Formation of neutrophil extracellular traps in the early stages exacerbate the healing process by regulating macrophage polarization in Achilles tendon-bone injury

Abstract

The influence of neutrophils and of neutrophil extracellular traps (NETs) on post-traumatic tendon-to-bone healing was studied in a murine model. The impact of neutrophil infiltration on macrophage polarization and peritendinous fibrosis in early-stage Achilles tendon injury is reported. Mice underwent Achilles tendon-bone injury and divided into four groups: sham operation, tendon injury (TI) treated with acetylcellulose (vehicle control), TI treated with a Protein arginine deiminase-4 (PAD4) inhibitor GSK484, and TI treated with a neutrophil elastase inhibitor Sivelestat. Each group was monitored for 21 days. Post-traumatic neutrophil infiltration and NET formation were assessed using flow cytometry and immunofluorescence. Immunohistochemistry, Western blot, and qPCR were used to evaluate macrophage polarization. Peritendinous fibrosis was assessed using Masson staining and Western blot. Neutrophil infiltration and NET formation increased significantly in the tendon following injury. A significant increase in M1-related markers and a decrease in M2-related markers were associated with NET formation. NET Inhibition using GSK484 or sivelestat reduced M1 markers and increased M2 markers. Furthermore, NET inhibition during the early stage suppressed peritendinous fibrosis and reduced inflammation during the healing process. In co-culture experiments, NETs induced proinflammatory cytokine secretion and upregulated M1 markers in bone marrow-derived macrophages while downregulating M2 markers. nlsNETs promote early-phase tendon-bone injury by inducing M1 macrophage polarization and peritendinous fibrosis. Targeting NETs during the initial phase of tendon injury could potentially facilitate the healing process.

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Introduction

Achilles tendon-bone injuries, such as Achilles tendinopathy and insertional Achilles tendonitis, are common musculoskeletal conditions that often result in pain, functional impairment, and a reduced quality of life. Fibroblast trans-differentiation and proliferation are key mediators of extracellular matrix (ECM) synthesis. Fibroblast activity is regulated by the secretion of cytokines within adhesive tendon tissues, including transforming growth factor (TGF)-β, fibroblast growth factor-2 (FGF-2), connective tissue growth factor (CTGF), and others. ¹ Additionally, resident tissue fibroblasts are highly sensitive to mechanical and biophysical cues present in tendon-bone injuries, which influence their behavior and function.^2,3 The healing process following these injuries involves a series of complex cellular and molecular events, with macrophages playing a key role in regulating tissue repair, inflammation, and remodeling.

Macrophages, essential immune cells, play a pivotal role in coordinating regenerative responses and facilitating tissue remodeling. As multifunctional phagocytic cells, macrophages exhibit diverse phenotypes and functions that are shaped by the surrounding microenvironment. In the context of tendon and bone injuries, macrophages contribute to critical processes such as inflammation, debris clearance, angiogenesis, matrix synthesis, and tissue remodeling. ⁴ Recent studies have highlighted their role in promoting tendon injury repair through the production of cytokines and growth factors. ⁵ These factors aid in resolving inflammation, enhancing cell proliferation and matrix synthesis, and promoting neovascularization and tissue reconstruction. ⁶ However, despite the recognized importance of macrophages in tendon injuries, the specific functions of macrophage subsets at different stages and types of injuries remain poorly understood. Further research is needed to elucidate how macrophage activity can be modulated to improve tendon injury repair and regeneration.

Neutrophils, the primary innate immune cells, play a critical role in both host defense and tissue damage. In addition to their well-known functions in phagocytosing pathogens and releasing antimicrobial agents, neutrophils can generate neutrophil extracellular traps (NETs). These structures consist of DNA, histones, and granular enzymes, forming intricate networks within the extracellular space. ⁷ Key enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO), which are embedded in NET fibers, play essential roles in regulating NET formation.^8–10 NETs are highly effective in capturing and eliminating pathogens, a function first identified in bacterial infections. However, they have since been implicated in various non-infectious conditions, including gout, type 1 diabetes, autoimmune disorders, and COVID-19.^11–14 Growing evidence suggests that excessive NET production can drive severe inflammation and contribute to tissue damage.^15,16 Despite their established roles in other conditions, the involvement of neutrophils and NETs in tendon-bone injuries remains poorly understood.

In this study, we investigated the influence of neutrophils on macrophage polarization in vitro using a mouse model of Achilles tendon injury, which was established through a chronic tendinopathy method. We evaluated the effects of NET inhibitors, GSK484 and sivelestat, to explore their therapeutic potential, and mechanisms of action in vivo, with a focus on the modulating macrophage polarization. Additionally, culture medium from PMA-treated neutrophils was collected to assess the impact of NETs on the balance between M1 and M2 macrophage phenotypes. We hypothesized that neutrophils could improve histological outcomes and enhance biomechanical strength by promoting M2 macrophage polarization in the treatment of Achilles tendon injuries.

Materials and methods

Reagents and antibodies

The cell culture medium, including Fetal bovine serum (26140-079), Dulbecco’s Modified Eagle’s Medium with low glucose and pyruvate (11885084), and supplements such as Penicillin Streptomycin (15140122), were sourced from ThermoFisher Scientific. Antibodies targeting Fibronectin (ab2413), Collagen I (ab34710), iNOS (ab178945), and IL-1 beta (ab315084) were procured from Abcam. The GAPDH antibody (2118) was acquired from Cell Signaling Technology. Additionally, α-SMA (55135-1-AP) and TLR4 polyclonal antibody (48-2300) were obtained from ThermoFisher. Macrophage colony-stimulating factor (M-CSF) was sourced from Miltenyi Biotec (130-094-129). Mouse INF-γ Recombinant Protein (300-02-100UG) and Mouse IL-4 Recombinant Protein (214-14-20UG) were obtained from PeproTech^® (ThermoFisher Scientific). Lipopolysaccharide (LPS; L2630) was purchased from Sigma-Aldrich. GSK484 hydrochloride (HY-100514) and sivelestat (HY-17443) were obtained from MedChemExpress.

Mice

The Animal Care and Use Committee of the Navy Medical University approved the methodology of this murine experiment. Standardized protocols were followed to minimize pain and discomfort. Six-week-old male mice were sourced from the SLRC laboratory in Shanghai, China. Anesthesia was administered via intraperitoneal injection of avertin at a dosage of 100 mg/kg. ¹⁷

Isolation of human peripheral neutrophils

Peripheral blood neutrophils were isolated as previously described. ¹⁸ Written informed consent was obtained from the human participants and, where applicable, their parents or legal guardians, under protocols approved by the Ethics Committee of the Naval Medical University Affiliated Shanghai Changzheng Hospital (Approval file number: 2024SL058). Heparinized blood from rotator cuff injury patients and healthy controls was separated by density gradient centrifugation using Polymorphprep (AS1114683, Axis-Shield, Dundee, UK) at 400×g for 40 min without braking. The neutrophils and red blood cells (RBCs) were suspended in RBC lysis buffer (40401ES76, Yeasen, Shanghai, China). Neutrophils were subsequently washed with sterile PBS and resuspended for further use.

NETs detection

Neutrophils were isolated from bone marrow and plated onto poly-D-lysine-coated coverslips (Beyotime Biotechnology, Jiangsu, China). NETs formation was induced according to an established protocol. ¹⁹ Cells were fixed with 1% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and blocked using a solution containing 1% bovine serum albumin (BSA) and 5% normal goat serum in PBS. Fixed cells were incubated overnight at 4°C with polyclonal rabbit anti-cit H3 and sheep anti-neutrophil myeloperoxidase (MPO) antibodies. After washing, cells were incubated with secondary antibodies (Life Technologies), diluted 1:1000, for 1 h at room temperature. Sytox Green (1:20,000 dilution) was applied for 15 min in the dark for nuclear staining. Imaging was performed using a Leca SP8 microscope at 20× magnification.

Tendon repair surgery

Tendon repair procedures were conducted with approval from the University Committee on Animal Research. A mouse tendon-bone reconstruction model was established as previously described. ²⁰ Male C57BL/6 mice (6–8 weeks old) were anesthetized via intraperitoneal injection of avertin (100 mg/kg). The Achilles tendon was detached above the calcaneus. The cartilage at the insertion point was excised. A hole was drilled in the posterior calcaneus using a 29-G syringe under magnification. A 6-0 suture was passed through the bone tunnel, and the distal Achilles tendon was sutured and fixed above the calcaneus. To reduce tension at the repair site, an additional transection was made proximally at the myotendinous junction. The skin was closed with continuous 4-0 nylon sutures. Post-surgery, mice were allowed unrestricted movement and weight-bearing after full recovery from anesthesia.

At 21 days post-repair, mice were euthanized for histological analysis (n = 6 repairs per time point, plus 6 age-matched contralateral sham specimens per time point), protein and RNA extraction (n = 6 repairs per time point), and biomechanical testing (n = 6 repairs per time point, with age-matched contralateral sham specimens collected from each mouse). Sham surgeries were performed on control groups using the same anesthesia and exposure procedures. Sample sizes were determined based on previous studies. ²¹ During sham surgeries, the distal Achilles tendon was isolated and secured with two horizontal 8-0 sutures in a modified Kessler pattern without transecting. The proximal Achilles tendon was released at the myotendinous junction, and the skin was closed with 4-0 nylon sutures.

Enzyme-linked immunosorbent assay (ELISA)

Levels of IL-10, TGF-β, TNF-α and IL-6 in cell culture supernatant were measured using commercial ELISA kits (RayBiotech, Peachtree Corners, GA) according to the manufacturer’s protocols.

Biomechanical testing

Tendon-bone junction specimens were analyzed for biomechanical properties using a microcomputer-controlled electronic universal testing machine (CTM9100, Xie Qiang Instrument Manufacturing, Shanghai, China), following a previously described methodology. ²¹ The cross-sectional area of the Achilles tendon at its insertion point into the calcaneus was measured with a vernier caliper. The calcaneus was clamped at the lower end, while the proximal Achilles tendon was sutured with 3-0 silk and fixed at the upper clamp. After a 1 N preload, samples were loaded to failure at 10 mm/min. Maximum force, stiffness, and elastic modulus were recorded using the Rigal Test Control System. Strength was calculated by dividing the maximum force by the cross-sectional area of the tendon-bone junction. ²²

Histological staining

Achilles tendon specimens were fixed in 4% PFA, decalcified in 0.5 M EDTA, embedded in paraffin, and sectioned into 7 μm slices. For M1 macrophages detection, sections were incubated with CD68 (14-0681-82; Invitrogen), CD11c (14-0114-82, Invitrogen), and iNOS (MAB9502; R&D systems) antibodies. For M2 macrophages, sections were stained with CD206 antibody (81525-1-RR; Proteintech, China) overnight at 4°C, followed by incubation with secondary antibodies (Jackson ImmunoResearch Laboratories, WestGrove, PA). Sections were visualized under a fluorescence microscope (Leica, Wetzlar, Germany).

Flow cytometry analysis

Freshly isolated tendons were processed for flow cytometry as previously described. ²³ Tissues were minced and digested in a solution containing type II collagenase (C2-22-1G, Sigma Aldrich), DNase I (10104159001, Sigma Aldrich), and EDTA (C0196, Beyotime, China) for 40 min at 37℃. Cell suspensions were filtered through a 70 µm filter and incubated with Fc receptor blocker (564219, BD) and Zombie Aqua™ Fixable Viability Kit (423101, Biolegend) for 30 min at 4°C. Cells were stained with CD11b (982614, Biolegend), F4/80 (123116, Biolegend), iNOS (696814, Biolegend), CD206 (321108, Biolegend) and TNF-α (11-7321-82, ThermoFisher) using the eBioscience™ Foxp3/Transcription Factor Staining Buffer (00-5523-00, ThermoFisher). Data were acquired using a BD Fortessa X20 and analyzed with FlowJo software.

For human peripheral blood flow cytometry, 200 μL of blood was treated with 5 mL RBC lysis buffer (40401ES76, Yeasen, Shanghai, China) for 10 min. The reaction was stopped with 100 μL PBS, and cells were collected by centrifugation at 3000 rpm for 6 min. Cells were stained with CD11b (982604, Biolegend), CD66b (392904, Biolegend), and citH3 (17939, Cayman Chemical) and analyzed as above.

Cell cultures

Bone marrow-derived macrophages (BMDMs) were isolated from euthanized mice. Femurs and tibias were flushed with PBS containing 3% FBS, and cell suspensions were filtered through a 70 μm filter. Erythrocytes were lysed, and remaining cells were resuspended in RPMI medium with 10% FBS. After overnight culture, contaminating cells were removed, and the remaining cells were treated with M-CSF (10 ng/mL) in 1640 medium (with 10% FBS and penicillin-streptomycin). Medium was replaced every 3 days, and cells were assessed for purity on day 7.

Western blotting

Cells and Achilles tendon species were lysed using RIPA lysis buffer (20114ES, Yeasen, China) with protease inhibitors. Protein concentration was measured using a BCA protein assay kit (Beyotime, P0012). Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies specified in the figure legends. Proteins were visualized using a ChemiDoc Imaging System (AI600), and data were analyzed with ImageJ software.

Statistical analyses

All experiments were performed at least three times unless specifically stated. Information about statistical details and methods is indicated in the figure legend. Statistical analysis was performed using GraphPad Prism nine software (La Jolla, CA, USA). For outliers, values exceeding ± 3 standard deviations from the group mean were considered extreme and assessed individually. If confirmed to be due to technical error (e.g. sample processing failure), they were excluded from analysis and the rationale documented. Data are expressed as mean ± SD (standard deviation). Unpaired two-tailed Student’s t test was used to analyze the differences between two groups. Comparisons among multiple groups were analyzed with one-way analysis of variance. p < 0.05 was considered statistically significant.

Results

An increase in neutrophil infiltration and NETs was observed in the early stage after mouse tendon injury

Previous studies have demonstrated that persistent immune cell activation, coupled with the upregulation of pro-fibrotic cytokines and ECM proteins, influence the pathogenesis of Achilles tendon-bone injuries.^24,25 In this study, we investigated the potential involvement of neutrophils and NETs in tendon injuries. A murine model of Achilles tendon injury was established by surgically transecting the calcaneal tendon. Mice were subsequently treated with the NET inhibitors GSK484 or Sivelestat for 3 days (Figure 1(a)). Analysis of immune cell infiltration revealed a significantly elevated population of neutrophils in the tendon injury (TI) group compared to the control group (Figure 1(b) and (c)). This increase in neutrophil infiltration was suppressed by treatment with GSK484 and Sivelestat. Additionally, we observed an increased population of F4/80⁺ macrophages following tendon injury, which was similarly reduced upon treatment with GSK484 and Sivelestat treatment. Neutrophils are short-lived immune cells that upon recruitment to injured tissue, undergo NETosis, a specialized form of cell death that facilitates the release of NETs. ²⁶ Immunofluorescence staining revealed co-localization of myeloperoxidase (MPO) and citrullinated histone H3 (cit-H3) at the tendon-bone interface following Achilles tendon injury, as indicated by white arrows in Figure 1(d). The expression of cit-H3 was significantly reduced in mice treated with GSK484 or Sivalestate (Figure 1(d) and (e)). However, while Sivelestat treatment reduced MPO intensity, indicating decreased neutrophil activation, GSK484 did not exhibit a significant effect on MPO levels Figure 1(f). In summary, our findings demonstrate a significant increase in neutrophil infiltration and NET formation in the TI mouse model. These results suggest that NETs contribute to the pathophysiology of tendon injury and that targeting NETosis may represent a potential therapeutic strategy.

Figure 1.

Inhibition of NETs with GSK484 or sivelestat reduces neutrophil infiltration and NET formation following tendon injury. (a) Schematic of the experimental mouse model. Mice underwent surgical transection of the Achilles tendon at the calcaneal insertion site, followed by daily treatment with vehicle (corn oil), GSK484, or sivelestat for three consecutive days. Tissues were harvested on day 3 post-surgery. (b and c) Flow cytometry analysis (b) and representative plots (c) of neutrophils (CD11b⁺Ly6G⁺) and macrophages (CD11b⁺F4/80⁺) from control, tendon injury (TI), TI + PAD4 inhibitor, and TI + NE inhibitor groups. n = 6 mice/group. (d) Confocal immunofluorescence images showing MPO (green), citrullinated histione H3 (cit-H3, purple), and Hoechst (blue) in tendon sections. Scale bars, 150 µm. (e and f) Quantification of MPO (e) and cit-H3 (f) fluorescence intensities. Data are presented as the mean ± SEM. n = 6 mice/group. Statistical significance determined by one-way ANOVA (c, e, and f). ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001.

Neutrophils from peripheral blood of patients with rotator cuff injury patient were more likely to produce NETs

Neutrophils play a critical role in the immune response to infection and acute injury. Neutrophils were recruited to the site of injury in the murine study, and they produce NETs. To further investigate the role of NETs in human tendon pathology, NET formation was examined in patients with rotator cuff injury (RCI). Peripheral blood neutrophils were isolated from RCI patients and healthy volunteers (HVs). The presence of extracellular DNA associated with citrullinated histone H3 (cit-H3) and myeloperoxidase (MPO) confirmed the formation of NETs. While low levels of NET release were detected in unstimulated neutrophils from RCI patients, phorbol 12-myristate 13-acetate (PMA)-induced NET formation was significantly elevated in RCI patient-derived neutrophils compared to those from HVs (Figure 2(a) and (b)). Consistent with these findings, the intensity of cit-H3 staining showed no significant difference between unstimulated neutrophils from HVs and RCI patients. However, cit-H3 levels were markedly increased in RCI patient-derived neutrophils following PMA stimulation (Figure 2(c)). Flow cytometry analysis revealed an increased percentage of CD11b⁺CD66b⁺ neutrophils in RCI patients compared to HVs (Figure 2(d) and (e)). To further quantify NETosis, a previously established flow cytometry protocol ²⁷ was adapted to analyze cit-H3 expression in CD11b⁺CD66b⁺ neutrophils following in vitro PMA stimulation. The results demonstrated a higher percentage of cit-H3-positive neutrophils in RCI patients compared to HVs after PMA treatment (Figure 2(f) and (g)). In summary, these findings indicate that neutrophils from RCI patients exhibit a heightened propensity for NET formation, particularly under stimulated conditions. This suggests that dysregulated NETosis may contribute to the pathophysiology of rotator cuff injury.

Figure 2.

NET formation is enhanced in neutrophils from RCI patients compared with healthy volunteers (HVs). (a) Immunofluorescence staining of Hoechst (Blue), Sytox Green (Green), cit-H3 (Red), and MPO (magenta) in neutrophils from rotator cuff injury (RCI) patients and HVs. Cells were treated with DMSO or PMA. Scale bar, 10 μm. (b) Quantification of extracellular DNA per field of view. (c) Quantification of cit-H3 intensity. (d) Flow cytometry analysis of CD11b⁺CD66b⁺ neutrophils from RCI patients and HVs. (e) Quantification of cell percentage. (f) Flow cytometry analysis of NETs formation in CD11b⁺CD66b⁺ neutrophils after PMA treatment. (g) Quantification of cell percentage of cit-H3. n = 6 HVs or RCI patients. Data are presented as the mean ± SEM. Significance was examined by unpaired two-sided Student’s t test (e) or one-way ANOVA (b, c, and g). ^****p < 0.0001.

PAD4 inhibitor and NE inhibitor protect against Achilles tendon bone injury

To investigate the role of neutrophils following tendon injury, NETs inhibitors (GSK484 or Sivelestat) were administered orally after the surgical procedure. Mice were divided into four experimental groups: sham operation, TI treated with acetylcellulose (vehicle control), TI treated with a PAD4 inhibitor (GSK484), and TI treated with a neutrophil elastase (NE) inhibitor (Sivelestat). Each group was monitored for 21 days (Figure 3(a)). Histological analysis demonstrated a significant reduction in adhesion formation, inflammatory cell infiltration, and fibroblast accumulation at the tendon-bone interface in the TI mice treated with PAD4 or NE inhibitors compared to the untreated TI group (Figure 3(b)). Hematoxylin and eosin (H&E) staining revealed disorganized cellular architecture and collagenous tissue at the tendon-bone junction in the TI group. In contrast, the PAD4 and NE inhibitor groups exhibited a more structured tendon-bone transition, characterized by an increased number of closely arranged chondrocytes and well-organized collagen fibers (Figure 3(b)). Quantitative histological healing scores were significantly lower in the PAD4 and NE inhibitor groups compared to the TI group, indicating improved tissue healing (Figure 3(c)). Furthermore, Safranin O-Fast Green staining revealed a larger Safranin O-positive area at the tendon-bone junction in the PAD4 and NE inhibitor groups compared to the TI group, suggesting enhanced proteoglycan deposition and cartilage-like tissue formation (Figure 3(d) and (e)). These findings suggest that inhibition of NET formation via PAD4 or NE targeting improves tendon-bone healing by reducing inflammation, promoting organized tissue regeneration, and enhancing extracellular matrix remodeling.

Figure 3.

GSK484 or sivelestat treatment attenuates tendon injury and enhances cartilage regeneration. (a) Mouse model schematic. Achilles tendon transection was followed by 3-day treatment and tissue harvesting at day 21. (b and c) H&E staining (b) and histological healing scores (c) from control, TI, TI + PAD4 inhibitor, and TI + NE inhibitor group. (d and e) Representative safranin O staining (d) and cartilage regeneration from the control, TI, TI + PAD4 inhibitor, and TI + NE inhibitor groups. n = 6 mice/group. Data are presented as the mean ± SEM. Significance was examined by one-way ANOVA (c and e). ^*p < 0.05, ^****p < 0.001, ^****p < 0.0001.

NETs inhibition suppresses peritendinous fibrosis after tendon injury

To further elucidate the role of neutrophils in peritendinous fibrosis following tendon injury, tissue from the mice within each of the four experimental groups was stained with Masson’s trichrome stain. A substantial increase in collagen deposition and fibrotic areas was apparent in the TI group, but significantly attenuated in mice treated with PAD4 or NE inhibitors (Figure 4(a) and (b)). Peritendinous fibrosis is characterized by the excessive production of ECM, including fibronectin (FN), collagen type I (Col-I), and alpha-smooth muscle actin (α-SMA), under TI conditions.^28,29 Western blot analysis was performed to evaluate the expression of these pro-fibrotic markers in tendon tissues. The results demonstrated a significant upregulation of FN, α-SMA, and Col-I in the TI group compared to the sham group. Notably, treatment with PAD4 or NE inhibitors markedly reduced the expression of these proteins in TI mice (Figure 4(c) and (d)). Additionally, other pro-fibrotic factors, such as Snail, matrix metalloproteinase-9 (MMP9), and tissue inhibitor of metalloproteinases-1 (TIMP1), were elevated in the TI group but showed reduced expression following PAD4 or NE inhibitor treatment (Figure 4(e)). Interleukin-1 beta (IL-1β), a pro-inflammatory cytokine implicated in fibrosis,^30,31 was also s upregulated in the TI group. However, its levels were significantly decreased after treatment with PAD4 or NE inhibitors (Figure 4(f)). In summary, these findings demonstrate that NET inhibition attenuates peritendinous fibrosis by reducing the expression of pro-fibrotic markers and inflammatory cytokines. This underscores the therapeutic potential of targeting NETs in the treatment of tendon injury.

Figure 4.

NETs inhibition alleviates peritendinous fibrosis. (a and b) Masson’s trichrome staining (a) and quantification of fibrotic area from control, TI, TI + PAD4 inhibitor, and TI + NE inhibitor groups. n = 6 mice/group. (c and d) Western blot (c) and quantitative analysis of fibronectin, collagen I, and α-SMA. n = 3 mice/group. (e) qPCR analysis of Snail, MMP9 and TIMP1 mRNA expression from control, TI, TI + PAD4 inhibitor, and TI + NE inhibitor groups. n = 3 mice/group. (f) ELISA analysis of IL-1β, TNF-α and TGF-β in serum from control, TI, TI + PAD4 inhibitor, and TI + NE inhibitor groups. n = 3 mice/group. Data are presented as the mean ± SEM. Significance was examined by unpaired two-sided Student’s t test (b) or one-way ANOVA (d–f). ^*p < 0.05, ^**p < 0.01. ^***p < 0.001, ^****p < 0.0001.

NETs promote M1-like macrophage polarization through regulating iNOS

The murine study demonstrated that NET formation influences the inflammatory response following TI. Furthermore, early inhibition of NETs post-TI attenuates peritendinous fibrosis and improve healing outcomes. To further investigate the mechanisms underlying these observations, mouse bone marrow (BM) neutrophils were treated with PMA to induce NET formation. Bone marrow-derived monocytes (BMDMs) were then categorized into five experimental groups (Figure 5(a)): the M0 group (unstimulated BMDMs), the NETs group (BMDMs stimulated with NETs for 24 h), the NETs + DNase I group (BMDMs stimulated with NETs and treated with DNase I for 24 h), the M1 group (BMDMs pre-stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) for 24 h), and the M2 group (BMDMs pre-stimulated with interleukin-2 (IL-2) for 24 h).

Figure 5.

PMA-induced NETs enhance M1 macrophage polarization. (a) Schematic of in vitro macrophage stimulation with NET-containing medium. (b) Flow cytometry analysis of iNOS M1 macrophages (CD11b⁺F4/80⁺) after stimulated with the cultured medium from control, NETs, NETs + DNaseI, LPS + IFN-γ (M1), and IL4 (M2) groups. (c) Quantification of iNOS expression. (d) Confocal analysis of iNOS (M1; green) and DAPI (blue) in neutrophils after stimulated with the cultured medium from control, NETs, and NETs + DNaseI groups. Scale bars, 5 µm. (e) Representative analysis of the iNOS intensity. (f) Flow cytometry analysis of the CD80 and CD86. (g and h) Quantification of Genomic mean of CD80 and CD86. (i) qRCR of iNOS mRNA from indicated groups. Each group included three technical replicates. The results were performed at least two-independent experiment. Data are presented as the mean ± SEM. Significance was examined by one-way ANOVA (c, e and g–i). ^*p < 0.05, ^**p < 0.01. ^***p < 0.001, ^****p < 0.0001.

Stimulation with NETs or LPS plus IFN-γ significantly increased the expression of inducible nitric oxide synthase (iNOS), with the most pronounced upregulation observed in the LPS plus IFN-γ group. The elevated iNOS expression induced by NETs was attenuated by DNase I treatment (Figure 5(b) and (c)), suggesting that NETs contribute to iNOS induction. Confocal microscopy confirmed a marked increase in iNOS intensity following NETs stimulation, which was reduced by DNase I treatment (Figure 5(d)). In addition to iNOS, CD80 and CD86-canonical markers of M1 macrophage polarization-were also examined (Figure 5(d) and (e)). Flow cytometry analysis revealed increased expression of CD80 and CD86 in M1 macrophages compared to M0 macrophages. This expression was further enhanced by NETs stimulation but reduced following DNase I treatment (Figure 5(f)–(h)). Moreover, DNase I treatment decreased the mRNA expression levels of iNOS in the M1 + NETs group (Figure 5(i)). Together, these findings demonstrate that NETs significantly promote M1 macrophage polarization, highlighting their role in modulating macrophage phenotype during inflammation.

NETs suppressed M2-like macrophage polarization through regulating CD206

Macrophages are highly versatile immune cells that play critical roles in maintaining tissue homeostasis, regulating inflammation, and facilitating tissue repair. Among macrophage subtypes, the M2 phenotype is particularly recognized for its involvement in tissue repair and immune modulation. ³² Flow cytometry analysis of bone marrow-derived macrophages (BMDMs) confirmed that CD11b⁺F4/80⁺ cells represent pan-BMDM markers (Figure 6(a)). To investigate the NET role in macrophage polarization, mouse bone marrow neutrophils were stimulated with PMA to induce NET formation. BMDMs were then divided into five experimental groups, as depicted in Figure 6(b): the M0 group (unstimulated BMDMs), the NETs group (BMDMs stimulated with NETs for 24 h), NETs + DNase I group (BMDMs stimulated with NETs and treated with DNase I for 24 h), the M1 group (BMDMs pre-stimulated with LPS and IFN-γ for 24 h), and the M2 group (BMDMs pre-stimulated with IL-4 for 24 h).

Figure 6.

NETs suppress IL-4–induced M2 macrophage polarization. (a) Flow cytometry of macrophages (CD11b⁺F4/80⁺). (b) Flow cytometry analysis of CD206 from macrophages (CD11b⁺F4/80⁺) after stimulated with the cultured medium from control, NETs, NETs + DNasel, LPS + IFN-γ (M1), and IL4 (M2) groups. (c) Quantification of CD206 . (d) M2 macrophage were induced after IL-4 treatment. Cultured medium from PMA-induced NETs or PMA-induced NETs with DNase I treatment were added. (e) Quantification of CD206. (f) qPCR analysis of Arginase 1 mRNA expression. (g) ELISA analysis of IL-10 and TGF-β from cultured medium in control, LPS, LPS + NETs, and LPS + NETs + DNaseI groups. (h) ELISA analysis of TNF-α and IL-6 from cultured medium in control, IL4, IL4 + NETs, and IL4 + NETs + DNaseI groups. Each group included three technical replicates. The results were performed at least two-independent experiment. Data are presented as the mean ± SEM. Significance was examined by one-way ANOVA (c, e and g, h). ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001.

The IL-4-stimulated M2 group exhibited the most significant increase in CD206 expression, a marker of M2 macrophages. However, CD206 expression was reduced following NETs treatment, and this reduction was reversed by DNase I (Figure 6(b) and (c)), suggesting that NETs negatively regulate CD206 expression. To further explore the role of NETs in M2 macrophage polarization, PMA-induced NETs were added to IL-4-induced M2 macrophages. Flow cytometry analysis revealed that NETs treatment blunted the increased expression of CD206, while pretreatment with DNase I restored CD206 levels (Figure 6(e) and (f)), indicating that NETs directly inhibit M2 polarization. Consistent with these findings, the expression of arginase 1, another canonical M2 marker, mirrored the changes observed in CD206 expression (Figure 6(g)). ELISA was used to measure the levels of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β), cytokines predominantly secreted by M2 macrophages. The elevated levels of IL-10 and TGF-β in M2 macrophages were reduced following NETs treatment. In contrast, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), markers associated with M1 macrophage activation, were also modulated by NETs (Figure 6(h)).

Inhibition of NETs by PAD4 inhibitor significantly suppressed M1 macrophage after tendon bone injury

Inhibition of NETs by PAD4 inhibitor significantly reduced the presence of M1 macrophages following tendon-bone injury. Building on our previous in vitro findings, which demonstrated that NETs promote M1 macrophage polarization, we sought to investigate whether early NETs inhibition after tendon injury (TI) could suppress M1 polarization in vivo. Mice were divided into three experimental groups: sham operation, TI treated with acetylcellulose (vehicle control), and TI treated with a PAD4 inhibitor, with observations conducted over 21 days.

To assess macrophage phenotype and distribution in injured Achilles tendons, immunohistochemistry (IHC) was performed using antibodies against CD68 and CD11c, markers associated with M1 macrophages. The results revealed that CD68⁺ and CD11c⁺ macrophages were predominantly localized around bone marrow-like tissues and persisted throughout the ossification process (Figure 7(a)–(d)). Quantitative analysis demonstrated a significant increase in CD68⁺ and CD11c⁺ cells in the TI group compared to the sham group, which was markedly reduced following PAD4 inhibitor treatment (Figure 7(b) and (d)). These findings suggest that iNOS, TLR4, and IL-1β influence M1 macrophage activation and function. iNOS generates nitric oxide, TLR4 recognizes pathogen-associated molecular patterns, and IL-1β drives inflammatory responses, collectively contributing to the immune response in inflammatory conditions.^33,34 Western blot analysis further confirmed that the expression of iNOS, TLR4, and cleaved IL-1β was significantly elevated in tendon tissues following TI. PAD4 inhibition suppressed the upregulation of these markers (Figure 7(e) and (f)). In summary, NET inhibition significantly attenuates M1 macrophage polarization after tendon injury, highlighting the therapeutic potential of targeting NETs to modulate macrophage-mediated inflammation and tissue repair.

Figure 7.

PAD4 inhibition reduces M1 macrophage polarization after tendon-bone injury. (a) Immunohistochemistry (IHC) analysis of CD68 in tendon-bone section from control, TI, and TI + PAD4 inhibitor groups. (b) Quantification of CD11c positive cells. n = 6 mice/group. (c) IHC analysis of CD11c in tendon section from control, TI, and TI + PAD4 inhibitor groups. (d) Quantitative analysis of CD11c positive cells in c. n = 6 mice/group. (e) Western blot of iNOS, TLR4 and IL-1β in tendon tissue from control, TI, and TI + PAD4 inhibitor groups. (f) Quantification of relative gray intensity/GAPDH in c. n = 3 mice/group. Data are presented as the mean ± SEM. Scale bar = 1 mm. Significance was examined by one-way ANOVA (b, d, and f). ^**P < 0.01, ^****p < 0.0001.

Inhibition of NETs by PAD4 inhibitor significantly promote M2 macrophage after tendon-bone injury

Following tendon injury, the inflammatory response activates macrophages, which are critical for tissue repair. Among macrophage subtypes, M2 macrophages are particularly recognized for their anti-inflammatory and reparative properties, playing a pivotal role in the later stages of tissue regeneration after tendon-bone injuries. Our previous in vitro experiments demonstrated that NET inhibition promotes M2 macrophage polarization. To explore whether early NETs inhibition after tendon injury (TI) enhances M2 polarization in vivo, we performed immunohistochemistry (IHC) using antibodies against CD206, a marker for M2 macrophages. The results revealed that CD206⁺ macrophages were predominantly localized around bone marrow-like tissues (Figure 8(a) and (b)). Quantitative analysis demonstrated an increase in CD206⁺ cells in the TI group compared to the sham control, with a further elevation following PAD4 inhibitor treatment (Figure 8(b) and (d)). To further elucidate the role of macrophage in TI, flow cytometry analysis was conducted. The total macrophage population CD11b⁺/F4/80⁺ within CD45⁺ cells increased significantly after TI but was reduced following PAD4 inhibitor treatment. Additionally, the levels of inducible nitric oxide synthase (iNOS) and CD206 were analyzed in CD11b⁺/F4/80⁺ macrophages. Both iNOS and CD206 expression were significantly elevated in the TI group compared to the sham control. However, PAD4 inhibitor treatment suppressed iNOS levels while enhancing CD206 expression (Figure 8(c)–(f)). In summary, these findings indicate that PAD4 inhibition significantly promotes M2 macrophage polarization, underscoring its potential to modulate macrophage-mediated tissue repair and inflammation following tendon injury.

Figure 8.

PAD4 inhibition promotes M2 macrophage polarization following tendon injury. (a) IHC analysis of CD206 in tendon-bone section from control, TI, and TI + PAD4 inhibitor groups (n = 6). (b) Quantification of CD206 positive cells. n = 6 mice/group. (c) Flow cytometry analysis of macrophages (CD11b⁺F4/80⁺) and iNOS, CD206 in control, TI, and TI + PAD4 inhibitor groups. (d) Quantification of the percentages of total macrophages. (e) Quantitative analysis of iNOS in macrophages (CD11b⁺F4/80⁺). (f) Quantitative analysis of CD206 in macrophages (CD11b⁺F4/80⁺). Each group included three technical replicates (d–f). Data are presented as the mean ± SEM. Significance was examined by one-way ANOVA (b and d–f). ^*p < 0.05. ^**p < 0.01, ^***p < 0.001.

Discussion

While the interaction between neutrophils and macrophages mediates the immune response, whether this interaction promotes the healing process following tendon-to-bone injury is unclear. The interest in targeting macrophage activity in tendon-bone injuries is increasing, through approaches such as macrophage-derived miRNA, exosomes, or chemokines.^35–37 Our research elucidates a significant role of neutrophils in macrophage polarization and peritendinous fibrosis. We found that NETs inhibition significantly reduced fibroblast activation and the expression of fibronectin, collagen I, and α-SMA. Additionally, treatment with a PAD4-inhibitor markedly reduced the expression of iNOS, TLR4, and the proinflammatory cytokines IL-1β in TI mice. These findings suggest the existence of a novel NETs-dependent pathway that regulates macrophage polarization, immune response, and fibrogenesis in tendon-bone injuries. NETs inhibitors such as GSK484 or sivelestat may serve as potential therapeutic agents for tendon-bone injuries.

The immune response influences tendon-to-bone healing. Macrophages are essential immune cell subsets needed for successful tendon repair. While macrophages are known to promote early inflammatory responses, which are crucial for fracture repair, their roles in later stages of healing remain poorly understood. Anterior cruciate ligament (ACL) reconstruction, for example, begins with an early influx of inflammatory cells and graft necrosis, followed by fibrovascular interface tissue formation, and concludes with intra-articular and intra-tunnel graft remodeling. Our study investigates the roles of neutrophils and macrophages in TI. In wild-type mice, CD11c-positive cells were present throughout the fracture healing process. Although the role of dendritic cells in bone development and repair remains largely unexplored, CD11c-positive dendritic cells have been shown to differentiate into functional osteoclasts.^38,39 Furthermore, CD11c is also recognized as a marker of M1 proinflammatory macrophages. ⁴⁰

For our in vivo study, we used a mouse model in which the Achilles tendon of the right hind paw was transected and repaired. This model induces peritendinous fibrosis around the injured tendon.^41,42 Our results revealed an increased presence of F4/80-positive macrophages adjacent to α-SMA-positive fibroblasts, suggesting a reciprocal activation between these cell types. Prior research has demonstrated the necessity of macrophages for peritendinous fibrosis development in this model, enabling macrophage depletion via clodronate liposomes. ³⁵ This approach allowed us to investigate the role of inflammatory cytokines and damage-associated molecular patterns (DAMPs) released by macrophages in TI mice. We observed significantly elevated levels of macrophage-derived IL-1β, IL-1α, and HMGB1 in healing tendons, reflecting an intensified inflammatory response that likely contributes to increased macrophage accumulation at the injury site. Immunofluorescence analysis further suggested heightened macrophage infiltration and activation of peritendinous fibroblasts in tendon-bone injuries, indicating that BMDMs may serve as critical mediators of peritendinous fibrosis. Moreover, the regulation of macrophage polarization into a proinflammatory phenotype may facilitate intercellular communication between immune cells and resident tenocytes or fibroblasts.

Our study demonstrated the significant role of NETs in exacerbating tendon-bone injury through macrophage polarization. NET-induced macrophage polarization primarily involves several established inflammatory and regulatory signaling pathways, including NF-κB, TGF-β, and IL-10 signaling. NETs have been reported to activate Toll-like receptor 4 (TLR4), subsequently triggering downstream NF-κB signaling cascades.^43,44 In our experiments, we observed elevated expression levels of TLR4 and its downstream effector, iNOS, indicating NF-κB activation in macrophages following exposure to NETs. Activation of NF-κB enhances pro-inflammatory gene transcription, promoting M1 macrophage polarization, which is consistent with the increased expression of pro-inflammatory cytokines (IL-1β and TNF-α) documented in our results. ⁴⁵ The IL-10 signaling pathway, a well-known regulator of macrophage polarization toward an M2 anti-inflammatory phenotype, appears to be inhibited by NETs as indicated by our in vitro ELISA data. This disruption of IL-10 signaling would favor sustained inflammation and delayed healing observed in tendon-bone injuries. ⁴⁶ Collectively, these molecular mechanisms involving NF-κB activation and IL-10 signaling pathways, support our conclusion that NETs contribute significantly to impaired healing and increased fibrosis in tendon-bone injuries. Additional studies using pathway-specific inhibitors or genetic models could further elucidate these intricate molecular interactions.

Macrophages, particularly polarized macrophage subsets, actively internalize NETs during the early inflammatory phase following tissue injury. Distinct macrophage subsets exhibit differential capacities to degrade NETs, with pro-inflammatory (M1-like) macrophages efficiently internalizing NET components, subsequently triggering robust inflammatory responses via the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) and activation of NF-κB signaling pathways. ⁴⁷ In line with this, macrophages internalizing NETs significantly exacerbate inflammatory diseases, such as rheumatoid arthritis, through Rab5a-dependent mechanisms, further amplifying inflammation and tissue damage. ⁴⁸ We have demonstrated that NETs significantly enhance M1 macrophage polarization, characterized by increased expression of iNOS, CD80, CD86, and inflammatory cytokines including IL-1β and TNF-α, thus confirming the link between NET internalization and macrophage-driven inflammation as described in the previous studies. Future studies should address macrophage internalization mechanisms of NETs, downstream signaling (e.g. NF-κB activation, Rab5a pathway), and subsequent inflammatory amplification by employing advanced microscopy, genetic manipulation, or pharmacological inhibition.

Although GSK484 has been primarily used as a specific inhibitor of NETs, recent studies have uncovered its additional biological effects, including antitumor, and anti-metabolic properties in various experimental models.^49,50 Based on our in vivo studies, both GSK484 and sivelestat mitigated peritendinous fibrosis following tendon injury. In vitro experiments further demonstrated that NETs inhibition significantly reduced fibroblast activation by regulating the expression of fibrotic markers such as fibronectin, collagen I, and α-SMA in fibroblasts treated with BMDM-derived exosomes. Exosomes derived from BMDMs have been shown to promote fibrosis in tendon-bone injury. ³⁵ Additionally, NETs inhibition markedly decreases fibroblast proliferation and migration following treatment with BMDM-derived exosomes. These findings suggest that NETs exert distinct and specific effects on macrophages and fibroblasts. Given that NETs disrupt the p97-NPL4-ubiquitin fusion degradation protein 1 (UFD1) pathway, their influence on fibroblast proliferation and migration may be linked to the accumulation of misfolded proteins, leading to subsequent cell death. Future studies are needed to explore the detailed mechanisms by which NETs regulate fibroblast behavior.

Our data showed NET inhibition exhibits a therapeutic approach, several key challenges should be considered for successful clinical translation including drug delivery and potential off-target effects. Drug delivery represents a critical challenge. NET inhibitors, such as PAD4 inhibitors (GSK484) and neutrophil elastase inhibitors (sivelestat), require targeted delivery to injury sites to maximize local therapeutic benefits while minimizing systemic side effects. ⁵¹ Advanced drug-delivery strategies such as nanoparticle-based formulations, biomaterial scaffolds, or hydrogel systems could facilitate localized, controlled release of NET inhibitors directly to the tendon-bone interface, thus enhancing treatment efficacy and safety. ⁵² Potential off-target effects are another significant consideration. PAD4 and neutrophil elastase have important physiological functions beyond NET formation, including roles in normal immune defense, apoptosis, and tissue remodeling. ⁵³ Therefore, systemic inhibition could inadvertently impair essential innate immune functions, potentially increasing susceptibility to infection or disrupting tissue homeostasis. To mitigate these risks, future research should focus on developing highly selective inhibitors or utilizing targeted local delivery methods, as discussed above.

In our current study, we observed elevated expression of these ECM proteins (fibronectin, collagen type I, and α-SMA) following NET exposure, indicating direct fibrogenic activation of fibroblasts. NET components such as histones, neutrophil elastase (NE), and myeloperoxidase (MPO) directly activate fibroblasts, leading to increased fibroblast proliferation, differentiation into myofibroblasts, and subsequent ECM deposition.^54,55 Previous studies demonstrated that NE released from NETs directly promotes fibroblast-to-myofibroblast transition via protease-activated receptor (PAR)-dependent signaling, subsequently increasing expression of fibrotic markers including α-SMA, fibronectin, and type I collagen. ⁵⁶ In addition, NETs may directly influence ECM organization by interacting with matrix components such as fibronectin, collagen, and proteoglycans, altering their arrangement, degradation, or synthesis. ⁵⁷ Previous literature demonstrated that NETs could physically entrap ECM proteins, impair normal tissue remodeling processes, and establish persistent inflammatory microenvironments, contributing to sustained fibrosis. ⁵⁸ Future research involving direct co-culture experiments or use of genetic models deficient in NET-forming pathways could further elucidate these fibrotic mechanisms.

In summary, our findings highlight the crucial role of NETs-mediated macrophage polarization in tendon injury. TI triggers the upregulation of multiple pro-inflammatory cytokines, and NETs play a pivotal role in the development of peritendinous fibrosis. Specifically, NETs enhance the production of IL-1β and TNF-α by M1 macrophages while suppressing the release of IL-10 and TGF-β. Current clinical management of tendon-bone injuries typically involves anti-inflammatory drugs (such as Nonsteroidal Antiinflammatory Drugs or corticosteroids), biological agents (e.g. platelet-rich plasma, growth factors) and stem cell.^59–61 NET inhibitors (e.g. PAD4 inhibitors such as GSK484, or neutrophil elastase inhibitors such as sivelestat) have already shown translational potential in other inflammatory conditions, indicating a clearer pathway for clinical application.^62,63 Given their specificity, NET inhibitors may complement or enhance existing treatments like biologics or scaffold-based regenerative therapies, providing a more tailored therapeutic strategy for tendon-bone injuries.

Supplemental Material

sj-docx-1-tej-10.1177_20417314251348038 – Supplemental material for Formation of neutrophil extracellular traps in the early stages exacerbate the healing process by regulating macrophage polarization in Achilles tendon-bone injury

Supplemental material, sj-docx-1-tej-10.1177_20417314251348038 for Formation of neutrophil extracellular traps in the early stages exacerbate the healing process by regulating macrophage polarization in Achilles tendon-bone injury by Yiqin Zhou, Xiaolei Yang, Dawei Niu, Peiliang Fu, Qirong Qian and Qi Zhou in Journal of Tissue Engineering