Figure 6. Adipocyte COX-2 promotes resident Treg proliferation through PGE₂.

(A) COX-2 deficiency suppressed the secretion levels of PGE₂ and PGI₂ in primary adipocytes. COX-2–KO and control (Ctrl) primary adipocytes were changed to fresh medium and cultured for 2 hours. Medium was collected and used to determine the levels of PGE₂ and PGI₂, using an ELISA kit. (B, C, E, and F) AT Tregs were isolated from AT for these studies. Treatment of PGE₂ but not PGD₂ and PGI₂ increased the population of Foxp3⁺ Tregs in a dose-dependent manner (B), and treatment of 100 nM PGE₂ induced proliferation of Tregs as indicated by the staining of Ki67 (C). Intracellular Ki67⁺ and Foxp3⁺ Tregs were determined by flow cytometry analysis. AT Tregs were treated with DMSO, PGD₂, PGE₂, or PGI₂, with indicated doses for 24 hours. *P < 0.05 compared with the group without treatment. (D) Treatment of PGE₂ stimulated activation of PKA and CaMKII in differentiated Tregs in a dose-dependent manner. CD4⁺-naive T cells were isolated from a single-cell suspension from lymph nodes and spleens and then differentiated into CD4⁺Foxp3⁺ Tregs. Differentiated Tregs were treated, or not, with PGE₂ for 1 hour. Representative data from 3 independent experiments are reported. (E) PGE₂ treatment stimulated phosphorylation of CaMKII in AT Tregs. n = 3/group. (F) Inhibiting PKA by 5 μM KT 5720 or inhibiting CaMKII by 5 μM TATCN21 suppressed PGE₂-treatment–induced proliferation of AT Tregs. AT Tregs were treated with KT 5720 or TATCN21 for 1 hour, followed by co-treatment with PGE₂ for 24 hours. n = 3/group. CaMKIIγ deficiency blocked PGE₂-stimulated CD4⁺Foxp3⁺ Treg proliferation, indicated by Ki67 expression (G) and total Treg fraction (H). Primary Tregs were isolated from AT of CaMKIIγ-KO and WT mice and treated with 100 nM PGE₂ for 24 hours, followed by flow cytometry analysis. (A–C) The t test was used for data analysis. (F–H) ANOVA was used for data analysis. Data are reported as mean ± SEM. *P < 0.05; **P < 0.01.