Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

Abstract

Background: Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers.

Methods: In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis.

Results: Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged.

Discussion: This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds.

Keywords: Adipose tissue; Adipose-derived stem cells; Inflammation; M1; M2; Macrophages; Polarization; Wound repair.

Figure 1. Histological section of HAT and IAT.

Histological sections from healthy adipose tissue (HAT) and inflammatory adipose tissue (IAT) samples adjacent to acute wound healing disorders were stained by hematoxylin/eosin (400× maginification). A HE staining of HAT B HE staining of IAT.

Figure 2. Messenger RNA expression levels of M1- and M2 markers in native HAT and IAT.

Messenger RNA was extracted from healthy adipose tissue (HAT, n = 20) and inflammatory adipose tissue (IAT, n = 20) samples adjacent to acute wound healing disorders . Expression of common pro-inflammatory M1 and anti-inflammatory M2 macrophage markers were measured by qRT-PCR. Relative mRNA expression levels are illustrated with mRNA expression of HAT set as 100%. (A–C) mRNA expression of M1 markers CD80, iNOS, and IL-1β; (D–F) mRNA expression of M2 markers CD163, TGF-β, and IL-1RA. Data are presented as mean mRNA expression ± SEM. Statistically significant differences are indicated by asterisks (^∗, p < 0.05; ^∗∗, p < 0.01; ^∗∗∗, p < 0.001).

Figure 3. Surface expression of CD80 and CD163 in native HAT and IAT.

Stromal vascular fraction (SVF) cells were collected by collagenase digestion of healthy adipose tissue (HAT, n = 20) and inflammatory adipose tissue (IAT, n = 20) samples and subjected to flow cytometry analysis. (A) ATM were gated by selecting cells which were positive for the pan-macrophage marker CD68. (B) ATMs in IAT show high expression of the M1 macrophage marker CD80 and low expression of the M2 macrophage marker CD163. (C) ATMs in HAT show high expression of CD163 but low expression of CD80. (D) The M1/M2 ratio was calculated as the ratio between CD80+ and CD163+ cells and show an increased M1/M2 ratio in IAT. Data are presented as mean mRNA expression ± SEM. Statistically significant differences are indicated by asterisks (^∗∗, p < 0.01).

Figure 4. Messenger RNA expression of stem cell markers in native HAT and IAT.

Messenger RNA was extracted from healthy adipose tissue (HAT, n = 20) and inflammatory adipose tissue (IAT, n = 20) samples. Expression of common stem cell markers were measured by qRT-PCR. Relative mRNA expression levels are illustrated with mRNA expression of HAT set as 100%. (A) mRNA expression of CD29 (B) mRNA expression of CD34 (C) mRNA expression of 73 (D) mRNA expression of 90 (E) mRNA expression of CD105. Data are presented in mean mRNA expression ± SEM. Statistically significant differences are indicated by asterisks (^∗∗∗, p < 0.001).