Sence of PGE2 suggests that endogenous PGE2 secretion was elevated in peritoneal macrophages, as compared with BMDCs. PGE2 also increased levels of Il1b and Il23 as well as Ccr7, a chemokine receptor that mediates migration of dendritic cells to lymph nodes [35] through EP4-dependent mechanisms (Fig 2E?H). The ability of PGE2 to stimulate IL-6 and inhibit TNF- in BMDCs was replicated at the protein level by ELISAs (Fig 2I and 2J). Furthermore, PGE2 inhibited Tnfa mRNA levels also in BMDMs through EP4 (wildtype basal Tnfa 0.97 ?0.03; wildtype PGE2 0.46 ?0.02; p<0.001 versus wildtype basal; EP4M-/- basal 1.08 ?0.09; EP4M-/- PGE2 0.90 ?0.10; mean ?SEM; n = 9?1), confirming this effect of PGE2 in second macrophage population. Levels of Il6 mRNA were not detected or were very low in the basal state or after PGE2 stimulation of BMDMs. We therefore focused our studies primarily on resident peritoneal macrophages, rather than BMDMs. An additional reason to study resident peritoneal macrophages was that the effect of diabetes on resident peritoneal macrophages could be assessed, as discussed below. Our findings suggest that the low concentration of PGE2 used (10 nmol/l) mediates its U0126-EtOH supplier effects primarily through EP4. However, we observed EP4-independent effects of a higherPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,6 /EP4, Diabetes, Inflammation and AtherosclerosisFig 2. PGE2 exerts divergent effects on inflammatory mediators through EP4 in myeloid cells. Bone marrow-derived dendritic cells (BMDCs) and resident peritoneal macrophages from EP4M-/- mice and WT littermates were stimulated with 10 nmol/l PGE2 or vehicle for 8 h. Il6 mRNA (A-B), Tnfa mRNA (C-D), Il1b mRNA (E-F), Ccr7 mRNA (G), and Il23 mRNA (H) were measured by real-time PCR. IL-6 (I) and TNF- (J) release was quantified by ELISA. The results are presented and mean ?SEM. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (real-time PCR data; 4?1; ELISA data; n = 4?). Statistical outliers were identified by Grubbs’ test and were excluded from analyses (one outlier in A, two outliers in E), * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.gconcentration of PGE2 (50 nmol/l). Indeed, 10 nmol/l PGE2 has been shown to increase IL-23 Anlotinib msds production in human monocyte-derived dendritic cells through EP4, similar to the results of the present study, but to downregulate IL-23 at higher concentrations (>50 nmol/l) through interaction with EP2 [36].PGE2 prevents inflammatory effects of LPS through EP4 in myeloid cellsIt is well-established that PGE2 signaling through EP4 suppresses the effects of LPS on cytokine production in macrophages by preventing LPS-mediated NF-B activation [17, 37, 38]. The mechanism of PGE2-EP4-mediated inhibition of cytokine production in macrophages involves the protein EPRAP [prostaglandin E receptor (EP) 4-associated protein; gene name Fem1a], which acts through a non-cyclic AMP-dependent pathway to inhibit macrophage activation through direct interaction and stabilization of NF-B1 p50/p105 following exposure to a proinflammatory stimulus, such as LPS [17, 39]. We therefore next investigated the effects of PGE2-EP4 on LPS-induced cytokine production in BMDCs and resident macrophages to further characterize our myeloid cell-targeted EP4-deficient model and to verify previous results. Contrary to the stimulatory effects of PGE2 on IL-6 production in the absence of LPS (Fig 2), PGE2 suppressed the effects of LPS on Il6 mRNA in BMD.Sence of PGE2 suggests that endogenous PGE2 secretion was elevated in peritoneal macrophages, as compared with BMDCs. PGE2 also increased levels of Il1b and Il23 as well as Ccr7, a chemokine receptor that mediates migration of dendritic cells to lymph nodes [35] through EP4-dependent mechanisms (Fig 2E?H). The ability of PGE2 to stimulate IL-6 and inhibit TNF- in BMDCs was replicated at the protein level by ELISAs (Fig 2I and 2J). Furthermore, PGE2 inhibited Tnfa mRNA levels also in BMDMs through EP4 (wildtype basal Tnfa 0.97 ?0.03; wildtype PGE2 0.46 ?0.02; p<0.001 versus wildtype basal; EP4M-/- basal 1.08 ?0.09; EP4M-/- PGE2 0.90 ?0.10; mean ?SEM; n = 9?1), confirming this effect of PGE2 in second macrophage population. Levels of Il6 mRNA were not detected or were very low in the basal state or after PGE2 stimulation of BMDMs. We therefore focused our studies primarily on resident peritoneal macrophages, rather than BMDMs. An additional reason to study resident peritoneal macrophages was that the effect of diabetes on resident peritoneal macrophages could be assessed, as discussed below. Our findings suggest that the low concentration of PGE2 used (10 nmol/l) mediates its effects primarily through EP4. However, we observed EP4-independent effects of a higherPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,6 /EP4, Diabetes, Inflammation and AtherosclerosisFig 2. PGE2 exerts divergent effects on inflammatory mediators through EP4 in myeloid cells. Bone marrow-derived dendritic cells (BMDCs) and resident peritoneal macrophages from EP4M-/- mice and WT littermates were stimulated with 10 nmol/l PGE2 or vehicle for 8 h. Il6 mRNA (A-B), Tnfa mRNA (C-D), Il1b mRNA (E-F), Ccr7 mRNA (G), and Il23 mRNA (H) were measured by real-time PCR. IL-6 (I) and TNF- (J) release was quantified by ELISA. The results are presented and mean ?SEM. Data were analyzed by two-way ANOVA with Tukey's multiple comparisons test (real-time PCR data; 4?1; ELISA data; n = 4?). Statistical outliers were identified by Grubbs' test and were excluded from analyses (one outlier in A, two outliers in E), * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.gconcentration of PGE2 (50 nmol/l). Indeed, 10 nmol/l PGE2 has been shown to increase IL-23 production in human monocyte-derived dendritic cells through EP4, similar to the results of the present study, but to downregulate IL-23 at higher concentrations (>50 nmol/l) through interaction with EP2 [36].PGE2 prevents inflammatory effects of LPS through EP4 in myeloid cellsIt is well-established that PGE2 signaling through EP4 suppresses the effects of LPS on cytokine production in macrophages by preventing LPS-mediated NF-B activation [17, 37, 38]. The mechanism of PGE2-EP4-mediated inhibition of cytokine production in macrophages involves the protein EPRAP [prostaglandin E receptor (EP) 4-associated protein; gene name Fem1a], which acts through a non-cyclic AMP-dependent pathway to inhibit macrophage activation through direct interaction and stabilization of NF-B1 p50/p105 following exposure to a proinflammatory stimulus, such as LPS [17, 39]. We therefore next investigated the effects of PGE2-EP4 on LPS-induced cytokine production in BMDCs and resident macrophages to further characterize our myeloid cell-targeted EP4-deficient model and to verify previous results. Contrary to the stimulatory effects of PGE2 on IL-6 production in the absence of LPS (Fig 2), PGE2 suppressed the effects of LPS on Il6 mRNA in BMD.
