Fig. 3.
Human umbilical cord mesenchymal stromal cells (hUC-MSCs) dose-dependently increase heme oxygenase-1 expression in peritoneal macrophages, while heme oxygenase-1 blockade ablates hUC-MSC enhancement of macrophage phagocytosis. (A) Representative Western blot demonstrating increased heme oxygenase-1 concentrations in peritoneal macrophage lysates from hUC-MSC–treated animals and (B) the effect of the heme oxygenase-1 inducer hemin. (C) Quantitative densitometry demonstrates the degree of heme oxygenase-1 increase (P = 0.015) and the effect of hemin in vehicle but not in umbilical cord mesenchymal stromal cell (UC-MSC)–treated samples (P = 0.616). (D) Quantitative densitometry of Western blots demonstrating dose-dependent hUC-MSC–induced increase of heme oxygenase-1 concentrations in the liver (P = 0.006) and (E) the spleen (P < 0.0001). (F) UC-MSC therapy increased macrophage phagocytic index, (G) phagosomal reactive oxygen species (ROS) production, and (H) fold change of phagocytosis in peritoneal macrophages isolated from animals after fecal peritonitis compared to macrophages from vehicle-treated and sham animals. (F and H) The direct heme oxygenase-1 inducer hemin increased phagocytosis, (G) but not ROS production in macrophages from vehicle-treated animals. (F and H) Hemin did not further increase phagocytosis in macrophages from hUC-MSC–treated animals. (F and H) The heme oxygenase-1 antagonist zinc protoporhyrin (ZnPP) abolished the hUC-MSC induced increase in phagocytosis and (G) reduced ROS production in macrophages from hUC-MSC–treated animals. (I) Representative images of peritoneal macrophages showing hUC-MSC therapy enhanced phagocytosis of serum opsonized zymosan (green fluorescence) and increased phagosomal ROS production (black dots) compared to vehicle, while ZnPP blocked these effects. The data are expressed as mean ± SD. All data from a minimum of three independent experiments were done in duplicate. (C, D, and E) #P < 0.05 versus sham group not treated with hemin; XP < 0.05 versus vehicle group not treated with hemin; *P < 0.05 versus vehicle-treated group; N = 4 for sham and 6 for all other groups, referring to the animal number/group. (F, G, and H) *P < 0.05 versus vehicle control group, #P < 0.05 versus sham control group, &P < 0.05 versus UC-MSC control group, ^P < 0.05 versus hemin-treated group. N = 4 for sham rats and 5 rats per group in all other groups. Samples of macrophages isolated from peritoneal fluid were taken from each animal and underwent treatment or not (controls). Two slides were prepared from each sample. Images were taken and analyzed in 8 to 10 randomly chosen fields/slide.

Human umbilical cord mesenchymal stromal cells (hUC-MSCs) dose-dependently increase heme oxygenase-1 expression in peritoneal macrophages, while heme oxygenase-1 blockade ablates hUC-MSC enhancement of macrophage phagocytosis. (A) Representative Western blot demonstrating increased heme oxygenase-1 concentrations in peritoneal macrophage lysates from hUC-MSC–treated animals and (B) the effect of the heme oxygenase-1 inducer hemin. (C) Quantitative densitometry demonstrates the degree of heme oxygenase-1 increase (P = 0.015) and the effect of hemin in vehicle but not in umbilical cord mesenchymal stromal cell (UC-MSC)–treated samples (P = 0.616). (D) Quantitative densitometry of Western blots demonstrating dose-dependent hUC-MSC–induced increase of heme oxygenase-1 concentrations in the liver (P = 0.006) and (E) the spleen (P < 0.0001). (F) UC-MSC therapy increased macrophage phagocytic index, (G) phagosomal reactive oxygen species (ROS) production, and (H) fold change of phagocytosis in peritoneal macrophages isolated from animals after fecal peritonitis compared to macrophages from vehicle-treated and sham animals. (F and H) The direct heme oxygenase-1 inducer hemin increased phagocytosis, (G) but not ROS production in macrophages from vehicle-treated animals. (F and H) Hemin did not further increase phagocytosis in macrophages from hUC-MSC–treated animals. (F and H) The heme oxygenase-1 antagonist zinc protoporhyrin (ZnPP) abolished the hUC-MSC induced increase in phagocytosis and (G) reduced ROS production in macrophages from hUC-MSC–treated animals. (I) Representative images of peritoneal macrophages showing hUC-MSC therapy enhanced phagocytosis of serum opsonized zymosan (green fluorescence) and increased phagosomal ROS production (black dots) compared to vehicle, while ZnPP blocked these effects. The data are expressed as mean ± SD. All data from a minimum of three independent experiments were done in duplicate. (C, D, and E) #P < 0.05 versus sham group not treated with hemin; XP < 0.05 versus vehicle group not treated with hemin; *P < 0.05 versus vehicle-treated group; N = 4 for sham and 6 for all other groups, referring to the animal number/group. (F, G, and H) *P < 0.05 versus vehicle control group, #P < 0.05 versus sham control group, &P < 0.05 versus UC-MSC control group, ^P < 0.05 versus hemin-treated group. N = 4 for sham rats and 5 rats per group in all other groups. Samples of macrophages isolated from peritoneal fluid were taken from each animal and underwent treatment or not (controls). Two slides were prepared from each sample. Images were taken and analyzed in 8 to 10 randomly chosen fields/slide.

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