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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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cAMP context-dependently regulates TCR activation and Th1 differentiation.(a) Expression of Il12rb2 mRNA in WT (left) and IFN-γR1−/− (right) T cells activated for 12 h without (unactivated) or with αCD3/CD28 in the presence or absence of db-cAMP, IFN-γ or anti-IFN-γ. (b) Surface expression of IL-12Rβ2 in WT or IFN-γR1−/− T cells activated for 48 h with αCD3/CD28. db-cAMP or vehicle was added from 24–48 h. (c) Expression of Il12rb2 mRNA in IFN-γR1–/– T cells activated for 24 h without (unactivated) or with αCD3/CD28 in the absence or presence of db-cAMP or anti-IL-2 or both. (d) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 36 h with addition of db-cAMP, anti-IFN-γ or anti-IL-2 for the last 12 h. (e) Expression of CD25 and production of IL-2 and IFN-γ by T cells activated with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or LY-294002 (LY) for 24 h. The percentages indicate the cAMP-mediated inhibition compared with each vehicle group. (f,g) T cells were activated for 48 h with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or PGE2 for indicated periods under Th1-priming conditions. Cells were then washed and reincubated for another 24 h under Th1-priming conditions followed by intracellular staining of IFN-γ (f). Effect of cell proliferation by cAMP is presented as a percentage relative to vehicle group (100%) under each αCD28 condition (g). Data shown as mean±s.e.m. are representative of two independent experiments with triplicates. MFI, mean fluorescence intensity; a.u., arbitrary units.
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f2: cAMP context-dependently regulates TCR activation and Th1 differentiation.(a) Expression of Il12rb2 mRNA in WT (left) and IFN-γR1−/− (right) T cells activated for 12 h without (unactivated) or with αCD3/CD28 in the presence or absence of db-cAMP, IFN-γ or anti-IFN-γ. (b) Surface expression of IL-12Rβ2 in WT or IFN-γR1−/− T cells activated for 48 h with αCD3/CD28. db-cAMP or vehicle was added from 24–48 h. (c) Expression of Il12rb2 mRNA in IFN-γR1–/– T cells activated for 24 h without (unactivated) or with αCD3/CD28 in the absence or presence of db-cAMP or anti-IL-2 or both. (d) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 36 h with addition of db-cAMP, anti-IFN-γ or anti-IL-2 for the last 12 h. (e) Expression of CD25 and production of IL-2 and IFN-γ by T cells activated with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or LY-294002 (LY) for 24 h. The percentages indicate the cAMP-mediated inhibition compared with each vehicle group. (f,g) T cells were activated for 48 h with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or PGE2 for indicated periods under Th1-priming conditions. Cells were then washed and reincubated for another 24 h under Th1-priming conditions followed by intracellular staining of IFN-γ (f). Effect of cell proliferation by cAMP is presented as a percentage relative to vehicle group (100%) under each αCD28 condition (g). Data shown as mean±s.e.m. are representative of two independent experiments with triplicates. MFI, mean fluorescence intensity; a.u., arbitrary units.

Mentions: As the above results suggest the involvement of IFN-γ and IL-2 in a part of cAMP-induced Il12rb2 expression by TCR-activated T cells, we next evaluated how and how much IFN-γ and IL-2 contribute to cAMP-induced Il12rb2 expression by stimulating T cells with or without TCR activation, with or without IFN-γ signalling, and in the presence or absence of cAMP for 12 h. In WT T cells, while db-cAMP, IFN-γ or TCR activation alone induced Il12rb2 expression at a similar level, cAMP synergistically induced Il12rb2 expression in the presence of IFN-γ or TCR or both (Fig. 2a). Addition of anti-IFN-γ did not affect the stimulation by db-cAMP in unactivated T cells, but blunted cAMP response in TCR-activated cells. Consistently, none of the above synergistic effects of IFN-γ or TCR with cAMP was seen in IFN-γR1−/− T cells at 12 h (Fig. 2a). Synergistic action of cAMP with IFN-γ or TCR activation on IL-12Rβ2 protein expression in T cells was also confirmed (Fig. 2b). We then evaluated the involvement of IL-2 signalling in cAMP-induced IL-12Rβ2 expression. We used IFN-γR1−/− T cells to exclude the effect of IFN-γ signalling. Although cAMP and TCR signalling had no synergistic action on Il12rb2 expression in IFN-γR1−/− T cells at 12 h (Fig. 2a), we noted that they synergistically induced Il12rb2 expression after 24 h and this synergistic action was eliminated by anti-IL-2 (Fig. 2c), suggesting that synergistic action on Il12rb2 expression with TCR activation and cAMP was mediated by IL-2 and this phenomenon did not appear until 24 h of stimulation. Consistently, blocking either IFN-γ or IL-2 signalling downregulated the basal, as well as the cAMP-induced Il12rb2 expression, and blocking both further reduced Il12rb2 expression in TCR-activated WT T cells at 36 h, but cAMP still enhanced Il12rb2 expression over the basal level under these conditions (Fig. 2d). These results demonstrate that there are direct and indirect mechanisms of cAMP-promoted Il12rb2 expression, and that IFN-γ and IL-2 are involved in the latter indirect mechanism.


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)

cAMP context-dependently regulates TCR activation and Th1 differentiation.(a) Expression of Il12rb2 mRNA in WT (left) and IFN-γR1−/− (right) T cells activated for 12 h without (unactivated) or with αCD3/CD28 in the presence or absence of db-cAMP, IFN-γ or anti-IFN-γ. (b) Surface expression of IL-12Rβ2 in WT or IFN-γR1−/− T cells activated for 48 h with αCD3/CD28. db-cAMP or vehicle was added from 24–48 h. (c) Expression of Il12rb2 mRNA in IFN-γR1–/– T cells activated for 24 h without (unactivated) or with αCD3/CD28 in the absence or presence of db-cAMP or anti-IL-2 or both. (d) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 36 h with addition of db-cAMP, anti-IFN-γ or anti-IL-2 for the last 12 h. (e) Expression of CD25 and production of IL-2 and IFN-γ by T cells activated with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or LY-294002 (LY) for 24 h. The percentages indicate the cAMP-mediated inhibition compared with each vehicle group. (f,g) T cells were activated for 48 h with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or PGE2 for indicated periods under Th1-priming conditions. Cells were then washed and reincubated for another 24 h under Th1-priming conditions followed by intracellular staining of IFN-γ (f). Effect of cell proliferation by cAMP is presented as a percentage relative to vehicle group (100%) under each αCD28 condition (g). Data shown as mean±s.e.m. are representative of two independent experiments with triplicates. MFI, mean fluorescence intensity; a.u., arbitrary units.
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Related In: Results  -  Collection

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f2: cAMP context-dependently regulates TCR activation and Th1 differentiation.(a) Expression of Il12rb2 mRNA in WT (left) and IFN-γR1−/− (right) T cells activated for 12 h without (unactivated) or with αCD3/CD28 in the presence or absence of db-cAMP, IFN-γ or anti-IFN-γ. (b) Surface expression of IL-12Rβ2 in WT or IFN-γR1−/− T cells activated for 48 h with αCD3/CD28. db-cAMP or vehicle was added from 24–48 h. (c) Expression of Il12rb2 mRNA in IFN-γR1–/– T cells activated for 24 h without (unactivated) or with αCD3/CD28 in the absence or presence of db-cAMP or anti-IL-2 or both. (d) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 36 h with addition of db-cAMP, anti-IFN-γ or anti-IL-2 for the last 12 h. (e) Expression of CD25 and production of IL-2 and IFN-γ by T cells activated with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or LY-294002 (LY) for 24 h. The percentages indicate the cAMP-mediated inhibition compared with each vehicle group. (f,g) T cells were activated for 48 h with αCD3 and indicated concentrations of αCD28 and treated with db-cAMP or PGE2 for indicated periods under Th1-priming conditions. Cells were then washed and reincubated for another 24 h under Th1-priming conditions followed by intracellular staining of IFN-γ (f). Effect of cell proliferation by cAMP is presented as a percentage relative to vehicle group (100%) under each αCD28 condition (g). Data shown as mean±s.e.m. are representative of two independent experiments with triplicates. MFI, mean fluorescence intensity; a.u., arbitrary units.
Mentions: As the above results suggest the involvement of IFN-γ and IL-2 in a part of cAMP-induced Il12rb2 expression by TCR-activated T cells, we next evaluated how and how much IFN-γ and IL-2 contribute to cAMP-induced Il12rb2 expression by stimulating T cells with or without TCR activation, with or without IFN-γ signalling, and in the presence or absence of cAMP for 12 h. In WT T cells, while db-cAMP, IFN-γ or TCR activation alone induced Il12rb2 expression at a similar level, cAMP synergistically induced Il12rb2 expression in the presence of IFN-γ or TCR or both (Fig. 2a). Addition of anti-IFN-γ did not affect the stimulation by db-cAMP in unactivated T cells, but blunted cAMP response in TCR-activated cells. Consistently, none of the above synergistic effects of IFN-γ or TCR with cAMP was seen in IFN-γR1−/− T cells at 12 h (Fig. 2a). Synergistic action of cAMP with IFN-γ or TCR activation on IL-12Rβ2 protein expression in T cells was also confirmed (Fig. 2b). We then evaluated the involvement of IL-2 signalling in cAMP-induced IL-12Rβ2 expression. We used IFN-γR1−/− T cells to exclude the effect of IFN-γ signalling. Although cAMP and TCR signalling had no synergistic action on Il12rb2 expression in IFN-γR1−/− T cells at 12 h (Fig. 2a), we noted that they synergistically induced Il12rb2 expression after 24 h and this synergistic action was eliminated by anti-IL-2 (Fig. 2c), suggesting that synergistic action on Il12rb2 expression with TCR activation and cAMP was mediated by IL-2 and this phenomenon did not appear until 24 h of stimulation. Consistently, blocking either IFN-γ or IL-2 signalling downregulated the basal, as well as the cAMP-induced Il12rb2 expression, and blocking both further reduced Il12rb2 expression in TCR-activated WT T cells at 36 h, but cAMP still enhanced Il12rb2 expression over the basal level under these conditions (Fig. 2d). These results demonstrate that there are direct and indirect mechanisms of cAMP-promoted Il12rb2 expression, and that IFN-γ and IL-2 are involved in the latter indirect mechanism.

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