M2b macrophages reduce early reperfusion injury after myocardial ischemia in mice: A predominant role of inhibiting apoptosis via A20  Yuan Yue, Xiao Yang, Kangni Feng, Lexun Wang, Jian Hou, Bo Mei, Han Qin, Mengya Liang, Guangxian Chen, Zhongkai Wu  International Journal of Cardiology  Volume 245, Pages 228-235 (October 2017) DOI: 10.1016/j.ijcard.2017.07.085 Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Identification of M2b macrophages. (A) BMDMs (M0) were stained to determine F4/80 (PE) and CD11b (PE-Cy7) expression and were analyzed by flow cytometry. Over 80% of the cells were F4/80+CD11b+. (B) BMDMs were incubated with LPS (M1), IL-4+IL-13 (M2a/c) or LPS+IC (M2b) for 48h. The mRNA levels of iNOS, MR, LIGHT (TNFSF14), IL-10 and IL-12p40 were detected by qPCR. Data are shown as fold changes relative to expression levels in untreated M0 cells. M2b macrophages can be identified by high levels of IL-10 and low levels of IL-12p40 as well as with the marker LIGHT. (C) CBA assays showed that M1 macrophages secrete high levels of IL-6, IL-12p70 and TNF and moderate levels of IL-10. M2b macrophages secrete high levels of IL-10 and low levels of IL-12p70, IL-6, and TNF. M2a/c macrophages only secreted low levels of IL-10 and almost no IL-6, IL-12p70 and TNF. Data were the mean of three independent experiments. *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Identification of M2b macrophages. (A) BMDMs (M0) were stained to determine F4/80 (PE) and CD11b (PE-Cy7) expression and were analyzed by flow cytometry. Over 80% of the cells were F4/80+CD11b+. (B) BMDMs were incubated with LPS (M1), IL-4+IL-13 (M2a/c) or LPS+IC (M2b) for 48h. The mRNA levels of iNOS, MR, LIGHT (TNFSF14), IL-10 and IL-12p40 were detected by qPCR. Data are shown as fold changes relative to expression levels in untreated M0 cells. M2b macrophages can be identified by high levels of IL-10 and low levels of IL-12p40 as well as with the marker LIGHT. (C) CBA assays showed that M1 macrophages secrete high levels of IL-6, IL-12p70 and TNF and moderate levels of IL-10. M2b macrophages secrete high levels of IL-10 and low levels of IL-12p70, IL-6, and TNF. M2a/c macrophages only secreted low levels of IL-10 and almost no IL-6, IL-12p70 and TNF. Data were the mean of three independent experiments. *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 Comparison of cTNI levels, the myocardial infarct area and echocardiographic data in each group. (A) The serum cTnI level in the SO group (n=10) was relatively lower, while that in the CK group (n=19) was significantly increased; however, cTnI level was reduced by M2b macrophages (n=19). (B) TTC-Evans blue staining. (C) The infarct area in the SO group was 0 (n=3), whereas in the CK group, the infarct area was significantly increased (n=5); the infarct area was decreased by M2b macrophage transplantation (n=5). (D) Echocardiography of mice. (E) Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) in the CK group (n=5) were significantly lower than those in the SO group (n=5). Both LVEF and LVFS were relatively higher in the MT group (n=5), but these differences were not statistically significant (p≥0.05). *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 Comparison of cTNI levels, the myocardial infarct area and echocardiographic data in each group. (A) The serum cTnI level in the SO group (n=10) was relatively lower, while that in the CK group (n=19) was significantly increased; however, cTnI level was reduced by M2b macrophages (n=19). (B) TTC-Evans blue staining. (C) The infarct area in the SO group was 0 (n=3), whereas in the CK group, the infarct area was significantly increased (n=5); the infarct area was decreased by M2b macrophage transplantation (n=5). (D) Echocardiography of mice. (E) Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) in the CK group (n=5) were significantly lower than those in the SO group (n=5). Both LVEF and LVFS were relatively higher in the MT group (n=5), but these differences were not statistically significant (p≥0.05). *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 Comparison of cTNI levels, the myocardial infarct area and echocardiographic data in each group. (A) The serum cTnI level in the SO group (n=10) was relatively lower, while that in the CK group (n=19) was significantly increased; however, cTnI level was reduced by M2b macrophages (n=19). (B) TTC-Evans blue staining. (C) The infarct area in the SO group was 0 (n=3), whereas in the CK group, the infarct area was significantly increased (n=5); the infarct area was decreased by M2b macrophage transplantation (n=5). (D) Echocardiography of mice. (E) Left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) in the CK group (n=5) were significantly lower than those in the SO group (n=5). Both LVEF and LVFS were relatively higher in the MT group (n=5), but these differences were not statistically significant (p≥0.05). *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 3 Effect of M2b macrophage transplantation on cardiomyocyte apoptosis induced by MI/R. (A) Double staining of the anterior left ventricular wall (400×). The TUNEL-stained cells (green) are apoptotic cells, the cardiac troponin I (cTNI)-stained cells (red) are cardiomyocytes, and the blue spots labeled all cells. (B) The apoptosis index in the SO group was quite low (n=3). The apoptosis index of the CK group (n=7) was significantly reduced by M2b macrophage transplantation (n=7). *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions

Fig. 4 A20 expression and NF-κB activation in each group. (A) A20 expression in the heart tissue was up-regulated in the CK group (n=7) and significantly increased by M2b macrophage transplantation (n=7) at the mRNA level, as detected by qPCR. (B) Western blot of A20, IκBα, P65, phospho-IκBα, and phospho-P65. (C) A significant increase in the levels of IκBα and P65 phosphorylation was observed in the heart tissue of the CK group (n=7) compared with that of the SO group (n=4); IκBα and P65 phosphorylation was inhibited by M2b macrophage transplantation (n=7). The expression of A20 in protein level was up-regulated in the CK group (n=7) compared with the SO group and significantly increased by M2b macrophage transplantation (n=7) *p<0.05. International Journal of Cardiology 2017 245, 228-235DOI: (10.1016/j.ijcard.2017.07.085) Copyright © 2017 The Authors Terms and Conditions