Cs (Fig 3A). This effect was not seen in resident macrophages

Cs (Fig 3A). This AM152 chemical information effect was not seen in resident get P144 Peptide macrophages (Fig 3B). PGE2 also markedly suppressed LPS-induced Tnfa and Il1b levels in both BMDCs and resident macrophages (Fig 3C?E), and this effect was dependentPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,7 /EP4, Diabetes, Inflammation and AtherosclerosisFig 3. PGE2 inhibits LPS-induced cytokines 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 2 h, and then for an additional 6 h in the presence or absence of 5 ng/ml LPS. Il6 mRNA (A-B), Tnfa mRNA (C-D), and Il1b mRNA (E) were measured by real-time PCR. TNF- (F-G) and IL-6 (H-I) release was quantified by ELISA. The results are presented as fold over WT cells incubated in the absence of LPS as mean ?SEM. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (n = 9?1). * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.gPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,8 /EP4, Diabetes, Inflammation and Atherosclerosison EP4, consistent with previous studies by the Libby laboratory [17]. The suppressive effects of PGE2 on LPS-induced IL-6 and TNF- in BMDCs were confirmed at the protein level by ELISAs (Fig 3F and 3H), although the effect of PGE2 was more marked for TNF-. Similarly, EP4 suppressed LPS-mediated TNF- in resident peritoneal macrophages, while IL-6 release was unaffected (Fig 3G and 3I). Together, these results show that whereas PGE2 alone promotes production of several inflammatory mediators, it suppresses the action of LPS, consistent with previous studies [17, 37]. Both actions of PGE2 are critically dependent on EP4. We next investigated regulation of the four PGE2 receptors (Ptger1-4) in both BMDCs and resident peritoneal macrophages from EP4M-/- mice and WT littermate controls in response to PGE2 and LPS, to further evaluate the possibility of compensatory regulation of EP1, EP2 and EP3. Levels of Ptger4 and Ptger1 mRNA were modestly reduced by LPS stimulation in both BMDCs and resident peritoneal macrophages (Fig 4A?D). Likewise, Ptger2 mRNA was suppressed by LPS in BMDCs, but this effect was not consistent with that on macrophages (Fig 4E and 4F). Interestingly, EP4-deficiency resulted in a significant downregulation of Ptger2 mRNA in both BMDCs and resident macrophages stimulated with a combination of LPS and PGE2 (Fig 4E and 4F). These results suggest that the expression of EP2 is dependent on EP4 expression under certain conditions. Finally, Ptger3 mRNA levels were significantly increased in LPS-stimulated BMDCs and resident macrophages (Fig 4G and 4H). Together, these data demonstrate that the four PGE2 receptors exhibit different regulation in myeloid cells, and that the effect of EP4-deficiency in cells stimulated by PGE2 in the presence of LPS might be due in part to downregulation of EP2, but that in the absence of LPS, there is no major compensatory regulation of other PGE2 receptors by EP4-deficiency.Myeloid cell-targeted EP4-deficiency does not alter diabetes induction, plasma lipid levels or white blood cell countsHematopoietic EP4-deficiency has been shown to prevent lesions of atherosclerosis [40] or to have no impact on lesion size [41] in non-diabetic fat-fed Ldlr-/- mice. We reasoned that the mouse model of T1DM might be more susceptible to differences in myeloid cell EP4-deficiency because plasma PGE metab.Cs (Fig 3A). This effect was not seen in resident macrophages (Fig 3B). PGE2 also markedly suppressed LPS-induced Tnfa and Il1b levels in both BMDCs and resident macrophages (Fig 3C?E), and this effect was dependentPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,7 /EP4, Diabetes, Inflammation and AtherosclerosisFig 3. PGE2 inhibits LPS-induced cytokines 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 2 h, and then for an additional 6 h in the presence or absence of 5 ng/ml LPS. Il6 mRNA (A-B), Tnfa mRNA (C-D), and Il1b mRNA (E) were measured by real-time PCR. TNF- (F-G) and IL-6 (H-I) release was quantified by ELISA. The results are presented as fold over WT cells incubated in the absence of LPS as mean ?SEM. Data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (n = 9?1). * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.gPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,8 /EP4, Diabetes, Inflammation and Atherosclerosison EP4, consistent with previous studies by the Libby laboratory [17]. The suppressive effects of PGE2 on LPS-induced IL-6 and TNF- in BMDCs were confirmed at the protein level by ELISAs (Fig 3F and 3H), although the effect of PGE2 was more marked for TNF-. Similarly, EP4 suppressed LPS-mediated TNF- in resident peritoneal macrophages, while IL-6 release was unaffected (Fig 3G and 3I). Together, these results show that whereas PGE2 alone promotes production of several inflammatory mediators, it suppresses the action of LPS, consistent with previous studies [17, 37]. Both actions of PGE2 are critically dependent on EP4. We next investigated regulation of the four PGE2 receptors (Ptger1-4) in both BMDCs and resident peritoneal macrophages from EP4M-/- mice and WT littermate controls in response to PGE2 and LPS, to further evaluate the possibility of compensatory regulation of EP1, EP2 and EP3. Levels of Ptger4 and Ptger1 mRNA were modestly reduced by LPS stimulation in both BMDCs and resident peritoneal macrophages (Fig 4A?D). Likewise, Ptger2 mRNA was suppressed by LPS in BMDCs, but this effect was not consistent with that on macrophages (Fig 4E and 4F). Interestingly, EP4-deficiency resulted in a significant downregulation of Ptger2 mRNA in both BMDCs and resident macrophages stimulated with a combination of LPS and PGE2 (Fig 4E and 4F). These results suggest that the expression of EP2 is dependent on EP4 expression under certain conditions. Finally, Ptger3 mRNA levels were significantly increased in LPS-stimulated BMDCs and resident macrophages (Fig 4G and 4H). Together, these data demonstrate that the four PGE2 receptors exhibit different regulation in myeloid cells, and that the effect of EP4-deficiency in cells stimulated by PGE2 in the presence of LPS might be due in part to downregulation of EP2, but that in the absence of LPS, there is no major compensatory regulation of other PGE2 receptors by EP4-deficiency.Myeloid cell-targeted EP4-deficiency does not alter diabetes induction, plasma lipid levels or white blood cell countsHematopoietic EP4-deficiency has been shown to prevent lesions of atherosclerosis [40] or to have no impact on lesion size [41] in non-diabetic fat-fed Ldlr-/- mice. We reasoned that the mouse model of T1DM might be more susceptible to differences in myeloid cell EP4-deficiency because plasma PGE metab.

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