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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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MAPK cascade–activated p38 inhibits T cell proliferation and effector function mediated by alternatively activated p38. (A and B) Naive CD4+ T cells were treated with 0.6 M sorbitol for 30 min or untreated, and then stimulated with anti-CD3/CD28 in neutral or Th17-skewing conditions. After 2 d, Irf4 and Rorc mRNA levels were analyzed by quantitative real-time PCR (A), and after 3 d, IL-17A expression was determined in cells restimulated with PMA and ionomycin for 4 h. (B) The numbers indicate the frequency of IL-17A–producing cells. The results are representative of two independent experiments. (C and D) Naive CD4+ T cells were treated as in A, and then stimulated with anti-CD3/CD28 either in neutral (Th0) or Th1 skewing conditions for 2 d, at which time Tbet mRNA was measured by real-time PCR (C), and after 3 d cells were restimulated with PMA and ionomycin for 4 h and intracellular IFN-γ expression was measured by flow cytometry (D). The numbers indicate the frequency of IFN-γ–producing cells. *, P < 0.05 (unpaired two-tailed Student’s t test). Data are representative of at least three independent experiments. (E) T cells were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (wells coated with 2 µg/ml) or PMA and ionomycin for 48 h and proliferation was assessed by [3H]thymidine incorporation. Data shown are representative of at least three independent experiments.
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fig4: MAPK cascade–activated p38 inhibits T cell proliferation and effector function mediated by alternatively activated p38. (A and B) Naive CD4+ T cells were treated with 0.6 M sorbitol for 30 min or untreated, and then stimulated with anti-CD3/CD28 in neutral or Th17-skewing conditions. After 2 d, Irf4 and Rorc mRNA levels were analyzed by quantitative real-time PCR (A), and after 3 d, IL-17A expression was determined in cells restimulated with PMA and ionomycin for 4 h. (B) The numbers indicate the frequency of IL-17A–producing cells. The results are representative of two independent experiments. (C and D) Naive CD4+ T cells were treated as in A, and then stimulated with anti-CD3/CD28 either in neutral (Th0) or Th1 skewing conditions for 2 d, at which time Tbet mRNA was measured by real-time PCR (C), and after 3 d cells were restimulated with PMA and ionomycin for 4 h and intracellular IFN-γ expression was measured by flow cytometry (D). The numbers indicate the frequency of IFN-γ–producing cells. *, P < 0.05 (unpaired two-tailed Student’s t test). Data are representative of at least three independent experiments. (E) T cells were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (wells coated with 2 µg/ml) or PMA and ionomycin for 48 h and proliferation was assessed by [3H]thymidine incorporation. Data shown are representative of at least three independent experiments.

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)

MAPK cascade–activated p38 inhibits T cell proliferation and effector function mediated by alternatively activated p38. (A and B) Naive CD4+ T cells were treated with 0.6 M sorbitol for 30 min or untreated, and then stimulated with anti-CD3/CD28 in neutral or Th17-skewing conditions. After 2 d, Irf4 and Rorc mRNA levels were analyzed by quantitative real-time PCR (A), and after 3 d, IL-17A expression was determined in cells restimulated with PMA and ionomycin for 4 h. (B) The numbers indicate the frequency of IL-17A–producing cells. The results are representative of two independent experiments. (C and D) Naive CD4+ T cells were treated as in A, and then stimulated with anti-CD3/CD28 either in neutral (Th0) or Th1 skewing conditions for 2 d, at which time Tbet mRNA was measured by real-time PCR (C), and after 3 d cells were restimulated with PMA and ionomycin for 4 h and intracellular IFN-γ expression was measured by flow cytometry (D). The numbers indicate the frequency of IFN-γ–producing cells. *, P < 0.05 (unpaired two-tailed Student’s t test). Data are representative of at least three independent experiments. (E) T cells were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (wells coated with 2 µg/ml) or PMA and ionomycin for 48 h and proliferation was assessed by [3H]thymidine incorporation. Data shown are representative of at least three independent experiments.
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fig4: MAPK cascade–activated p38 inhibits T cell proliferation and effector function mediated by alternatively activated p38. (A and B) Naive CD4+ T cells were treated with 0.6 M sorbitol for 30 min or untreated, and then stimulated with anti-CD3/CD28 in neutral or Th17-skewing conditions. After 2 d, Irf4 and Rorc mRNA levels were analyzed by quantitative real-time PCR (A), and after 3 d, IL-17A expression was determined in cells restimulated with PMA and ionomycin for 4 h. (B) The numbers indicate the frequency of IL-17A–producing cells. The results are representative of two independent experiments. (C and D) Naive CD4+ T cells were treated as in A, and then stimulated with anti-CD3/CD28 either in neutral (Th0) or Th1 skewing conditions for 2 d, at which time Tbet mRNA was measured by real-time PCR (C), and after 3 d cells were restimulated with PMA and ionomycin for 4 h and intracellular IFN-γ expression was measured by flow cytometry (D). The numbers indicate the frequency of IFN-γ–producing cells. *, P < 0.05 (unpaired two-tailed Student’s t test). Data are representative of at least three independent experiments. (E) T cells were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (wells coated with 2 µg/ml) or PMA and ionomycin for 48 h and proliferation was assessed by [3H]thymidine incorporation. Data shown are representative of at least three independent experiments.
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