GRAPHICAL ABSTRACT

Highlights

• 4-OI promotes diabetic wound healing via macrophage-dependent and independent actions.

• 4-OI enhanced macrophage function through MCT1-mediated lactate release after efferocytosis.

• Extracellular lactate signals are transmitted through G-protein–coupled receptor 132.

INTRODUCTION

In recent years, the prevalence of diabetes mellitus (DM) has been increasing, and patients with diabetes are susceptible to multi-system damage, including diabetic kidney disease, cardiovascular disease, and retinopathy [1]. Among these complications, diabetic foot ulcers have emerged as one of the most common and serious complications in patients with diabetes, with a lifetime risk of 19% to 34% [2]. They are characterized by high recurrence rates, a high incidence of lower-extremity amputation, and high 5-year mortality. As a result, they are costly and seriously affect quality of life in individuals with diabetes [3]. Consequently, considerable effort has been dedicated to exploring the pathogenesis of diabetic wounds and developing novel therapeutics.

Classically-activated macrophages, which exhibit pro-inflammatory effects and are defined as the M1 phenotype in vitro, play a central role in the late inflammatory phase. In contrast, alternatively activated macrophages, characterized by their pro-resolving effects and defined as the M2 phenotype in vitro, are important for promoting tissue repair. Even though recent studies have identified multiple macrophage subtypes at wound sites, a fine-tuned balance between classically and alternatively activated macrophages provides a fundamental basis for normal wound healing. In non-healing diabetic wounds, impaired function of infiltrating macrophages leads to a hyper-inflammatory phenotype at the wound site and consequently results in delayed wound healing. Efferocytosis is defined as the process by which apoptotic cells are cleared by professional and non-professional phagocytes, such as macrophages, dendritic cells (DCs), fibroblasts, and epithelial cells [4]. Clearance of dead and dying cells through efferocytosis plays a crucial role in regulating inflammatory resolution by preventing secondary necrosis [5]. Furthermore, efferocytosis can trigger a pro-resolving macrophage phenotype through metabolic reprogramming [6], proliferation of pro-resolving macrophages [7], and M2 macrophage polarization [8]. Consequently, dysregulated pro-resolution macrophage functions can impair resolution of inflammatory responses and delay wound healing.

Monocarboxylate transporter 1 (MCT1) is a membrane transporter protein in the solute carrier protein family. It is encoded by the solute carrier family 16 member 1 (SLC16A1) gene, and is mainly responsible for transporting lactate, pyruvate, and other monocarboxylic acids. Recently, several studies have shown that MCT1 is critical for efferocytosis-induced lactate release which subsequently promotes macrophages toward anti-inflammatory/M2 polarization, continual efferocytosis, and pro-resolving macrophage proliferation [7,9]. Additionally, MCT1 has been identified as an important determinant of macrophage phenotype by promoting phagocytosis of apoptotic cells [10,11]. Based on these findings, we wondered whether MCT1 is involved in the regulation of macrophage function during diabetic wound healing.

Itaconate is a metabolite produced when the tricarboxylic acid cycle is bypassed during energy production [12]. In mammals, it is mainly produced by decarboxylation of cis-aconitic acid catalyzed by cis-aconitic acid decarboxylase, which is encoded by immune responsive gene 1 (Irg1) [13]. In addition to its antimicrobial effect, recent research has highlighted the immunoregulatory properties of itaconate because of its critical effects in regulating inflammatory immune responses and oxidative stress [14]. Itaconate has been proposed as a potential therapeutic agent for inflammatory diseases, autoimmune diseases, and cancer [15–20]. As a carboxylic acid itaconate, is highly polar and exhibits limited cell membrane permeability, which makes it unsuitable for mechanistic studies [21]. Therefore, many studies of itaconate use its derivative, 4-octyl itaconate (4-OI), which is more permeable to cell membranes than its parent compound. Compared with other derivatives, 4-OI shares similar thiol reactivity with itaconate and is converted into itaconate intracellularly [12,22]. Furthermore, both 4-OI and itaconic acid consistently inhibited activation of the non-obese diabetic (NOD)-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome [23]. Some studies have also demonstrated that 4-OI is a promising therapeutic substance for diabetic wound repair [24–26]. However, the mechanism through which itaconate effects diabetic wound healing remains unknown. In the current study, we first confirmed the effect of 4-OI on diabetic wound healing. Then, we observed its ameliorative effects on apoptotic neutrophil efferocytosis, M2 macrophage polarization, inflammatory status, and angiogenesis at diabetic wound sites. In addition, we further investigated whether 4-OI promotes wound healing and rescues dysregulated macrophage function through upregulated MCT1 expression and subsequent lactate release.

METHODS

Animals

Five to 8-week-old C57BL/6J male mice were supplied by Chengdu Yaokang Biotechnology Co. Ltd. and Luzhou Yinhui Biotechnology Co., Ltd. in China. All mice were kept in an environment with a 12-hour light/dark cycle and given free access to food and water. Mice were randomly divided into six groups: negative control (NC; treated with solvent), DM (diabetic group; no treatment), 4-OI (diabetic group, treated with 4-OI), 4-OI+MCT1 inhibitor-AZD3965 (diabetic group treated with 4-OI and AZD), 4-OI+telmisartan (diabetic group treated with 4-OI and telmisartan), and 4-OI+clodronate-liposomes (diabetic group treated with 4-OI and CLD-Lipo). To introduce type 1 DM, mice were intraperitoneally injected with low-dose streptozotocin (STZ; 50 mg/kg, Beijing Solarbio Technology Co., Beijing, China) for 5 consecutive days. Mice with random blood glucose levels that exceeding 16.7 mmol/L were considered diabetic. One month after blood glucose stabilization, a 1-cm-diameter full-thickness wound was created on the back of each mouse.

To determine the effect of 4-OI on diabetic wound healing, mice were treated with 4-OI (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 10 mg/kg by intraperitoneal injection after successful wound modeling. Moreover, to examine the role of MCT1 in 4-OI-promoted diabetic wound healing, mice were treated with AZD3965 (MedChemExpress) at a dose of 100 mg/kg by intraperitoneal injection for 7 days after successful wound modeling. To further investigate the effect of the G protein-coupled receptor 132 (GPR132) inhibitor telmisartan on macrophage function and diabetic wound healing after 4-OI treatment, mice were treated with telmisartan (MedChemExpress) at a dose of 5 mg/kg by intraperitoneal injection for 7 days after successful wound modeling. To determine the role of macrophages in the response to 4-OI treatment, mice were treated with CLD-Lipo (YEASEN Biotechnology, Shanghai, China) at a dose of 10 μg/g intraperitoneally three times within 1 week after successful wound modeling. Wound images were collected on days 3, 7, 11, and 14 after wound modeling, and three mice in each group were randomly selected for euthanasia and sampling.

Histopathological examination

Skin tissues were fixed in 4% paraformaldehyde for 24 hours at room temperature, dehydrated through a graded ethanol series, embedded in paraffin, and cut into 5-μm-thick sections. The tissue sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome (Beijing Solarbio Technology Co.) for morphometric measurements. The sections were observed under a microscope (Leica, Wetzlar, Germany).

Tissue immunofluorescence staining

Wound tissue sections were deparaffinized and then blocked with 10% goat serum for 2 hours at room temperature. Tissues were then incubated overnight at 4°C with primary antibodies against F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-377009), inducible nitric oxide synthase (iNOS; Cell Signaling Technology, Danvers, MA, USA; 13120), arginase 1 (Arg-1; Cell Signaling Technology; #93668), CD31 (Beyotime Biotechnology, Shanghai, China; AF6408), and MCT1 (Proteintech, Rosemont, IL, USA; 20139-1-AP), followed by 1 hour of incubation with fluorescent dye-labeled secondary antibodies (Beyotime Biotechnology; A0428 or A0453). Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). For double staining experiments, the TSAPLus Fluorescent Triple Staining Kit (Wuhan Xavier Biotechnology Co. Ltd., Wuhan, China) was used according to the manufacturer’s instructions. Immunofluorescent images were obtained using a fluorescence microscope (Olympus, Tokyo, Japan).

Quantitative polymerase chain reaction

Total RNA from skin wound tissue was extracted with TRIzol. cDNA was synthesized using the ReverTra Ace quantitative polymerase chain reaction (qPCR) RT Master Mix (TOYOBO, Osaka, Japan; FSQ-201). qRT-PCR analyses were performed using cDNA from the reverse transcription reactions, gene-specific primers, and 2× SYBR Premix Ex Taq II (Takara Bio, Kusatsu, Japan). All primers for qRT-PCR are listed in Supplementary Table 1.

Cell culture

RAW264.7 cells and Jurkat cells were purchased from the cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences. Both cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (Sciencell, Carlsbad, CA, USA). Cells were incubated at 37°C with 5% CO₂. According to the intervention conditions, 4-OI, AZD3965, and telmisartan were added to RAW264.7 cells induced by high glucose.

Cellular immunofluorescence

RAW264.7 cells were seeded on 24-well chamber slides, with additional stimuli added at appropriate times as needed for each experiment. The cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Fixed cells were rinsed with phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature, and blocked with 10% goat serum for 1 hour. Cells were then incubated overnight at 4°C with primary antibodies against MCT1, Arg-1, iNOS (Santa Cruz Biotechnology; sc-7271), Ki67 (Cell Signaling Technology; 9129). The cells were then rinsed with PBS and incubated with fluorescent dye-labeled secondary antibodies for 1 hour at room temperature in the dark. Cell nuclei were labeled with DAPI and immunofluorescence was observed and captured using a live cell workstation microscope (Olympus).

Measurement of cell viability

Cell viability was measured using the cell counting kit-8 (CCK8; Beyotime Biotechnology). RAW264.7 cells at a density of 1.92× 10⁴ cells per well were cultured in 96-well plates at 37°C and 5% CO₂. After different intervention stimuli, the supernatants were discarded, medium containing 10% CCK8 was added to each well, and after incubation at 37°C for 2 hours, the absorbance of each well was measured at 450 nm. Each independent experiment was performed at least three times.

Efferocytosis assay

Jurkat cells were treated with staurosporine (2 μM) to obtain apoptotic Jurkat cells. Next, a sufficient number of apoptotic Jurkat cells was added to serum-free 1640 medium containing carboxytetramethylrhodamine (TAMRA) dye and incubated for 15 minutes for staining. A 0.3 μM 5-chloromethylfluorescein diacetate (CMFDA) probe working solution (YEASEN Biotechnology) was prepared in serum-free 1640 medium and preheated for 15 minutes at 37°C. The pre-warmed probe working solution was added to RAW264.7 cells and incubated for 30 minutes, after which the medium was replaced with fresh culture medium for another 30 minutes. Finally, apoptotic Jurkat cells were co-cultured with RAW264.7 cells at a ratio of 3:1 for 3 hours; then, rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After rinsing again with PBS, the macrophages were observed and captured using a fluorescence microscope.

Efferocytosis-induced lactate release assay

RAW264.7 cells were inoculated in six-well plates at a density of 1.25×10⁶ cells/well and treated with different intervention stimuli for 48 hours. Jurkat cells were treated with staurosporine (2 μM) to obtain apoptotic Jurkat cells. Next, apoptotic Jurkat cells were co-cultured with RAW264.7 cells at a ratio of 3:1 for 3 hours, 1 mL of culture supernatant was collected, and the extracellular lactate content was detected using the lactate assay kit (Rexin Bio; article No. RXWB0476-96). Lactate extract (200 μL) was added to the cell precipitate to detect the intracellular lactate content. The protein content of the cells in each group was detected and used to correct the lactate content.

Statistical analyses

Statistical analysis was performed using GraphPad Prism version 8.0 software (GraphPad Software Inc., San Diego, CA, USA), and data are expressed as mean±standard deviation. Multiple group comparisons were performed using one-way analysis of variance (ANOVA). For all tests, P<0.05 was considered statistically significant.

RESULTS

4-OI hastens wound healing in STZ-induced diabetic mice

To investigate the effect of 4-OI on diabetic wound healing, we used an STZ-induced diabetic mouse model. After successful modeling, we created a 1-cm-diameter full-thickness wound on the back of each mouse, and then administered 4-OI intraperitoneally for 2 weeks. Photographs were taken and wound healing rates were calculated on days 3, 7, 11, and 14 after wound model generation. As shown in Fig. 1A and B, diabetic mice exhibited prolonged wound healing time and reduced wound closure rates compared with the control group. In contrast, 4-OI administration significantly promoted wound healing, with enhanced closure rates compared with those in diabetic mice. Furthermore, histological analysis showed that 4-OI treatment improved the wound tissue structure, with fewer infiltrating neutrophils, better collagen deposition, and denser arrangement compared with diabetic mice (Fig. 1C). With immunofluorescence staining for CD31, we also observed that 4-OI treatment promoted angiogenesis in the wound tissue of diabetic mice (Fig. 1D). Additionally, we also analyzed blood glucose levels in mice. In 4-OI-treated mice at 3 days, there was no significant difference in random blood glucose compared with diabetic mice (Fig. 1E). These results suggest that 4-OI treatment promotes wound healing in STZ-induced diabetic mice.

4-OI promotes M2 macrophage polarization, and enhances inflammatory resolution in diabetic mice

The repair process of acute wounds is closely related to the orchestrated balance of M1/M2 macrophage polarization. Neutrophil clearance by efferocytosis is an important function of M2 macrophages. Therefore, we explored whether the promotion of diabetic wound healing by 4-OI is associated with macrophage polarization. We evaluated macrophage infiltration at the wound site and observed significant infiltration of macrophages (F4/80⁺), with an increase in the number of M1-type macrophages (F4/80⁺iNOS⁺) and a decreased number of M2-type macrophages (F4/80⁺Arg-1⁺) in diabetic wounds. In contrast, macrophage infiltration was significantly reduced in 4-OI-treated wounds at 3 days (Fig. 2A and B) and 7 days (Supplementary Fig. 1), with a decreased number of M1-type macrophages and an increase in M2-type macrophages. In vitro, high glucose stimulation promoted M1 macrophage polarization. However, 4-OI treatment promoted M2 macrophage polarization (Fig. 2C). Consistent with the enhanced infiltration of pro-inflammatory M1 macrophages, 4-OI intervention resulted in a significant reduction in the expression of the pro-inflammatory factors interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and an increasing tendency but no significant alteration in the expression of the inflammation-resolving factor transforming growth factor-β (TGF-β) in the wounds at 3 and 7 days (Fig. 2D). These data suggest that 4-OI treatment promotes the conversion of M1-type macrophages to M2-type macrophages and reduces the expression of pro-inflammatory factors in the wound tissue of diabetic mice.

Next, we further examined the role of macrophages in 4-OI promoted diabetic wound healing. Mice were treated with CLD-Lipo to deplete macrophages (Supplementary Fig. 2A). Interestingly, 4-OI treatment further promoted wound healing in diabetic mice even under macrophage depletion conditions (Supplementary Fig. 2B). Histopathologic examination revealed that macrophage depletion reduced inflammatory cell infiltration and promoted collagen deposition in the wound tissue (Supplementary Fig. 2C). Compared with diabetic mice, CLD-Lipo intervention resulted in a significant reduction in the expression of the pro-inflammatory factors IL-1β, IL-6, and TNF-α, and a tendency toward increased expression of the inflammation-resolving factor TGF-β in the wounds at 7 days. However, no significant alterations were observed compared with the 4-OI group (Supplementary Fig. 2D).

These results suggest that both macrophage-dependent and pathways contribute to the acceleration of wound healing induced by 4-OI. Apart from this, the findings may reflect the complex function of macrophages, with pro-inflammatory M1 macrophages predominating in diabetic wounds. In the current study, we focused on the mechanisms involved in M2 macrophage polarization, and subsequent inflammatory resolution after 4-OI intervention in diabetic mice.

4-OI ameliorates neutrophil infiltration and promotes apoptotic neutrophil efferocytosis

Persistent chronic inflammation is associated with delayed wound healing in diabetes, and should be partially attributed to excessive neutrophil infiltration and decreased clearance [27]. We explored whether 4-OI could play a role in neutrophil infiltration and clearance at diabetic wound sites. Therefore, we detected neutrophils and apoptotic cells in the wound tissues, and the results showed that the numbers of neutrophil-infiltrating (lymphocyte antigen 6 family member G [Ly6G]⁺) cells and apoptotic cells (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]-positive) in the diabetic group were higher than those in the control group, and were suppressed by 4-OI treatment (Fig. 3A and B). These findings suggest that 4-OI treatment reduces neutrophil infiltration in wound tissue, and promotes clearance of apoptotic cells.

During wound regeneration, apoptotic neutrophils should be removed by macrophages through efferocytosis, which is considered an anti-inflammatory and pro-resolving event. This process is impaired in chronic wounds, including those caused by diabetes [28]. We explored whether 4-OI intervention promotes apoptotic neutrophil efferocytosis by macrophages. As expected, we found that many macrophages (F4/80⁺) contained Ly6G⁺ remnants, consistent with macrophage efferocytosis. The frequency of these efferocytic F4/80⁺Ly6G⁺ macrophages was lower in diabetic wounds, and increased after 4-OI treatment (Fig. 3C). In vitro, we co-cultured apoptotic Jurkat cells (TAMRA-labeled fluorescent dye) with RAW264.7 mouse macrophages (CellTracker fluorescent dye-labeled). Consistently, the ability of macrophages to phagocytose apoptotic neutrophils decreased in vitro after high glucose (HG, 30 mM) stimulation (Fig. 3D). We found that high glucose intervention significantly reduced efferocytosis of apoptotic cells (TAMRA⁺CellTracker⁺) by murine RAW264.7 macrophages. In contrast, 4-OI administration ameliorated the phagocytotic ability of macrophages (Fig. 3E).

4-OI improves macrophage function and diabetic wound healing through MCT1-mediated lactate release triggered by efferocytosis

Efferocytosis-derived lactate plays an important role in regulating macrophage function, including pro-resolving macrophage proliferation, continual efferocytosis and consequent inflammatory resolution [9,10,29]. MCT1 has been reported to act as a critical membrane transporter that facilitates lactate release during efferocytosis [10]. Interestingly, MCT1 expression was significantly decreased in macrophages (MCT1⁺F4/80⁺) in diabatic wounds (Fig. 4A). In vitro experiments showed that, high glucose stimulation decreased MCT1 expression in cultured murine RAW264.7 macrophages, and MCT1 was mainly distributed in the cytoplasm, with an inconspicuous distribution on the cell membranes. In contrast, 4-OI administration upregulated MCT1 expression, and MCT1 aggregated on macrophage cell membranes (Fig. 4B). Consistently, high glucose stimulation led to decreased lactate release from macrophages after challenge with apoptotic cells, and 4-OI treatment led to increased lactate release (Fig. 4C). In addition, we evaluated the effect of 4-OI on macrophage proliferation and cell viability using CCK8 and Ki67 staining. After co-culture with apoptotic cells, decreased proliferation and cell viability were observed under high glucose conditions, and 4-OI significantly promoted macrophage proliferation and cell viability (Fig. 4E and F). These results suggest that 4-OI rescues dysregulated macrophage function through upregulated MCT1 expression and subsequent lactate release.

To confirm this hypothesis, MCT1 inhibitor AZD3965 was used (Fig. 4H). In vivo, AZD3965 administration blocked 4-OI-induced M2 macrophage polarization and impaired diabetic wound healing (Fig. 4I–K), qPCR assays also revealed that AZD3965 administration elevated the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α and reduced the expression of the inflammatory-resolving factor TGF-β in wounds at 7 days (Fig. 4L). Histological analysis showed that AZD3965 treatment counteracted the effects of 4-OI, increasing neutrophil infiltration and reducing collagen deposition (Fig. 4M). In vitro, AZD3965 administration blocked 4-OI-induced lactate release and continual efferocytosis (Fig. 4C and D), and blocked the effects of 4-OI on macrophage proliferation and pro-resolving macrophage polarization (Fig. 4E–G).

4-OI-induced lactate release promotes macrophage proliferation and M2 macrophage polarization through GPR132 receptors

Efferocytosis-derived lactate has been observed to be released and to promote macrophage proliferation through cell-surface GPR132 [7]. We next explored the effect of the GPR132 inhibitor telmisartan on macrophage function and diabetic wound healing after 4-OI treatment. As expected, telmisartan blocked 4-OI-induced macrophage proliferation and cell viability (Fig 5A–C), resulting in decreased pro-resolving macrophage polarization (Fig. 5D). Consistently, in vivo, telmisartan administration blocked 4-OI-induced M2 macrophage polarization and impaired diabetic wound healing (Fig. 5E–G). In addition, telmisartan treatment blocked the effect of 4-OI on the resolution of diabetic wound inflammation (Fig. 5H). H&E and Masson staining showed that telmisartan administration counteracted the beneficial effects of 4-OI on wound tissue structure, increased infiltrating neutrophils, and decreased collagen deposition (Fig. 5I).

DISCUSSION

Recently, 4-OI, a derivative of itaconate, has been observed to ameliorate wound healing [24]. However, the mechanism of action of itaconate in diabetic wound healing is unknown. In the current study, we also found a protective effect of 4-OI on diabetic wound healing. In diabetic mice, excessive accumulation of apoptotic neutrophils and sustained M1 macrophage activation were observed at the wound site, with delayed clearance of apoptotic neutrophils and impeded resolution of inflammation. As expected, 4-OI intervention promoted efferocytosis of apoptotic neutrophils, M2 macrophage polarization and inflammatory resolution. Moreover, administration of 4-OI upregulated MCT1 expression and consequently led to increased lactate release. Finally, we confirmed that 4-OI enhanced pro-resolving macrophage function through MCT1-mediated lactate release and consequent activation of GPR132 by lactate.

Wound healing is a complex process involving the coordinated actions of various tissues and cellular lineages, including immune cells, epidermal cells, and connective tissue cells. Immunocytes, especially macrophages and neutrophils, play an important role in the early inflammatory response, whereas keratinocytes and fibroblasts play a key role in the repair process. Regulatory T cells facilitate healing process through immunomodulatory effects. Skin stem cells support tissue repair and regeneration through regenerative actions [30]. In recent years, emerging evidence has shown that itaconate plays an important role in regulating macrophage function and subsequently results in protective effects in acute liver failure [31], myocardial ischemia-reperfusion [32], and some chronic inflammatory diseases [33]. In our study, we observed a significant decrease in the number of M1-type macrophages and an increase in M2-type macrophages in 4-OI-treated wounds, indicating an important role of macrophages in 4-OI-promoted wound healing. To clarify the role of macrophages in 4-OI promoting diabetic wound healing, diabetic mice were treated with CLD-Lipo to deplete macrophages. Wound healing was shown to be faster in diabetic mice with and without 4-OI treatment under macrophage depletion conditions. The complexity of macrophage function may account for these results. In diabetic wounds, pro-inflammatory M1-type macrophages are the predominant phenotype, with few anti-inflammatory M2-type macrophages present [33]. This phenotypic imbalance of macrophages causes a persistent inflammatory state in diabetic wounds, hindering the pro-healing activities of endothelial cells, keratinocytes, and fibroblasts [32]. Therefore, in diabetes, the most significant effect of removing macrophages may be to eliminate the pro-inflammatory effect of macrophages, thereby promoting wound healing. Consistent with our results, CLD-Lipo-mediated macrophage depletion ameliorated corneal nerve involvement [34], postponed the development of diabetic neuropathic pain [35], and improved systemic glucose homeostasis and insulin sensitivity [36]. Furthermore, these data also suggest that, in addition to macrophage-dependent mechanisms, macrophage-independent pathways may be involved. In this current study, we also found that 4-OI treatment significantly reduced neutrophil infiltration and improved angiogenesis in diabetic wounds. This indicates that, in addition to macrophages, neutrophils and endothelial cells were also involved in the protective effect of 4-OI in diabetic wound healing. Consistently, it has been reported that 4-OI promotes angiogenesis by activating extracellular signal-regulated kinase (ERK) in endothelial cells and alleviates myocardial ischemia-reperfusion injury [37]. Furthermore, DCs [38] and fibroblast-like synoviocytes [39] have also been reported to be target cells of itaconate, resulting in enhanced immunotherapy sensitivity and ameliorated rheumatoid arthritis. Therefore, further studies are warranted to clarify whether additional cell types and signaling pathways also contribute to the protective effect of 4-OI in diabetic wound healing.

Recently, emerging evidence has highlighted the roles of impaired efferocytosis in a variety of chronic inflammatory diseases, including diabetic foot ulcers [40], diabetic periodontitis [41], non-alcoholic steatohepatitis [42], and atherosclerosis [43,44]. Macrophages isolated from the peritoneum of NOD mice or wounds of obese diabetic (db/db) mice showed significant impairment in the phagocytosis of apoptotic cells [27,45]. Moreover, it is well known that efferocytosis of apoptotic cells is critical for promoting M2 polarization of macrophages and establishing a pro-resolving microenvironment [44,46]. Consistently, we observed decreased efferocytosis of apoptotic neutrophils and reduced numbers of M2 macrophages at diabetic wound sites. Additionally, previous studies and our current study showed that impaired efferocytosis was associated with a significantly higher burden of apoptotic cells, as well as higher expression of pro-inflammatory cytokines and lower expression of anti-inflammatory cytokines [41]. As expected, 4-OI treatment improved the efferocytosis function of macrophages, and consequently promoted the conversion of M1-like macrophages to M2-like macrophages and reduced the expression of pro-inflammatory factors in the wound tissue of diabetic mice.

A recent study uncovered that efferocytosis of apoptotic cells modified multiple transcriptional programs, including 33 genes for solute carriers (SLCs) [10]. Among these SLCs, genes coding for carbohydrate metabolism and amino acid transport were upregulated. SLC2A1, a glucose transporter, has been proven to contribute to engulfment of apoptotic cells by phagocytes by promoting glucose uptake and subsequent aerobic glycolysis. Upregulated expression of SLC7A11, also referred to as the cystine-glutamate antiporter (xCT), which forms a heteromeric cystine-glutamate antiporter system with SLC3A2, was also found in DCs during efferocytosis [40]. However, SLC7A11 was surprisingly found to act as a negative regulator of efferocytosis. Loss of SLC7A11 expression or inhibition of its activity improves efferocytosis by DCs and promotes diabetic wound healing. As mentioned above, intracellular metabolism and metabolic by-products, such as glucose, cysteine, and glutamate, have profound effects on macrophage function. Moreover, a metabolic switch from oxidative phosphorylation to glycolysis is required for efferocytosis [9,10]. Recent evidence shows that efferocytosis-induced release of lactate, the end product of aerobic glycolysis, is important for promoting secretion of pro-resolving factors and driving continual efferocytosis by macrophages. Released lactate promotes the proliferation of pro-resolving macrophages, termed efferocytosis-induced macrophage proliferation, through the protein kinase A/AMP-activated protein kinase (PKA-AMPK) signaling pathway and the apoptotic cell-derived oligonucleotide-activated PKA-mammalian target of rapamycin (mTOR)-protein kinase B (Akt) pathway [7,46]. SLC16A1, also known as MCT1, was observed to be upregulated in engulfing phagocytes and to mediate lactate release during efferocytosis. In our current research, we found that 4-OI improved diabetic wound healing by promoting macrophage proliferation and M2 macrophage polarization, which were triggered by enhanced efferocytosis and subsequent MCT1-mediated lactate release. However, limited studies have investigated the mechanisms underlying on how 4-OI upregulates MCT1 expression. Studies have found that, in human colon cancer cells, the levels of MCT1 mRNA and protein were increased with enhanced expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, silenced expression of Nrf2 can reduce MCT1 expression [47]. Nrf2 is an important multifunctional intracellular antioxidant transcription factor that regulates cellular immune mechanisms [48]. 4-OI has been shown to efficiently activate Nrf2 signaling in mammalian cells [16,49,50]. Although direct evidence is limited, prior studies suggest that 4-OI may enhance MCT1 expression via Nrf2 activation. Further work is needed to validate this regulatory pathway in the context of wound healing.

Both pro-inflammatory macrophages and pro-resolving macrophages can proliferate after polarization. However, pro-inflammatory macrophages undergo proliferation mediated by colony-stimulating factor 1 (CSF1), a cytokine also known as macrophage colony-stimulating factor, but not by lactate [9,51,52]. Lactate has been proven to be required for efferocytosis-induced macrophage proliferation but not for the proliferation of pro-inflammatory macrophages [7,51]. Extracellular lactate signals for proliferation can be transduced by GPR132, a receptor for lactate. Furthermore, GPR132 has been reported to be downregulated in pro-inflammatory macrophages and upregulated in pro-resolving macrophages [29]. Consistent with these previous studies, we found that the GPR132 inhibitor telmisartan blocked 4-OI-induced macrophage proliferation and M2-macrophage polarization in vitro. Furthermore, telmisartan administration inhibited the function of 4-OI in rescuing dysregulated macrophages and promoting diabetic wound healing.

Collectively, this study demonstrates that 4-OI intervention promotes efferocytosis of apoptotic neutrophils increases the number of M2 macrophages, and promotes inflammatory resolution during diabetic wound healing. Moreover, 4-OI promoted efferocytosis of apoptotic cells by macrophages and rescued dysregulated macrophage function through MCT1-mediated lactate release triggered by efferocytosis. However, this study has a few limitations. First, further studies are warranted to clarify whether additional cell types and signaling pathways also contribute to the protective effect of 4-OI in diabetic wound healing, and how 4-OI upregulates MCT1 expression. Furthermore, we have demonstrated the effectiveness of 4-OI in various animal models and in vitro cell experiments; however, its potential for clinical applications should be considered in future studies. Clinical efficacy could be evaluated through trials or by investigating alternative macrophage-related mechanisms. Furthermore, identifying and validating biomarkers that reflect the mechanism of 4-OI, such as cytokine levels and gene expression, would be crucial. However, translating 4-OI into human applications may present certain challenges. For example, as a fat-soluble compound, 4-OI may have limited absorption and distribution in the body. Ensuring its effective concentration in topical or systemic administration will require optimization of drug delivery systems, such as nanocarriers or gels, to improve their bioavailability. In addition, the choice of 4-OI dose and route of administration requires careful consideration of its biological activity, safety, and efficacy. Starting with a low dose is recommended to assess tolerability and safety. Addressing these challenges will enhance the practical impact of the study. Taken together, human studies or trials would also reinforce the translational value of the research.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: Y.L., Y.X.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: M.T., X.Z., Y.L., Y.X.

Final approval of the manuscript: all authors.

FUNDING

This study received financial support from the Natural Science Foundation of China (Grant No. U22A20286, 82470854, 8217 1860, 82371882), the collaborative project between Sichuan Province and Luzhou-Southwest Medical University (Grant No. 2022YFS0617), the Sichuan Science and Technology Program (Grant No. 2023ZYD0095), and the Office of Science, Technology, and Talent Work of Luzhou (Grant No. 2021LZX NYD-P02).

ACKNOWLEDGMENTS

None

Fig. 1

4-Octyl itaconate (4-OI) promotes wound healing in diabetic mice. (A) Representative skin wound images from the negative control (NC), diabetes mellitus (DM), and 4-OI groups on day 0, day 3, day 7, and day 14. (B) Wound healing rate of mice on days 3 and 7 after wound modeling. (C) Representative photomicrographs of hematoxylin and eosin (H&E) and Masson staining of skin wounds in each group on day 7 (20×). (D) Immunofluorescence staining for CD31 in wounds on day 7 after wound modeling (100×). (E) Random blood glucose levels in each group of mice. NS, no statistical significance; DAPI, 4′,6-diamidino-2-phenylindole. ^aP<0.01, ^bP<0.001.

Fig. 2

4-Octyl itaconate (4-OI) promotes M2 macrophage polarization and attenuates the wound inflammatory response. (A, B) Immunofluorescence staining for inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), and F4/80 in wounds on day 3 after wound modeling (40×). (C) Immunofluorescence for iNOS, and Arg-1 in RAW264.7 cells (40×). (D) The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1β, and transforming growth factor-β (TGF-β) in the wound tissue of mice were detected by quantitative polymerase chain reaction on days 3 and 7 after wound model generation by surgical excision. NC, negative control; DM, diabetes mellitus; NS, no statistical significance. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 3

4-Octyl itaconate (4-OI) promotes apoptotic neutrophil efferocytosis. (A) Immunofluorescence staining for lymphocyte antigen 6 family member G (Ly6G) in wounds on day 3 after wound modeling (100×). (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed in wounds from mice on day 3 after wound modeling (40×). (C) Immunofluorescence staining for Ly6G, and F4/80 in wounds on day 3 after wound modeling (100×). (D) TUNEL staining and immunofluorescence staining for F4/80 were performed in wounds on day 3 after wound modeling (40×). (E) Jurkat cells were labeled with the fluorescent dye carboxytetramethylrhodamine (TAMRA; red) after induction of apoptosis; RAW264.7 cells were labeled with CellTracker fluorescent dye (green). After co-culturing apoptotic Jurkat cells with RAW264.7 cells for 3 hours, phagocytosis of Jurkat cells by RAW264.7 cells was observed by fluorescence microscopy (40×). NC, negative control; DM, diabetes mellitus; DAPI, 4′,6-diamidino-2-phenylindole; HG, high glucose 30 mM; AC, normal glucose 5.6 mM.

Fig. 4

4-Octyl itaconate (4-OI) improves diabetic wound healing and rescues dysregulated macrophage function through monocarboxylate transporter 1 (MCT1)-mediated lactate release. (A) Immunofluorescence staining for MCT1 and F4/80 in wounds on day 3 after wound modeling (40×). (B) Immunofluorescence staining for MCT1 in RAW264.7 cells (40×). (C) Extracellular lactate was measured in RAW264.7 cells treated with high glucose (HG; 30 mM), 4-OI, and/or the MCT1 inhibitor-AZD3965 (AZD). (D) Immunofluorescence detection of phagocytosis of Jurkat cells by RAW264.7 cells under different intervention conditions (40×). (E) Cell viability was assessed by cell counting kit-8 (CCK8) assay. (F) Immunofluorescent staining of Ki67 in RAW264.7 cells (40×). (G) Immunofluorescence staining for inducible nitric oxide synthase (iNOS), and arginase 1 (Arg-1) in RAW264.7 cells (40×). (H) Immunofluorescence staining for MCT1 and F4/80 in wounds on day 7 after wound modeling (40×). (I) Representative skin wound images and wound healing rates of the diabetes mellitus (DM), 4-OI, and 4-OI+AZD3965 groups on day 0, day 3, and day 7. (J, K) Immunofluorescence staining for iNOS, Arg-1, and F4/80 in wounds on day 7 after wound modeling (40×). (L) The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1β, and transforming growth factor-β (TGF-β) in the wound tissue of mice were detected by quantitative polymerase chain reaction on day 7 after wound model generation by surgical excision. (M) Representative photomicrographs of hematoxylin and eosin (H&E) and Masson staining of skin wounds in each group on day 7 (20×). NC, negative control; DAPI, 4′,6-diamidino-2-phenylindole; TAMRA, carboxytetramethylrhodamine; AC, normal glucose 5.6 mM; NS, no statistical significance. ^aP<0.05, ^bP<0.01, ^cP<0.001.

Fig. 5

4-Octyl itaconate (4-OI)-mediated lactate release promotes macrophage proliferation and M2 macrophage polarization through G protein-coupled receptor 132 (GPR132). (A) Extracellular lactate abundance in RAW264.7 cells treated with high glucose (HG; 30 mM), 4-OI, and/or telmisartan (Telm). (B) Cell viability was assessed by cell counting kit-8 (CCK8) assay. (C) Immunofluorescent staining of Ki67 in RAW264.7 cells (40×). (D) Immunofluorescence staining for inducible nitric oxide synthase (iNOS), and arginase 1 (Arg-1) in RAW264.7 cells (40×). (E) Representative skin wound images and wound healing rates of the diabetes mellitus (DM), 4-OI, and 4-OI+Telm groups on day 0, day 3, and day 7. (F, G) Immunofluorescence staining for iNOS, Arg-1, and F4/80 in wounds on day 7 after wound modeling (40×). (H) The mRNA levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), IL-1β, and transforming growth factor-β (TGF-β) in the wound tissue of mice were detected by quantitative polymerase chain reaction on day 7 after wound model generation by surgical excision. (I) Representative photomicrographs of hematoxylin and eosin (H&E) and Masson staining of skin wounds in each group on day 7 (20×). NC, negative control; NS, no statistical significance. ^aP<0.05, ^bP<0.01, ^cP<0.001.

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• Roles of efferocytosis in wound repair: Process, cells, and signals
  Yilin Sun, Haiying Guo, Yang Bai, Jin Chen, Yuhong Li
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About this article

Tu M, Zou X, Tan X, Liu Y, Ge X, Hu Y, Peng Q, Huang L, Zeng Y, Jia C, Guo M, Chen J, Long Y, Xu Y. 4-Octyl Itaconate Promotes Diabetic Wound Healing by Enhancing Pro-Resolving Macrophages via the Efferocytosis-MCT1-Lactate-GPR132 Pathway and Macrophage-Independent Synergistic Effects. Diabetes Metab J. 2026;50(4):707-723.

Received: Sep 21, 2024; Accepted: Jul 02, 2025

DOI: https://doi.org/10.4093/dmj.2024.0579.

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