Figure 1.
Anti-C5a treatment ameliorated sepsis.
(A) Anti-C5a antibody blocked the C5a effect. 100 nM mouse C5a and 100 nM preimmune IgG or anti-C5a were incubated for 2 hours at room temperature. Peripheral blood cells (PBMC) were collected from 7-week-old mice and diluted 2 times the volume of whole blood. PBMC were stimulated for 15 min at 37°C with 100 nM mouse C5a or preincubated mixture (100 nM mouse C5a and 100 nM preimmune IgG, or 100 nM mouse C5a and 100 nM anti-C5a). Fluorescence was determined by an enzyme reaction processed from cultured supernatants described in Materials and Methods. The results were analyzed based on three independent experiments (**P<0.01, ***P<0.001). (B) Anti-C5a antibody ameliorated sepsis. Peritonitis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Immediately after CLP operations, each mouse was intravenously injected with 40 µg of anti-C5a IgG in 100 µL Dulbecco’s phosphate buffered saline solution (DPBS). Control animals received similar a amount of normal rabbit IgG. Data are presented as the percentage of survival (n = 9/group). Overall survival rates were analyzed by the Kaplan–Meier method. Compared to isotype IgG antibody-treated littermates, the anti-C5a antibody-treated mice showed a higher survival rate (p<0.05).
Figure 2.
Anti-C5a reduced IL-12+DCs in PBMC and LN.
(A and B) IL-12+DCs cells decreased in PBMC and LN from anti-C5a-treated sepsis. (A) Mononuclear cells were collected by Ficoll solution from PBMC and LN in sham, preimmune IgG-treated CLP, or anti-C5a-treated CLP mice (described as Fig. 1) on day 3 after sepsis induction. Cells stained with anti-mouse CD11c and IL-12 with numbers in quadrants indicating the percentage of IL12+DC cells. (B) The statistical analysis was done based on four independent experiments (**P<0.01, ***P<0.001). (C) IL-12 level decreased in PBMC from anti-C5a-treated sepsis. The serum was collected from sham, preimmune IgG-treated CLP, or anti-C5a-treated CLP mice (described as Fig. 1) on day 3 after sepsis induction. IL-12 level in the serum was determined by ELISA. The statistical analysis was done based on three independent experiments (**P<0.01, ***P<0.001).
Figure 3.
IL-12+DCs increased in peritoneal cavity of anti-C5a-treated CLP mice.
Peritonitis was induced in C57BL/6 mice by CLP. We chose 2 time points: 48 hours and 1 week after induction of sepsis. Normal saline was injected to the peritoneal cavity of mice and washed several times. Cell-containing fluid was collected. Peritoneal cells were stained with PE-anti-IL-12 mAb to detect the percentage of IL-12 positive cells on the gate of monocytes population. The percentage of IL-12 positive cells was analyzed (upper panel). The peritoneal fluid was collected and IL-12 level was determined by ELISA (lower panel). The statistical analysis was done based on three independent experiments (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Figure 4.
Sepsis reduced IL-12-expressing cells in peritoneal cavity.
(A) Sepsis dramatically and time-dependently reduced IL-12+ cells in peritoneal cavity. Peritonitis was induced in C57BL/6 mice by CLP. We chose 4 time points: 0, 24, 48, 72 hours. Normal saline was injected into the peritoneal cavity of mice and washed several times. Cell-containing fluid was collected. The peritoneal cells were stained with PE-anti-IL-12 mAb to detect percentage of IL-12 positive cells on the gate of lymphocyte and monocyte populations (*P<0.05, **P<0.01). (B-C) Sepsis dramatically and time-dependently reduced IL-12+CD11c+ cells in the peritoneal cavity. The peritoneal cells were also stained with PE-anti-IL-12 mAb and FITC-anti-CD11c mAb to detect whether DC cells expressed IL-12. The percentage of IL-12 positive cells was analyzed and compared between the groups. (B) The statistical analysis was done based on four independent experiments (*P<0.05, **P<0.01). (C) showed the percentage of IL-12+CD11c+ cells.
Figure 5.
DC-depletion exacerbated the sepsis disease.
(A) IL-12-expressing cells were mainly DCs. Conditional DCs-depleted mice B6.FVB-Tg (Itgax-DTR/EGFP)57Lan/J mice were developed by administering 8ng/g of diphtheria toxin (DT) by i.p. injection. Normal saline was injected into the peritoneal cavity of mice and washed several times. Cell-containing fluid was collected. Peritoneal cells were stained with PE-anti-IL-12 mAb to detect the percentage of IL-12 positive cells on the gate of monocyte population. The percentage of IL-12 positive cells was analyzed. The data represents at least four independent experiments (**P<0.01). (B) DCs-depletion exacerbated the sepsis induced by CLP. Conditional DCs-depleted mice were injected i.p. with 8ng/g of DT. On day 2, peritonitis were induced in DC-depleted mice by CLP. Data is presented as percentage survival (n = 9/group). Overall survival rates were analyzed by the Kaplan–Meier method. Compared with the wild type group, DCs-depleted mice have a higher mortality rate (P<0.05).
Figure 6.
CD11c+DC cells induced Th1 and Th17 cells in sepsis.
Lymphocytes were collected from PBMC in sham, CLP, CLP/DCs-depletion mice on day 3 after sepsis induction. Cells were stained with anti-mouse CD4, IFNγ, and IL-17 with numbers in quadrants indicating percentage of IFNγ+Th1 and IL-17+Th17 cells. Right panel showed the statistical analysis based on four independent experiments ((**P<0.01, ***P<0.001).
Figure 7.
Injection of IL-12 in peritoneal cavity ameliorated sepsis.
Conditional DC-depleted mice were injected i.p. with 8 ng/g of DT. On day 2, peritonitis was induced in DC-depleted mice by CLP. DC-depleted CLP mice were divided into two groups: one was given recombinant mouse IL-12 i.p.with injection, while the other group received normal saline as control. Data is presented as the percentage of survival (n = 9/group). Overall survival rates were analyzed by the Kaplan–Meier method. Compared with PBS-treated group, IL-12-treated group had a higher survival rate (P<0.05).