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. 2011 Apr 7;184(1):37–49. doi: 10.1164/rccm.201010-1637OC

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Figure 5. — Cyclooxygenase (COX)-2–derived prostaglandins (PGs) regulate Th17 cell differentiation in vitro. (A) Stat3 phosphorylation during Th17 differentiation of CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice was assayed using anti–phospho-stat3 (pY705)-PE by flow cytometry. (B) Ratio of phospho-Stat3/total Stat3 during Th17 cell differentiation of COX-2^{\\plus/\plus+/+} and COX-2^−/− naive CD4⁺ T cells. (C) RORγt mRNA expression in differentiated CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice was analyzed by real-time polymerase chain reaction. (D) Ratio of RORγt to β-actin mRNA during Th17 cell differentiation of COX-2^+/+ and COX-2^−/− naive CD4⁺ CD62L⁺T cells. All data are representative of three independent experiments; ^#P < 0.05 versus COX-2^+/+ naive CD4⁺ T cells; *P < 0.05 versus COX-2^+/+. (E) Eicosanoid levels in supernatants of naive CD4⁺ T cells and in vitro differentiated Th17 cells from COX-2^+/+ and COX-2^−/− mice were measured by liquid chromatography/tandem mass spectroscopy. Results are representative of five independent experiments. Effect of synthetic PGs on Th17 cell differentiation of (F) and IL-17A production by (G) naive CD4⁺ T cells isolated from COX-2^+/+ and COX-2^−/− mice in vitro. PGs were added at 1 μM on Days 2 and 3 of culture, and culture supernatants were collected on Day 4. IL-17A was measured by ELISA. Results are representative of three independent experiments; *P < 0.05 versus vehicle. (H) Dose dependency of PGI₂, PGF_2α, and PGE₂ (50 nM to 1 μM) on Th17 cell differentiation was examined in vitro; n = 4; *P < 0.05 versus no PG. (I and J) Phospho-Stat3 and total Stat3 levels were determined by immunoblotting before and 5–60 minutes after treatment of naive CD4⁺ T cells with synthetic PGs (1 μM each). Results are representative of three independent experiments; * P < 0.05 versus time 0. (K and L) RORγt mRNA levels measured by real-time polymerase chain reaction in in vitro differentiated Th17 cells treated with or without synthetic PGs (1 μM each). Results are representative of three independent experiments; * P < 0.05 versus vehicle.

Figure 5. — Cyclooxygenase (COX)-2–derived prostaglandins (PGs) regulate Th17 cell differentiation in vitro. (A) Stat3 phosphorylation during Th17 differentiation of CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice was assayed using anti–phospho-stat3 (pY705)-PE by flow cytometry. (B) Ratio of phospho-Stat3/total Stat3 during Th17 cell differentiation of COX-2^{\\plus/\plus+/+} and COX-2^−/− naive CD4⁺ T cells. (C) RORγt mRNA expression in differentiated CD4⁺ T cells from COX-2^+/+ and COX-2^−/− mice was analyzed by real-time polymerase chain reaction. (D) Ratio of RORγt to β-actin mRNA during Th17 cell differentiation of COX-2^+/+ and COX-2^−/− naive CD4⁺ CD62L⁺T cells. All data are representative of three independent experiments; ^#P < 0.05 versus COX-2^+/+ naive CD4⁺ T cells; *P < 0.05 versus COX-2^+/+. (E) Eicosanoid levels in supernatants of naive CD4⁺ T cells and in vitro differentiated Th17 cells from COX-2^+/+ and COX-2^−/− mice were measured by liquid chromatography/tandem mass spectroscopy. Results are representative of five independent experiments. Effect of synthetic PGs on Th17 cell differentiation of (F) and IL-17A production by (G) naive CD4⁺ T cells isolated from COX-2^+/+ and COX-2^−/− mice in vitro. PGs were added at 1 μM on Days 2 and 3 of culture, and culture supernatants were collected on Day 4. IL-17A was measured by ELISA. Results are representative of three independent experiments; *P < 0.05 versus vehicle. (H) Dose dependency of PGI₂, PGF_2α, and PGE₂ (50 nM to 1 μM) on Th17 cell differentiation was examined in vitro; n = 4; *P < 0.05 versus no PG. (I and J) Phospho-Stat3 and total Stat3 levels were determined by immunoblotting before and 5–60 minutes after treatment of naive CD4⁺ T cells with synthetic PGs (1 μM each). Results are representative of three independent experiments; * P < 0.05 versus time 0. (K and L) RORγt mRNA levels measured by real-time polymerase chain reaction in in vitro differentiated Th17 cells treated with or without synthetic PGs (1 μM each). Results are representative of three independent experiments; * P < 0.05 versus vehicle.