GRAPHICAL ABSTRACT

Highlights

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

• 4-OI enhanced macrophage function by 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 rising year by year, and diabetic patients are susceptible to multi-system damage, including diabetic kidney disease, cardiovascular disease, retinopathy and so on [1]. Among these, diabetic foot ulcers have emerged as one of the most common and serious complications for diabetic patients, with lifetime risk of 19% to 34% [2]. It is characterized by high recurrence rates, high lower-extremity amputation incidence, high 5-year mortality. As a result, it becomes costly and seriously affects the quality of life for individuals with diabetes [3]. Consequently, considerable effort has been dedicated to exploring the pathogenesis of diabetic wounds and developing novel therapeutics.

Classically-activated macrophage, which exhibit pro-inflammatory effect and are defined as M1 phenotype in vitro, paly a central role in the late inflammatory phase. In contrast, alternative-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 found multiple subtypes of macrophage at wound site, a fine-tuned balance between classical and alternative-activated macrophages provide a fundamental guarantee for normal wound healing. In non-healing diabetic wounds, impaired function of infiltrating macrophages leads to a hyper-inflammatory phenotype at wound site and consequently results in delayed healing of wound. Efferocytosis is defined as the process by which apoptotic cells are cleared by professional and non-professional phagocytes, such as macrophage, dendritic cells (DCs), fibroblasts and epithelial cells [4]. Clearance of dead and dying cells through efferocytosis play a crucial role in regulating inflammatory resolution by preventing secondary necrosis [5]. Furthermore, efferocytosis can trigger a pro-resolving macrophage phenotype by metabolic reprogramming [6], inducing proliferation of pro-resolving macrophage [7] and M2 polarization of macrophage [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 belonging to solute carrier protein family, which is encoded by the solute carrier family 16 member 1 (SLC16A1) gene, and is mainly responsible for the transport of lactate, pyruvate and other monocarboxylic acids. Recently, several studies have shown that MCT1 is critical in 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 the immune responsive gene 1 (Irg1) [13]. Except for its antimicrobial effect, recent researches have shed light on immunoregulatory properties of itaconate because of its critical effects in regulating the inflammatory immune response and oxidative stress [14]. Itaconate has been shown proposed as a potential therapeutic agent for inflammatory disease, autoimmune disease and cancer [15-20]. Itaconate, as a carboxylic acid, is highly polar and exhibits limited permeability to cell membranes, which makes it unsuitable for mechanistic study [21]. Therefore, many studies on itaconate utilize its derivative, 4-octyl itaconate (4-OI), which is more permeable to cell membranes than its parent compound. Compared to 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 the activation of non-obese diabetic (NOD)-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome [23]. Some studies have also demonstrated that 4-OI was a promising therapeutic substance for diabetic wound repair [24-26]. However, the mechanism through which itaconate impacts diabetic wound healing remain unknown. In the current study, we first confirmed the effect of 4-OI on diabetic wound healing. Then, we observed its ameliorative effect on apoptotic neutrophil efferocytosis, M2 macrophage polarization, inflammatory state and angiogenesis at diabetic wound sites. In addition, we further investigated whether 4-OI promotes wound healing and rescues the dysregulated macrophage function through upregulated expression of MCT1 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 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, not 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), 4-OI⁺clodronate-liposomes (diabetic group, treated with 4-OI and CLD-Lipo). For induction of type 1 DM, mice were intraperitoneally injected with low-dose streptozotocin (STZ; 50 mg/kg, Beijing Soleilbao Technology Co., Beijing, China) for 5 consecutive days. Mice with random blood glucose levels that exceeded 16.7 mmol/L were considered to be diabetic. One month after blood glucose stabilization, a 1-cm-diameter full-thickness wound was created on the back of each group of mice.

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

Histopathological examination

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

Tissue immunofluorescence staining

Wound tissue sections were deparaffinized, then blocked with 10% goat serum for 2 hours at room temperature. Tissues were then incubated 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), MCT1 (Proteintech, Rosemont, IL, USA; 20139-1-AP) overnight at 4°C. followed by 1 hour of incubation with fluorescent dye-labeled secondary antibodies (Beyotime Biotechnology; A0428 or A0453). Cell nuclei were labeled with 4ʹ,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 taken 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 the cDNAs from the reverse transcription reactions, gene-specific primers, and 2xSYBR 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 respectively.

Cellular immunofluorescence

RAW264.7 cells were seeded on 24-well chamber slides, with additional stimuli added as needed for the experiment at appropriate times. The cells were fixed for 10 minutes at room temperature in 4% paraformaldehyde. Fixed cells were rinsed with phosphate-buffered saline (PBS) and then permeabilized with 0.2% TritonX-100 for 10 minutes at room temperature, and blocked with 10% goat serum for 1 hour. Cells were then incubated with primary antibodies against MCT1, Arg-1, iNOS (Santa Cruz Biotechnology; sc-7271), Ki67 (Cell Signaling Technology; 9129) overnight at 4°C. 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 by 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, a 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

The Jurkat cells were treated with staurosporine (2 μM) to obtain apoptotic Jurkat cells. Next, a sufficient amount of apoptotic Jurkat cells were taken and added to serum-free 1640 medium containing carboxytetramethylrhodamine (TAMRA) dye to continue incubation for staining for 15 minutes. Configure 0.3 μM 5-chloromethylfluorescein diacetate (CMFDA) probe working solution (YEASEN Biotechnology) 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, and then changed to 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 fluorescence microscope.

Efferocytosis-induced lactate release assay

RAW264.7 was inoculated on six-well plates at a density of 1.25×10⁶ cells/well and treated with different intervention stimuli for 48 hours. The 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 taken, and the extracellular lactate content was detected in accordance with the lactate assay kit (Rexin Bioarticle No. RXWB0476-96). A 200 μL of lactate extract was added to the cell precipitate to detect the intracellular lactate content, and the protein content of the cells in each group was detected, and the protein content was used to lactate content correction was performed.

Statistical analyses

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

RESULTS

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

To investigate the effect of 4-OI on diabetic wound healing, we used a STZ-induced diabetic mouse model. After successful modeling, we constructed a 1-cm-diameter full-thickness wounds on the back of the mice, and then administered 4-OI intraperitoneally for 2 weeks. Photographs were taken and wound healing rates were calculated at days 3, 7, 11, and 14 after wound model generation. As shown in Fig. 1A and B, the 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 to those of 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 the 4-OI-treated wounds 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 wound is closely related to orchestrated balance of M1/M2 macrophage polarization. Neutrophil clearance by efferocytosis is an important function of M2 macrophages. Then, we explored whether the promotion of diabetic wound healing by 4-OI is associated with macrophage polarization. We evaluated the infiltration of macrophage at the wound site, observing significant infiltration of macrophage (F4/80⁺) with an increasing in the number of M1-type macrophages (F4/80⁺iNOS⁺) and decreased number of M2-type macrophages (F4/80⁺Arg-1⁺) in diabetic wound. In contrast, macrophage infiltration was significantly reduced in the 4-OI-treated wounds at 3 (Fig. 2A and B) and 7 days (Supplementary Fig. 1), with a decreasing number of M1-type macrophages and an increasing in M2-type macrophages. In vitro, high glucose stimulation promoted M1 macrophage polarization. However, 4-OI treatment promoted M2 macrophage polarization (Fig. 2C). In consistent with the enhanced infiltration of pro-inflammatory M1 macrophage, 4-OI intervention resulted in a significantly reduction in the expression of 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 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 wound tissue of diabetic mice.

Next, we further examined the role of macrophages in 4-OI promoting 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 macrophages depletion reduced inflammatory cell infiltration and promoted collagen deposition in the wound tissue (Supplementary Fig. 2C). Compared to diabetic mice, CLD-Lipo intervention resulted in a significantly reduction in the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and a tendency toward increased expression of inflammation resolving factor TGF-β in the wounds at 7 days. However, no significant alterations were observed compared with 4-OI group (Supplementary Fig. 2D).

These results suggest that macrophage-dependent and independent pathways both contributes to acceleration of wound healing induced by 4-OI. Apart from this, it may also be accounted for the complex function of macrophage, with pro-inflammatory M1 macrophages being dominated in diabetic wounds. In the current study, we focused on the mechanisms which are involved in M2 macrophage polarization, and subsequent inflammatory resolution by 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 number 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 that in the control group, and were suppressed by 4-OI treatment (Fig. 3A and B). These findings suggest that itaconic acid treatment reduces neutrophil infiltration in wound tissue, promotes clearance of apoptotic cells.

In the progression of wound regeneration, apoptotic neutrophils should be removed by macrophages through efferocytosis, which is considered an anti-inflammatory and pro-resolving event. It is impaired in chronic wounds, including those due to diabetes [28]. We explored whether 4-OI intervention promote apoptotic neutrophils efferocytosis by macrophages. As expected, we revealed 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 wound, and enhanced after 4-OI treatment (Fig. 3C). In vitro, we co-cultured apoptotic Jurkat cells (fluorescent dye TAMRA-labeled) with RAW264.7 mouse macrophages (CellTracker fluorescent dye-labeled). Consistently, the ability of macrophages to phagocytize apoptotic neutrophils decreased in vitro after high glucose (HG, 30 mM) stimulation (Fig. 3D). We found that high glucose intervention significantly reduced the efferocytosis of murine macrophages RAW264.7 cells on apoptotic cells (TAMRA⁺CellTracker⁺). 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 the function of macrophage, 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 to facilitate the release of lactate during efferocytosis [10]. Interestingly, the expression of MCT1 was significantly decreased in macrophage (MCT1⁺F4/80⁺) in the diabetic wound (Fig. 4A). In vitro experiments, high glucose stimulation decreased the expression of MCT1 in cultured murine macrophage cells RAW264.7, and MCT1 was mainly distributed in the cytoplasm with an inconspicuous distribution on the cell membranes. Whereas the administration of 4-OI upregulated the expression of MCT1, which were aggregated on the cell membranes of macrophage (Fig. 4B). Consistently, high glucose stimulation led to decreased lactate release from macrophage after challenged with apoptotic cells and 4-OI treatment led to increased release of lactate (Fig. 4C). In addition, we evaluated the effect of 4-OI on macrophage proliferation and cell viability by using CCK8 and Ki67 staining. After co-cultured with apoptotic cells, decreased proliferation and cell viability were observed under the condition of high glucose, and 4-OI significantly promoted macrophage proliferation and cell viability (Fig. 4E and F). These results suggest that 4-OI rescues the dysregulated macrophage function through upregulated expression of MCT1 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 assay also revealed that AZD3965 administration elevated the expression of pro-inflammatory factors IL-1β, IL-6, and TNF-α, and reduced the expression of inflammation resolving factor TGF-β in the 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 functions of 4-OI in promoting 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

It has been observed that efferocytosis-derived lactate released and promoted macrophage proliferation through the 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), resulted in decreased pro-resolving macrophage (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, a derivative of itaconate 4-OI have 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, the 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 promotes efferocytosis of apoptotic neutrophils, M2 macrophage polarization and inflammatory resolution. Moreover, the administration of 4-OI upregulated the expression of MCT1 and consequently led to increased release of lactate. Finally, we confirmed that 4-OI encouraged the function of pro-resolving macrophage through MCT1-mediated lactate release and consequent activation of GPR132 receptor by lactate.

Wound healing is a complex process involving the coordinated actions of various tissues and cellular lineages, including immune cells, epidermal cells, 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 the healing process through immunomodulatory effects. Skin stem cells support tissue repair and regeneration through regenerative actions [30]. In recent years, emerging evidences show that itaconate plays an important role in regulating the function of macrophage, and subsequently results in protective effects in acute liver failure [31], myocardial ischemia-reperfusion [32] and in some chronic inflammatory disease [33]. In our study, we observed that there was a significant decrease in the number of M1-type macrophages and increase in M2-type macrophages in 4-OI treated wound, indicating an important role of macrophage 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. It was shown that wound healing was still faster in diabetic mice with and without 4-OI treatment under macrophage depletion conditions. The complexity in macrophage function may be accounted for these results. In diabetic wounds, pro-inflammatory M1-type macrophages is the predominant phenotype, with few anti-inflammatory M2-type macrophages existing [33]. This phenotypic imbalance of macrophages causes a persistent inflammatory state in diabetic wounds, hindering the prohealing activities of endothelial cells, keratinocytes and fibroblasts [32]. Therefore, in the state of diabetes, the more significant effect of removing macrophages should be to eliminate the pro-inflammatory effect of macrophages, thereby promoting wound healing. In 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 mechanism, macrophage-independent pathways should be involved. In this current study, we also found that 4-OI treatment resulted in significantly neutrophil infiltration, and improved angiogenesis in the diabetic wounds. It was indicated that, other than macrophage, neutrophil and endothelial cells were also involved and account for 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], fibroblast-like synoviocytes [39] have also been reported to be the targeted 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 of 4-OI in diabetic wound healing.

Recently, emerging evidence highlights 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 peritoneal of NOD mice or wounds of obese diabetic (db/db) mice showed significant impairment in phagocytosis of apoptotic cells [27,45]. Moreover, it is well known that efferocytosis of apoptotic cells is critical in promoting M2 polarization of macrophages and leading to pro-resolving microenvironment [44,46]. Consistently, we observed decreased efferocytosis of neutrophil apoptotic cells and reduced M2 macrophages at the diabetic wound site. Additionally, previous and our current studies showed that impaired efferocytosis was associated with significantly higher burden of apoptotic cells as well as higher expression of pro-inflammatory and lower expression of anti-inflammatory cytokines [41]. As expected, 4-OI treatment improved efferocytosis function of macrophage, and consequently promoted the conversion of M1-like macrophages to M2-like macrophages and reduced the expression of pro-inflammatory factors in wound tissue of diabetic mice.

A recent study uncovered that efferocytosis of apoptotic cell modified multiple transcriptional programs, including 33 genes for solute carrier (SLC) [10]. Among these SLCs, genes coding for carbohydrate metabolism and amino acid transport were upregulated. SLC2A1, a glucose transporter, has been proved to contribute to engulfment of apoptotic cells by phagocytes through promoting glucose uptake and subsequent aerobic glycolysis. Upregulated expression of SLC7A11, also referred as cystine-glutamate antiporter (xCT) and form a heteromeric cysteine-glutamate antiporter system with SLC3A2, were also found in DCs during efferocytosis [40]. However, it was surprisingly uncovered that SLC7A11 acts as a negative regulator of efferocytosis. Loss of SLC7A11 expression or activity inhibition improves efferocytosis by DCs and promotes diabetic wound healing. As mentioned above, intracellular metabolism and metabolic by-products, such as glucose, cysteine and glutamate, play profound effects in macrophage function. Moreover, it has been shown that a metabolic switch from oxidative phosphorylation to glycolysis is required for efferocytosis [9,10]. Some recent evidences show that efferocytosisinduced release of lactate, the end product of aerobic glycolysis, is important for promoting secretion of pro-resolving factor and driving continual efferocytosis by macrophages. Released lactate promote proliferation of pro-resolving macrophages, termed efferocytosis-induced macrophage proliferation, through protein kinase A/AMP-activated protein kinase (PKA-AMPK) signaling pathway and apoptotic cells-derived oligoneucleotide actived 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 mediate release of lactate during efferocytosis. In our current research, we found that 4-OI improved diabetic wound healing through promoting macrophage proliferation and M2 macrophage polarization, which were triggered by enhanced efferocytosis and subsequent MCT1-mediated lactate release. However, there are limited studies investigating the mechanisms underlying on how 4-OI upregulate the expression of MCT1. 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 the expression of MCT1 [47]. Nrf2 is an important multifunctional intracellular antioxidant transcription factor that regulates cellular immune mechanism [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]. It has been proved that lactate is required for efferocytosis-induced macrophage proliferation but not for the proliferation of pro-inflammatory macrophage [7,51]. The signals of extracellular lactate 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 GPR132 inhibitor telmisartan blocked 4-OI-induced macrophage proliferation and M2-macrophage polarization in vitro. Furthermore, telmisartan administration inhibited the function of 4-OI to rescue dysregulated macrophages and promote diabetic wound healing.

Collectively, this study demonstrates that 4-OI intervention promotes efferocytosis of apoptotic neutrophils and enhanced numbers of M2 macrophage, and inflammatory resolution in progression of diabetic wound healing. Moreover, 4-OI promoted apoptotic cells efferocytosis by macrophage and rescued the dysregulated macrophage function through MCT1-mediated lactate release triggered by efferocytosis. However, there are a few limitations in this current studies. First, further studies are warranted to clarify whether additional cell types and signaling pathways also contribute to the protective of 4-OI in diabetic wound healing, and how 4-OI upregulate the expression of MCT1. 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 trails 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 the 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. It is recommended to start with a low dose to assess its 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 negative control (NC), diabetes mellitus (DM), and 4-OI group 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) The representative photomicrographs of hematoxylin & eosin (H&E) and Masson staining of skin wound in each group on day 7 (20×). (D) Immunofluorescence staining for CD31 in wounds at days 7 after wound modeling (100×). (E) Random blood glucose level in each group of mice. NS, no statistical significance; DAPI, 4ʹ,6-diamidino-2phenylindole. ^aP<0.01, ^bP<0.001.

Fig. 2.

4-Octyl itaconate (4-OI) promotes M2 macrophage polarization and attenuates wound inflammatory response. (A, B) Immunofluorescence staining for inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), F4/80 in wounds at days 3 after wound model (40×). (C) It show the immunofluorescence for iNOS, 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 was detected by quantitative polymerase chain reaction on day 3 and day 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 at days 3 after wound modeling (100×). (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed in the wounds from mice at days 3 after wound modeling (40×). (C) Immunofluorescence staining for Ly6G, F4/80 in wounds at days 3 after wound modeling (100×). (D) TUNEL staining and immunofluorescence staining for F4/80 were performed in wounds at days 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, the phagocytosis of Jurkat cells by RAW264.7 cells was observed by fluorescence microscope (40×). NC, negative control; DM, diabetes mellitus; DAPI, 4ʹ,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 the dysregulated macrophage function through monocarboxylate transporter 1 (MCT1)-mediated lactate release. (A) Immunofluorescence staining for MCT1 and F4/80 in wounds at days 3 after wound modeling (40×). (B) Immunofluorescence staining for MCT1 in RAW264.7 cells (40×). (C) Extracellular lactate were measured in RAW264.7 cells treated with high glucose (HG; 30 mM), 4-OI, and/or 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), arginase 1 (Arg-1) in RAW264.7 cells (40×). (H) Immunofluorescence staining for MCT1 and F4/80 in wounds at days 7 after wound modeling (40×). (I) Representative skin wound images and wound healing rate of diabetes mellitus (DM), 4-OI, 4-OI+AZD3965 group on day 0, day 3, and day 7. (J, K) Immunofluorescence staining for iNOS, Arg-1, F4/80 in wounds at days 7 after wound model (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 was detected by quantitative polymerase chain reaction on day 7 after wound model generation by surgical excision. (M) The representative photomicrographs of hematoxylin & eosin (H&E) and Masson staining of skin wound in each group on day 7 (20×). NC, negative control; DAPI, 4ʹ,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) receptors. (A) extracellular lactate abundance lactate 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), arginase 1 (Arg-1) in RAW264.7 cells (40×). (E) Representative skin wound images and wound healing rate of diabetes mellitus (DM), 4-OI, 4-OI+Telm group on day 0, day 3, and day 7. (F, G) Immunofluorescence staining for iNOS, Arg-1, F4/80 in wounds at days 7 after wound model (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 was detected by quantitative polymerase chain reaction on day 7 after wound model generation by surgical excision. (I) The representative photomicrographs of hematoxylin & eosin (H&E) and Masson staining of skin wound 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
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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. 2025 Nov 3. doi: 10.4093/dmj.2024.0579. Epub ahead of print.

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

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

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