Olites were elevated. Furthermore, the two previous studies were performed in mice that lacked EP4 in all bone marrow-derived cells, whereas our model of EP4-deficiency targeted to myeloid cells is the first to investigate effects on atherosclerosis. The study plan is shown in Fig 5A. EP4M-/- mice and WT littermate controls were used as donors for bone marrow transplants into female Ldlr-/-; GpTg mice (the model of T1DM). The mice were allowed to recover for 7 weeks following the bone marrow transplant, and were then injected with LCMV to induce diabetes or saline as control (Fig 5A). All mice were fed a lowfat semi-purified diet, described previously [27], for an additional 12 weeks after induction of diabetes. Diabetic mice were hyperglycemic at 4 weeks after induction of diabetes, and maintained hyperglycemia throughout the study (Fig 5B). Myeloid cell EP4-deficiency did not alter hyperglycemia or diabetes induction in this model of diabetes, consistent with data on global EP4deficiency in the streptozotocin-induced diabetes model [42]. Plasma cholesterol levels and triglyceride levels were not significantly different in the four groups of mice (Fig 5C and 5D), although triglyceride levels tended to be increased in diabetic mice. Because EP4 has been shown to regulate bone marrow progenitor cells, we next evaluated numbers of blood leukocytes. There were no significant differences between the groups in total leukocytes, neutrophils, Nutlin-3a chiral supplement monocytes (Fig 5E?G) or lymphocytes (ND WT 9.6 ?0.5 x 103 cells/l blood; ND EP4M-/8.0 ?1.6; D WT 8.0 ?1.1 and D EP4M-/- 7.5 ?1.4 x 103 cells/l; mean ?SEM; n = 5?). Plasma levels of IL-6 and TNF- were below the detection limit of the assay (18.6 pg/ml for IL-6 andPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,9 /EP4, Diabetes, Inflammation and AtherosclerosisFig 4. PGE2 receptors are differentially regulated by LPS and PGE2 in myeloid cells. Bone marrowderived dendritic cells (BMDCs) and resident Nutlin-3a chiral site 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. Ptger4 mRNA (A-B), Ptger1 mRNA (C-D), Ptger2 mRNA (E-F) and Ptger3 mRNA (G-H) were measured by real-time PCR. The results are presented and mean ?SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (n = 7?1). * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.g004 PLOS ONE | DOI:10.1371/journal.pone.0158316 June 28, 2016 10 /EP4, Diabetes, Inflammation and AtherosclerosisFig 5. Myeloid cell EP4-deficiency does not alter diabetes induction, plasma lipid levels or WBC counts. The study plan in shown in A. Blood glucose levels were measured at week 0 (prior to injection of LCMV), 4, 8 and 12 by a stick test (B). Plasma cholesterol (C) and triglycerides (D) were measured by kits from Wako. Blood leukocyte counts were determined by a Hemavet (E-G). Leukocyte Ptger4 mRNA levels were measured by realtime PCR (H). The results are presented and mean ?SEM. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test (n = 5?1 in B-C; n = 9?4 in D; 4? in E-G and 14?1 in H). * p<0.05; ** p<0.01; *** p<0.001; ND, non-diabetic; D, diabetic; LCMV, lymphocytic choriomeningitis virus; LFD, low-fat diet. doi:10.1371/journal.pone.0158316.g005 PLOS ONE | DOI:10.1371/journal.pone.0158316 June 28, 2016 11 /EP4, Diabetes, Inflammation and Atherosclerosis1.1 pg/ml for TNF-). Leukocyte mRNA.Olites were elevated. Furthermore, the two previous studies were performed in mice that lacked EP4 in all bone marrow-derived cells, whereas our model of EP4-deficiency targeted to myeloid cells is the first to investigate effects on atherosclerosis. The study plan is shown in Fig 5A. EP4M-/- mice and WT littermate controls were used as donors for bone marrow transplants into female Ldlr-/-; GpTg mice (the model of T1DM). The mice were allowed to recover for 7 weeks following the bone marrow transplant, and were then injected with LCMV to induce diabetes or saline as control (Fig 5A). All mice were fed a lowfat semi-purified diet, described previously [27], for an additional 12 weeks after induction of diabetes. Diabetic mice were hyperglycemic at 4 weeks after induction of diabetes, and maintained hyperglycemia throughout the study (Fig 5B). Myeloid cell EP4-deficiency did not alter hyperglycemia or diabetes induction in this model of diabetes, consistent with data on global EP4deficiency in the streptozotocin-induced diabetes model [42]. Plasma cholesterol levels and triglyceride levels were not significantly different in the four groups of mice (Fig 5C and 5D), although triglyceride levels tended to be increased in diabetic mice. Because EP4 has been shown to regulate bone marrow progenitor cells, we next evaluated numbers of blood leukocytes. There were no significant differences between the groups in total leukocytes, neutrophils, monocytes (Fig 5E?G) or lymphocytes (ND WT 9.6 ?0.5 x 103 cells/l blood; ND EP4M-/8.0 ?1.6; D WT 8.0 ?1.1 and D EP4M-/- 7.5 ?1.4 x 103 cells/l; mean ?SEM; n = 5?). Plasma levels of IL-6 and TNF- were below the detection limit of the assay (18.6 pg/ml for IL-6 andPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,9 /EP4, Diabetes, Inflammation and AtherosclerosisFig 4. PGE2 receptors are differentially regulated by LPS and PGE2 in myeloid cells. Bone marrowderived 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. Ptger4 mRNA (A-B), Ptger1 mRNA (C-D), Ptger2 mRNA (E-F) and Ptger3 mRNA (G-H) were measured by real-time PCR. The results are presented and mean ?SEM. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test (n = 7?1). * p<0.05; ** p<0.01; *** p<0.001. doi:10.1371/journal.pone.0158316.g004 PLOS ONE | DOI:10.1371/journal.pone.0158316 June 28, 2016 10 /EP4, Diabetes, Inflammation and AtherosclerosisFig 5. Myeloid cell EP4-deficiency does not alter diabetes induction, plasma lipid levels or WBC counts. The study plan in shown in A. Blood glucose levels were measured at week 0 (prior to injection of LCMV), 4, 8 and 12 by a stick test (B). Plasma cholesterol (C) and triglycerides (D) were measured by kits from Wako. Blood leukocyte counts were determined by a Hemavet (E-G). Leukocyte Ptger4 mRNA levels were measured by realtime PCR (H). The results are presented and mean ?SEM. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test (n = 5?1 in B-C; n = 9?4 in D; 4? in E-G and 14?1 in H). * p<0.05; ** p<0.01; *** p<0.001; ND, non-diabetic; D, diabetic; LCMV, lymphocytic choriomeningitis virus; LFD, low-fat diet. doi:10.1371/journal.pone.0158316.g005 PLOS ONE | DOI:10.1371/journal.pone.0158316 June 28, 2016 11 /EP4, Diabetes, Inflammation and Atherosclerosis1.1 pg/ml for TNF-). Leukocyte mRNA.
