PMC

PGE2 suppresses IL-17, but not IL-22, induction by TGF-β and IL-6. (A) IL-17 mRNA (left panel) and protein (right panel) three days after stimulation of naïve CD4⁺ T cells in the presence of TGF-β and IL-6 with PGE2 titration. (B) IL-22 mRNA (left panel) and protein (right panel) three days after stimulationas in (A). (C) Supernatant IL-17 and IL-22 from naive CD4⁺ T cells activated for 3 days with TGF-β, IL-1β, IL-23, and IL-6 with or without PGE2 (1μM). (D) IL-17 and IL-22 protein (top panels) and transcript (bottom panels) after activation of naive CD4⁺ T cells for 3 days in the presence of IL-6 and TGF-β (0.1, 1.0, 10, or 100 ng/ml) with or without 1 μM PGE2. Data shown as mean +/− SD.

PGE2 produced from Cryptococcus suppresses T cell IRF4 and IL-17 expression. (A–C) Naïve CD4⁺ T cells were exposed to Th17 differentiation conditions for three days in the presence of wild-type H99 or Δlac1Cryptococcus. The ratio of cryptococci/T cells is indicated. T cells were then harvested to determine relative mRNA expression of IRF4 (A), IL-17 (B), and IL-22 (C). (D–E) Naive CD4⁺ T cells activated as above in the presence of H99 Cryptococcus were concomitantly cultured with the EP2 antagonist AH6809 or the EP4 antagonist AH23848. Expression of IRF4 (D) and IL-17 (E) mRNA is shown relative to TGF-β+IL-6 activation. Data shown as mean +/− SD. See also .

IRF4-deficiency inhibits IL-17 expression and enhances IL-22 expression. (AB) Relative mRNA expression of IL-17, IL-22, and IRF4 after Th17 differentiation of naïve CD4⁺ T cells from Rorc^−/−(A) and Irf4^−/−(B) mice (“KO” in figure). (C) siRNA gene targeting of IRF4 expression in naive T cells stimulated with anti-CD3+anti-CD28+IL-6+TGF-β was confirmed by flow cytometry (left panel). Shaded histogram shows cells transfected with a scrambled siRNA; open histogram shows cells transfected with siRNA against IRF4. Supernatant IL-17 and IL-22 were detected after three days of stimulation (right panels). (D) Supernatant IL-17 and IL-22 from wild-type or Irf4^−/− (“KO” in figure) naive CD4⁺ T cells activated in the presence of anti-CD3+anti-CD28, TGF-β (1 ng/ml), IL-1β, IL-23, and IL-6 for three days. Data shown as mean +/− SD. See also .

PGE2 suppresses the development of Th17 cells. (A) Intracellular IL-17 and IFN-γ staining in naive CD4⁺ T cells activated for three days in the presence of TGF-β (1 ng/ml), IL-6, and PGE2 (top panels). Cells initially activated with TGF-β and IL-6 were restimulated for an additional three days in the presence of PGE2, IL-23, or both (bottom panels). (B) IL-17 protein expression (left panel) and relative IL-17 mRNA expression (right panel) after three days of stimulation with 0.1, 1.0, or 10.0 μM of PGE2, the EP2 agonist butaprost, the EP4 agonist misoprostol, or the EP1 and EP3 agonist sulprostone. (C) IL-17 protein and RNA expression after three days of stimulation under the indicated conditions with PGE2 (1 μM) or forskolin (10 μM). Data shown as mean +/− SD. See also .

PGE2 alters expression of Th17-associated transcription factors. (A) Unphosphorylated and phosphorylated STAT3 (pY705) detected by immunoblot at 10 and 30 minutes post-activation with anti-CD3+anti-CD28 magnetic beads and the cytokines indicated. (B) Intracellular IRF4 detected by flow cytometry in naive T cells after Th17 differentiation for three days in the absence (shaded histogram) or presence (open histogram) of PGE2. (C) Relative IRF4 mRNA expression 24 and 48 hours after stimulation under the indicated conditions with 0.1 and 1.0 μM PGE2. (D–F) Relative mRNA expression of Rorγt (D), Rorα (E), and AHR (F) three days after activation with TGF-β and IL-6, with or without PGE2. Results shown are relative to activation (anti-CD3+anti-CD28) in the absence of TGF-β and IL-6. Data shown as mean +/− SD. See also .

PGE2 blockade in vivo increases T cell IL-17 production and prolongs survival after Cryptococcus infection. (A) PGE metabolites in BALF 13 days after infection with Cryptococcus and treatment with either PBS or indomethacin. (B) Percent survival of mice infected with Cryptococcus and treated with either PBS or indomethacin (n=10 mice per group). (C) Percent survival with and without CD4 depletion of mice infected with Cryptococcus and treated with either PBS or indomethacin (n=9–10 mice per group). (D) IL-17, IL-22, IFN-γ, and IL-4 from splenic CD4⁺ T cells isolated from mice 13 days post-infection (n=5 mice per group). Control mice (left bar) were not infected and received no treatment. (E and F) Number of IL-17-producing CD45⁺, CD4⁺, and γδ⁺ cells in BALF from mice infected with Cryptococcus and treated with PBS or indomethacin harvested one (E) or two (F) weeks post-infection. (G) Colony forming units from whole lung one and two weeks after infection (n=4 mice per group). (H) Percent survival with and without IL-17 neutralization for mice infected with Cryptococcus and treated with either PBS or indomethacin (n=8–10 mice per group). Data shown as mean +/− SD.

PGE2 inhibits IRF4 function. (A) Relative mRNA expression of FoxP3, IL-23R, IL-21 and Rorγt three days after activation of wild-type or Irf4^−/−(“ KO” in figure) naive CD4⁺ T cells with TGF-β+IL-6 (top panels), or wild type cells with TGF-β+IL-6 in the absence or presence of PGE2 (bottom panels). (B) Relative expression of IL-17 and IL-22 after activation of wild type or Irf4^−/− naive CD4⁺ T cells in the presence of TGF-β (1 ng/ml) and IL-6 plus PGE2 (0.1 or 1.0 μM). (C) Retroviral overexpression of IRF4 (right panels) or vector (left panels) in naive cells differentiated under Th17 conditions with (bottom panels) or without (top panels) PGE2. Intracellular IL-22 and IL-17 were detected by flow cytometry in GFP⁺ cells. (D) Relative chromatin binding of IRF4 in naive CD4⁺ T cells after Th17-inducing conditions with (white bars) or without (black bars) PGE2 displayed as percent of input DNA after immunoprecipitation with IRF4 (left) or isotype control (right) antibody. A CD11b coding region is shown as a negative control for IRF4 binding. IRF4 binding was assessed at three IL-17 promoter regions and one IL-22 promoter region. Data shown as mean +/− SD. See also .