EP4-deficiency in diabetic mice. However, we also observed increased Tnfa in EP4-deficient macrophages from diabetic mice, demonstrating the complexity of PGE2’s effects on inflammatory pathways. In the present study, myeloid cell EP4-deficiency did not result in increased necrotic core formation in lesions, suggesting that macrophage apoptosis was not affected in this model of T1DM-accelerated atherosclerosis. The second study used a similar method to induce hematopoietic EP4-deficiency in fat-fed Ldlr-/- mice [41]. No differences in lesion size were observed at 5 or 10 weeks after initiation of fat-feeding, consistent with the lack of effects of EP4-deficiency on atherosclerosis in our study. However, hematopoietic EP4-deficiency resulted in increased lesional macrophages and T cells, with no effect on apoptosis in lesional macrophages [41]. It is possible that the differences in atherosclerosis observed in fat-fed Ldlr-/- mice with hematopoietic EP4-deficiency, as compared with our study, was due to the fat-feeding or that the inhibition of atherosclerosis was mediated by hematopoietic cells other than myeloid cells. The present study clearly demonstrates that myeloid cell-targeted EP4-deficiency alters cytokine production but has no effect on atherosclerosis in non-diabetic or diabetic mice. It is however possible that a greater effect would have been observed if both EP4 and EP2 had been deleted in myeloid cells. Since myeloid cell EP4 expression does not impact diabetes-accelerated atherogenesis and there was no correlation between plasma PGE metabolites and lesion area in diabetic mice, what then is the mechanism whereby diabetes promotes atherosclerotic lesion initiation? The present study and published studies offer some insights into this question. For example, our study demonstrates that the increased atherogenesis in diabetic mice was not due to elevated cholesterol levels, as compared to non-diabetic mice, consistent with previous studies [3, 27]. Furthermore, we did not observe myelopoiesis and neutrophilia in diabetic mice in this study, suggesting that diabetes-accelerated lesion initiation was not due to elevated levels of circulating myeloid cells. Moreover, we have recently shown that increased glucose flux in myeloid cells is not sufficient to stimulate atherosclerosis in Ldlr-/- mice [33], but it is quite possible that hyperglycemia plays a role in increasing myeloid cell accumulation in lesions through other mechanisms [9]. It is clear that diabetes leads to a relative increase in accumulation of myeloidPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,16 /EP4, Diabetes, Inflammation and Atherosclerosiscells in the TAK-385 molecular weight artery wall, in both atherosclerosis progression models like the one used in the present study and in atherosclerosis regression models [9]. The mechanism appears to involve altered fatty acid handling in myeloid cells [3], increased activation of the receptor for advanced endproducts [9, 56], increased oxidative stress through NADPH oxidase 1 [57], increased cholesterol accumulation in bone marrow progenitor cells [58], and most likely increased adhesion molecule expression by purchase Vesatolimod endothelial cells [59, 60]. While the current study adds an important missing piece to the puzzle, further studies are needed to elucidate the mechanisms of diabetes-accelerated atherogenesis. In summary, in this mouse model of T1DM increased myeloid cell PGE2-EP4 signaling contributes significantly to some aspects of diabetes-.EP4-deficiency in diabetic mice. However, we also observed increased Tnfa in EP4-deficient macrophages from diabetic mice, demonstrating the complexity of PGE2’s effects on inflammatory pathways. In the present study, myeloid cell EP4-deficiency did not result in increased necrotic core formation in lesions, suggesting that macrophage apoptosis was not affected in this model of T1DM-accelerated atherosclerosis. The second study used a similar method to induce hematopoietic EP4-deficiency in fat-fed Ldlr-/- mice [41]. No differences in lesion size were observed at 5 or 10 weeks after initiation of fat-feeding, consistent with the lack of effects of EP4-deficiency on atherosclerosis in our study. However, hematopoietic EP4-deficiency resulted in increased lesional macrophages and T cells, with no effect on apoptosis in lesional macrophages [41]. It is possible that the differences in atherosclerosis observed in fat-fed Ldlr-/- mice with hematopoietic EP4-deficiency, as compared with our study, was due to the fat-feeding or that the inhibition of atherosclerosis was mediated by hematopoietic cells other than myeloid cells. The present study clearly demonstrates that myeloid cell-targeted EP4-deficiency alters cytokine production but has no effect on atherosclerosis in non-diabetic or diabetic mice. It is however possible that a greater effect would have been observed if both EP4 and EP2 had been deleted in myeloid cells. Since myeloid cell EP4 expression does not impact diabetes-accelerated atherogenesis and there was no correlation between plasma PGE metabolites and lesion area in diabetic mice, what then is the mechanism whereby diabetes promotes atherosclerotic lesion initiation? The present study and published studies offer some insights into this question. For example, our study demonstrates that the increased atherogenesis in diabetic mice was not due to elevated cholesterol levels, as compared to non-diabetic mice, consistent with previous studies [3, 27]. Furthermore, we did not observe myelopoiesis and neutrophilia in diabetic mice in this study, suggesting that diabetes-accelerated lesion initiation was not due to elevated levels of circulating myeloid cells. Moreover, we have recently shown that increased glucose flux in myeloid cells is not sufficient to stimulate atherosclerosis in Ldlr-/- mice [33], but it is quite possible that hyperglycemia plays a role in increasing myeloid cell accumulation in lesions through other mechanisms [9]. It is clear that diabetes leads to a relative increase in accumulation of myeloidPLOS ONE | DOI:10.1371/journal.pone.0158316 June 28,16 /EP4, Diabetes, Inflammation and Atherosclerosiscells in the artery wall, in both atherosclerosis progression models like the one used in the present study and in atherosclerosis regression models [9]. The mechanism appears to involve altered fatty acid handling in myeloid cells [3], increased activation of the receptor for advanced endproducts [9, 56], increased oxidative stress through NADPH oxidase 1 [57], increased cholesterol accumulation in bone marrow progenitor cells [58], and most likely increased adhesion molecule expression by endothelial cells [59, 60]. While the current study adds an important missing piece to the puzzle, further studies are needed to elucidate the mechanisms of diabetes-accelerated atherogenesis. In summary, in this mouse model of T1DM increased myeloid cell PGE2-EP4 signaling contributes significantly to some aspects of diabetes-.
