Interleukin 10 Gene-Modified Bone Marrow-Derived Dendritic Cells Attenuate Liver Fibrosis in Mice by Inducing Regulatory T Cells and Inhibiting the TGF-β/Smad Signaling Pathway

Figure 5

(a) BMDCs were transfected with LV-IL10-GFP or LV-mock-GFP to acquire DC-IL10 and DC-mock cells, respectively. Mice were randomly divided into G1 (control group), G2 (model group), G3 (DC-mock-treated group), and G4 (DC-IL10-treated group). Liver fibrosis (G2, G3, and G4) was induced by twice a week intraperitoneal injection of 40% CCl4 as a 2 ∶ 3 mixture with olive oil for 8 weeks. The control group was administered with the same volume of olive oil only, twice a week for 8 weeks. In the 6th week of CCl4 injection, the mice in the G3 and G4 groups were treated with DC-mock or DC-IL10 (1 × 10⁶/0.2 ml) suspended in RPMI 1640 into the tail vein (twice a week for 2 weeks) till the 8th week, while the control group and model group received equivalent volumes of RPMI 1640 tail vein injection instead of DC treatment. All mice were sacrificed 72 h following the final CCl4 or olive oil injection at the 8th week. Splenocytes were isolated from the spleen in each group of mice to analyze the immune-regulatory effect of DC-IL10 on Th17 cells. The lymphocytes were stimulated for 5 h with PMA and BFA in RPMI 1640 containing 10% FBS and then stained with anti-CD4-FITC. They were fixed and permeabilized with Fix/PERM kit and stained intracellularly with anti-IL-17-PE. The percentages of Th17 cells were determined by flow cytometry. Cells stained with IgG isotype control were used as controls. (b) DC-IL10 suppresses the generation of Th17 cells. The percentages of Th17 cells in CD4⁺T were quantified. Following DC-IL10 administration, the percentages of Th17 cells were significantly decreased when compared with the model group and DC-mock treatment group ( for all). Data represent the of 8 mice in each group. G1: control group (); G2: model group (); G3: DC-mock-treated group (); G4 DC-IL10-treated group (). (c) Mice were randomly divided into G1 (control group), G2 (model group), G3 (DC-mock treated group), and G4 (DC-IL10 treated group). Liver fibrosis (G2, G3, and G4) was induced by twice a week intraperitoneal injection of 40% CCl4 as a 2 ∶ 3 mixture with olive oil for 8 weeks. The control group was administered with the same volume of olive oil only, twice a week for 8 weeks. In the 6th week of CCl4 injection, the mice in the G3 and G4 groups were treated with DC-mock or DC-IL10 (1 × 10⁶/0.2 ml) suspended in RPMI 1640 into the tail vein (twice a week for 2 weeks) till the 8th week, while the control group and model group received equivalent volumes of RPMI 1640 tail vein injection instead of DC treatment. All mice were sacrificed for 72 h following the final CCl4 or olive oil injection at the 8th week. Splenocytes were isolated from the spleen in each group of mice to analyze the immune-regulatory effect of DC-IL10 on Treg cells. The lymphocytes from the spleen were surface-stained with anti-CD4-FITC and anti-CD25-APC, then fixed, permeabilized, and stained with anti-FoxP3-PE. The percentages of Treg cells were determined by flow cytometry. Cells stained with IgG isotype control were used as controls. (d) DC-IL10 increases the generation of Treg cells. The percentages of Treg cells in CD4⁺T were quantified. Following DC-IL10 administration, the percentages of Treg cells were significantly increased when compared with the model group and DC-mock treatment group ( for all). Data represent the of 8 mice in each group. G1: control group (); G2: model group (); G3: DC-mock- treated group (); G4: DC-IL10-treated group ().

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