Exosome-Guided Phenotypic Switch of M1 to M2 Macrophages for Cutaneous Wound Healing

Abstract

Macrophages (Mϕs) critically contribute to wound healing by coordinating inflammatory, proliferative, and angiogenic processes. A proper switch from proinflammatory M1 to anti-inflammatory M2 dominant Mϕs accelerates the wound healing processes leading to favorable wound-care outcomes. Herein, an exosome-guided cell reprogramming technique is proposed to directly convert M1 to M2 Mϕs for effective wound management. The M2 Mϕ-derived exosomes (M2-Exo) induce a complete conversion of M1 to M2 Mϕs in vitro. The reprogrammed M2 Mϕs turn Arginase (M2-marker) and iNOS (M1-marker) on and off, respectively, and exhibit distinct phenotypic and functional features of M2 Mϕs. M2-Exo has not only Mϕ reprogramming factors but also various cytokines and growth factors promoting wound repair. After subcutaneous administration of M2-Exo into the wound edge, the local populations of M1 and M2 Mϕs are markedly decreased and increased, respectively, showing a successful exosome-guided switch to M2 Mϕ polarization. The direct conversion of M1 to M2 Mϕs at the wound site accelerates wound healing by enhancing angiogenesis, re-epithelialization, and collagen deposition. The Mϕ phenotype switching induced by exosomes possessing the excellent cell reprogramming capability and innate biocompatibility can be a promising therapeutic approach for various inflammation-associated disorders by regulating the balance between pro- versus anti-inflammatory Mϕs.

Keywords: cutaneous wound healing; direct cell reprogramming; exosomes; macrophage phenotype switch.

Conflict of interest statement

The authors declare no conflict of interest.

© 2019 The Authors. Published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1

Schematic illustration of exosome‐guided macrophage reprogramming. M2 Mϕ‐derived exosomes (M2‐Exo) can induce a conversion of M1 to M2 Mϕs and accelerate cutaneous wound healing.

Figure 2

Establishment of M1 and M2 Mϕs and characterization of M2‐Exo. A) Schematic diagram of Mϕs and exosomes preparation. B) Western blot analysis demonstrating differences in expression of Mϕ markers by polarization time. C) Representative TEM image of exosomes after negatively staining with uranium acetate. D) Size distribution diagram of M2‐Exo measured using dynamic light scattering. E) Western blot analysis of exosomes. Equal amounts of total proteins extracted from exosomes were immunoblotted for CD63, Alix, iNOS, and Arginase. F) Confocal images of M1 Mϕs after 1 or 4 h incubation with 10, 25, 50, and 100 µg mL−1 of DiD‐labeled M2‐Exo, respectively. Images of DiD‐labeled M2‐Exo (red) with DAPI (blue) were visualized by merging the confocal images. G) Relative fluorescence intensity of DiD‐labeled M2‐Exo internalized in M1 Mϕs.

Figure 3

M2‐Exo‐guided direct reprogramming of M1 Mϕs to M2 Mϕs. A) Illustration of M2‐Exo‐induced Mϕ reprogramming. B) Immunostaining of iNOS and Arginase in M1 Mϕs after 24 h incubation with 10, 25, 50, and 100 µg mL−1 of M2‐Exo, respectively. C) Western blot analysis of M1 Mϕs treated with 50 µg mL−1 of M2‐Exo over time. D) FACS histogram showing reprogramming efficiency of M1 Mϕs treated with 50 and 100 µg mL−1 of M2‐Exo.

Figure 4

Cytokine expression profile of exosomes determined by antibody array and identification of major molecules of macrophage reprogramming. A) The representative images of cytokine antibody array. B) Comparison of mean pixel density measurement between cytokines of M1‐Exo and M2‐Exo. C) FACS histogram showing reprogramming efficiency of M1 Mϕs treated with M2‐Exo (25 µg mL−1) and each cytokine (100 ng mL−1).

Figure 5

In vitro therapeutic effects of RM2 Mϕs. A) Illustration of cocultures of fibroblasts and Mϕs. B) Representative phase‐contrast images of wounded fibroblasts cocultured with Mϕs. C) Quantification of wound closure among the three types of Mϕ groups. n = 3; **p < 0.01, and ***p < 0.001 versus saline. D) Levels of MMP‐2 in Mϕ/fibroblast coculture supernatant at 24 h after inflicting the wounds. E) Representative pictures of tube formation assay of vascular endothelial cells cocultured with Mϕs. F,G) Quantitative evaluation of total number of branches and tube length at 24 h after coculturing endothelial cells and Mϕs. n = 5; *p < 0.05, **p < 0.01, and ***p < 0.001 versus saline.

Figure 6

In vivo biodistribution of exosomes. A) Real‐time in vivo imaging of Cy5.5‐NHS labeled exosome. The mice were analyzed at the indicated times after subcutaneous injection of phosphate‐buffered saline (PBS) and 100 µg per 100 µL of exosomes. B) Ex vivo imaging of skins and major organs at day 2 after mice had been treated with exosomes. C) Tissue distribution of exosomes at day 2 after subcutaneous injection of exosomes.

Figure 7

M2 Mϕs‐derived exosomes promoted wound healing in vivo. A) Representative images of wound closure after local injection of saline, M1‐Exo, and M2‐Exo. B) Wound area at every 4 d postwounding. n = 4; *p < 0.05, **p < 0.01, and ***p < 0.001 versus saline. C) Representative photoimages of whole wound sections injected with saline, M1‐Exo, and M2‐Exo at day 24 postwounding. The black arrows indicate the wound margins.

Figure 8

In vivo therapeutic effects of RM2 Mϕs. A) Representative immunostaining images of M1 (iNOS) and M2 (Arginase) Mϕs in mice skin tissue at 8 d postwounding. B) Western blot analysis of the expression of iNOS and Arginase in mouse skin tissues with PBS, M1‐Exo, and M2‐Exo (100 µg per 100 µL). C) Representative magnified photographs of wounds (H&E staining) 24 d after injury and subcutaneous injection of PBS, M1‐Exo, and M2‐Exo. The dotted line indicates the dermal region and the black arrow represents collagen fibers. D) Fluorescence image of MMP‐2 activity surrounding the wound bed using MMP‐2‐activatable probe. E) Representative flow plots of M1 and M2 Mϕs population in the wound 5 d after injury and subcutaneous injection of PBS, M1‐Exo, and M2‐Exo.