Topical Aspirin Administration Improves Cutaneous Wound Healing in Diabetic Mice Through a Phenotypic Switch of Wound Macrophages Toward an Anti-inflammatory and Proresolutive Profile Characterized by LXA₄ Release

Diabetes is one of the most common diseases in the world, affecting >420 million people worldwide. Nonhealing diabetic wounds, such as diabetic foot ulcers, are major clinical complications because their management shows limited results, raising the need for more effective therapeutic approaches.

The chronic low-grade inflammation observed in the diabetic context has been related to an alteration of endogenous programs for macrophage resolution (1,2) that lead to the formation of chronic wounds (3–5). Several strategies that dampen proinflammatory macrophages or promote their proresolutive phenotype have been considered to help the healing of chronic wounds (6,7). Among these interventions, cell therapy using anti-inflammatory exogenous macrophage delivery were developed. As previously reported in humans, the injection of blood-derived macrophages stimulated by hypo-osmotic shock increased the expression of genes related to wound repair, resulting in healing of a majority of pressure and diabetic ulcers compared with a weak healing with standard treatment (8,9). Interestingly, mesenchymal stromal cells infused in murine wound healing induced an anti-inflammatory phenotype in surrounding macrophages, leading to an accelerated wound healing (10).

Pharmacological approaches to promote an anti-inflammatory or prorepair phenotype of endogenous macrophages have also been developed. Goren et al. (11) showed that endogenous proinflammatory macrophage attenuation by anti–tumor necrosis factor-α (TNF-α) and anti-F4/80 therapies in obese diabetic mouse models accelerated the healing of diabetic wounds. Moreover, platelet-derived growth factor treatment in patients with chronic neuropathic diabetic ulcers increased the incidence of complete wound closure and significantly reduced the time to complete closure through its ability to stimulate the production of tumor growth factor-β (TGF-β) by macrophages (7,12). Likewise, granulocyte macrophage colony-stimulating factor injected perilesionally has also shown benefits in the treatment of chronic venous leg ulcers in humans through its activity on macrophage activation of the wound bed (13,14).

Macrophages directly contribute to the control of inflammation in the wound area by secreting pro/anti-inflammatory cytokines, arachidonic acid (AA) metabolites, and reactive oxygen or nitrogen species. Although strategies developed in tissue repair mainly focus on cytokines, specifically targeting the macrophage production of proresolutive AA-derived eicosanoids, such as lipoxins, represents an attractive strategy (15). Among the pharmacological modulators that address this AA metabolic pathway, aspirin by acetylating cyclooxygenase 2 (COX-2) allows the formation of 15R hydroxyeicosatetraenoic acid (15R-HETE) (16), which is then metabolized via 5-lipoxygenase (5-LOX) to generate 15-epi-lipoxin A4 (LXA₄) anti-inflammatory eicosanoid (17). In this context, aspirin administration could improve cutaneous wound healing through its impact on macrophage lipoxin production, leading to a phenotypic switch of wound macrophages toward an anti-inflammatory and proresolutive profile. In the current study, we demonstrate in a murine experimental model of excisional skin injury in the type 2 diabetic context that topical aspirin treatment improves cutaneous wound healing by orienting the phenotype of wound macrophages toward an anti-inflammatory and proresolutive profile characterized by LXA₄ release.

Mice were bred and handled by the protocols approved by Conseil Scientifique du Centre de Formation et de Recherche Experimental Medico Chirurgical and the ethics board of Midi-Pyrénées for animal experimentation (experimentation permit no. 31-067, approval no. B3155503).

Eight-week-old C57BL/6J, Alox5tm1Fun/J (5-LOX knockout [KO]) and 12/15 Alox^−/− (12/15-LOX KO) male mice were purchased from Janvier Labs. Animals were housed in a temperature-controlled animal facility (23°C) with a 12-h light/dark cycle and provided with HFD diet (Research Diets Inc.) or normal chow diet (SSNIFF, GMGH), and water ad libitum. All cages were changed twice weekly, and all manipulations of animals were done in a laminal flow hood under aseptic conditions. The model of type 2 diabetes is described in the Supplementary Materials.

A 0.8-cm² circular full-thickness skin excision wound was performed on the back of high-fat diet (HFD)–induced diabetic mice and normal chow (NC)–fed lean mice, and a healing chamber was noninvasively implanted on the injury (18,19). This model is detailed in the Supplementary Materials.

The wounds were photographed at day 0 postinjury and daily until complete healing, and the percentage of wound closure was calculated as described in the Supplementary Materials. The mice were topically treated with aspirin (36 μg/wound in 200 μL; Sigma-Aldrich), with vehicle (NaCl 0.9%, 200 μL; Baxter), or with leukotriene (LT) A₄m (140 ng; Cayman Chemical).

On days 7 and 14 postinjury, the wounds were removed and then fixed in paraformaldehyde, dehydrated, and embedded in paraffin. Sections were stained with hematoxylin-eosin for cellular diagnosis or Sirius red for the visualization of mature collagen fibers (type I).

For histology scoring, all slides were examined double blinded. The four parameters measured were epidermal and dermal regeneration, presence and size of scar crust, and inflammatory status (granulation tissue). The samples were scored from 1 to 3, with 1 indicating the lowest healing score and 3 the highest (20). Wound merges from each histological section and normal wounds were used as controls for the evaluation.

Collection of exudate was performed by the administration of 200 μL of PBS directly into the healing chamber, and the wash was recovered. Consistent with the earlier disappearance of exudates in NC-fed mice, the wash was recovered for analysis from day 1 to day 5 postinjury in NC-fed mice and from day 1 to day 7 for HFD-fed mice.

The cells of wound exudate were labeled with the following antibodies: Ly6G-antigen-presenting cell (APC) (BD Biosciences), 7/4-FITC (Serotec), F4/80-APC (BD Biosciences), and CD36-phycoerythrin (Santa Cruz Biotechnology). For efferocytosis quantification, wound macrophages were labeled with F4/80-phycoerythrin antibody (AbD Serotec), permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and marked with Ly6G-APC antibody. The percentage of positive cells for each marker was compared with isotype control antibodies. A total of 10,000 events were acquired for each sample on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest Pro software. All analyses were gated on viable cells after live/dead staining (Molecular Probes LIVE/DEAD Fixable Violet Dead Cell Stain Kit; Life Technologies). The gating strategy is described in Supplementary Fig. 1.

Wound macrophages from exudate were allowed to adhere for 2 h at 37°C and 5% CO₂. Nonadherent cells were removed by washing. Neutrophils were isolated using Neutrophil Isolation Kit (MACS; Miltenyi) and then treated with staurosporine (1 μmol/L; Sigma-Aldrich) during 4 h to induce apoptosis. The apoptotic neutrophils were added to the wound macrophages for 1 h at 37°C. Efferocytosis of apoptotic neutrophils by macrophages was analyzed using flow cytometry by monitoring the percentage of F4/80⁺ macrophages in wound exudates that were colabeled with LY6G⁺.

Efferocytosis was also evaluated by the quantification of myeloperoxidase (MPO) activity of wound macrophages. To measure MPO activity, 100 μmol/L of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) and 0.1 mol/L of H₂O₂ (Sigma-Aldrich) were injected. Chemiluminescence was measured using a thermostatically (37°C) controlled luminometer (EnVision; PerkinElmer).

Quantitative real-time PCR was performed on a LightCycler 480 system (Roche Diagnostics) using LightCycler SYBR Green I Master (Roche Diagnostics). GAPDH mRNA was used as invariant control. Serially diluted samples of pooled cDNA were used as external standards in each run for the quantification. Primer sequences are listed in Supplementary Table 1.

Tissue was crushed and homogenized in Lysing Matrix Tubes containing 15% methanol. Exudates were concentrated and purified by solid phase extraction. The concentrations of LXA₄, 15-epi-LXA₄, and LTB₄ in the wounded skin tissue were measured by enzyme immunoassay (Oxford Biomedical Research, Cayman Chemical) according to the manufacturer’s instructions. TNF-α, interleukin-1β (IL-1β), and IL-10 quantification in exudates was evaluated using the OptEIA Mouse Set (Becton Dickinson France) according to the manufacturer’s instructions.

In vivo data are expressed as mean ± SD, and in vitro and ex vivo data as mean ± SEM. Statistical analysis was performed using the means multiple comparison method of Bonferroni-Dunnett test. P < 0.05 was considered statistically significant.

All data generated or analyzed during this study are included in the published article (and its online supplementary files. The resource generated and/or analyzed during the current study is available from the corresponding author upon reasonable request.

To determine whether topical treatment with aspirin improves wound healing in the context of type 2 diabetes, we first evaluated wound closure after an excisional skin injury in a diet-induced diabetic mouse model (Fig. 1 and Supplementary Fig. 2). From day 4 postinjury, the wound closure was significantly delayed in mice fed an HFD compared with NC-fed mice (Fig. 1B). This delay is reflected by a complete closure of the wound that required 5 additional days in HF-fed mice (Fig. 1B). Interestingly, although topical low-dose aspirin treatment (36 μg/wound/day) did not affect the wound healing process in NC-fed mice, it improved wound closure from day 1 posttreatment (day 4 postinjury) in HFD-fed mice (Fig. 1B). The treatment of wounds with aspirin in HFD-fed mice restored the wound closure kinetics observed in NC-fed mice. Likewise, topical low-dose aspirin treatment improved wound closure in db/db mice (Supplementary Fig. 3A). Interestingly, the topical administration of a high dose of aspirin (200 μg/wound/day) did not improve wound healing in db/db mice (Supplementary Fig. 3B).

Moreover, low-dose aspirin treatment improved wound contraction in HFD-fed mice from day 4 postinjury to a level similar to NC-fed mice at day 5 postinjury (Supplementary Fig. 4). Taken together, our results show that topical application of aspirin accelerated the repair of excisional wounds both in genetic and nutritional type 2 diabetic murine models.

Skin histology demonstrated that unlike the wounds from NC-fed mice, which showed a structured tissue fiber, a cell architecture, and a continuous epidermis and dermis, wounds from HFD-fed mice still presented at day 14 postinjury a granulation tissue with numerous inflammatory cells at the wound site and a discontinuous epidermis and dermis (Fig. 1C). The inflammatory cell infiltration was confirmed by a robust MPO activity in granulation tissue at day 7 postinjury (Fig. 1D). Interestingly, the wound from HFD-fed mice treated with aspirin showed a complete formation of a new structured epithelium with less inflammatory cell infiltration, as reflected by lower MPO activity (Fig. 1C and D), demonstrating the ability of topical aspirin treatment to restore the histological features observed in NC-fed mice. Consistent with these results, the histological score based on the state of crust on the wound surface, the degree of epithelialization, and the inflammatory status (20) strongly increased in HF-fed mice treated with aspirin, supporting improved wound healing by aspirin in HF-fed mice (Fig. 1E).

To evaluate whether the aspirin treatment affected leukocyte recruitment within the wound, we studied neutrophil and macrophage infiltrations into injury exudates during the early stage of wound healing by flow cytometry using Ly6B (7/4) and Ly6G (Gr-1) antibodies for neutrophil staining and F4/80 for macrophage staining (Fig. 2A and B and Supplementary Fig. 1). Consistent with the delayed wound closure observed in mice fed an HFD, the exudation disappeared earlier in NC-fed mice. In this context, the exudates were aseptically collected from day 1 to day 5 postinjury in NC-fed mice and from day 1 to day 7 for HFD-fed mice.

In accordance with delayed wound closure in mice fed an HFD, the number of neutrophils is strongly increased and persisted until day 7 postinjury in exudate from HF-fed mice compared with NC-fed mice, in which the neutrophils disappeared from J2 postinjury (Fig. 2A). Aspirin treatment did not modify the neutrophil recruitment in exudate from NC-fed mice. However, this topical treatment reduced significantly the number of neutrophils in exudate from HF-fed mice until a complete disappearance of neutrophils in the exudate at day 7 postinjury (Fig. 2A).

Concomitantly with the rapid recruitment and disappearance of neutrophils in wound exudate of NC-fed mice from day 1 postinjury, the number of macrophages strongly increased from day 1 postinjury and then declined drastically from day 2 postinjury until their complete disappearance on day 3 postinjury (Fig. 2A and B), suggesting a great capacity of macrophages to eliminate the neutrophils. In HFD-fed mice, the recruitment of macrophages into the exudate was delayed (day 3 postinjury) compared with NC-fed mice (day 1 postinjury), correlating with a higher number of neutrophils at day 2 postinjury and a later neutrophil decline (Fig. 2A and B). From day 3 postinjury, while the number of macrophages in the exudate from HFD-fed mice decreased, the number of neutrophils persisted, indicating an impaired neutrophil clearance by macrophages in this diabetic context (Fig. 2A and B). Interestingly, aspirin treatment promoted the persistence of macrophages in the exudate of HFD-fed mice (Fig. 2B). Consistently, the neutrophils in exudate of aspirin-treated HFD-fed mice decreased from day 3 postinjury and disappeared at day 7 postinjury, supporting that aspirin treatment can improve the capacity of macrophages to clear the neutrophils.

To dissect the mechanism triggered by aspirin treatment to enhance the capacity of macrophages to clear neutrophils, we evaluated whether aspirin modulates the efferocytosis of apoptotic neutrophils by macrophages, a critical event involved in the resolution of inflammation and in the initiation of wound repair (2,21). At days 3 and 5 postinjury, the percentage of F4/80⁺/Ly6G⁺ cells, which corresponded to the F4/80⁺ macrophages that engulfed Ly6G⁺ neutrophils, decreased significantly in wound exudate from HFD-fed mice compared with the NC-fed mice, demonstrating an impaired efferocytosis by macrophages during type 2 diabetes (Fig. 2C). Although aspirin treatment had no effect on the efferocytosis capacity of wound macrophages from NC-fed mice at day 5 postinjury, it restored the capacity of macrophages from mice fed an HFD to engulf apoptotic neutrophils (Fig. 2C).

To further support that aspirin treatment promotes the efferocytosis of macrophages from HFD-fed mice, we evaluated the MPO activity of macrophages from HFD-fed mice treated or not with aspirin in the presence of apoptotic neutrophils (Fig. 2D). Aspirin treatment significantly increased MPO activity of macrophages from HFD-fed mice, reinforcing the ability of aspirin to improve efferocytosis in type 2 diabetes.

We then assessed the percentage of F4/80⁺ macrophages that expressed CD36, a major macrophage receptor involved in efferocytosis (22,23). In HFD-fed mice, the number of wound macrophages expressing CD36 was significantly decreased compared with macrophages from NC-fed mice (Fig. 2E). Interestingly, aspirin treatment restored the number of wound CD36⁺ macrophages in HFD-fed mice. Altogether, these data suggest that the improvement of wound healing in diabetic mice by aspirin treatment is mediated by increased CD36-dependent efferocytosis.

To investigate whether macrophages are involved in the prolongation of the inflammatory phase and impaired wound healing, we evaluated the polarization of macrophages in exudates at day 3 and day 5 postinjury (after 2 days of aspirin treatment) (Fig. 3). At day 3 postinjury, the macrophages from NC-fed mice were characterized by a weak mRNA expression of proinflammatory markers, such as Fcgr1, CD11b, inducible nitric oxide synthase (iNOS), IL-12, TNF-α, IL-1β, chemokine ligand 22 (CCL22), and C-C chemokine receptor type 2 (CCR2), and a robust expression of Mrc1, CD36, YM1, arginase-1, IL-10, IL-1 receptor agonist, and TGF-β1 anti-inflammatory markers (Fig. 3A). Interestingly, the expression of mRNA encoding anti-inflammatory markers was lower in exudate macrophages from HFD-fed mice than from NC-fed mice. Concomitantly, the proinflammatory mRNA markers, such as Fcgr1, CD11b, iNOS, IL-12, TNF-α, IL-1β, CCL22, and CCR2, were highly expressed (Fig. 3A). These data show that the persistence of the inflammatory phase in HFD-fed mice is associated with a proinflammatory phenotype of the exudate macrophages, whereas in NC-fed mice, the exudate macrophages exhibited an anti-inflammatory and proresolutive phenotype.

Aspirin treatment of HFD-fed mice decreased significantly the mRNA expression of IL-12, TNF-α, and IL-1β proinflammatory cytokines, which was accompanied by the upregulation of IL-10 and TGF-β anti-inflammatory cytokines (Fig. 3B). This finding was further supported by the induction of Mrc1, CD36, YM1, and arginase-1 anti-inflammatory markers and the reduction of Fcgr1, CD11b, iNOS, and CCR2 proinflammatory markers (Fig. 3B). In accordance, the analysis of the protein levels of TNF-α, IL-1β, IL-10, and CD36 is in agreement with their gene expression level (Fig. 3C).

Interestingly, macrophages from wound tissues display similar gene expression patterns, hence demonstrating that exudate macrophages are representative of all macrophages in the wound (Supplementary Fig. 5). These results clearly demonstrate that aspirin treatment shifts the phenotype of wound macrophages in diabetic mice from a proinflammatory profile toward an anti-inflammatory and proresolutive status involved in tissue repair and remodeling.

Because the main pathways for AA metabolism include COX-1 and COX-2 involvement in the production of pro- and anti-inflammatory prostanoids and 5-LOX, 12-LOX, 15-LOX, and LTA₄ hydrolase (LTA₄h) involvement in proinflammatory LTB₄ and anti-inflammatory LXA₄ synthesis (24), their expression was evaluated in macrophages from wounds of NC- and HFD-fed mice treated or not with aspirin. The mRNA level of COX-2 was not differentially expressed in macrophages from NC- and HFD-fed mice (Fig. 4A). However, the mRNA expression of 5-LOX was induced in macrophages from HFD-fed mice, while that of 12/15-LOX was reduced (Fig. 4A). Moreover, the mRNA level of LTA₄h was robustly increased in macrophages from HFD-fed mice. In line with enhanced 5-LOX and LTA₄h gene expression and decreased 12/15-LOX gene expression in macrophages, the level of LXA₄ in wound exudate was reduced, while the LTB₄ level was significantly increased (Fig. 4B). Taken together, these data demonstrate the impact of diabetes on the orientation of AA metabolism toward LT synthesis. Consistent with the improved LTB₄ level in wound exudate from HFD-fed mice, the mRNA expression of LTB₄ receptor BLT2 increased in macrophages from mice fed an HFD (Fig. 4A). Interestingly, aspirin treatment reduced 5-LOX and LTA₄h gene expression in macrophages from HFD-fed mice and increased the mRNA level of 12/15-LOX (Fig. 4A). These findings correlated with an augmentation of LXA₄ level in wound exudate from aspirin-treated HFD-fed mice and a decline of LTB₄ (Fig. 4B). According to this altered LTB₄ level, the mRNA level of BLT2 was poorly expressed in macrophages from HFD-fed mice treated with aspirin (Fig. 4A).

In wound tissues between 3 and 14 days postinjury, the mRNA level of COX-2 was similar in NC- and HFD-fed mice, and the aspirin treatment had no impact on this gene expression (Fig. 4C). At day 3 and day 14 postinjury, the 12/15-LOX mRNA expression was strongly decreased in tissues from HFD-fed mice compared with NC-fed mice (Fig. 4C). Conversely, from day 3 until day 14 postinjury, the 5-LOX and LTA₄h gene expressions were induced in wound tissues from HFD-fed mice. Consistent with these findings, from day 3 postinjury, the level of LTB₄ in injury tissue of HFD-fed mice was robustly increased, while the level of LXA₄ was decreased (Fig. 4D). Remarkably, within 2 days after aspirin treatment (day 5 postinjury), while the 12/15-LOX gene expression in tissue from HFD-fed mice was strongly augmented, the expression of 5-LOX and LTA₄h was reduced (Fig. 4C). In line, a significant decrease of LTB₄ in injury tissue of HFD-fed mice associated with an increase of LXA₄ was observed under aspirin treatment (Fig. 4D).

Consistent with the activity of aspirin on COX-2 acetylation to produce the 15-epi-LXA₄ anti-inflammatory mediator (25–27), we evaluated its levels in wound tissue from NC- and HFD-fed mice treated with aspirin at day 7 postinjury. Aspirin treatment increased 15-epi-LXA₄ levels in tissue from NC- and HFD-fed mice (Fig. 4E). Taken together, these data suggest that delayed wound closure observed in HFD-fed mice is correlated with the generation of proinflammatory LTB₄ through the 5-LOX, LTA₄h axis and with the inhibition of anti-inflammatory LXA₄ release related to a weak 12/15-LOX expression. The topical application of aspirin accelerates the repair of excisional wounds in diabetic mice through the decrease of LTB₄ production correlated with an increase of LXA₄ release induced by 12/15-LOX overexpression.

To further explore the involvement of 5-LOX and 12/15-LOX in the delayed wound closure observed in HFD-fed mice and in prohealing effects of topical aspirin treatment, we monitored the rate of wound closure in NC- and in HFD-fed 5-LOX– or 12/15-LOX–deficient mice (Fig. 5A–E and Table 1). NC- and HFD-fed mice deficient for 5-LOX exhibited a delay of 4 days to obtain a 100% wound closure compared with wild-type (WT) littermates (Fig. 5A and B), demonstrating that 5-LOX is required for complete wound closure.

As expected, topical aspirin treatment improved the wound healing process only in HFD-fed WT mice. Moreover, improved wound healing following topical aspirin treatment in HFD-fed WT mice was not observed in 5-LOX–deficient HFD-fed mice (Fig. 5B), highlighting that 5-LOX is required to mediate the prohealing effects of aspirin (Fig. 5B). Consistently, in wound tissue from 5-LOX–deficient HFD-fed mice, aspirin treatment did not change LTB₄ and LXA₄ production (Fig. 5F and G). Interestingly, in HFD-fed mice invalidated for 5-LOX, we demonstrated that LTA₄m topical treatment restored the positive effect of aspirin on wound healing (Fig. 5C). In line, the LTB₄/LXA₄ balance in wound tissue of 5-LOX–deficient HFD-fed mice treated with aspirin and LTA₄m was in favor of LXA₄ release (Fig. 5F and G). In contrast, in the absence of aspirin, LTA₄m treatment delayed the wound closure in HFD-fed mice (Fig. 5C) and increased the production of LTB₄ (Fig. 5F), confirming the preponderant activation of the 5-LOX, LTA₄h, LTB₄ axis in the diabetic context.

Moreover, wound closure was delayed in NC- and HFD-fed mice invalidated for 12/15-LOX (Fig. 5D and E). Similar to HFD-fed mice invalidated for 5-LOX, the deletion of 12/15-LOX suppressed the amelioration of wound healing induced by aspirin treatment (Fig. 5E) and did not modify LXA₄ and LTB₄ release in wound tissue of HFD-fed mice (Fig. 5F and G), demonstrating that 12/15-LOX is also essential for the beneficial effect of aspirin on wound healing in the diabetic context. These data associated with the restoration of the prohealing effects of aspirin upon LTA₄ treatment in 5-LOX–deficient mice support that the 5-LOX, LTA₄, 12/15-LOX/LXA₄ axis is critical for the beneficial effects of aspirin.

In patients with diabetes, the persistent inflammatory process is responsible of impaired wound healing (28). Indeed, it has been shown that neutrophil infiltration is strongly increased during the inflammatory phase of wound healing in the diabetic context (29). Consistently, we demonstrate here that the number of neutrophils in exudate from diabetic mice was higher than in nondiabetic mice and that the level of LTB₄, a neutrophil chemotactic eicosanoid (30), was increased in the injury exudate and in wound tissue of diabetic animals. We also establish that the greater number of neutrophils in wound exudate of the diabetic mice was correlated with a low proportion of CD36-expressing macrophages and, hence, with an impaired efferocytosis. These data are consistent with in vitro studies showing that macrophages from diabetic db/db mice have a deficiency of apoptotic cell clearance (2) and that efferocytosis is defective in humans with metabolic disorders (31). In addition to the loss of capacity of macrophages to engulf neutrophils, previous data showed a phenotypic and functional deregulation of diabetic monocytes/macrophages (2,32). In line with this, we show here in injury exudate of HFD-fed mice that the persistence of proinflammatory macrophages is characterized by the orientation of their AA metabolism toward LTB₄ production. Thus, given that during wound healing the macrophages switch from a proinflammatory toward an anti-inflammatory and proresolutive phenotype, we demonstrate that the impaired wound healing in diabetic mice is associated with a default of macrophage polarization toward an anti-inflammatory and proresolutive phenotype.

To restore the phenotypic switch of macrophages toward an anti-inflammatory and proresolutive phenotype, our strategy has been to topically treat the wounds with low-dose aspirin, which is known to inhibit prostanoid production and to promote the pathway involved in proresolving lipid mediator production (27,33). In this study, the topical application of 36 μg/wound/day of aspirin promoted the repair of excisional wounds in type 2 diabetic mice through its activity both on contraction and re-epithelialization. A special feature of aspirin is linked to its dose-dependent mechanisms. Indeed, we demonstrate here that the topical administration of 200 μg/day of aspirin did not improve wound healing (Supplementary Fig. 3B). In line, the intraperitoneal administration of aspirin at high doses (625 μg/day or 5 mg/day) in mice slowed cutaneous wound healing (34,35). Conversely, consistent with enhanced wound repair with a low dose of aspirin in our murine model, human studies have shown that low-dose oral administration of aspirin improves healing of chronic venous leg ulcers (36,37). Interestingly, the topical administration in humans of ibuprofen in a foam dressing showed a beneficial effect on venous leg ulcer healing (38), supporting the beneficial use of local nonsteroidal anti-inflammatory drugs in chronic wound healing. Altogether, these studies on wound repair in both rodent and human revealed a different efficacy of COX inhibitors, depending on the wound (27). Indeed, there is a great difference in the microenvironment of a chronic wound compared with an acute wound, with a persistence of active macrophages and neutrophils only in chronic wounds, reinforcing the topical administration of low-dose aspirin mainly in chronic wound treatment. Interestingly, we demonstrate here that the enhanced wound healing by topical aspirin treatment in diabetic mice is associated with the persistence of macrophages in wound exudates and their orientation toward an anti-inflammatory and proresolutive phenotype. In line with previous data showing that aspirin increases CD36 expression in human macrophages (26), we demonstrated that wound macrophages from aspirin-treated mice are characterized by increased CD36 expression and consequently by their higher efferocytosis capacity. Consistently, the neutrophils in exudate of aspirin-treated HFD-fed mice quickly disappear. Thus, aspirin treatment shortens the inflammatory phase and thereby improves wound healing by enhancing the intrinsic ability of macrophages to eliminate the neutrophils (Fig. 6).

In addition, the topical application of aspirin accelerates the repair of excisional wounds in diabetic mice through the orientation of macrophage AA metabolism toward the production of proresolutive LXA₄ metabolite (Fig. 6). Although aspirin decreases the expression of 5-LOX in wound macrophages of HFD-fed mice, its level remains equivalent to the wound macrophages from NC-fed mice, and in this context, the induction of 12/15-LOX and the decrease of LTA₄h by aspirin are sufficient to orient the LXA₄/LTB₄ balance in favor of LXA₄. The role of the 5-LOX, 12/15-LOX, LXA₄ axis in the beneficial effect of aspirin was supported by the lack of its beneficial effect in mice invalidated for 5-LOX and 12/15-LOX and by the restoration of its positive effect by LTA₄ topic treatment in HFD-fed mice invalidated for 5-LOX. Moreover, the impaired wound closure in HFD-fed mice invalidated for 5-LOX treated by LTA₄ without aspirin reinforces that this default of wound healing in HFD-fed mice is related to the increase of LTB₄ production and confirms that aspirin is essential to induce 12/15-LOX expression and, thus, the production of LXA₄.

Interestingly, we demonstrate that topical aspirin treatment slightly increases the level of 15-epi-LXA₄ anti-inflammatory mediator in wound tissue from HFD-fed mice. All these data show that aspirin induces the production of both LXA₄ and 15-epi-LXA₄. However, the absence of a beneficial effect of aspirin on wound healing in HFD-fed mice invalidated for 12/15-LOX and the restoration of this effect by addition of LTA₄ in the HFD-fed mice invalidated for 5-LOX treated with aspirin suggest that in the context of diabetes, the anti-inflammatory and proresolutive effects of aspirin are preferentially mediated by 5-LOX, LTA₄, 12/15-LOX, LXA₄ signaling and not by the COX-2, 15R-HETE, 5-LOX, 15-epi-LXA₄ axis. Although the main studies on the mechanism of action of aspirin identified the COX-2, 15R-HETE, 5-LOX pathway to generate the 15-epi-LXA₄ anti-inflammatory and proresolutive mediator (16,25,37), we highlight alternative aspirin enzymatic targets that lead to the production of the proresolutive LXA₄.

The AA metabolites from 12/15-LOX are essential for peroxisome proliferator–activated receptor γ (PPARγ) endogenous activation (39–41). PPARγ is involved in the balance of macrophage differentiation in favor of anti-inflammatory and proresolutive macrophages (42). In this context, in diabetic mice, the switch of wound macrophage phenotype toward an anti-inflammatory and proresolutive status induced by aspirin treatment could depend on the production of PPARγ endogenous ligands through 12/15-LOX induction. Consistent with these results, in diabetic mice invalidated for 12/15-LOX, PPARγ is not sufficient to improve wound healing because of the absence of its endogenous ligands. Conversely, in diabetic WT mice, aspirin treatment restores the induction of 12/15-LOX and, thus, promotes PPARγ activation through the production of LXA₄ endogenous ligands. This hypothesis was reinforced by reports showing that low doses of aspirin increase PPARγ protein expression (43).

In conclusion, this work provides evidence of the beneficial effect of topical administration of low-dose aspirin in cutaneous wound healing in type 2 diabetic mice. Given that diabetic mice have many fundamental similarities on the tissue and cellular level as humans and that this is a widely accepted model in current wound healing research (44), the restoration of the anti-inflammatory and proresolutive macrophage phenotype by aspirin represents a promising therapeutic approach in patients with diabetes. Since many chronic wounds do not improve with standard care (7), the introduction of advanced therapies targeting and correcting the aberrations that occur in macrophage populations in chronic wounds may be an effective method to stop the sustained inflammation in the diabetic context and to return to a healing state. In this context, the topical administration of low-dose U.S. Food and Drug Administration–approved aspirin appears to be an effective therapy in chronic wounds. This pharmacological approach highlights a new mechanism of resolution of wound inflammation in the diabetic context and novel enzymatic targets promoting the healing process.

Acknowledgments. The authors thank Alexia Zakaroff-Girard and Elodie Riant (TRI Imaging Platform, IFR150/I2MC) for flow cytometry technical assistance and Philippe Batigne (Université Paul Sabatier) for technical support in animal experimentation. The authors also thank Coralie Sengenes for helpful discussion and kind assessment.

Funding. This research was supported by a grant from CIFRE-Association Nationale de la Recherche et de la Technologie to C.D. and from Laboratoires URGO.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.D., M.S., M.B., L.L., B.P., and A.C. designed the study and analyzed the data. M.S., L.L., B.P., and A.C. wrote the manuscript. C.D., M.S., H.A., E.M., M.A., and J.B. performed the experiments and analyzed the data. A.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.