Ick test (A). Plasma PGE metabolites were RP54476MedChemExpress Dalfopristin AZD-8055 structure measured by ELISA (B). The pups from the Ptger4fl/fl x Lyz2-CreTg/Tg cross were genotyped as described in Materials and Methods (C). Resident peritoneal macrophages (rpMac) (D) and hearts (E) were harvested from EP4M-/- mice and WT littermate controls, and Ptger1-4 mRNA levels were measured by real-time PCR. The results are presented and mean ?SEM. Data in A-B (n = 9?2) were analyzed by unpaired two-tailed Student’s t-test and data in D-E were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test (n = 5?). Statistical outliers were identified by Grubbs’ test and were excluded from the analysis (one outlier in B), * p<0.05; *** p<0.001; NS, nonsignificant; ND, non-diabetic; D, diabetic. doi:10.1371/journal.pone.0158316.gPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,5 /EP4, Diabetes, Inflammation and Atherosclerosissubset of diabetic mice exhibited elevated plasma PGE metabolite levels, as compared with non-diabetic littermate controls, while others did not (Fig 1B). Together, the diabetic group had significantly higher PGE metabolite levels than non-diabetic littermates (Fig 1B). This mouse model of T1DM is therefore characterized by increased PGE status, consistent with our previous studies in which macrophages from diabetic mice were shown to secrete more PGE2 than macrophages from non-diabetic controls [3].The generated myeloid cell-targeted EP4-deficient (EP4M-/-) mouse exhibits a specific loss of Ptger4 in myeloid cellsBecause the PGE2 receptor EP4 (Ptger4) has been implicated in inflammatory activation associated with diabetes, we generated a mouse model of myeloid cell-targeted EP4-deficiency by taking advantage of the EP4-floxed mouse and Lyz2-CreTg mice, which express Cre recombinase in myeloid cells. Following backcrossing for 10 generations into the C57BL/6 background, Lyz2-CreTg/Tg Ptger4fl/fl mice and Lyz2-CreTg/Tg Ptger4wt/wt littermate controls (Fig 1C) were used to measure Ptger mRNA levels in resident peritoneal macrophages and heart, to evaluate specificity of the EP4-deficiency. Ptger4 mRNA was absent in macrophages from EP4M-/(Lyz2-CreTg/Tg Ptger4fl/fl) mice, as compared with wildtype (WT; Lyz2-CreTg/Tg Ptger4wt/wt) littermates (Fig 1D). There was no significant compensatory regulation of Ptger1, Ptger2 or Ptger3 mRNA. Furthermore, levels of Ptger4 mRNA were not significantly reduced in hearts of EP4M-/- mice (Fig 1E), demonstrating cell-specific Ptger4 deletion.PGE2 exerts divergent effects on inflammatory mediators through EP4-dependent pathways in myeloid cellsHaving confirmed that the loss of EP4 is selective for myeloid cells and does not lead to compensatory changes in other PGE2 receptors under baseline conditions, we explored the role of EP4 in mediating effects of PGE2 on several inflammatory mediators in four types of myeloid cells; resident peritoneal macrophages, bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMDMs), and bone marrow neutrophils. Ptger4 was poorly expressed in neutrophils, and these cells were therefore not studied further. PGE2 (10 nmol/l) stimulated Il6 mRNA levels in both isolated peritoneal resident macrophages and BMDCs at 8 h and this effect was largely mediated by EP4 (Fig 2A and 2B). Conversely, the same concentration of PGE2 inhibited Tnfa mRNA levels through EP4 (Fig 2C and 2D). The fact that EP4deficiency increased macrophage Tnfa to levels higher than those in WT macrophages in the ab.Ick test (A). Plasma PGE metabolites were measured by ELISA (B). The pups from the Ptger4fl/fl x Lyz2-CreTg/Tg cross were genotyped as described in Materials and Methods (C). Resident peritoneal macrophages (rpMac) (D) and hearts (E) were harvested from EP4M-/- mice and WT littermate controls, and Ptger1-4 mRNA levels were measured by real-time PCR. The results are presented and mean ?SEM. Data in A-B (n = 9?2) were analyzed by unpaired two-tailed Student's t-test and data in D-E were analyzed by one-way ANOVA followed by Tukey's multiple comparison test (n = 5?). Statistical outliers were identified by Grubbs' test and were excluded from the analysis (one outlier in B), * p<0.05; *** p<0.001; NS, nonsignificant; ND, non-diabetic; D, diabetic. doi:10.1371/journal.pone.0158316.gPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,5 /EP4, Diabetes, Inflammation and Atherosclerosissubset of diabetic mice exhibited elevated plasma PGE metabolite levels, as compared with non-diabetic littermate controls, while others did not (Fig 1B). Together, the diabetic group had significantly higher PGE metabolite levels than non-diabetic littermates (Fig 1B). This mouse model of T1DM is therefore characterized by increased PGE status, consistent with our previous studies in which macrophages from diabetic mice were shown to secrete more PGE2 than macrophages from non-diabetic controls [3].The generated myeloid cell-targeted EP4-deficient (EP4M-/-) mouse exhibits a specific loss of Ptger4 in myeloid cellsBecause the PGE2 receptor EP4 (Ptger4) has been implicated in inflammatory activation associated with diabetes, we generated a mouse model of myeloid cell-targeted EP4-deficiency by taking advantage of the EP4-floxed mouse and Lyz2-CreTg mice, which express Cre recombinase in myeloid cells. Following backcrossing for 10 generations into the C57BL/6 background, Lyz2-CreTg/Tg Ptger4fl/fl mice and Lyz2-CreTg/Tg Ptger4wt/wt littermate controls (Fig 1C) were used to measure Ptger mRNA levels in resident peritoneal macrophages and heart, to evaluate specificity of the EP4-deficiency. Ptger4 mRNA was absent in macrophages from EP4M-/(Lyz2-CreTg/Tg Ptger4fl/fl) mice, as compared with wildtype (WT; Lyz2-CreTg/Tg Ptger4wt/wt) littermates (Fig 1D). There was no significant compensatory regulation of Ptger1, Ptger2 or Ptger3 mRNA. Furthermore, levels of Ptger4 mRNA were not significantly reduced in hearts of EP4M-/- mice (Fig 1E), demonstrating cell-specific Ptger4 deletion.PGE2 exerts divergent effects on inflammatory mediators through EP4-dependent pathways in myeloid cellsHaving confirmed that the loss of EP4 is selective for myeloid cells and does not lead to compensatory changes in other PGE2 receptors under baseline conditions, we explored the role of EP4 in mediating effects of PGE2 on several inflammatory mediators in four types of myeloid cells; resident peritoneal macrophages, bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMDMs), and bone marrow neutrophils. Ptger4 was poorly expressed in neutrophils, and these cells were therefore not studied further. PGE2 (10 nmol/l) stimulated Il6 mRNA levels in both isolated peritoneal resident macrophages and BMDCs at 8 h and this effect was largely mediated by EP4 (Fig 2A and 2B). Conversely, the same concentration of PGE2 inhibited Tnfa mRNA levels through EP4 (Fig 2C and 2D). The fact that EP4deficiency increased macrophage Tnfa to levels higher than those in WT macrophages in the ab.
