TGF-β expressed by M2 macrophages promotes wound healing by inhibiting TSG-6 expression by mesenchymal stem cells

Abstract

Wound healing involves the collaboration of multiple cells, including macrophages and fibroblasts, and requires the coordination of cytokines, growth factors, and matrix proteins to regulate the repair response. In this study, we investigated how M2 macrophages regulate expression of the anti-fibrotic and anti-inflammatory regulator tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) secreted by adipose tissue-derived stem cells (ASCs) during wound healing. Interleukin (IL)-4/IL-13, which is used to differentiate macrophage M2 phenotypes, increases TSG-6 in ASCs; however, M2 macrophages significantly decrease TSG-6 in ASCs. Transforming growth factor (TGF)-β expression was increased, and TNF-α expression was decreased in M2 macrophages. TGF-β inhibited IL-4/IL-13-induced ASC TSG-6 expression. In addition, TSG-6 suppressed TGF-β-triggered wound closure and fibrogenic responses in LX-2 cells. Collectively, TSG-6 inhibited wound healing, but M2 macrophage-expressed TGF-β prevented TSG-6 production from ASCs, which ultimately helped wound healing. Our results indicate that the balance of TNF-α and TGF-β levels during wound healing regulates TSG-6 production from ASCs, which may ultimately modulate the healing process. Our study findings could contribute to novel therapeutic strategies that manipulate the delicate balance between TNF-α and TGF-β to enhance wound repair and mitigate fibrosis.

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.