Fig 1.
TIPE2 promotes M2 but inhibits M1 macrophage differentiation.
Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were untreated (M0), treated with IFN-γ (50 ng/ml) and LPS (10 ng/ml) (M1) or IL-4 (10 ng/ml) (M2) for 24 h. (A, B & C) To detect the expression of markers specific for M1 macrophages, mRNA level of iNOS was examined by real-time RT-PCR (A). The production of NO (B) and IL-12 (C) in the culture supernatant were examined by a commercial NO detection kit and ELISA, respectively. (D, E & F) To detect the expression of markers specific for M2 macrophages, mRNA level of Arg-1 was examined by real-time RT-PCR (D). The amount of urea in the protein extract was examined by a commercial urea detection kit (E) and the production of IL-10 in the culture supernatant was examined by ELISA (F). For A and D, data shown are fold increase over the group with the lowest expression level. For all figures, data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05, ** p<0.01.
Fig 2.
Activation of PI3K-AKT but not JAK-STAT6 signaling pathway is defective in Tipe2-deficient macrophages.
Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were either untreated (UT) or treated with IL-4 (10 ng/ml) (T) for 20 min. (A) Cells were stained with anti-Phospho-AKT (T308) and analyzed by flow cytometry. (B) The MFI (mean fluorescence intensity) of phosphorylated AKT (pAKT) was determined using FlowJo software. (C) Cells were stained with anti-Phospho-PDK1 (Ser241) and analyzed by flow cytometry. (D) The MFI of phosphorylated PDK1 (pPDK1) was determined using the same method as shown in (B). (E) Cells were stained with anti-Phospho-STAT6 (Tyr641) and analyzed by flow cytometry. (F) The MFI of phosphorylated STAT6 (pSTAT6) was determined using the same method as shown in (B). Results are representative of three independent experiments. For B, D and F, Data shown are mean±SD of one representative experiment. * p<0.05.
Fig 3.
Expression levels of M2 macrophage markers regulated by PI3K-AKT signaling pathway are significantly decreased by Tipe2-deficient macrophages.
Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were untreated (M0), treated with IFN-γ (50 ng/ml) and LPS (10 ng/ml) (M1) or IL-4 (10 ng/ml) (M2) for 24 h. Expression levels of Fizz1 (A), Ym1 (B), Mgl-1 (C) and Mgl-2 (D) were determined by real time RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05, ** p<0.01.
Fig 4.
Expression levels of M2 macrophage markers regulated by JAK-STAT6 signaling pathway are either increased or not affected by Tipe2-deficient macrophages.
Bo Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were untreated (M0), treated with IFN-γ (50 ng/ml) and LPS (10 ng/ml) (M1) or IL-4 (10 ng/ml) (M2) for 24 h. Expression levels of Atgl (A), Fabp4 (B) and Cd36 (C) were determined by RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05.
Fig 5.
Cellular levels of PIP2 and PIP3 were significantly decreased by Tipe2-deficient macrophages.
Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were treated with IL-4 (10 ng/ml) for 20 min. (A) Cellular level of PIP2 was estimated by dot blot with anti-PIP2 antibody. (B) Relative expression level of PIP2 was determined using β-actin as the control and quantified by densitometry using ImageJ software. (C) Cellular level of PIP3 was estimated by dot blot with anti-PIP3 antibody. (D) Relative expression level of PIP3 was determined using same method as shown in (B). For A and C, results are representative of three independent experiments. For B and D, data shown are mean±SD for cells from three independent experiments. ** p<0.01.
Fig 6.
Inhibition of PI3K reduces the difference of Arg-1 expression between WT and Tipe2-deficient M2 macrophages.
Bone marrow derived macrophages from WT and Tipe2-deficient mice (n = 3) were treated with or without PI3K inhibitor NSC23766 (10 μM) for 30 min before stimulated with IL-4 (10 ng/ml) for 24 h. Expression level of Arg-1 was determined by real time RT-PCR and normalized to the expression level of GAPDH. Data shown are mean±SD of one representative experiment. The experiments were repeated three times with similar results. * p<0.05.