Fig 1.
Tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) expression in adipose tis-sue-derived stem cells (ASCs) co-cultured with macrophages.
ASCs were treated with interferon (IFN)-γ/lipopolysaccharide (LPS) or interleukin (IL)-4/IL-13 or co-cultured with macrophages for two days. TSG-6 mRNA (A) and protein (B) expression in ASCs under treatment with IFN-γ/LPS or IL-4/IL-13 and/or co-culture with macrophages. In panels A and B, IFN-γ + LPS treatment in the 5th sample induced macrophages to differentiate to M1 macrophages and IL-4 + IL-13 treatment in the 6th sample induced macrophages to differentiate to M2 macrophages. Immunoblotting was used to analyze TSG-6 expression in ASCs. Relative expression was normalized with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Data are presented as the mean ± standard deviation (SD) of three independent experiments. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. MΦ, macrophages (PMA-treated THP-1 cells).
Fig 2.
Inhibition of tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) expression via transforming growth factor (TGF)-β in adipose tissue-derived stem cells (ASCs) treated with interleukin (IL)-4 and IL-13.
Macrophages and ASCs were seeded in the lower and upper chamber (transwell insert) of 6-well plate, respectively, and the TGF-β1 and tumor necrosis factor (TNF)-α expression in macrophages was analyzed using quantitative polymerase chain reaction (PCR). (A) TGF-β or (B) TNF-α mRNA in macrophages treated with IFN-γ/LPS or IL-4/IL-13 or co-cultured with ASCs. Relative expression was normalized with respect to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Data are presented as the mean ± standard deviation (SD) of four independent experiments. *p ≤ 0.05 and **p ≤ 0.01. To observe TNF-α and TGF-β-induced alterations of TSG-6 production from ASCs, ASCs were treated with 1 ng/mL of TGF-β or 10 ng/mL of TNF-α for two days. (C) TSG-6 expression in ASCs treated with TNF-α or TGF-β. (D) TSG-6 expression in ASCs treated with TGF-β (0.4–2.5 ng/mL). Immunoblotting was used to analyze TSG-6 expression in ASCs. Relative expression was normalized with respect to GAPDH expression. Data are presented as the mean ± SD of three independent experiments. *p ≤ 0.05 and **p ≤ 0.01. MΦ, macrophages (PMA-treated THP-1 cells).
Fig 3.
Effects of tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) on the fibrosis, proliferation, and migration of LX-2 cells.
To determine the anti-fibrotic effect of TSG-6 and its effect on fibrosis, proliferation, and migration, LX-2 cells were treated with transforming growth factor (TGF)-β (1 ng/mL) and/or TSG-6 (40 ng/mL). (A) Anti-fibrotic effects of TSG-6 in LX-2 cells. (B) Effects of TSG-6 on LX-2 cell proliferation after 48 h. Data are presented as the mean ± SD of four replicates. (C) Effects of TSG-6 on LX-2 cell migration. Representative images are shown from three independent experiments and the cell-free region (outlined wound area) was analyzed using ImageJ. Values of percentage wound closure were shown as the mean ± standard deviation (SD). *p ≤ 0.05. and **p ≤ 0.01.
Fig 4.
Effects of adipose tissue-derived stem cells (ASCs) treated with interleukin (IL)-4/IL-13 or co-cultured with M2 macrophages on LX-2 cell migration.
Representative images from wound healing assay of LX-2 cells are shown from three independent experiments and the cell-free region (outlined wound area) was analyzed using ImageJ. Values of percentage wound closure were shown as the mean ± standard deviation (SD). *p ≤ 0.05 and **p ≤ 0.01. M2, M2 macrophage.
Fig 5.
Schematic representation of the therapeutic actions of mesenchymal stem cell (MSC) on fibrosis and wound healing.
Pro-inflammatory M1 macrophages induce overexpression of tumor necrosis factor-α (TNF-α) stimulated gene 6 (TSG-6) via TNF-α, which induces M2 polarization of macrophages and exerts anti-fibrotic actions. On the other hand, M2 macrophages can inhibit TSG-6 expression on MSCs via TGF-β, leading to fibrosis and wound healing.