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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 amplifies IFN-γ and IL-2 signalling through induction of IFN-γR1 and IL-2Rβ.(a) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 24 h with addition of db-cAMP or cycloheximide (CHX) or both for the last 12 h. (b) Profile of db-cAMP-dependent expression of cytokines and their receptors by WT or IFN-γR1−/− T cells activated for 12 h with αCD3/CD28 followed by treatment with db-cAMP or vehicle for another 3 h. Data shown in bar graphs represents fold change (db-cAMP versus vehicle) in mean intensity from each probe in biological duplicates. The probe for Ifngr1 in this array is targeted to a segment of sequence in the exon VII, while The IFN-γR1−/− mouse that we used has Ifngr1 gene disrupted by inserting the neomycin resistance gene into exon V. (c,d) Expression of IFN-γR1 mRNA (c) and protein (d) in T cells stimulated with or without αCD3/CD28 in the absence or presence of db-cAMP for 12 h. (e) Immunoblot for p-STAT1 (Y701) and STAT1 in T cells pretreated with db-cAMP for 12 h, washed and restimulated with 1 ng ml−1 IFN-γ for another 30 min. (f) Expression of Il2rb mRNA in T cells treated with db-cAMP for 12 and 24 h. (g) IL-2Rβ protein expression in T cells activated with αCD3/CD28 for 2 days, allowed to rest for another 2 days, then restimulated with db-cAMP in the presence of IL-2 for 24 h. Data shown as mean±s.e.m. are representative of two independent experiments with triplicates (a,c–g) or are from one experiment (b). a.u., arbitrary units.
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f3: cAMP amplifies IFN-γ and IL-2 signalling through induction of IFN-γR1 and IL-2Rβ.(a) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 24 h with addition of db-cAMP or cycloheximide (CHX) or both for the last 12 h. (b) Profile of db-cAMP-dependent expression of cytokines and their receptors by WT or IFN-γR1−/− T cells activated for 12 h with αCD3/CD28 followed by treatment with db-cAMP or vehicle for another 3 h. Data shown in bar graphs represents fold change (db-cAMP versus vehicle) in mean intensity from each probe in biological duplicates. The probe for Ifngr1 in this array is targeted to a segment of sequence in the exon VII, while The IFN-γR1−/− mouse that we used has Ifngr1 gene disrupted by inserting the neomycin resistance gene into exon V. (c,d) Expression of IFN-γR1 mRNA (c) and protein (d) in T cells stimulated with or without αCD3/CD28 in the absence or presence of db-cAMP for 12 h. (e) Immunoblot for p-STAT1 (Y701) and STAT1 in T cells pretreated with db-cAMP for 12 h, washed and restimulated with 1 ng ml−1 IFN-γ for another 30 min. (f) Expression of Il2rb mRNA in T cells treated with db-cAMP for 12 and 24 h. (g) IL-2Rβ protein expression in T cells activated with αCD3/CD28 for 2 days, allowed to rest for another 2 days, then restimulated with db-cAMP in the presence of IL-2 for 24 h. Data shown as mean±s.e.m. are representative of two independent experiments with triplicates (a,c–g) or are from one experiment (b). a.u., arbitrary units.

Mentions: As the above findings suggest that cAMP can induce IL-12Rβ2 expression in T cells partly through IFN-γ and IL-2, the next question was how cAMP amplified IFN-γ and IL-2 signalling. We first noted that the induction of Il12rb2 by cAMP was partially reduced by treatment of the cells with cycloheximide, suggesting the involvement of new protein synthesis in this process (Fig. 3a). To identify protein(s) newly synthesized in response to cAMP, we profiled gene expression in TCR-activated WT and IFN-γR1−/− T cells stimulated with or without db-cAMP. Consistent with previous reports8, cAMP inhibited gene expression of several cytokines such as Il2, Ifng, Tnf and Lta in TCR-activated T cells (Fig. 3b). In congruence with our results (Fig. 2a), Il12rb2 expression was upregulated by db-cAMP in both WT and IFN-γR1−/− T cells (Fig. 3b), verifying an IFN-γ signalling-independent induction of Il12rb2 by cAMP. Additionally, we noted enhanced expression of genes encoding receptors for IFN-γ and IL-2, such as Ifngr1 and Il2rb, in T cells (Fig. 3b). We confirmed by real-time PCR and flow cytometry that cAMP increased both Ifngr1 mRNA and IFN-γR1 protein expression in naive T cells (Fig. 3c). This action of cAMP was also observed in TCR-activated T cells, although TCR itself strongly downregulated IFN-γR1 expression (Fig. 3c). IFN-γ-induced STAT1 activation in naive T cells was also enhanced by pretreatment with db-cAMP, suggesting that cAMP amplifies IFN-γ signalling (Fig. 3e). Similarly, cAMP also upregulated Il2rb mRNA expression in T cells after 24 h, but not 12 h, stimulation (Fig. 3f) and upregulation of IL-2Rβ protein expression by cAMP was also confirmed using flow cytometry (Fig. 3g). These data suggested that cAMP amplifies IFN-γ and IL-2 signalling in T cells through the upregulation of IFN-γR1 and IL-2Rβ, respectively.


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 amplifies IFN-γ and IL-2 signalling through induction of IFN-γR1 and IL-2Rβ.(a) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 24 h with addition of db-cAMP or cycloheximide (CHX) or both for the last 12 h. (b) Profile of db-cAMP-dependent expression of cytokines and their receptors by WT or IFN-γR1−/− T cells activated for 12 h with αCD3/CD28 followed by treatment with db-cAMP or vehicle for another 3 h. Data shown in bar graphs represents fold change (db-cAMP versus vehicle) in mean intensity from each probe in biological duplicates. The probe for Ifngr1 in this array is targeted to a segment of sequence in the exon VII, while The IFN-γR1−/− mouse that we used has Ifngr1 gene disrupted by inserting the neomycin resistance gene into exon V. (c,d) Expression of IFN-γR1 mRNA (c) and protein (d) in T cells stimulated with or without αCD3/CD28 in the absence or presence of db-cAMP for 12 h. (e) Immunoblot for p-STAT1 (Y701) and STAT1 in T cells pretreated with db-cAMP for 12 h, washed and restimulated with 1 ng ml−1 IFN-γ for another 30 min. (f) Expression of Il2rb mRNA in T cells treated with db-cAMP for 12 and 24 h. (g) IL-2Rβ protein expression in T cells activated with αCD3/CD28 for 2 days, allowed to rest for another 2 days, then restimulated with db-cAMP in the presence of IL-2 for 24 h. Data shown as mean±s.e.m. are representative of two independent experiments with triplicates (a,c–g) or are from one experiment (b). a.u., arbitrary units.
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f3: cAMP amplifies IFN-γ and IL-2 signalling through induction of IFN-γR1 and IL-2Rβ.(a) Expression of Il12rb2 mRNA in T cells activated with αCD3/CD28 for 24 h with addition of db-cAMP or cycloheximide (CHX) or both for the last 12 h. (b) Profile of db-cAMP-dependent expression of cytokines and their receptors by WT or IFN-γR1−/− T cells activated for 12 h with αCD3/CD28 followed by treatment with db-cAMP or vehicle for another 3 h. Data shown in bar graphs represents fold change (db-cAMP versus vehicle) in mean intensity from each probe in biological duplicates. The probe for Ifngr1 in this array is targeted to a segment of sequence in the exon VII, while The IFN-γR1−/− mouse that we used has Ifngr1 gene disrupted by inserting the neomycin resistance gene into exon V. (c,d) Expression of IFN-γR1 mRNA (c) and protein (d) in T cells stimulated with or without αCD3/CD28 in the absence or presence of db-cAMP for 12 h. (e) Immunoblot for p-STAT1 (Y701) and STAT1 in T cells pretreated with db-cAMP for 12 h, washed and restimulated with 1 ng ml−1 IFN-γ for another 30 min. (f) Expression of Il2rb mRNA in T cells treated with db-cAMP for 12 and 24 h. (g) IL-2Rβ protein expression in T cells activated with αCD3/CD28 for 2 days, allowed to rest for another 2 days, then restimulated with db-cAMP in the presence of IL-2 for 24 h. Data shown as mean±s.e.m. are representative of two independent experiments with triplicates (a,c–g) or are from one experiment (b). a.u., arbitrary units.
Mentions: As the above findings suggest that cAMP can induce IL-12Rβ2 expression in T cells partly through IFN-γ and IL-2, the next question was how cAMP amplified IFN-γ and IL-2 signalling. We first noted that the induction of Il12rb2 by cAMP was partially reduced by treatment of the cells with cycloheximide, suggesting the involvement of new protein synthesis in this process (Fig. 3a). To identify protein(s) newly synthesized in response to cAMP, we profiled gene expression in TCR-activated WT and IFN-γR1−/− T cells stimulated with or without db-cAMP. Consistent with previous reports8, cAMP inhibited gene expression of several cytokines such as Il2, Ifng, Tnf and Lta in TCR-activated T cells (Fig. 3b). In congruence with our results (Fig. 2a), Il12rb2 expression was upregulated by db-cAMP in both WT and IFN-γR1−/− T cells (Fig. 3b), verifying an IFN-γ signalling-independent induction of Il12rb2 by cAMP. Additionally, we noted enhanced expression of genes encoding receptors for IFN-γ and IL-2, such as Ifngr1 and Il2rb, in T cells (Fig. 3b). We confirmed by real-time PCR and flow cytometry that cAMP increased both Ifngr1 mRNA and IFN-γR1 protein expression in naive T cells (Fig. 3c). This action of cAMP was also observed in TCR-activated T cells, although TCR itself strongly downregulated IFN-γR1 expression (Fig. 3c). IFN-γ-induced STAT1 activation in naive T cells was also enhanced by pretreatment with db-cAMP, suggesting that cAMP amplifies IFN-γ signalling (Fig. 3e). Similarly, cAMP also upregulated Il2rb mRNA expression in T cells after 24 h, but not 12 h, stimulation (Fig. 3f) and upregulation of IL-2Rβ protein expression by cAMP was also confirmed using flow cytometry (Fig. 3g). These data suggested that cAMP amplifies IFN-γ and IL-2 signalling in T cells through the upregulation of IFN-γR1 and IL-2Rβ, respectively.

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