Table 1.
Primary antibodies used for immunofluorescence and Western blot.
Table 2.
The Primer sequences for real-time PCR.
Fig 1.
The infiltrated macrophages in liver tissues exhibit M2 polarization in schistosomiasis mice.
After eight weeks of cercariae or PBS administration liver sections of mice were stained with antibodies against F4/80+, Arg-1 and CD206. Nuclei were stained with DAPI. Images were taken by confocal fluorescent microscopy. Arrows indicate F4/80+/Arg-1 or F4/80+/CD206 double positive cells, and the bars represent 20 μm.
Fig 2.
SEA-induced macrophage M2 polarization in vitro.
RAW264.7 cells were stimulated with SEA (40 μg/ml) or PBS for two hours. (A) The expression of Arg-1 and NOS-2 in the RAW264.7 cells was assessed by Western blot. (B) The production of IL-10 and IL-12 in the supernatants of RAW264.7 cells was assessed by ELISA. Data represents the results of three independent experiments. In compared with the control group, *: p < 0.05.
Fig 3.
SEA upregulates Notch1/Jagged1 signaling in M2 macrophages in vitro.
(A) RAW264.7 cells were treated with SEA (40 μg/ml) or PBS for two hours. The mRNA expression of Notch receptors, Jagged1 and Jagged2 in the RAW264.7 cells were assessed by real-time PCR. (B and C) The mRNA levels of Notch1, Jagged1 and Hes1 in RAW264.7 cells stimulated with SEA (40 μg/ml) for the indicated time or with increasing concentration of SEA for two hours was assayed by real-time PCR. (D and E) The protein expression of Notch1, Jagged1 and Hes1 in RAW264.7 cells treated with SEA (40 μg/ml) for the indicated time was determined by Western blot analysis. Data shown are representative the three independent experiments. In compared with the control group, *: p < 0.05, **: p < 0.01.
Fig 4.
DAPT blocks SEA-induced macrophage M2 polarization by inhibiting Notch signaling.
RAW264.7 cells were administrated with DAPT (0.4 μmol/l) or DMSO (0.1%) for twelve hours, and then stimulated with SEA (40 μg/ml) or PBS for two hours. (A) The expression of Notch1, Jagged1 and Hes1 in the RAW264.7 cells was determined by Western blot. (B) The levels of IL-10 and IL-12 in the supernatants were assessed by ELISA. (C) The expression of Arg-1 and NOS-2 in the RAW264.7 cells was assessed by Western blot. All data represent the results of three independent experiments. In compared with the control group, *: p < 0.05. 1 indicates DAPT+SEA; 2 indicates DMSO+SEA.
Fig 5.
DAPT reverses macrophage M2 polarization in murine schistosomiasis.
Mice were infected with cercariae or PBS for twelve weeks. Eight weeks after the initial administration, the mice were treated with DAPT or vehicle control for another four weeks. Twelve weeks after the initial treatment, mice were sacrificed, and the liver sections were stained with antibodies against F4/80+ and Arg-1. Nuclei were stained with DAPI. Images were taken by confocal fluorescent microscopy. Arrows indicate F4/80+/Arg-1 double positive cells, and the bars represent 20 μm.
Fig 6.
DAPT attenuates hepatic granulomata and fibrosis in murine schistosomiasis.
(A) The pathological changes and collagen deposition were assessed by Hematoxylin and eosin and Masson’s trichrome staining. (B) The result of semi-quantitative analysis of the Masson’s trichrome staining. (C) Assay of hydroxyproline content. (D) Mean hepatic granuloma diameter (mm). Data from the three independent experiments represent the results of eight mice in each group. *: P < 0.05 versus mice in the model group.