Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing

Abstract

Increasing evidence has supported the important role of mesenchymal stem cells (MSCs) in wound healing, however, the underlying mechanism remains unclear. Recently, we have isolated a unique population of MSCs from human gingiva (GMSCs) with similar stem cell-like properties, immunosuppressive, and anti-inflammatory functions as human bone marrow-derived MSCs (BMSCs). We describe here the interplay between GMSCs and macrophages and the potential relevance in skin wound healing. When cocultured with GMSCs, macrophages acquired an anti-inflammatory M2 phenotype characterized by an increased expression of mannose receptor (MR; CD206) and secretory cytokines interleukin (IL)-10 and IL-6, a suppressed production of tumor necrosis factor (TNF)-α, and decreased ability to induce Th-17 cell expansion. In vivo, we demonstrated that systemically infused GMSCs could home to the wound site in a tight spatial interaction with host macrophages, promoted them toward M2 polarization, and significantly enhanced wound repair. Mechanistically, GMSC treatment mitigated local inflammation mediated by a suppressed infiltration of inflammatory cells and production of IL-6 and TNF-α, and an increased expression of IL-10. The GMSC-induced suppression of TNF-α secretion by macrophages appears to correlate with impaired activation of NFκB p50. These findings provide first evidence that GMSCs are capable to elicit M2 polarization of macrophages, which might contribute to a marked acceleration of wound healing.

Figure 1

GMSCs promote the polarization of M2 macrophages. Monocytes isolated from PBMCs using human monocyte isolation kit were seeded in 6-well plates (2 × 10⁵ per well) and cultured in macrophage growth medium for 7 days, followed by coculture with the same number of GMSCs for 3 days. Cocultured cells were collected and immunostained with PerCp/Cy5.5-CD90, PE-CD14, and FITC-CD206 and analyzed by flow cytometry. (A): Strategy of gating CD14-positive cells from the coculture with GMSCs (CD90-PerCp/Cy5.5). (B, C): Comparison of CD206 expression on macrophages cultured alone (Control) and cocultured in direct contact with GMSCs or in a transwell system (GMSCs/TW). (D): After coculture with GMSCs in the transwell for 72 hours, macrophages were incubated with 25 μg/ml FITC-Zymosan for 1 hour at 4°C (nonspecific binding) and 37°C, respectively, and analyzed by flow cytometry. Macrophages cultured alone were used as controls. (E): The average MFI of phagocytosed particles is shown for three different cultures. The results represent three independent experiments (mean ± SEM). **, p < .01. Abbreviations: FITC, fluorescein isothiocyanate; FSC, forward-scattered light; GMSC, mesenchymal stem cells from human gingival; MFI, mean fluorescence intensity; ns, no significance; PE, phycoerythrin; SSC, side-scattered light; TW, transwell.

Figure 2

Cytokine expression profiles in human macrophages cocultured with GMSCs. (A–C): After coculture with GMSCs in the transwell for 72 hours, macrophages were stained with FITC-CD206, followed by intracellular cytokine staining with PE-conjugated antibodies human IL-10 (A), IL-6 (B), and TNFα (C) and subjected to flow cytometry analysis, wherein cells stained with FITC- and PE-conjugated isotype control antibodies and macrophages cultured alone were used as controls. The graphs showed the average values from three independent experiments (mean ± SEM). (D–F): The secretion of IL-10 (D), IL-6 (E), and TNFα (F) in the supernatants of cocultured macrophages/GMSCs (2 × 10⁵) was determined using enzyme-linked immunosorbent assay as compared with GMSCs and macrophages cultured alone. (G): Peripheral blood monocytes (PBMCs) (2 × 10⁵) were cultured alone or cocultured with the same number of GMSCs in the presence of macrophage-colony stimulating factor (M-CSF; 30 ng/ml) for 72 hours. Then PBMCs were collected and immunostained with isotype-matched IgGs or CD4-PerCP/Cy5.5 and IL-17-FITC and analyzed by flow cytometry. PBMCs cultured alone in the absence of M-CSF were used as controls. The results represent three independent experiments (mean ± SEM). *, p < .05; **, p < .01. Abbreviations: FITC, fluorescein isothiocyanate; GMSC, mesenchymal stem cells from human gingival; IL, interleukin; PE, phycoerythrin; TNF, tumor necrosis factor.

Figure 3

GMSCs regulate the immunophenotype and function of human monocyte leukemic cells (THP-1) during differentiation. (A–D): THP-1 cells (2 × 10⁵) were differentiated on stimulation with PMA (10 ng/ml) in the absence or presence of IL-4 (100 ng/ml) or coculture with GMSCs in the transwell for 96 hours. Cells were stained with isotype-matched control IgGs (A), FITC-CD11a, and PE-conjugated CD14 (B), CD86 (C), or DC-SIGN (D) antibodies (A) and then analyzed by flow cytometry. THP-1 cells without any treatment were used as controls. (E–F): After coculture with GMSCs for 72 hours, THP-1 cells were stimulated with 0.1 and 1 μg/ml LPS for 4 hours (TNF-α) or 24 hours (IL-10), and the secretion of TNF-α (E) and IL-10 (F) in the supernatants was determined by enzyme-linked immunosorbent assay. (G): After coculture with GMSCs for 72 hours, THP-1 cells were stimulated with 0.1 and 1 μg/ml LPS for 1 hour, and the expression of NFκB p50 and p65 were determined by western blot, where the graphs represent the relative densities to the band of β-actin as the internal control. The results were representative of four independent experiments (mean ± SEM). *, p < .05; **, p < .01. Abbreviations: FITC, fluorescein isothiocyanate; GMSC, mesenchymal stem cells from human gingival; IL, interleukin; LPS, lipopolysaccharide; NFκB, nuclear factor kappa B; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; THP-1, human acute monocytic leukemia cell line; TNF, tumor necrosis factor.

Figure 4

Cytokine expression profile determined by antibody array. The cytokine expression profile in the conditioned media collected from macrophage, GMSC, and their coculture were detected using the RayBio Human Cytokine Antibody Array 3 (RayBiotech, Inc., Norcross, GA), which allows the detection of 42 cytokines, chemokines, and growth factors in one experiment. The fresh medium without cell culture was used as a background control. (A): The representative image of cytokine antibody array. (B): The graphs show the relative intensity of spots of individual protein, whereby the intensity of the medium control was arbitrarily set as 1.0. The results were representative of three independent arrays. *, p < .05; **, p < .01; ***, p < .0001. Abbreviations: EGF, epithelial growth factor; ENA, epithelial neutrophil-activating peptide; GMSC, mesenchymal stem cells from human gingival; GRO, growth-related oncogene; IL, interleukin; MCP-1, macrophage chemotactic protein-1.

Figure 5

Blocking IL-6 and GM-CSF synergistically inhibit GMSC-mediated induction of M2 macrophages. Macrophages were cocultured with GMSCs in transwells for 72 hours in the presence or absence of COX-2 inhibitor NS398 (10 μM) or specific neutralizing antibodies for IL-6, GM-CSF, CCL-2/MCP-1, or IL-10 (10 μg/ml). An isotype-matched rat IgG was used as negative controls. (A, C): Cells were immuno-stained with PE-CD14 and FITC-CD206 antibodies and subjected to flow cytometry analysis. (B, D): The graphs show the average values from three independent experiments (mean ± SEM). *, p < .05; **, p < .01. Abbreviations: FITC, fluorescein isothiocyanate; GM-CSFAb, granulocyte macrophage-colony stimulating factor neutralizing antibody; GMSC, mesenchymal stem cells from human gingival; IL, interleukin; MCP, macrophage chemotactic protein-1; ns, no significance; PE, phycoerythrin.

Figure 6

Systemic administration of GMSCs accelerates wound closure and suppress local inflammatory responses in C57BL/6 mice. One day after excisional skin wound, GMSCs (2 × 10⁶ per mice) were systemically infused by tail vein (i.v.) into mice and wound closure was daily observed. (A): Representative photographs of wounds at different time postwounding with or without GMSC treatment. (B): Measurement of wound closure at different time points (n = 4). The percentage of wound closure was calculated as: (area of original wound – area of measured wound)/area of original wound × 100. (C): Representative H&E-stained paraffin-embedded sections of full-thickness incisional skin wounds from mice with or without receiving systemic administration of GMSCs (n = 4). Mice were sacrificed at different days post-wounding. Scale bar = 100 μm. (D): At different time points after wounding, skin samples were collected and tissue lysates were prepared for further analysis: (A): MPO activity assay. (B–D): Enzyme-linked immunosorbent assay on inflammatory cytokines, including TNF-α (B), IL-6 (C) and anti-inflammatory cytokine IL-10 (D). The results were representative of three independent experiments (mean ± SEM). *, p < .05; **, p < .01. Abbreviations: GMSC, mesenchymal stem cells from human gingival; IL, interleukin; MPO, myeloperoxidase; TNF, tumor necrosis factor.

Figure 7

Interactions of homed GMSCs with macrophages during wound healing. (A): GMSCs prelabeled with CM-DiI were systemically infused by tail vein (i.v.) into mice one day after skin wounding. Seven days after cell injection, skin tissues were frozen sectioned and observed under a fluorescence microscope, whereby normal skin on the other side of the same mice were used as controls. (B): Frozen sections of wounded skins from mice after injection with CM-DiI pre-labeled GMSCs were immunostained with fluorescein isothiocyanate-conjugated antibody for mice CD11b. Scale bar = 50 μm. The results were representative of at least three independent experiments. (C): Frozen sections of full-thickness incisional skin wounds from mice after treatment with GMSCs for different days were dual-color immunostained with specific antibodies for F4/80 (Green) and RELM-α (Red). Scale bar = 50 μm. (D): Quantification of M2 macrophages positive for RELM-α. (E): Western blot analysis of arginase-1 (Arg-1) and RELM-α expression in skin wounds 7 days post GMSC treatment. (F): Time-dependent increases in Arg-1 and RELM-α expression induced by GMSC treatment. The results are representative of three independent experiments. *, p < .05; **, p < .01; ***, p < .001. Abbreviations: DAPI, 4′, 6-diamidino-2-phenylindole; GMSC, mesenchymal stem cells from human gingival; RELM, resistin-like molecule.