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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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EP4-cAMP signalling in T cells modulates expression of IL-12Rβ2 and IFN-γR1, and Th1 response in vivo.(a) Expression of Ptger4 mRNA in CD4+ T cells of mice of indicated genotypes. (b) IFN-γ and IL-2 production by dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5 and then stimulated with αCD3/CD28 for 24 h. (c) mRNA expression of Th1-related cytokine receptor genes in dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5. Il12rb2 and Cd69 mRNA are shown in dLN CD4+ T cells stimulated with αCD3/CD28 for 24 h. (d) Numbers of IL-12Rβ2-, IFN-γR1-, or CD69-positive CD4+ T cells in dLNs of DNFB-sensitized mice on day 5. (e) Ear swelling of WT recipient mice given intravenous transfer of dLN cells from DNFB-sensitized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice. (f) Change in body weight of Rag2−/− mice (8–10 mice per group) given intravenous transfer of Lck-Cre+EP4+/+ or Lck-Cre+EP4fl/+ naive T cells. (g) Hematoxylin and eosin staining of the colon of Rag2−/− mice 42 days after transfer. Scale bar, 200 μM. (h,i) IFN-γ and IL-2 production (h) and mRNA expression of indicated genes (i) in CD4+ T cells isolated from the mLNs of Rag2–/– mice 42 days after transfer and stimulated with αCD3 for 3 days. Horizontal and error bars represent the mean and s.e.m., respectively. Statistical significance in e was examined by two-way ANOVA. *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
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f7: EP4-cAMP signalling in T cells modulates expression of IL-12Rβ2 and IFN-γR1, and Th1 response in vivo.(a) Expression of Ptger4 mRNA in CD4+ T cells of mice of indicated genotypes. (b) IFN-γ and IL-2 production by dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5 and then stimulated with αCD3/CD28 for 24 h. (c) mRNA expression of Th1-related cytokine receptor genes in dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5. Il12rb2 and Cd69 mRNA are shown in dLN CD4+ T cells stimulated with αCD3/CD28 for 24 h. (d) Numbers of IL-12Rβ2-, IFN-γR1-, or CD69-positive CD4+ T cells in dLNs of DNFB-sensitized mice on day 5. (e) Ear swelling of WT recipient mice given intravenous transfer of dLN cells from DNFB-sensitized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice. (f) Change in body weight of Rag2−/− mice (8–10 mice per group) given intravenous transfer of Lck-Cre+EP4+/+ or Lck-Cre+EP4fl/+ naive T cells. (g) Hematoxylin and eosin staining of the colon of Rag2−/− mice 42 days after transfer. Scale bar, 200 μM. (h,i) IFN-γ and IL-2 production (h) and mRNA expression of indicated genes (i) in CD4+ T cells isolated from the mLNs of Rag2–/– mice 42 days after transfer and stimulated with αCD3 for 3 days. Horizontal and error bars represent the mean and s.e.m., respectively. Statistical significance in e was examined by two-way ANOVA. *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.

Mentions: To study whether PGE2-EP4-cAMP signalling in T cells regulates cytokine receptors and Th1 response in vivo, we generated mice with deletion of EP4 in T cells by crossing Lck-Cre mice37 with EP4-floxed mice38. Loss of EP4 in CD4+ T cells was confirmed in Lck-Cre+EP4fl/+ and Lck-Cre+EP4fl/fl mice (Fig. 7a). To investigate whether EP4 deficiency in T cells affects in vivo Th1 differentiation and expression of Th1-related cytokine receptors, we used the CHS model and immunized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice by painting the shaved abdomen with dinitrofluorobenzene (DNFB) on day 0 and purified CD4+ T cells in draining lymph nodes (dLNs) on day 5. CD4+ T cells from Lck-Cre+EP4fl/fl mice produced less amounts of IFN-γ and IL-2 (Fig. 7b) and exhibited less mRNA expression of Th1 cytokine receptors, for example, Il12rb2, Ifngr1, Il2rb and CREB/CRTC2-targeted gene Icer than T cells from littermate control Lck-Cre−EP4fl/fl mice (Fig. 7c). Consistent with the above results, the numbers of IL-12Rβ2- and IFN-γR1-expressing CD4+ T cells in dLNs from Lck-Cre+EP4fl/fl mice were also lower than those from control mice (Fig. 7d). We also found that Cd69 gene expression and the number of CD4+CD69+ T cells were decreased in dLNs from DNFB-sensitized Lck-Cre+EP4fl/fl mice (Fig. 7c). Moreover, when we adoptively transferred dLN cells from DNFB-sensitized Lck-Cre+EP4fl/fl or Lck-Cre−EP4fl/fl mice into naive C57BL/6 mice on day 5 and immediately challenged the recipient mice by painting the same antigen on the ear, transfer of EP4-deficient T cells induced less ear swelling (Fig. 7e).


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)

EP4-cAMP signalling in T cells modulates expression of IL-12Rβ2 and IFN-γR1, and Th1 response in vivo.(a) Expression of Ptger4 mRNA in CD4+ T cells of mice of indicated genotypes. (b) IFN-γ and IL-2 production by dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5 and then stimulated with αCD3/CD28 for 24 h. (c) mRNA expression of Th1-related cytokine receptor genes in dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5. Il12rb2 and Cd69 mRNA are shown in dLN CD4+ T cells stimulated with αCD3/CD28 for 24 h. (d) Numbers of IL-12Rβ2-, IFN-γR1-, or CD69-positive CD4+ T cells in dLNs of DNFB-sensitized mice on day 5. (e) Ear swelling of WT recipient mice given intravenous transfer of dLN cells from DNFB-sensitized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice. (f) Change in body weight of Rag2−/− mice (8–10 mice per group) given intravenous transfer of Lck-Cre+EP4+/+ or Lck-Cre+EP4fl/+ naive T cells. (g) Hematoxylin and eosin staining of the colon of Rag2−/− mice 42 days after transfer. Scale bar, 200 μM. (h,i) IFN-γ and IL-2 production (h) and mRNA expression of indicated genes (i) in CD4+ T cells isolated from the mLNs of Rag2–/– mice 42 days after transfer and stimulated with αCD3 for 3 days. Horizontal and error bars represent the mean and s.e.m., respectively. Statistical significance in e was examined by two-way ANOVA. *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
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f7: EP4-cAMP signalling in T cells modulates expression of IL-12Rβ2 and IFN-γR1, and Th1 response in vivo.(a) Expression of Ptger4 mRNA in CD4+ T cells of mice of indicated genotypes. (b) IFN-γ and IL-2 production by dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5 and then stimulated with αCD3/CD28 for 24 h. (c) mRNA expression of Th1-related cytokine receptor genes in dLN CD4+ T cells isolated from DNFB-sensitized mice on day 5. Il12rb2 and Cd69 mRNA are shown in dLN CD4+ T cells stimulated with αCD3/CD28 for 24 h. (d) Numbers of IL-12Rβ2-, IFN-γR1-, or CD69-positive CD4+ T cells in dLNs of DNFB-sensitized mice on day 5. (e) Ear swelling of WT recipient mice given intravenous transfer of dLN cells from DNFB-sensitized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice. (f) Change in body weight of Rag2−/− mice (8–10 mice per group) given intravenous transfer of Lck-Cre+EP4+/+ or Lck-Cre+EP4fl/+ naive T cells. (g) Hematoxylin and eosin staining of the colon of Rag2−/− mice 42 days after transfer. Scale bar, 200 μM. (h,i) IFN-γ and IL-2 production (h) and mRNA expression of indicated genes (i) in CD4+ T cells isolated from the mLNs of Rag2–/– mice 42 days after transfer and stimulated with αCD3 for 3 days. Horizontal and error bars represent the mean and s.e.m., respectively. Statistical significance in e was examined by two-way ANOVA. *P<0.05; **P<0.01; ***P<0.001. a.u., arbitrary units.
Mentions: To study whether PGE2-EP4-cAMP signalling in T cells regulates cytokine receptors and Th1 response in vivo, we generated mice with deletion of EP4 in T cells by crossing Lck-Cre mice37 with EP4-floxed mice38. Loss of EP4 in CD4+ T cells was confirmed in Lck-Cre+EP4fl/+ and Lck-Cre+EP4fl/fl mice (Fig. 7a). To investigate whether EP4 deficiency in T cells affects in vivo Th1 differentiation and expression of Th1-related cytokine receptors, we used the CHS model and immunized Lck-Cre−EP4fl/fl or Lck-Cre+EP4fl/fl mice by painting the shaved abdomen with dinitrofluorobenzene (DNFB) on day 0 and purified CD4+ T cells in draining lymph nodes (dLNs) on day 5. CD4+ T cells from Lck-Cre+EP4fl/fl mice produced less amounts of IFN-γ and IL-2 (Fig. 7b) and exhibited less mRNA expression of Th1 cytokine receptors, for example, Il12rb2, Ifngr1, Il2rb and CREB/CRTC2-targeted gene Icer than T cells from littermate control Lck-Cre−EP4fl/fl mice (Fig. 7c). Consistent with the above results, the numbers of IL-12Rβ2- and IFN-γR1-expressing CD4+ T cells in dLNs from Lck-Cre+EP4fl/fl mice were also lower than those from control mice (Fig. 7d). We also found that Cd69 gene expression and the number of CD4+CD69+ T cells were decreased in dLNs from DNFB-sensitized Lck-Cre+EP4fl/fl mice (Fig. 7c). Moreover, when we adoptively transferred dLN cells from DNFB-sensitized Lck-Cre+EP4fl/fl or Lck-Cre−EP4fl/fl mice into naive C57BL/6 mice on day 5 and immediately challenged the recipient mice by painting the same antigen on the ear, transfer of EP4-deficient T cells induced less ear swelling (Fig. 7e).

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