Oral administration of EPA-rich oil impairs collagen reorganization due to elevated production of IL-10 during skin wound healing in mice

Abstract

Wound healing is an essential process for organism survival. Some fatty acids have been described as modulators of wound healing. However, the role of omega-3 fatty acids is unclear. In the present work, we investigate the effects of oral administration of eicosapentaenoic acid (EPA)-rich oil on wound healing in mice. After 4 weeks of EPA-rich oil supplementation (2 g/kg of body weight), mice had increased serum concentrations of EPA (20:5ω-3) (6-fold) and docosahexaenoic acid (DHA; 22:6ω-3) (33%) in relation to control mice. Omega-3 fatty acids were also incorporated into skin in the EPA fed mice. The wound healing process was delayed at the 3^rd and 7^th days after wounding in mice that received EPA-rich oil when compared to control mice but there was no effect on the total time required for wound closure. Collagen reorganization, that impacts the quality of the wound tissue, was impaired after EPA-rich oil supplementation. These effects were associated with an increase of M2 macrophages (twice in relation to control animals) and interleukin-10 (IL-10) concentrations in tissue in the initial stages of wound healing. In the absence of IL-10 (IL-10^-/- mice), wound closure and organization of collagen were normalized even when EPA was fed, supporting that the deleterious effects of EPA-rich oil supplementation were due to the excessive production of IL-10. In conclusion, oral administration of EPA-rich oil impairs the quality of wound healing without affecting the wound closure time likely due to an elevation of the anti-inflammatory cytokine IL-10.

Figure 1

Fatty acid composition of serum and unwounded skin throughout experiments in the control group (C, black bar) and EPA-group (EPA, grey bar). (A) Omega-3 and omega-6 concentrations in serum. (B) Omega-6/omega-3 ratio in serum. (C) Omega-3 and omega-6 content of skin phosphatidylcholine (PC). (D) Omega-6/omega-3 ratio in skin phosphatidylcholine (PC). (E) Omega-3 and omega-6 content of skin phosphatidylethanolamine (PE). (F) Omega-6/omega-3 ratio in skin phosphatidylethanolamine (PE). (n = 5–13 animals/group). Healthy mice were supplemented daily with EPA-rich oil (2 g of EPA-rich oil/kg bw) for 4 weeks and the serum and skin were sampled immediately prior to induction of the skin lesion and during the wound healing process. The percentage contribution of each fatty acid to the total fatty acid pool in each fraction was determined by gas chromatography. Values are expressed as mean ± SD. *p < 0.05; **p < 0.01, ***p < 0.001 indicates significant differences in relation to the control as indicated by Two-Way analysis of variance (ANOVA) and Bonferroni post-test (A,C,E) or test t and Mann Whitney post-test (B,D,F). The fractions analyzed were: 18:2 (ω-6) – Linoleic acid (LA); 18:3 (ω-6) – Gamma-linolenic acid (GLA); 20:2 (ω-6) – Eicosadienoic acid; 20:3 (ω-6) – Dihomo-gamma-linolenic acid (DGLA); 20:4 (ω-6) – Arachidonic acid (AA); 18:3 (ω-3) – Alpha linolenic acid (ALA); 20:4 (ω-3) – Eicosatetrenoic acid (ETA); 20:5 (ω-3) – Eicosapentaenoic acid (EPA); 22:5 (ω-3) – Docosapentaenoi acid (DPA); 22:3 (ω-3) – Docosahexaenoic acid (DHA).

Figure 2

Wound closure and dermal architecture of late granulation tissue (21 days after lesion) in the control group (C, Black bar) and EPA-group (EPA, grey bar). (A) Wound area percentages during the experimental period and representative photos of wounds during the experiment (n = 7–9 animals/group). Values are expressed as mean ± SEM. *p < 0.05 indicates significant differences in relation to the control as indicated by two-way analysis of variance (ANOVA) and Bonferroni post-test. (B) Histological sections were stained with hematoxylin and eosin. Progression of the re-epithelium is indicated by arrows and graphs of wound diameter (mm) on skin harvested at 3 and 10 days (n = 4–5 animals/group). Scale bar: 1 mm. Values are expressed as mean ± SD. *p < 0.05 indicates significant differences in relation to the control as indicated by test t and Mann Whitney post-test. (C) Representative photomicrographs of skin stained with picrosirius and hematoxylin. The examination without (left) and with (right) polarized light revealed the organization and heterogeneity of collagen fiber orientation, squamous stratified epithelium (asterisk) and bulbs of the hair follicles (black arrowhead) and sebaceous glands (white arrowhead) in 21^st day after lesion induction (n = 3–5 animals/group). Scale bar = 50 µm.

Figure 3

Immunophenotyping and cytokine profile of wound tissue in the control group (C, Black bar) and EPA-group (EPA, grey bar). (A–C) Percentage of positive neutrophils (CD45⁺Ly6G⁺), M1 macrophages (CD45⁺F4/80⁺CD11c⁺), M2 macrophages (CD45⁺F4/80⁺CD206⁺), T helper lymphocytes (CD45⁺TCRb⁺CD4⁺) and T cytotoxic lymphocytes (CD45⁺TCRb⁺CD8⁺) were quantified by flow cytometry in scar tissue harvested: (A) before skin lesion (unwounded); (B) 3 days after wounding and; (C) 10 days after wounding. (D) Concentrations of interleukin 1-β (IL- β), tumor necrosis factor-α (TNF-α), keratinocyte chemoattractant (CXCL1), interleukin-6 (IL-6), interleukin-10 (IL-10), vascular endothelial growth factor (VEGF), metalloproteinase-9 (MMP-9) and tissue inhbitor of metalloproteinase-1 (TIMP-1) in wound tissue was analyzed by ELISA in tissue collected 1, 3 and 7 days after wounding (n = 5–12 animals/group). Values are expressed as mean ± SD. *p < 0.05; **p < 0.01, ***p < 0.001 indicates significant differences in relation to control as indicated by t Test and Mann Whitney posttest.

Figure 4

Wound healing, immunophenotyping, MMP9 and TIMP-1 production of scar tissue on 10^th day after wounding in IL-10^−/− mice and IL-10^−/− supplemented daily with EPA-rich oil. (A) Wound area percentages during the experimental period in IL-10^−/− mice (blue line) and IL-10^−/− mice supplemented daily with EPA-rich oil (red line) (n = 5–7 animals/group). Values are expressed as mean ± SEM. The comparison between the groups was made through two-way analysis of variance (ANOVA) and Bonferroni post-test. (B) Percentage of positive M2 macrophages (CD45⁺F4/80⁺CD206⁺), were quantified by flow cytometry in scar tissue harvested 10 days after wound induction. Values are expressed as mean ± SD (n = 3–4 animals/group). The comparison between the groups was made through t Test and Mann Whitney posttest. (C) MMP9 and TIMP-1 quantification of scar tissue collected 10 days after wound induction. Values are expressed as mean ± SD (n = 5–8 animals/group). *p < 0.05 indicates significant differences in relation to control and ^#p < 0.05 indicates significant differences in relation to IL-10^−/−. (D) Representative photomicrographs of skin stained with picrosirius and hematoxylin. The examination without (left) and with (right) polarized light revealed the organization and heterogeneity of collagen fiber orientation, squamous stratified epithelium (asterisk) and bulbs of the hair follicles (black arrowhead) and sebaceous glands (white arrowhead) in 21^st day after lesion induction (n = 2–5 animals/group). Scale bar = 50 µm.

Figure 5

Effects of EPA on the wound healing process. The intake of EPA-enriched oil leads to an increment in the incorporation of omega-3 in cell membranes, an increase of M2 macrophages and an increase of a key anti-inflammatory cytokine produced by this cell population, interleukin-10, in the scar tissue. This anti-inflammatory effect of EPA is associated with delayed of wound closure and affects the reorganization of collagen.