Proresolution therapy for the treatment of delayed healing of diabetic wounds

Abstract

Obesity and type 2 diabetes are emerging global epidemics associated with chronic, low-grade inflammation. A characteristic feature of type 2 diabetes is delayed wound healing, which increases the risk of recurrent infections, tissue necrosis, and limb amputation. In health, inflammation is actively resolved by endogenous mediators, such as the resolvins. D-series resolvins are generated from docosahexaenoic acid (DHA) and promote macrophage-mediated clearance of microbes and apoptotic cells. However, it is not clear how type 2 diabetes affects the resolution of inflammation. Here, we report that resolution of acute peritonitis is delayed in obese diabetic (db/db) mice. Altered resolution was associated with decreased apoptotic cell and Fc receptor-mediated macrophage clearance. Treatment with resolvin D1 (RvD1) enhanced resolution of peritonitis, decreased accumulation of apoptotic thymocytes in diabetic mice, and stimulated diabetic macrophage phagocytosis. Conversion of DHA to monohydroxydocosanoids, markers of resolvin biosynthesis, was attenuated in diabetic wounds, and local application of RvD1 accelerated wound closure and decreased accumulation of apoptotic cells and macrophages in the wounds. These findings support the notion that diabetes impairs resolution of wound healing and demonstrate that stimulating resolution with proresolving lipid mediators could be a novel approach to treating chronic, nonhealing wounds in patients with diabetes.

Figures

FIG. 1.

Resolution of acute inflammation is altered in db/db mice. A: Resident peritoneal leukocytes in WT and db/db mice (n = 4–5). B: Time course of total leukocyte accumulation during the course of acute peritonitis stimulated by zymosan (0.04 mg ⋅ g−1) challenge (n = 4–7). C: Representative flow cytometry dot plots of Ly6G+ cells and F4/80+ cells in peritoneal exudates obtained 48 h post–zymosan challenge in WT and db/db mice. D and E: Time course of Ly6G+ PMNs (D) and F4/80+ macrophages (MΦ) (E) during acute peritonitis (n = 3–7). F: Time course of apoptotic annexin V+ PMN accumulation in the inflamed peritoneum in WT and db/db mice (n = 3–4). G: Accumulation of FITC–zymosan (left panel) and total MΦ numbers (right panel) in the peritoneum 72 h post–zymosan challenge (n = 3). Data are means ± SEM. *P < 0.05 by two-way ANOVA (B and D–F) or Student t test (A and G).

FIG. 2.

Resolvin D1 restores resolution of acute inflammation and promotes the clearance of apoptotic cells in obese diabetic mice. A: Schematic of treatment protocol of mice challenged with zymosan and then treated with RvD1 and leukocyte differentials (n = 8–9). B: Quantification of apoptotic cells in the thymus of WT and db/db mice challenged with dexamethasone (15 mg ⋅ kg−1) and treated without or with RvD1 (1 μg) (n = 3–4). Top panel: Representative images of TUNEL staining per high power field (HPF), scale bars = 50 µm. Sections were counterstained with methyl green. Data are means ± SEM. *P < 0.05 by one-way ANOVA (B) or Student t test (A). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Resolvin D1 rescues diabetic defects in macrophage phagocytosis. A: Phagocytosis of IgG-opsonized FITC–zymosan by peritoneal macrophages isolated from WT and db/db mice (n = 7–8). B: Phagocytosis in db/db macrophages stimulated without or with RvD1 (0.1 nmol/L; 15 min) in the presence or absence of PI3K inhibitor, wortmannin (200 nmol/L) (n = 3–9). C: Blockade of RvD1-stimulated macrophage phagocytosis in db/db macrophages by Fpr2-antagonist peptide, WRW4 (10 µmol/L) (n = 3–4). D: Levels of cAMP in db/db peritoneal macrophages stimulated with RvD1 (1 nmol/L, 15 min) (n = 4). Data are means ± SEM. *P < 0.05 by one-way ANOVA (B and C) or Student t test (A and D).

FIG. 4.

Diabetes impairs wound healing and metabolism of DHA. A: Progressive changes in cutaneous wound closure in WT and db/db mice. B: Quantitative analysis of wound closure in WT and db/db mice (n = 5). C: Quantification of DHA and downstream metabolites, 17-HDHA, 14-HDHA, and 4-HDHA by liquid chromatography–tandem mass spectrometry analysis using MRM in cutaneous wounds isolated from WT and db/db mice at day (d) 5 (n = 4). The MRM transitions that were used are as follows: DHA, 327 > 283; 17-HDHA, 343 > 147; 14-HDHA, 343 > 161; and 4-HDHA, 343 > 101. Schematic of downstream mediators generated from DHA is also shown. Data are means ± SEM. *P < 0.05 by two-way ANOVA (B) or Student t test (C). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

RvD1 accelerates wound closure and granulation tissue formation in db/db mice. A: Representative images of vehicle (0.1% ethanol in saline) and RvD1-treated (100 ng per day [d] per wound) splinted wounds in db/db mice. Lower panel: Quantitative analysis of wound closure in db/db mice treated without or with RvD1, with the structure of RvD1 shown in the inset (n = 5–9). B: Assessment of granulation tissue area in vehicle or RvD1-treated wounds in db/db mice. Representative histological sections of hematoxylin-eosin staining (top panels, scale bar = 1,000 µm; lower panels, scale bar = 200 µm). Lower panel: Quantification of granulation tissue area (day 27; n = 5). C: Histological analysis of RvD1 receptor, Fpr2, in wounds isolated from WT and db/db mice at day 5 (n = 5). Sections were counterstained with hematoxylin-eosin; scale bars = 200 μm (left two panels) and 50 µm (right two panels) for each group. Quantification of Fpr2 receptor density is shown as pixels per low power field (LPF). Data are means ± SEM. *P < 0.05 by two-way ANOVA (A) or Student t test (B and C). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Apoptotic cell and macrophage levels are decreased in diabetic wounds treated with resolvin D1. A: Representative images of TUNEL staining in db/db wounds treated without or with RvD1 (day 5; n = 4/group). Scale bars = 200 μm (left panel) and 50 μm (right panel). B: Immunofluorescence imaging of CD68+ macrophages in db/db wounds treated without or with RvD1 and harvested at day 5. Scale bars = 200 µm (upper panel) and 50 μm (lower panel) for each group. C: Quantification of TUNEL+ cells and CD68+ cells in db/db wounds treated without or with RvD1 (n = 4) per high power field (HPF). Data are means ± SEM. *P < 0.05 by Student t test. (A high-quality digital representation of this figure is available in the online issue.)