Ly understood. In the present study, we explored the effects of NE on macrophage differentiation, maturation and function. Using the ex vivo bone marrow-derived macrophage culturing system, our study demonstrates that NE has comprehensive regulatory effects on macrophage differentiation, maturation and function. First, we showed that NE regulates BMM proliferation and maturation. Both high and low doses of NE inhibit BMM proliferation. However, only low dose of NE enhances BMM maturation as determined by MHC II and F4/80 expression. Secondly, high dose of NE (1 x 10-6 M) regulates macrophage migration by decreasing CCRexpression. The migration of macrophages from circulation into tissue plays an D to play a role in maintaining immune homeostasis in the essential role for wound healing. Our data suggest that NE may regulate tissue immune response by regulating CCR2-dependent monocyte tissue infiltration in severe burn and sepsis. We also showed that both 1 x 10-8 M and 1 x 10-6 16574785 M of NE Title Loaded From File enhanced macrophage phagocytosis. Since increased macrophage phagocytosis is essential for a faster wound healing, NE may promote wound healing in this regard. Fourth, both high and low doses of NE enhanced TNF production. Macrophages are major producers of proinflammatory mediators following thermal injury and hyperactivity is of critical importance to the development of post-burn immune dysfunction, such as systemic inflammatory response syndrome (SIRS) [10]. Our results show that concentrations as low as 1 x 10-8 M NE significantly increase TNF- production from BMM, under the stimulation of LPS. Furthermore, it indicates that catecholamines play a role in acute inflammation during burn. We also found that epinephrine has similar effects on macrophage but with a lessNorepinephrine Inhibits MigrationFigure 7. NE regulates MafB expression of BMM. At the end of the 7 day BMM culture, cells were harvested and stained with antibodies for phenotypic markers CD11b and F4/80 followed by sequential intracellular staining with primary MafB antibody and FITC-conjugated secondary antibodies. Representative dot plot data of percentage of MafB+ BMM were shown in (A) and their corresponding graphic format in (B). The MFI of MafB+ BMM were shown in (C). Data represent samples in triplicate for each time of two independent determinations. * p<0.05, ** p<0.01, compared to non-hormone treated control (0 M).doi: 10.1371/journal.pone.0069167.gNorepinephrine Inhibits Migrationefficient mode (Figure S2). In summary, our study suggests that catecholamines may play a significant role in disturbed immune response during severe burn and sepsis by regulating macrophage differentiation and function. The modulatory effects of NE on macrophage function are particularly prominent in phagocytosis and cytokine secretion. For macrophage phagocytosis, our results are in contrast to previous studies [12,30]. One of those studies using macrophage isolated from burn wound showed that NE inhibited macrophage phagocytosis of Escherichia coli [12]. This discrepancy may be due to the activation status and source of macrophage used in the experiments. A study on the effects of stress on macrophage behaviours found that stress caused a decrease in phagocytosis mediated by Fc- or mannose receptors in resting peritoneal macrophage, whereas increased phagocytosis in peritoneal macrophage isolated from mice that had been intraperitoneally injected with LPS 4 days before the experiment [31]. The increased cytokine secretion presently shown in our study is cons.Ly understood. In the present study, we explored the effects of NE on macrophage differentiation, maturation and function. Using the ex vivo bone marrow-derived macrophage culturing system, our study demonstrates that NE has comprehensive regulatory effects on macrophage differentiation, maturation and function. First, we showed that NE regulates BMM proliferation and maturation. Both high and low doses of NE inhibit BMM proliferation. However, only low dose of NE enhances BMM maturation as determined by MHC II and F4/80 expression. Secondly, high dose of NE (1 x 10-6 M) regulates macrophage migration by decreasing CCRexpression. The migration of macrophages from circulation into tissue plays an essential role for wound healing. Our data suggest that NE may regulate tissue immune response by regulating CCR2-dependent monocyte tissue infiltration in severe burn and sepsis. We also showed that both 1 x 10-8 M and 1 x 10-6 16574785 M of NE enhanced macrophage phagocytosis. Since increased macrophage phagocytosis is essential for a faster wound healing, NE may promote wound healing in this regard. Fourth, both high and low doses of NE enhanced TNF production. Macrophages are major producers of proinflammatory mediators following thermal injury and hyperactivity is of critical importance to the development of post-burn immune dysfunction, such as systemic inflammatory response syndrome (SIRS) [10]. Our results show that concentrations as low as 1 x 10-8 M NE significantly increase TNF- production from BMM, under the stimulation of LPS. Furthermore, it indicates that catecholamines play a role in acute inflammation during burn. We also found that epinephrine has similar effects on macrophage but with a lessNorepinephrine Inhibits MigrationFigure 7. NE regulates MafB expression of BMM. At the end of the 7 day BMM culture, cells were harvested and stained with antibodies for phenotypic markers CD11b and F4/80 followed by sequential intracellular staining with primary MafB antibody and FITC-conjugated secondary antibodies. Representative dot plot data of percentage of MafB+ BMM were shown in (A) and their corresponding graphic format in (B). The MFI of MafB+ BMM were shown in (C). Data represent samples in triplicate for each time of two independent determinations. * p<0.05, ** p<0.01, compared to non-hormone treated control (0 M).doi: 10.1371/journal.pone.0069167.gNorepinephrine Inhibits Migrationefficient mode (Figure S2). In summary, our study suggests that catecholamines may play a significant role in disturbed immune response during severe burn and sepsis by regulating macrophage differentiation and function. The modulatory effects of NE on macrophage function are particularly prominent in phagocytosis and cytokine secretion. For macrophage phagocytosis, our results are in contrast to previous studies [12,30]. One of those studies using macrophage isolated from burn wound showed that NE inhibited macrophage phagocytosis of Escherichia coli [12]. This discrepancy may be due to the activation status and source of macrophage used in the experiments. A study on the effects of stress on macrophage behaviours found that stress caused a decrease in phagocytosis mediated by Fc- or mannose receptors in resting peritoneal macrophage, whereas increased phagocytosis in peritoneal macrophage isolated from mice that had been intraperitoneally injected with LPS 4 days before the experiment [31]. The increased cytokine secretion presently shown in our study is cons.
