Dendritic Cells Derived MMP9 Fuels Sepsis-Induced Cardiac Dysfunction Through JNK/AP1 Driven Pro-Inflammatory Cytokine and Nitric Oxide Storm

Figures

• 

  Fig. 1. MMP9 promoted inflammation in SIMD.A, Survival of WT and MMP9−/− mice (all male) intraperitoneally injected with LPS(25 µg/g), was monitored every 3 h (n = 20 for WT mice group, n = 20 for MMP9−/− mice group). The experiment was repeated independently three times (the same sex (either all male or all female) each time). B, C, Analysis of the serum myocardial damage markers (CK-MB, cTnT) and inflammatory factor (IL-6, IL-17 A, IL-1β and TNF-α) 8 h after intraperitoneal injection of LPS were measured by Elisa. These experiments were repeated independently three times. (n = 5 for WT mice group, n = 6 for MMP9−/− mice group, all male). The experiment was repeated independently three times, the same sex (either all male or all female) each time. D, Flow cytometry analysis of the cardiac infiltrated immune cells (CD45+cells, DCs (CD11c+Iab+), and neutrophils (CD11b+Ly6G+)) 8 h after intraperitoneal injection of LPS (n = 8 for WT mice group, n = 6 for MMP9−/− mice group, all male) was shown. The experiment was repeated independently three times, the same sex (either all male or all female) each time. the log-rank test was performed; B, C, D data were shown as means ± SD and analyzed…

• 

  Fig. 2. MMP9 inhibitor alleviated the inflammation in SIMD. A, The survival of C57 mice which were pretreated with PBS or MMP9 inhibitor and then intraperitoneally injected with LPS(25 µg/g) was monitored every 3 h. (n = 10 for PBS group, n = 10 for MMP9 inhibitor group, all male). The experiment was repeated independently three times, the same sex (either all male or all female) each time. B, Analysis of the serum myocardial damage markers (CK-MB, cTnT, n = 5 for each group, all male) 8 h after intraperitoneal injection of LPS were measured by Elisa. These experiments were independently repeated three times, the same sex (either all male or all female) each time. C, Representative echocardiography images were obtained 8 h after intraperitoneal injection of LPS from C57 mice which were pretreated with PBS or MMP9 inhibitor, as measured by EF (%) and FS (%) (n = 5 for PBS group, n = 6 for MMP9 inhibitor group, all male). The experiment was repeated independently three times, the same sex (either all male or all female) each time. EF, ejection fraction; FS, fraction shortening. D, Analysis of inflammatory factors (IL-6, IL-17 A, IL-1β and TNF-α, n = 8 for each group, all male) 8 h after…

• 

  Fig. 3. The protective effect of MMP9−/− BMDCs against SIMD was associated with decreased inflammatory cell infiltration and inflammatory cytokine production.A, WT BMDCs, or MMP9−/− BMDCs (2 × 106 cells per mouse) or PBS was transferred to the mice with LPS-induced sepsis respectively. The survival of C57 mice was monitored every 3 h (n = 9 for PBS group, n = 10 for LPS + WT BMDCs group, n = 15 for LPS + MMP9−/−BMDCs group, all male). *P < 0.05, **P < 0.001. These experiments were repeated independently three times, the same sex (either all male or all female) each time. B, The Cd11c.DOG mice, which were pretreated with DT (8ng/g) every day for one week, were transferred with WT BMDCs or MMP9−/− BMDCs (5 × 106 cells per mouse) by caudal vein injection. 8 h later the mice were intraperitoneally injected with PBS or LPS. The survival of these groups of mice was monitored every 3 h (n = 3 for DT + PBS group, n = 5 for DT + LPS group, n = 6 for LPS + WT BMDCs group, n = 7 for LPS + MMP9−/− BMDCs group, all male). These experiments were repeated independently three times, the same sex (either all male or all female) each time. C, Analysis of the serum myocardial damage markers (CK-MB, cTnT, n…

• 

  Fig. 4. Transfer of MMP9−/− BMDCs protected cardiomyocytes from pyroptosis and apoptosis. A, WT mice were transferred with normal BMDCs or Cell-VueBurgandy-labelled BMDCs (5 × 106 cells per mouse) through the caudal vein. 12 h later, ex vivo fluorescence reflectance imaging (FRI) of the extracted organs (the lymph nodes and the hearts) was performed. The dissection image of the fluorescence showed that the Cell-VueBurgandy-labelled BMDCs could migrate to the lymph nodes and the heart. n = 4 for each group, all male. These experiments were independently repeated three times, the same sex (either all male or all female) each time. B, C Western Blot detection of pyroptosis-related protein levels (NLRP3, GSDMD-N, c-Caspase1) and apoptosis-related protein level (c-Caspase3)in cardiac tissue 8 h after LPS (25 µg/g) intraperitoneal injection (n = 2 for DT + PBS group, n = 2 for DT + LPS group, n = 3 for LPS + WT BMDCs group, n = 4 for LPS + MMP9−/− BMDCs group, all male). These experiments were independently repeated three times, the same sex (either all male or all female) each time. Ctrl, Cd11c.DOG mice pretreated with DT after PBS injection group; LPS, Cd11c.DOG mice pretreated with DT after…

• 

  Fig. 5. DCs-Specific MMP9 deletion ameliorates SIMD via attenuated inflammation and cardiomyocyte protection. A, Survival of MMP9fl/fl CD11ccre− and MMP9fl/flCD11ccre+ mice intraperitoneally injected with LPS(25 µg/g), was monitored every 3 h (n = 11 for MMP9fl/flCD11ccre− mice group, n = 10 for MMP9fl/flCD11ccre+ mice group, all female). These experiments were independently repeated three times, the same sex (either all male or all female) each time. B, Analysis of the serum myocardial damage markers (CK-MB, cTnT, IL-6) 8 h after intraperitoneal injection of LPS were measured by Elisa. (n = 11 for MMP9fl/flCD11ccre− mice group, n = 10 for MMP9fl/flCD11ccre+ mice group, all female). These experiments were independently repeated three times, the same sex (either all male or all female) each time. C, Representative echocardiography images were obtained 8 h after LPS (25 µg/g)intraperitoneal injection(n = 6 for MMP9fl/flCD11ccre− mice group, n = 8 for MMP9fl/flCD11ccre+ mice group, all female), as measured by EF (%). D, Western Blot detection of pyroptosis-related protein levels (NLRP3, GSDMD-N, c-Caspase1) and apoptosis-related protein level(c-Caspase3)in cardiac tissue 8 h after LPS ༈25…

• 

  Fig. 6. MMP9−/− BMDCs protected cardiomyocytes from death by attenuating inflammatory factor secretion. A, Flow cytometry analysis of the AnexinV+ and/or PI+ cell proportion of cardiomyocytes from neonatal mouse myocardium cocultured with WT BMDCs or MMP9−/− BMDCs with 24 h-LPS (1 µg/ml) treatment was shown. B, Western Blot detection and analysis of pyroptosis-related protein levels (NLRP3, GSDMD-N, c-Caspase1) and apoptosis-related protein c-Caspase3 in neonatal mouse myocardium cocultured with WT BMDCs or MMP9−/− BMDCs with 24 h-LPS treatment (1 µg/ml) was shown. C, Levles of inflammatory factors (IL-6, IL-1β, IL-18) in the cell supernatant of BMDCs and cardiomyocytes coculture system with 24 h-LPS treatment (1 µg/ml) were measured. D, Analysis of NO production in cell supernatant with 24 h-LPS treatment (1 µg/ml) in BMDCs and H9c2 co-culture system was shown. E, Flow cytometry analysis of real-time ROS response in WT or MMP9−/− BMDCs with LPS treatment for different time (1 µg/ml) was shown. ctrl, Cardiomyocyte from Neonatal mouse myocardium or H9c2; +LPS, Cardiomyocyte from Neonatal mouse myocardium or H9c2 with 24 h LPS treatment; +WT, Cardiomyocyte from Neonatal mouse myocardium or…

• 

  Fig. 7. MMP9 regulated BMDCs function through JNK signaling pathway. A, Flow cytometric analysis of the expression level (CD86, Iab, CD80, CD40) of WT or MMP9−/−BMDCs 24 h after LPS stimulation was displayed. B, Elisa analysis of inflammatory factors (IL-6, TNF-α) in the cell supernatant of BMDCs with 24 h LPS treatment at different concentration were measured. C, Western Blot detection of the JNK phosphorylation in WT BMDCs or MMP9−/− BMDCs with LPS stimulation was measured. D, Western Blot detection of the c-Jun phosphorylation in WT BMDCs or MMP9−/− BMDCs with LPS stimulation was measured. In all panels, data were shown as means ± SD and analyzed with one-way and two-way analysis of variance (ANOVA) and *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. The experiments were repeated independently three times