Mesenchymal stem cells inhibit cutaneous radiation-induced fibrosis by suppressing chronic inflammation

Abstract

Exposure to ionizing radiation (IR) can result in the development of cutaneous fibrosis, for which few therapeutic options exist. We tested the hypothesis that bone marrow-derived mesenchymal stem cells (BMSC) would favorably alter the progression of IR-induced fibrosis. We found that a systemic infusion of BMSC from syngeneic or allogeneic donors reduced skin contracture, thickening, and collagen deposition in a murine model. Transcriptional profiling with a fibrosis-targeted assay demonstrated increased expression of interleukin-10 (IL-10) and decreased expression of IL-1β in the irradiated skin of mice 14 days after receiving BMSC. Similarly, immunoassay studies demonstrated durable alteration of these and several additional inflammatory mediators. Immunohistochemical studies revealed a reduction in infiltration of proinflammatory classically activated CD80(+) macrophages and increased numbers of anti-inflammatory regulatory CD163(+) macrophages in irradiated skin of BMSC-treated mice. In vitro coculture experiments confirmed that BMSC induce expression of IL-10 by activated macrophages, suggesting polarization toward a regulatory phenotype. Furthermore, we demonstrated that tumor necrosis factor-receptor 2 (TNF-R2) mediates IL-10 production and transition toward a regulatory phenotype during coculture with BMSC. Taken together, these data demonstrate that systemic infusion of BMSC can durably alter the progression of radiation-induced fibrosis by altering macrophage phenotype and suppressing local inflammation in a TNF-R2-dependent fashion.

Keywords: Chronic inflammation; Dermal fibrosis; Ionizing radiation; Mesenchymal stem cell.

Figure 1.

Phenotype and multipotency of bone marrow-derived mesenchymal stem cells (BMSC). (A): Cell surface expression of Sca1, CD29, CD44, CD106, CD45, and CD11b (red) or isotype (blue). (B): BMSC induced toward osteogenic (Alizarin red; ×20 magnification bar= 50 μm), adipogenic (oil red O/hematoxylin; ×40 magnification, bar = 50 μm), or (C) chondrogenic phenotypes (alginate bead section, alcian blue/nuclear fast red; ×10 magnification, bar = 100 μm).

Figure 2.

BMSC suppress radiation-induced skin contracture, thickening, and collagen deposition. (A): Hind limb extension, (B) skin thickness, and (C) collagen content were improved in BMSC-infused mice. Brackets show differences, p < .05. (D): Partial improvement of skin architecture in BMSC-treated mice. Masson trichrome stain, ×20 magnification, bar shows 150 μm. Abbreviation: BMSC, bone marrow-derived mesenchymal stem cells.

Figure 3.

BMSC alter fibrosis-associated gene expression in irradiated skin. Unsupervised hierarchical clustering of 84 fibrosis-associated transcripts, comparing unirradiated skin to the irradiated skin of mice receiving either vehicle or sBMSC. IL-10, IL-1β, PDGF-A, and Serpine1 were significantly greater than twofold differentially expressed between the vehicle and BMSC-infused mice. Abbreviation: BMSC, bone marrow-derived mesenchymal stem cells.

Figure 4.

Temporal alteration of immunoregulatory cytokine levels by BMSC infusion. Tissue homogenates prepared for enzyme-linked immunosorbent assay to determine levels (A) IL-1α, (B) IL-1β, (C) TNF-α, (D) IL-1RN, (E) IL-10, and (F) total TGF-β1. Abbreviations: BMSC, bone marrow-derived mesenchymal stem cells; IL-1α, interleukin-1α; IR, ionizing radiation; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Figure 5.

BMSC suppress macrophage infiltration in irradiated skin. (Left) Representative immunohistochemistry and (right) positive cell counts for (A) CD68, (B) CD80/B7.1, and (C) CD163. DAB histochemistry with Gill’s hematoxylin counterstain; ×20 magnification, bar shows 150 μm, and brackets p ≤ .05. Abbreviation: BMSC, bone marrow-derived mesenchymal stem cells.

Figure 6.

Cytokine secretion by LPS-stimulated bone marrow-derived mesenchymal stem cells (BMSC)-MΦ cultures. Mono- and cocultures of BMSC or MΦ were exposed to either PBS (control) or LPS for 24 hours, and enzyme-linked immunosorbent assay interrogated media supernatants for levels of secreted (A) TNFα, (B) IL-10, (C) IL-1RN, and (D) total TGF-β1. Abbreviations: IL-10, interleukin-10; IL-1RN, interleukin-1 receptor antagonist; LPS, lipopolysaccharides; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α.

Figure 7.

TNF-R2 expression is increased by BMSC, and mediates IL-10 production. IL-10 release by (A) cocultures exposed to a TNF-R2 neutralizing antibody or (B), mixed cocultures of MΦ and BMSC from wild-type and TNF-R2 mice. (C): ELISA of TNF-R2 levels in skin tissue. Abbreviations: BMSC, bone marrow-derived mesenchymal stem cells; IL-10, interleukin-10; IR, ionizing radiation; LPS, lipopolysaccharides.