4+ T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4+ T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig1: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4+ T cells. (A) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4+ T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4+ T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4+ T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).
Mentions: The finding that up-regulation of several NFAT-dependent genes was decreased in TCR-signaled DKI T cells prompted us to first ask if expression of NFATc1, the only family member that is induced at the transcriptional level, and IRF4, also upstream of cytokine expression, are regulated by p38 in T cells. Anti-CD3 induced NFATc1 and IRF4 up-regulation in CD4+ T cells was prevented by SB203580, a p38α and p38β catalytic inhibitor (Fig. 1 A). The effect was specific in that up-regulation of another inducible IRF family member, IRF8, was not prevented by inhibiting p38. To determine if p38-dependent up-regulation of IRF4 is independent, or downstream, of NFAT, CD4+ T cells were stimulated via the TCR in the presence of the cell-permeable, NFAT-specific inhibitor 11R-VIVIT. Induction of IRF4 mRNA (Fig. 1 B) and protein (Fig. 1 C) was prevented by 11R-VIVIT but not the inactive peptide 11R-VEET. The contribution of alternatively activated as opposed to MAPK cascade-activated p38 was addressed with CD4+ T cells from DKI mice. Induction of NFATc1 and IRF4 was markedly impaired in DKI CD4+ T cells at both the mRNA (Fig. 1 D) and protein (Fig. 1 E) levels. In contrast, expression of other NFAT (Nfatc2, Nfatc3, and Nfatc5) and IRF (Irf8) family members did not differ between TCR-stimulated WT and DKI mice (unpublished data). Remarkably, stimulation with PMA and ionomycin, which bypasses the TCR and activates p38 via the MAPK cascade, induced much less NFATc1 and IRF4 compared with stimulation via the TCR (Fig. 1 E). Blockade of NFAT pathway by CsA impairs IFN-γ and IL-17 expression and Th17 development (Granelli-Piperno et al., 1984; Tsuda et al., 2012). Moreover, CsA inhibits the expression of the transcription factor IRF4, and Th17 development is blocked in IRF4 knockout mice (Cristillo and Bierer, 2002; Brüstle et al., 2007; Tsuda et al., 2012). Therefore, the ability of stimulation via the TCR versus PMA plus ionomycin to skew CD4+ T cells was investigated. As shown in Fig. 1 F, naive CD4+ T cells cultured under neutral (Th0) or Th17-skewing (Th17) conditions expressed more IFN-γ and IL-17A (both in terms of numbers of positive cells and the amount of cytokine produced), respectively, when skewing was performed in the presence of anti-CD3/CD28 compared with PMA and ionomycin at a concentration that results in robust T cell activation (see also Fig. 4 C, right). Together, these results indicate that activation of p38 via the TCR, but not the MAPK cascade, is required for induction of NFATc1 and IRF4 and efficient cytokine production.

Bottom Line: Unlike the MAP kinase (MAPK) cascade that phosphorylates p38 on the activation loop, T cell receptor (TCR) signaling results in phosphorylation on Tyr-323 (pY323, alternative pathway).Notably, UVB treatment of human psoriatic lesions reduced skin-infiltrating p38 pY323(+) T cell IRF4 and IL-17 production.Thus, distinct mechanisms of p38 activation converge on NFATc1 with opposing effects on T cell immunity, which may underlie the beneficial effect of phototherapy on psoriasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immune Cell Biology, Center for Cancer Research; Dermatology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
Unlike the MAP kinase (MAPK) cascade that phosphorylates p38 on the activation loop, T cell receptor (TCR) signaling results in phosphorylation on Tyr-323 (pY323, alternative pathway). Using mice expressing p38α and p38β with Y323F substitutions, we show that alternatively but not MAPK cascade-activated p38 up-regulates the transcription factors NFATc1 and IRF4, which are required for proliferation and cytokine production. Conversely, activation of p38 with UV or osmotic shock mitigated TCR-mediated activation by phosphorylation and cytoplasmic retention of NFATc1. Notably, UVB treatment of human psoriatic lesions reduced skin-infiltrating p38 pY323(+) T cell IRF4 and IL-17 production. Thus, distinct mechanisms of p38 activation converge on NFATc1 with opposing effects on T cell immunity, which may underlie the beneficial effect of phototherapy on psoriasis.

Show MeSH
Related in: MedlinePlus