Fig. 1

eCIRP impairs macrophage phagocytosis in vitro and in vivo. RAW 264.7 cells (A) and murine peritoneal macrophages (B) were treated with rmCIRP (1 µg/ml) for 24 h. The cells were incubated with pHrodo green-labeled E. coli for 1.5 h. Bacterial phagocytosis was measured using a fluorescence microplate reader. At the end of the phagocytosis period, the cells were fixed, and microscopy images were acquired. Scale bar, 200 µm. The experiment was repeated 3–5 times. The data presented were combined from two independent experiments and are expressed as the mean ± SD (n = 9–11/group). The groups were compared by a two-tailed Student’s t test. *p < 0.05 vs. PBS control. RAW 264.7 cells were stimulated either with different doses of rmCIRP for 24 h (C) or with rmCIRP (1 µg/ml) for different times (D). The cells were then incubated with pHrodo green-labeled E. coli for 1.5 h. Bacterial phagocytosis was measured using a fluorescence microplate reader. Data are expressed as the mean ± SD (n = 4–11/group). The PBS control was set to 100% for normalization. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS control. E, F rmCIRP (5 mg/kg body weight) in saline or saline alone (control) was injected i.p. into mice. At 20 h after rmCIRP injection, 1 ml of pHrodo green-labeled E. coli (3 × 10⁸) was administered into the peritoneal cavity via i.p. injection, and mice were rested for 1 h to allow phagocytosis. Then, peritoneal lavage cells were extracted, peritoneal macrophages were labeled with a Pacific blue-conjugated anti-F4/80 Ab, and phagocytosis was assessed using flow cytometry. The efficiency of phagocytosis is indicated as the median fluorescence intensity (MFI). G, H RAW 264.7 cells were treated with rmCIRP (1 µg/ml) in the presence of IgG (10 µg/ml) or anti-CIRP Abs (10 µg/ml) for 24 h. In another group, cells were treated with denatured rmCIRP (1 µg/ml, rmCIRP was boiled for 15 min). The cells were incubated with pHrodo green-labeled E. coli for 1.5 h. Bacterial phagocytosis was measured using a fluorescence microplate reader. At the end of the phagocytosis period, the cells were fixed, and microscopy images were acquired. Scale bar, 100 µm. The data presented are expressed as the mean ± SD (n = 4–5/group), and the experiment was performed twice. The groups were compared by one-way ANOVA and the SNK method. *p < 0.05 vs. PBS; ^#p < 0.05 vs. rmCIRP + IgG