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Prostaglandin E₂ promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase.

Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S - Nat Commun (2013)

Bottom Line: Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28.Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo.These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606 8501, Japan.

ABSTRACT
T helper 1 (Th1) cells have critical roles in various autoimmune and proinflammatory diseases. cAMP has long been believed to act as a suppressor of IFN-γ production and Th1 cell-mediated immune inflammation. Here we show that cAMP actively promotes Th1 differentiation by inducing gene expression of cytokine receptors involved in this process. PGE2 signalling through EP2/EP4 receptors mobilizes the cAMP-PKA pathway, which induces CREB- and its co-activator CRTC2-mediated transcription of IL-12Rβ2 and IFN-γR1. Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28. Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo. These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

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PGE2-cAMP signalling induces IL-12Rβ2 expression in TCR-activated T cells.(a) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for indicated times with antibody to CD3 and antibody to CD28 (αCD3/CD28) in the absence or presence of PGE2 under Th1-priming conditions. A portion of cells were restimulated with PMA and ionomycin for the last 4 h (72R). (b) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 or selective agonists to EP1 to EP4 under Th1-priming conditions. (c) Surface expression of IL-12Rβ2 in T cells activated for 48 h with αCD3/CD28 in the absence or presence of PGE2 or agonists selective to EP1 to EP4 under Th1-priming conditions. Grey-filled histogram represents isotype control. ΔMFI (mean fluorescence intensity) indicates the differences between MFI of IL-12Rβ2 and MFI of isotype control (right). (d) Time-course of Il12rb2 mRNA expression by PGE2 in T cells activated with αCD3/CD28. (e) PGE2 induces IL-12Rβ2 protein expression in T cells activated with αCD3/CD28 for 48 h. (f,g) Expression of Il12rb2 mRNA in WT T cells (f) or IFN-γR1–/– T cells supplemented with anti-IL-2 (g), activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with or without Wortmannin, LY-294002 or H-89. (h) Expression of Il12rb2 mRNA in T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with H-89, Rp-8-Br-CAMPS or Rp-8-CPT-CAMPS. (i,j) Expression of IL-12Rβ2 mRNA (i) and protein (j) in T cells activated with αCD3/CD28 in the presence of db-cAMP, forskolin or 3-isobutyl-1- methylxanthine (IBMX) for 12 h (i) or 48 h (j). (k) Expression of Il12rb2 mRNA in IFN-γR1−/− CD4+ T cells activated for 24 h with αCD3/CD28 with or without db-cAMP in the absence or presence of anti-IL-2 or LY-294002 (LY) or both. Data shown as mean±s.e.m. are representative of two or more independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant, veh, vehicle; a.u., arbitrary units.
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f1: PGE2-cAMP signalling induces IL-12Rβ2 expression in TCR-activated T cells.(a) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for indicated times with antibody to CD3 and antibody to CD28 (αCD3/CD28) in the absence or presence of PGE2 under Th1-priming conditions. A portion of cells were restimulated with PMA and ionomycin for the last 4 h (72R). (b) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 or selective agonists to EP1 to EP4 under Th1-priming conditions. (c) Surface expression of IL-12Rβ2 in T cells activated for 48 h with αCD3/CD28 in the absence or presence of PGE2 or agonists selective to EP1 to EP4 under Th1-priming conditions. Grey-filled histogram represents isotype control. ΔMFI (mean fluorescence intensity) indicates the differences between MFI of IL-12Rβ2 and MFI of isotype control (right). (d) Time-course of Il12rb2 mRNA expression by PGE2 in T cells activated with αCD3/CD28. (e) PGE2 induces IL-12Rβ2 protein expression in T cells activated with αCD3/CD28 for 48 h. (f,g) Expression of Il12rb2 mRNA in WT T cells (f) or IFN-γR1–/– T cells supplemented with anti-IL-2 (g), activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with or without Wortmannin, LY-294002 or H-89. (h) Expression of Il12rb2 mRNA in T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with H-89, Rp-8-Br-CAMPS or Rp-8-CPT-CAMPS. (i,j) Expression of IL-12Rβ2 mRNA (i) and protein (j) in T cells activated with αCD3/CD28 in the presence of db-cAMP, forskolin or 3-isobutyl-1- methylxanthine (IBMX) for 12 h (i) or 48 h (j). (k) Expression of Il12rb2 mRNA in IFN-γR1−/− CD4+ T cells activated for 24 h with αCD3/CD28 with or without db-cAMP in the absence or presence of anti-IL-2 or LY-294002 (LY) or both. Data shown as mean±s.e.m. are representative of two or more independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant, veh, vehicle; a.u., arbitrary units.

Mentions: To clarify how PGE2 facilitates Th1 differentiation, we examined effects of PGE2 on expression of IFN-γ (Ifng), T-bet (Tbx21), and IL-12Rβ2 (Il12rb2), three genes critical for Th1 differentiation, in T cells cultured under the Th1-priming conditions. PGE2 considerably enhanced expression of Il12rb2 and Ifng mRNA from 12 and 48 h, respectively, while enhancement of Tbx21 expression was not seen until 72 h (Fig. 1a). Enhanced expression of Il12rb2 mRNA at 24 h was mimicked by agonists selective to EP2 (ONO-AE1-259) or EP4 (ONO-AE1-329) but not by agonists to EP1 (ONO-DI-004) or EP3 (ONO-AE-248) (ref. 28) (Fig. 1b). Induction of IL-12Rβ2 protein by PGE2, EP2 or EP4 agonist during Th1 differentiation was confirmed by flow cytometry (Fig. 1c). These data suggested that promotion of Th1 differentiation by PGE2 is likely to be initiated through induction of IL-12Rβ2 via EP2 and EP4 receptors.


Prostaglandin E₂ promotes Th1 differentiation via synergistic amplification of IL-12 signalling by cAMP and PI3-kinase.

Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S - Nat Commun (2013)

PGE2-cAMP signalling induces IL-12Rβ2 expression in TCR-activated T cells.(a) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for indicated times with antibody to CD3 and antibody to CD28 (αCD3/CD28) in the absence or presence of PGE2 under Th1-priming conditions. A portion of cells were restimulated with PMA and ionomycin for the last 4 h (72R). (b) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 or selective agonists to EP1 to EP4 under Th1-priming conditions. (c) Surface expression of IL-12Rβ2 in T cells activated for 48 h with αCD3/CD28 in the absence or presence of PGE2 or agonists selective to EP1 to EP4 under Th1-priming conditions. Grey-filled histogram represents isotype control. ΔMFI (mean fluorescence intensity) indicates the differences between MFI of IL-12Rβ2 and MFI of isotype control (right). (d) Time-course of Il12rb2 mRNA expression by PGE2 in T cells activated with αCD3/CD28. (e) PGE2 induces IL-12Rβ2 protein expression in T cells activated with αCD3/CD28 for 48 h. (f,g) Expression of Il12rb2 mRNA in WT T cells (f) or IFN-γR1–/– T cells supplemented with anti-IL-2 (g), activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with or without Wortmannin, LY-294002 or H-89. (h) Expression of Il12rb2 mRNA in T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with H-89, Rp-8-Br-CAMPS or Rp-8-CPT-CAMPS. (i,j) Expression of IL-12Rβ2 mRNA (i) and protein (j) in T cells activated with αCD3/CD28 in the presence of db-cAMP, forskolin or 3-isobutyl-1- methylxanthine (IBMX) for 12 h (i) or 48 h (j). (k) Expression of Il12rb2 mRNA in IFN-γR1−/− CD4+ T cells activated for 24 h with αCD3/CD28 with or without db-cAMP in the absence or presence of anti-IL-2 or LY-294002 (LY) or both. Data shown as mean±s.e.m. are representative of two or more independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant, veh, vehicle; a.u., arbitrary units.
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f1: PGE2-cAMP signalling induces IL-12Rβ2 expression in TCR-activated T cells.(a) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for indicated times with antibody to CD3 and antibody to CD28 (αCD3/CD28) in the absence or presence of PGE2 under Th1-priming conditions. A portion of cells were restimulated with PMA and ionomycin for the last 4 h (72R). (b) Expression of Ifng, Tbx21 and Il12rb2 mRNA by T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 or selective agonists to EP1 to EP4 under Th1-priming conditions. (c) Surface expression of IL-12Rβ2 in T cells activated for 48 h with αCD3/CD28 in the absence or presence of PGE2 or agonists selective to EP1 to EP4 under Th1-priming conditions. Grey-filled histogram represents isotype control. ΔMFI (mean fluorescence intensity) indicates the differences between MFI of IL-12Rβ2 and MFI of isotype control (right). (d) Time-course of Il12rb2 mRNA expression by PGE2 in T cells activated with αCD3/CD28. (e) PGE2 induces IL-12Rβ2 protein expression in T cells activated with αCD3/CD28 for 48 h. (f,g) Expression of Il12rb2 mRNA in WT T cells (f) or IFN-γR1–/– T cells supplemented with anti-IL-2 (g), activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with or without Wortmannin, LY-294002 or H-89. (h) Expression of Il12rb2 mRNA in T cells activated for 24 h with αCD3/CD28 in the absence or presence of PGE2 with H-89, Rp-8-Br-CAMPS or Rp-8-CPT-CAMPS. (i,j) Expression of IL-12Rβ2 mRNA (i) and protein (j) in T cells activated with αCD3/CD28 in the presence of db-cAMP, forskolin or 3-isobutyl-1- methylxanthine (IBMX) for 12 h (i) or 48 h (j). (k) Expression of Il12rb2 mRNA in IFN-γR1−/− CD4+ T cells activated for 24 h with αCD3/CD28 with or without db-cAMP in the absence or presence of anti-IL-2 or LY-294002 (LY) or both. Data shown as mean±s.e.m. are representative of two or more independent experiments with triplicates. Statistical significance was examined by unpaired two-tailed Student’s t-test, *P<0.05; **P<0.01; ***P<0.001. NS, not significant, veh, vehicle; a.u., arbitrary units.
Mentions: To clarify how PGE2 facilitates Th1 differentiation, we examined effects of PGE2 on expression of IFN-γ (Ifng), T-bet (Tbx21), and IL-12Rβ2 (Il12rb2), three genes critical for Th1 differentiation, in T cells cultured under the Th1-priming conditions. PGE2 considerably enhanced expression of Il12rb2 and Ifng mRNA from 12 and 48 h, respectively, while enhancement of Tbx21 expression was not seen until 72 h (Fig. 1a). Enhanced expression of Il12rb2 mRNA at 24 h was mimicked by agonists selective to EP2 (ONO-AE1-259) or EP4 (ONO-AE1-329) but not by agonists to EP1 (ONO-DI-004) or EP3 (ONO-AE-248) (ref. 28) (Fig. 1b). Induction of IL-12Rβ2 protein by PGE2, EP2 or EP4 agonist during Th1 differentiation was confirmed by flow cytometry (Fig. 1c). These data suggested that promotion of Th1 differentiation by PGE2 is likely to be initiated through induction of IL-12Rβ2 via EP2 and EP4 receptors.

Bottom Line: Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28.Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo.These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto 606 8501, Japan.

ABSTRACT
T helper 1 (Th1) cells have critical roles in various autoimmune and proinflammatory diseases. cAMP has long been believed to act as a suppressor of IFN-γ production and Th1 cell-mediated immune inflammation. Here we show that cAMP actively promotes Th1 differentiation by inducing gene expression of cytokine receptors involved in this process. PGE2 signalling through EP2/EP4 receptors mobilizes the cAMP-PKA pathway, which induces CREB- and its co-activator CRTC2-mediated transcription of IL-12Rβ2 and IFN-γR1. Meanwhile, cAMP-mediated suppression of T-cell receptor signalling is overcome by simultaneous activation of PI3-kinase through EP2/EP4 and/or CD28. Loss of EP4 in T cells restricts expression of IL-12Rβ2 and IFN-γR1, and attenuates Th1 cell-mediated inflammation in vivo. These findings clarify the molecular mechanisms and pathological contexts of cAMP-mediated Th1 differentiation and have clinical and therapeutic implications for deployment of cAMP modulators as immunoregulatory drugs.

Show MeSH
Related in: MedlinePlus