Neutrophil Extracellular Traps Exacerbate Liver Ischemia-Reperfusion Injury by Promoting Macrophage M1 Polarization via the cGAS-STING Pathway

Neutrophil Extracellular Traps Exacerbate Liver Ischemia-Reperfusion Injury by Promoting Macrophage M1 Polarization via the cGAS-STING Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Neutrophil Extracellular Traps Exacerbate Liver Ischemia-Reperfusion Injury by Promoting Macrophage M1 Polarization via the cGAS-STING Pathway Dingheng Hu, Yunhai Luo, Liangxu Wang, Zhengli Tan, Qi Li, Denghui Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8822760/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Hepatic ischemia-reperfusion injury (IRI) is a serious complication during liver transplantation, which triggers a strongrepresents a significant complication in the context of liver transplantation, characterized by the induction of a robust non-specific inflammatory response throughmediated by damage-associated molecular patterns (DAMPs) and leads to, ultimately resulting in dysfunction of the transplanted liverorgan. The pathogenic pro-inflammatory (M1) polarization of hepatic macrophages is a driver of this hepatocyte injury, but the exact upstream trigger factor for this remains unclear. We investigated thecritical contributor to this hepatocellular damage; however, the precise upstream trigger remains unidentified. This study explores the potential role of neutrophil extracellular traps (NETs) as an upstream regulator of M1-type macrophage polarization. Utilizing the in vivo H-IRI mouse model alongside an in vitro macrophage co-culture system, we elucidated that the interaction between neutrophil extracellular traps (NETs) and macrophages is facilitated by the cGAS-STING signaling pathway. Our findings indicate that the double-stranded DNA generated by NETs is internalized by macrophages, subsequently activating the cytoplasmic sensor cGAS and the adaptor protein 3wSTING. This activation serves as a driver for the M1 macrophage phenotype. Our study unveils a novel and critical pathway, "NETs → cGAS-STING → M1 polarization," which plays a significant role in hepatic ischemia-reperfusion injury (HIRI). This pathway presents a promising therapeutic target for mitigating graft damage following liver transplantation. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Gastroenterology Biological sciences/Immunology Health sciences/Medical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hepatic ischemia-reperfusion injury (HIRI) is an unavoidable pathological consequence following liver resection and transplantation, and it represents a primary cause of graft dysfunction and biliary complications. The underlying mechanism of HIRI can be delineated into two distinct phases: during the ischemic phase, there is a disruption of the mitochondrial electron transport chain accompanied by the accumulation of abnormal metabolites. Subsequently, the reperfusion phase, characterized by the restoration of blood flow, precipitates a surge in reactive oxygen species (ROS), which in turn activates sterile inflammatory responses and programmed cell death pathways, including necrosis, apoptosis, and ferroptosis. Current therapeutic strategies emphasize mechanical perfusion techniques as a pivotal advancement. Hypothermic oxygenated perfusion (HOPE) mitigates oxidative damage by restoring mitochondrial metabolism, whereas normothermic mechanical perfusion (NMP) emulates the physiological environment to evaluate organ function. Both techniques have demonstrated superiority over traditional cold preservation methods, significantly enhancing transplant outcomes. Furthermore, adjunctive approaches such as antioxidants and ischemic preconditioning are subjects of ongoing investigation.Nevertheless, ischemia-reperfusion injury (IRI) remains the predominant factor contributing to the failure of transplanted organs, underscoring the urgent necessity for novel molecularly targeted therapies. The involvement of the inflammatory cascade in liver IRI underscores inflammation as the principal factor in this condition. The pathogenesis of liver IRI is driven by a robust sterile inflammatory cascade, in which neutrophils play a crucial role. These neutrophils are recruited to the injured liver, where they inflict direct tissue damage by releasing reactive oxygen species and proteases, forming neutrophil extracellular traps (NETs), and obstructing microvessels, thereby exacerbating the injury.Neutrophil extracellular traps (NETs) are a complex network structure consisting of loosely condensed chromatin (double-stranded DNA) and cytotoxic proteins released by neutrophils. They are recognized as an independent pathogenic factor contributing to liver cell damage [8]. NETs are rapidly deployed during reperfusion, facilitating the formation of "immune thrombosis" by capturing platelets, which leads to microcirculation obstruction and "reperfusion failure," thereby exacerbating tissue hypoxia [9]. Furthermore, the extracellular histones and proteases released by NETs exhibit direct cytotoxic effects, further damaging the liver sinusoidal endothelium and hepatocytes, thereby amplifying the local sterile inflammatory response [10]. During hepatic ischemia-reperfusion injury (HIRI), resident macrophages, known as Kupffer cells, in the liver significantly promote the polarization of necrotic hepatocytes towards a pro-inflammatory M1 phenotype. This process occurs through the phagocytosis of damage-associated molecular patterns (DAMPs) released by the necrotic hepatocytes. The M1 macrophages serve as the primary effectors of tissue destruction, releasing substantial quantities of reactive oxygen species and cytotoxic cytokines [11]. While it is established that neutrophils and macrophages engage in functional interactions, and the HMGB1-TLR4 pathway may contribute partially to this process [12, 13], the precise synergistic mechanism that connects the upstream formation of neutrophil extracellular traps (NETs) with the downstream polarization of M1 macrophages remains understood. Recent substantial evidence has unequivocally demonstrated that the activation of the cGAS-STING pathway serves as a potent and predominant driving force for M1 macrophage polarization [14]. The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway has emerged as the principal sensor for cytoplasmic double-stranded DNA, initiating an inflammatory response upon detecting the mislocalization of endogenous DNA [15]. Ischemia-reperfusion injury (IRI) leads to the overactivation of the cGAS-STING pathway, and extensive recent evidence has confirmed that this activation is a significant driving factor for M1 macrophage polarization. This reprogramming occurs through the activation of the interferon signaling pathway regulated by IRF3, coupled with a metabolic shift towards glycolysis to sustain the pro-inflammatory state [14]. Herein, we propose a theory: in the context of liver ischemia-reperfusion injury (IRI), double-stranded DNA generated by neutrophil extracellular traps (NETs) is internalized by macrophages and acts as the primary extracellular danger signal. We posit that this DNA from NETs serves as one of the significant contributing factors for the activation of the cGAS-STING pathway within macrophages, functioning as an upstream molecular switch that drives pathogenic M1 polarization. This process, in turn, exacerbates and sustains liver cell damage. The present study seeks to validate this hypothesis through a comprehensive analysis involving clinical transplantation samples, in vitro co-culture systems, and murine models of liver ischemia-reperfusion injury. Furthermore, the study aims to elucidate the role of the NETs-cGAS-STING-M1 axis, with the ultimate goal of identifying potential therapeutic targets for liver ischemia-reperfusion injury. Materials 3.1 Human Subjects and Sample Collection This study adhered to the principles outlined in the Declaration of Helsinki and received approval from the Institutional Review Board of the Human Experiment Ethics Committee at the First Affiliated Hospital of Chongqing Medical University (IIT: KX2025-KYC0751-01). Written informed consent was obtained from all participants. Peripheral blood samples were collected from patients who had undergone orthotopic liver transplantation (n = 5) within 24 hours post-operation. Additionally, samples were obtained from age- and gender-matched healthy volunteers (n = 5), serving as the control group. Blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) for the purpose of neutrophil separation, or in serum separation tubes for subsequent enzyme-linked immunosorbent assay (ELISA) analysis. Item Healthy Controls (n = 5) Positive Control (n = 5) Liver Transplant 24h (n = 5) P Value) Demographics Age,years 48 (42–54) 54 (45–63) 52 (48–59) 0.682 Male Sex, n (%) Female Sex, n (%) 3 (60%) 2 (40%) 3 (60%) 2 (40%) 4 (80%) 1 (20%) > 0.999 > 0.999 BMI(kg/m²) 23.5 (22.1–24.8) 24.1 (22.8–25.5) 27.2 (25.5–29.8) 0.038* Hepatic Biochemistry ALT(U/L) 22 (18–28) 145 (112–188) 703 (679–780) < 0.001* AST(U/L) 24 (20–31) 110 (95–135) 891 (680–934) < 0.001* AST/ALT (Ratio) 1.1 (0.9–1.2) 0.75 (0.71–0.84) 1.26 (1.00–1.19) < 0.001* ALT(U/L) 65 (55–78) 82 (70–95) 145 (110–190) 0.021* Total Bilirubin(mg/dL) 0.8 (0.6–1.0) 1.1 (0.8–1.4) 4.8 (3.2–6.5) < 0.001* Synthetic & Renal Function Albumin(g/dL) 4.2 (4.0–4.5) 4.1 (3.9–4.3) 2.9 (2.6–3.2) 0.002* (INR Serum Creatinine(mg/dL) 0.9 (0.8–1.1) 0.9 (0.8–1.0) 1.4 (1.2–1.8) 0.015* Hematology WBC(×10 9 /L) 5.8 (4.9–6.5) 6.1 (5.2–7.0) 12.5 (10.1–15.8) < 0.001* Platelets(×10 9 /L) 250 (210–285) 245 (220–270) 95 (75–120) < 0.001* 3.2 Isolation and Culture of Peripheral Blood Mononuclear Cells (PBMCs) Peripheral blood was freshly collected and diluted at a 1:1 ratio with phosphate-buffered saline (PBS). The diluted blood was then gently layered onto the upper surface of Ficoll-Paque PLUS (GE Healthcare, USA). The sample underwent centrifugation at 400×g for 30 minutes at 20°C, with both acceleration and deceleration settings adjusted to zero. Following centrifugation, the distinct white membrane layer containing the PBMCs was carefully aspirated and subjected to two wash cycles with PBS, each involving centrifugation at 300×g for 10 minutes. To achieve a high degree of purity in the mononuclear cell population, CD14 + cells were isolated from the total PBMCs using anti-CD14 magnetic beads, in accordance with the manufacturer's protocol (Miltenyi Biotec, Germany) [ 16 ] .Following flow cytometry verification, the purity of the sorted CD14 + monocytes consistently exceeded 95%. The freshly isolated CD14 + monocytes were resuspended in RPMI 1640 medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. To promote differentiation into resting macrophages (M0 phenotype), the cells were seeded at a density of 1×10^6 cells/mL onto culture plates and incubated with 50 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF) (PeproTech, USA) for a duration of 7 days [ 17 ] . The medium, containing fresh rhM-CSF, was partially replaced on the 3rd and 5th days. On the 7th day, the differentiated mature macrophages (M0 macrophages) were confirmed via phase-contrast microscopy to exhibit adherent growth and characteristic morphology prior to their use in subsequent experiments. 3.3 Isolation and Quantification of Human Neutrophil Extracellular Traps (NETs) Human neutrophils were isolated from the peripheral blood of healthy donors using the Human Bone Marrow Neutrophil Isolation Kit (Solarbio), in accordance with the manufacturer's protocol. The isolation process involved density gradient centrifugation. The purity of the isolated neutrophils was evaluated via flow cytometry, revealing a purity level exceeding 95%. To induce NETosis, the purified neutrophils were resuspended at a concentration of 3×10⁶ cells/mL in RPMI-1640 medium and stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) at 37°C for a duration of 4 hours [ 18 ] . Following incubation, the culture medium was carefully aspirated, and the wells were washed with 500 µL of pre-cooled Ham's Balanced Salt Solution (HBSS) to collect the released NETs. The suspension was subjected to centrifugation at 4°C for 5 minutes at 400 g to remove any residual cells and large fragments. The supernatant, containing the purified NETs, was collected and stored at -80°C. The concentration of NETs was quantified by measuring the double-stranded DNA (dsDNA) content using the Quant-iT™ PicoGreen™ dsDNA assay kit (Thermo Fisher Scientific), following the manufacturer's instructions. 3.4 Animals and Ethical Statement All animal experimental protocols were evaluated and approved by the Animal Care and Ethics Committee of Chongqing Medical University (IACUCapproval number: IACUC-CQMU-2025-03088). All experiments were performed in accordance with the relevant guidelines and regulations. Furthermore, we confirm that this study is reported in accordance with the ARRIVE guidelines. At the endpoint of the experiments, mice were euthanized by cervical dislocation to minimize animal suffering. The euthanasia procedures were strictly conducted in compliance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). 3.5 Partial Liver Ischemia-Reperfusion Injury Mouse Model All animal experiments were conducted following approval from the Animal Care and Use Committee of ChongqingMedical University (IACUCapproval number: IACUC-CQMU-2025-03088). Male C57BL/6 mice, aged 8 to 12 weeks and weighing between 20 and 25 grams, were utilized in this study. A 70% hepatic warm ischemia non-lethal model was established in accordance with previously documented methodologies [ 19 ] . Briefly, anesthesia was induced in the mice via intraperitoneal injection of 1.25% trichloroethanol (125 mg/kg) and intravenous administration of pentobarbital sodium (150 mg/kg). A median laparotomy was performed, and the hepatic portal triad, consisting of the hepatic artery, portal vein, and bile duct supplying the left and middle lobes of the liver, was occluded using non-invasive microvascular clips for a duration of 60 minutes. Following the removal of the clips, reperfusion was initiated. In the sham operation group, mice underwent identical surgical procedures without the application of vascular clamping. The mice were euthanized at intervals of 6, 12, and 24 hours post-reperfusion, and samples of blood and liver tissues were collected for subsequent analysis. 3.6 In Vivo Inhibitory Effect Study The experimental mice were randomly assigned to four groups, each consisting of four mice, with endpoint assessments conducted 12 hours post-reperfusion. The groups were as follows: (1) sham operation group; (2) ischemia-reperfusion with blank drug control; (3) ischemia-reperfusion with cytidine deaminase I (Dnase I) treatment; and (4) ischemia-reperfusion with RU.521 treatment. In the NETs inhibitor group, recombinant human cytidine deaminase I (Solarbio) was administered intraperitoneally at a dose of 10 mg/kg, 15 minutes prior to reperfusion. In the cGAS inhibitor group, RU.521 was administered intraperitoneally at a dose of 5 mg/kg, 30 minutes before the onset of ischemia. Mice in the drug control group received an equivalent volume of sterile normal saline. 3.7 In Vitro Co-Culture Experiment For the co-culture experiment, differentiated and matured macrophages (M0 macrophages) were utilized. In the direct co-culture setup, highly purified neutrophil extracellular traps (NETs) were introduced directly into the culture medium of M0 macrophages at a final double-stranded DNA (dsDNA) concentration of 5 µg/mL, and the cells were co-cultured for a duration of 12 hours. In the indirect co-culture setup, a Transwell system with a pore size of 0.4 µm (Corning) was employed. M0 macrophages were cultured in the lower chamber of a 6-well plate, while NETs, also at a dsDNA concentration of 5 µg/mL, were placed in the upper chamber. This configuration prevented direct cell-to-cell contact while allowing the diffusion of soluble molecules. The co-culture period was maintained for 12 hours. As a control, M0 macrophages were exposed to an equivalent volume of Hank's Balanced Salt Solution (HBSS). 3.8 In Vitro Pharmacological Inhibition Experiment The inhibition assay targeting the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and stimulator of interferon genes (STING) signaling pathway was conducted using the specific cGAS inhibitor RU.521 (Corning). Prior to the introduction of neutrophil extracellular traps (NETs) into the direct co-culture system, differentiated M0 macrophages were pre-treated with RU.521 at a concentration of 2.5 µg/mL or with the solvent dimethyl sulfoxide (DMSO) for a duration of 1 hour. Subsequently, the cells were incubated for an additional 12 hours. The experimental groups were as follows: (1) M0 macrophage control group; (2) M0 macrophage + NETs group; (3) M0 macrophage + RU.521 group; and (4) M0 macrophage + NETs + RU.521 group. 3.9 Biochemical Analysis Using a commercial colorimetric assay kit (Solarbio) in accordance with the manufacturer's instructions, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were determined. 3.10 Histology and Cytology Immunofluorescence (IF) The liver tissue was initially fixed in 10% neutral buffered formalin, subsequently embedded in paraffin, and sectioned into slices of 5 microns in thickness. For histological evaluation, these sections were stained using hematoxylin and eosin (H&E). The severity of hepatic injury was assessed by scoring the degree of sinusoidal vessel congestion, hepatocyte necrosis, and the extent of inflammatory cell infiltration. Apoptotic cells were identified utilizing the Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) kit (Roche). During the immunofluorescence analysis, the sections, following dewaxing, underwent antigen retrieval.The primary antibodies utilized in this study include anti-mastoperoxidase (MPO; Zenbio), anti-neutrophil elastase (NE; Proteintech), and anti-citrullinated histone H3 (CitH3; Zenbio). For the purpose of cell staining, neutrophils or co-cultured macrophages were fixed with 4% formaldehyde and permeabilized using 0.1% Triton X-100. The sections were incubated with the primary antibodies overnight at 4°C, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen). The cell nuclei were counterstained with DAPI. Images were acquired using either a fluorescence microscope or a confocal fluorescence microscope (Leica). 3.11 Enzyme-Linked Immunosorbent Assay (ELISA) The quantitative assessment of double-stranded DNA concentrations in human and mouse serum samples was conducted utilizing the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Thermo Fisher Scientific). The detection of interleukin-6 (IL-6) in human cell suspensions was performed using the Human IL-6 ELISA Kit (Abcam). Similarly, the presence of tumor necrosis factor-alpha (TNF-α) in human cell suspensions was identified using the Human TNF alpha ELISA Kit (Abcam), and interleukin-1 beta (IL-1β) was detected employing the Human IL-1 beta ELISA Kit (Abcam). 3.12 Protein Western Blotting Total protein extraction from cultured cells or frozen liver tissues was conducted using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified utilizing the BCA assay. Equivalent protein amounts (30 micrograms) were subjected to separation via SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat milk protein or BSA and incubated overnight at 4°C with the following antibodies: MPO, histone H3cit, cGAS, STING, p-STING, TBK1, p-TBK1 (serine 172), IRF3, p-IRF3, NF-κB p65, iNOS, IL-6, CD206, IL-10 (sourced from Zenbio or Abmart), and GAPDH, which served as a loading control. Following incubation with an HRP-conjugated secondary antibody, protein bands were visualized using the enhanced chemiluminescence (ECL) detection system. Optical density analysis was conducted using ImageJ software. 3.13 Flow Cytometry Macrophages in the M0 state, derived from in vitro inhibition experiments, were subjected to staining with fluorescently labeled antibodies. Specifically, antibodies targeting CD11b were labeled with FITC, those targeting CD86 (a marker for M1 macrophages) were labeled with PE, and those targeting CD206 (a marker for M2 macrophages) were labeled with APC (Elabscience). Data acquisition was performed using a FACSCanto II flow cytometer (BD Biosciences), and subsequent data analysis was conducted with FlowJo software. The macrophages were sorted based on CD11b expression, and the proportions of CD86-positive and CD206-positive cells within this population were quantified. 3.14 Statistical Analysis Data are expressed as the mean ± standard error of the mean (SEM), obtained from a minimum of three independent experiments. Statistical analyses were conducted utilizing GraphPad Prism 9 software. For comparisons between two groups, an unpaired two-tailed Student's t-test was employed. In cases involving more than two groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test. A p-value of less than 0.05 was deemed to indicate statistical significance. Results 4.1 After liver transplantation, increased neutrophil infiltration and elevated neutrophil extracellular traps (NETs) worsen liver damage. Initially, we conducted an analysis of the transcriptomic dataset (GSE14951) pertaining to mouse liver ischemia-reperfusion to identify differentially expressed genes. The findings indicated a significant upregulation of NETs expression in the ischemia-reperfusion injury (IRI) group (Figs. 1 A, 1 B, 1 C). To further substantiate the involvement of NETs in liver ischemia-reperfusion injury, we assessed NETs levels in the peripheral circulation of patients who had recently undergone liver transplantation. Immunofluorescence staining was performed on neutrophils isolated from the peripheral blood of these patients, revealing a marked increase in NETs formation in the transplant group compared to the healthy control group. These structures exhibited the characteristic net-like morphology of NETs, with key components such as myeloperoxidase (MPO), neutrophil elastase (NE), and histone H3 showing pronounced co-localization. The morphology and abundance of NETs in the patient group closely resembled those in the positive control group stimulated by phorbol 12-myristate 13-acetate (PMA), a potent inducer of NET formation.The quantitative analysis of immunofluorescence images demonstrated a statistically significant increase in neutrophil extracellular traps (NETs) in the patient cohort post-transplantation compared to the healthy control group (P < 0.01) (Figs. 1 D, 1 E). Furthermore, we assessed the concentration of circulating free double-stranded DNA (dsDNA) in the serum. In alignment with the imaging findings, the serum dsDNA levels in post-transplantation patients were markedly elevated relative to the healthy controls (Fig. 1 F). To further investigate the relationship between circulating serum NETs content in post-liver transplantation patients and the severity of liver injury, we quantified serum dsDNA levels and measured the classic liver injury biomarker, alanine aminotransferase (ALT). The analysis revealed a positive correlation between elevated serum NETs content and increased severity of liver injury (Fig. 1 G). In conclusion, these clinical data suggest a significant activation of neutrophils and subsequent release of NETs in the serum following liver transplantation, which is closely associated with post-transplantation liver injury. 4.2 In the mouse liver IRI model, neutrophil extracellular traps (NETs) worsen acute liver injury. To elucidate the role of NETs in liver injury, we developed a model of 70% warm liver ischemia-reperfusion injury in mature mice. Histological analyses, employing hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, demonstrated that prolonged reperfusion time correlated with increased inflammation and apoptosis in the mouse liver. The most pronounced coagulative necrosis and hepatocyte apoptosis were observed at 12 hours post-reperfusion (Fig. 2 A). Concurrently, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicative of liver cell injury, showed significant elevation at the I1R6 time point and peaked at I1R12 (Fig. 2 B). Furthermore, we investigated the temporal dynamics of NETs deposition and its association with the severity of liver injury. Immunofluorescence staining of liver sections revealed a time-dependent aggregation of NETs, confirmed by the co-localization of neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (CitH3) (Fig. 2 C).In the sham operation group, neutrophil extracellular traps (NETs) were scarcely detected; however, they began to appear significantly at 6 hours post-reperfusion (I1R6). By 12 hours post-reperfusion (I1R12), there was a marked increase in NET deposition, reaching a peak and forming extensive aggregations within the hepatic sinusoids. At 24 hours post-reperfusion (I1R24), the quantity of NETs began to decline. This pattern was corroborated by serum double-stranded DNA (dsDNA) level measurements, which also peaked at 12 hours (Fig. 2 D). Notably, the dynamic progression of liver injury closely paralleled the deposition process of NETs. The strong temporal correlation between the peak of NET infiltration and the peak of liver injury suggests that NETs play a pivotal role in the pathogenic process. The subsequent reduction in NETs and injury indicators after 24 hours may indicate the activation of endogenous clearance and repair mechanisms, underscoring the critical therapeutic intervention window within the first 12 hours post-reperfusion. 4.3 In the mouse liver ischemia-reperfusion injury model, NETs infiltration induces macrophage polarization to the pro-inflammatory M1 phenotype. To further investigate the association between NETs infiltration and hepatic injury, we examined the heatmap of immune cell infiltration proportions within the GSE14951 dataset. The findings revealed a significant increase in the polarization level of M1 macrophages in the ischemia-reperfusion (IR) group compared to the control group (Fig. 3 A). Pearson correlation analysis demonstrated a positive correlation between NETs and pro-inflammatory M1 polarization, and a negative correlation with anti-inflammatory M2 polarization (Fig. 3 B). By constructing murine IRI models at various reperfusion time points and performing Western blot analysis, it was observed that the expression of pro-inflammatory M1 polarization markers, such as inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6), in macrophages exhibited similar temporal kinetics to NET deposition and liver injury, peaking at 12 hours post-reperfusion (Fig. 3 C).To elucidate the impact of neutrophil extracellular trap (NET) components on macrophage polarization, we employed the NET inhibitor DNase I within the hepatic ischemia-reperfusion injury (HIRI) group. The findings indicated that DNase I markedly suppressed NET formation and reduced M1 polarization levels in macrophages, thereby confirming that NET infiltration is one of the significant contributing factors in promoting M1 polarization (Fig. 3 D). Furthermore, an in vitro co-culture experiment involving NETs (comprising polymorphonuclear leukocytes [PMN] and phorbol 12-myristate 13-acetate [PMA]) with macrophages (Mø) revealed that activated neutrophils releasing NETs significantly elevated M1 polarization levels (Fig. 3 E). Flow cytometry analysis corroborated these results, demonstrating a substantial increase in the proportion of macrophages expressing the M1 marker CD86 following NET exposure (Fig. 3 F). Collectively, these data provide compelling evidence that, within the murine liver ischemia-reperfusion injury model, NET infiltration drives macrophage polarization towards the pro-inflammatory M1 phenotype. 4.4 Neutrophil extracellular traps (NETs) promote M1-type polarization of macrophages via direct intercellular interactions. It has been established that the accumulation of neutrophil extracellular traps (NETs) following liver ischemia-reperfusion can promote M1 polarization of macrophages. In our study, we sought to elucidate the specific mechanisms by which NETs influence macrophage function in vitro. Utilizing the Transwell system, we co-cultured Mø macrophages with purified human NETs, either directly or indirectly (Fig. 4 A). Immunofluorescence imaging of the direct co-culture revealed that macrophages were in close proximity to the NETs. Double staining with histone H3 and CMFDA demonstrated that the reticular NETs not only adhered to the macrophage surface but also exhibited significant co-localization signals within the cytoplasm, suggesting internalization of the NETs by the macrophages (Fig. 4 B). Conversely, such intimate interactions were absent in the indirect Transwell co-culture system.Moreover, Western blot analysis demonstrated that the M1 markers, iNOS and IL-6, were significantly upregulated exclusively in macrophages that had direct contact with NETs, while the M2 markers, CD206 and IL-10, were downregulated (Fig. 4 C). Conversely, macrophages isolated from the Transwell membrane, which were physically separated from NETs, exhibited no significant alterations in polarization when compared to the control group. These findings suggest that NETs-induced macrophage polarization is contingent upon direct interaction between NETs and macrophages. The mere presence of soluble factors released by NETs is insufficient to induce M1 polarization. This direct physical contact suggests that macrophages may recognize and internalize the NETs structure, thereby activating intracellular signaling pathways rather than those at the cell surface. 4.5 NETs enhance M1 macrophage polarization by activating the cGAS-STING pathway. In order to investigate how NETs activate the M1 polarization of macrophages, we examined the activation status of classical signaling pathways using the GSE14951 dataset, comparing the ischemia-reperfusion injury (IRI) group with the control group. The analysis revealed significant differences in the activation of four pathways: NF-κB, MAPK, cGAS-STING, and mitochondrial oxidative stress (Fig. 5 A). Considering that NETs are characterized by double-stranded DNA (dsDNA) and their interaction with macrophages necessitates direct contact, and given that the cGAS-STING pathway is the primary cytoplasmic dsDNA sensor, its activation is recognized as a factor in driving M1 polarization of macrophages. Consequently, we postulated that the intracellular cGAS-STING DNA sensing pathway is the principal mediator of NETs-induced M1 polarization. To test this hypothesis, we assessed the expression of the cGAS-STING pathway in a mouse hepatic ischemia-reperfusion injury (HIRI) model at various reperfusion time points.The Western blot analysis revealed that the expression level of the cGAS-STING pathway peaked at 12 hours post-reperfusion, aligning with the kinetics of NET deposition (Fig. 5 B). This observation suggests that the activation of the cGAS-STING pathway during mouse liver ischemia-reperfusion injury (IRI) predominantly occurs through post-transcriptional modifications, such as phosphorylation, rather than alterations in protein abundance. In vitro experiments further corroborated these findings, demonstrating that the presence of NETs (composed of PMN and PMA) released by activated neutrophils in conjunction with macrophages resulted in an upregulation of the cGAS-STING pathway (Fig. 5 C) and a marked enhancement of M1 macrophage polarization. Collectively, these data provide compelling evidence that the activation of the cGAS-STING pathway is one of the significant contributing factors in the NETs-mediated M1 polarization of macrophages. 4.6 Inhibiting NETs or cGAS pharmacologically reduces M1 macrophage polarization and liver damage in mouse models of ischemia-reperfusion injury. To elucidate the role of the NETs-cGAS-STING-M1 macrophage axis in liver IRI, we performed an in vitro co-culture experiment involving NETs and macrophages. The findings indicated that NETs markedly activated the cGAS-STING signaling pathway in macrophages, thereby promoting M1 polarization. Furthermore, the administration of the cGAS inhibitor RU.521 significantly attenuated the activation of cGAS-STING pathway proteins and reduced M1 polarization levels (Figs. 6 A, 6 B). In the mouse hepatic ischemia-reperfusion injury (HIRI) model, administration of NETs inhibitors, specifically DNase I, resulted in a reduction in downstream cGAS-STING expression levels and a decrease in M1 macrophage polarization (refer to Figs. 6 C and 6 D). This intervention significantly mitigated liver injury, restoring it to levels comparable to the control group, thereby suggesting that NETs inhibition can effectively alleviate hepatic damage (refer to Fig. 6 E). Furthermore, the application of RU.521 in the mouse HIRI model demonstrated that, despite elevated NETs expression, there was a marked inhibition of the cGAS-STING pathway activation (refer to Figs. 6 C and 6 D). These findings imply that NETs primarily contribute to liver injury through the activation of the macrophage cGAS-STING pathway. Inhibition of this pathway can reduces the impact of NETs on liver damage and M1 polarization, thereby delineating a distinct pathogenic axis. Discussion Hepatic ischemia-reperfusion injury (IRI) represents a significant complication associated with hemorrhagic shock, liver resection, and transplantation [ 22 ] . This condition is characterized by severe hepatic damage resulting from oxidative stress, inflammation, and mitochondrial dysfunction [ 23 ] . The critical role of IRI is primarily attributed to its nature as a sterile inflammatory response, driven by damage-associated molecular patterns (DAMPs) that activate innate immune cells, such as macrophages [ 24 ] . Previous research has established that neutrophils release neutrophil extracellular traps (NETs), which can independently exacerbate liver IRI [ 25 ] , while the M1 polarization of macrophages serves as a pivotal factor in the progression of this injury [ 26 ] . In this context, we propose a more intricate scenario: NETs derived from neutrophils may compensate for the insufficiency of other DAMP signals and represent a crucial upstream mechanism for the M1 polarization of macrophages in hepatic ischemia-reperfusion. While other damage-associated molecular patterns (DAMPs), such as mitochondrial DNA (mtDNA) [ 27 ] and high mobility group protein B1 (HMGB1) [ 28 ] , have been extensively studied, their effects appear to be cell type-dependent. In contrast, neutrophil extracellular traps (NETs), as potent amplifiers of inflammation, may exert a more pervasive influence [ 29 ] . Through direct measurements of the liver immune microenvironment in an in vivo hepatic ischemia-reperfusion injury (HIRI) model, the results demonstrated that, compared to the control group, the levels of neutrophil extracellular traps (NETs), specifically neutrophil elastase (NE), myeloperoxidase (MPO), and histone H3, were elevated in the livers of HIRI mice. This finding indicates that both macrophages and neutrophils are significantly involved in the inflammatory response induced by HIRI. However, in vitro experiments revealed that stimulating macrophages with the supernatant of NETs containing only soluble factors resulted in a markedly reduced effect compared to stimulation with the purified NETs structure. This suggests that the initiation of the M1 polarization signal is likely dependent on the DNA backbone or related components of NETs, rather than on the soluble chemotactic factors. Considering the role of the NETs-macrophage axis in both in vivo and in vitro models, our study demonstrates that hepatic function is compromised following reperfusion, as hepatic ischemia-reperfusion injury (HIRI) results in extensive hepatocyte apoptosis and a pronounced inflammatory response. The application of DNase I to degrade neutrophil extracellular traps (NETs) has been shown to markedly enhance liver function and ameliorate the immune microenvironment. Notably, a recent investigation utilizing macrophage-specific cGAS knockout mice in the context of HIRI revealed a similar protective phenotype [ 30 ] . This phenomenon is contingent upon the cytoplasmic DNA sensing mechanism [ 31 ] , underscoring the critical role of inhibiting the cGAS-STING pathway in macrophages during HIRI [ 32 ] . Concurrently, bioinformatics analyses indicate that, relative to the control group, the cGAS-STING pathway is significantly upregulated in the HIRI group. Consequently, we identified the cGAS-STING signaling pathway as the principal downstream mechanism by which macrophages detect DNA from neutrophil extracellular traps (NETs) and undergo M1 polarization, addressing the research question. It is important to note that previous studies have shown that cGAS-STING-deficient mice do not exhibit injury under basal conditions [ 33 ] , suggesting that the activation of the STING signal alone is insufficient to induce liver injury. However, pre-treatment could potentially increase cytoplasmic DNA levels (originating from either mitochondrial DNA or NETs) to a certain threshold [ 34 ] , at which point the cGAS-STING pathway acts merely as an amplifier. Nonetheless, we acknowledge that while cGAS-STING inhibitors can prevent NETs-induced M1 polarization in vitro [ 35 ] , the precise contribution of DNA from NETs compared to mitochondrial DNA in the hepatic ischemia-reperfusion injury (HIRI) model requires further in vivo validation [ 36 ] . Having established that the "NETs-cGAS-STING" axis is instrumental in the tissue damage associated with ischemia/reperfusion, targeting this specific immune cell interaction may facilitate advancements in mitigating early graft dysfunction (EAD) in liver transplantation. Consequently, it is pertinent to explore the functional relationship between the NETs-cGAS-STING axis and other inflammatory signaling pathways, such as Toll-like receptors (TLRs) and NLRP3 inflammasomes. We highlighted that while STING inhibitors have historically been employed to elucidate the role of the cGAS-STING pathway in hepatic ischemia-reperfusion injury (HIRI), their potential to mitigate damage via unidentified targets or upstream ligands suggests that numerous early investigations may have overlooked the NET-driven M1 polarization pathway [ 33 ] . Consequently, the discovery that this signaling cascade can be activated both in vitro and in vivo through pharmacological interventions, such as DNase I or STING inhibition, presents novel opportunities for the development of immunomodulatory therapies. Furthermore, this study establishes a theoretical framework for future research aimed at understanding how the cytoplasmic DNA of neutrophil extracellular traps (NETs) communicates with macrophages, influencing epigenetic and metabolic reprogramming to induce a stable M1 phenotype. Declarations Funding The authors received no specific funding for this work Author Contributions Zhongjun Wu conceived the project. Dingheng Hu and Yunhai Luo performed the experiments and collected the data. Dingheng Hu, Liangxu Wang, and Zhengli Tan performed the statistical analysis. Dingheng Hu, Qi Li, and Denghui Wang prepared the figures and drafted the manuscript (Dingheng Hu prepared Figs. 1, 2, 3, and 6; Qi Li prepared Fig. 4; Denghui Wang prepared Fig. 5). Dingheng Hu and Yunhai Luo provided critical feedback and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript. References LIU, Z. et al. Molecular Mechanisms of Ischemia/Reperfusion Injury and Graft Dysfunction in Liver Transplantation: Insights from Multi-Omics Studies in Rodent Animal Models [J]. Int. J. Biol. Sci. 21 (5), 2135–2154 (2025). NEMES, B. & GáMáN, G. Biliary complications after liver transplantation [J]. Expert Rev. Gastroenterol. Hepatol. 9 (4), 447–466 (2015). CHOUCHANI E T, PELL V R, G. A. U. D. E. E. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS [J]. Nature 515 (7527), 431–435 (2014). DE OLIVEIRA T H C & GONçALVES G K N. Liver ischemia reperfusion injury: Mechanisms, cellular pathways, and therapeutic approaches [J]. Int. Immunopharmacol. 150 , 114299 (2025). SCHLEGEL, A. & MULLER, X. Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation [J]. EBioMedicine 60 , 103014 (2020). KIM, S. H. Machine perfusion in liver transplantation: A step forward, but still on the runway [J]. World J. Gastroenterol. 31 (40), 112408 (2025). JAESCHKE, H. & WOOLBRIGHT, B. L. Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species [J]. Transpl. Rev. (Orlando) . 26 (2), 103–114 (2012). HUANG, H. et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury [J]. Hepatology 62 (2), 600–614 (2015). JORCH S K, K. U. B. E. S. P. An emerging role for neutrophil extracellular traps in noninfectious disease [J]. Nat. Med. 23 (3), 279–287 (2017). KAWAI, C. et al. Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury [J]. Am. J. Pathol. 186 (4), 829–843 (2016). HIRAO, H., NAKAMURA, K. & KUPIEC-WEGLINSKI J, W. Liver ischaemia-reperfusion injury: a new understanding of the role of innate immunity [J]. Nat. Rev. Gastroenterol. Hepatol. 19 (4), 239–256 (2022). TADIE, J. M. et al. HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4 [J]. Am. J. Physiol. Lung Cell. Mol. Physiol. 304 (5), L342–L349 (2013). TSUNG, A. et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion [J]. J. Exp. Med. 201 (7), 1135–1143 (2005). LUO, X. & LI, H. MA L, et al. Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice [J]. Gastroenterology , 155 (6): (2018). 1971-84.e4. SUN, L. et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway [J]. Science 339 (6121), 786–791 (2013). Mosher, B. S., Fulkerson, H. L. & Yurochko, A. D. Collection and Isolation of CD14+ Primary Human Monocytes Via Dual Density Gradient Centrifugation as a Model System to Study Human Cytomegalovirus Infection and Pathogenesis. Methods Mol Biol. ;2244:103–113. (2021). 10.1007/978-1-0716-1111-1_6 . PMID: 33555584. Hamidzadeh, K., Belew, A. T., El-Sayed, N. M. & Mosser, D. M. The transition of M-CSF-derived human macrophages to a growth-promoting phenotype. Blood Adv. 4 (21), 5460–5472. 10.1182/bloodadvances.2020002683 (2020). PMID: 33166408; PMCID: PMC7656919. Najmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J. & Ferri, L. E. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J. Vis. Exp. (98), 52687. 10.3791/52687 (2015). PMID: 25938591; PMCID: PMC4541576. Lei, D. et al. MAFF alleviates hepatic ischemia-reperfusion injury by regulating the CLCF1/STAT3 signaling pathway. Cell. Mol. Biol. Lett. 30 (1), 39. 10.1186/s11658-025-00721-x (2025). PMID: 40169936; PMCID: PMC11963299. Liu, Y. et al. Neutrophil Extracellular Traps Regulate HMGB1 Translocation and Kupffer Cell M1 Polarization During Acute Liver Transplantation Rejection. Front. Immunol. 13 , 823511. 10.3389/fimmu.2022.823511 (2022). PMID: 35603144; PMCID: PMC9120840. Stoimenou, M., Tzoros, G., Skendros, P. & Chrysanthopoulou, A. Methods for the Assessment of NET Formation: From Neutrophil Biology to Translational Research. Int. J. Mol. Sci. 23 (24), 15823. 10.3390/ijms232415823 (2022). PMID: 36555464; PMCID: PMC9781911. ZHAI, Y. & PETROWSKY, H. Ischaemia-reperfusion injury in liver transplantation–from bench to bedside [J]. Nat. Rev. Gastroenterol. Hepatol. 10 (2), 79–89 (2013). DE OLIVEIRA T H C & GONçALVES G K N. Liver ischemia reperfusion injury: Mechanisms, cellular pathways, and therapeutic approaches [J]. Int. Immunopharmacol. 150 , 114299 (2025). DERY K J, Y. A. O. S. et al. New therapeutic concepts against ischemia-reperfusion injury in organ transplantation [J]. Expert Rev. Clin. Immunol. 19 (10), 1205–1224 (2023). LIU, Y. et al. Neutrophil extracellular traps and complications of liver transplantation [J]. Front. Immunol. 13 , 1054753 (2022). WANG, H. & XI, Z. Macrophage Polarization and Liver Ischemia-Reperfusion Injury [J]. Int. J. Med. Sci. 18 (5), 1104–1113 (2021). ZHANG, F. et al. The role of extracellular traps in ischemia reperfusion injury [J]. Front. Immunol. 13 , 1022380 (2022). ZHAO, J. et al. NETs Promote Inflammatory Injury by Activating cGAS-STING Pathway in Acute Lung Injury [J]. Int. J. Mol. Sci. , 24 (6). (2023). YOKOYAMA A P H et al. Neutrophil extracellular traps (NETs), transfusion requirements and clinical outcomes in orthotopic liver transplantation [J]. J. Thromb. Thrombolysis . 56 (2), 253–263 (2023). LI X J, QU J R, ZHANG, Y. H. et al. The dual function of cGAS-STING signaling axis in liver diseases [J]. Acta Pharmacol. Sin . 45 (6), 1115–1129 (2024). XIONG, Y. et al. Dimethyl fumarate alleviate hepatic ischemia-reperfusion injury through suppressing cGAS-STING signaling [J]. MedComm 2025, 6(2): e70077. (2020). XU, D. et al. The cGAS-STING Pathway: Novel Perspectives in Liver Diseases [J]. Front. Immunol. 12 , 682736 (2021). CHEN, R. et al. The role of cGAS-STING signalling in liver diseases [J]. JHEP Rep. 3 (5), 100324 (2021). LEI Z, DENG, M. et al. cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING [J]. Am. J. Physiol. Gastrointest. Liver Physiol. 314 (6), G655–g67 (2018). COUILLIN I, RITEAU N. STING Signaling and Sterile Inflammation [J]. Front. Immunol. 12 , 753789 (2021). BRYANT, J. D. et al. Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells [J]. Curr. Protoc. 2 (2), e372 (2022). Additional Declarations No competing interests reported. Supplementary Files WesternBlotSupplementaryInfoFile.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 13 May, 2026 Reviews received at journal 31 Mar, 2026 Reviewers agreed at journal 31 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 23 Feb, 2026 Reviewers invited by journal 20 Feb, 2026 Editor assigned by journal 20 Feb, 2026 Editor invited by journal 18 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 16 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8822760","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":595615926,"identity":"609bdb34-a4ba-4b86-a84a-4be5b0e522cd","order_by":0,"name":"Dingheng Hu","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dingheng","middleName":"","lastName":"Hu","suffix":""},{"id":595615927,"identity":"2443b5ff-b547-43e3-91a8-d48e75bda972","order_by":1,"name":"Yunhai Luo","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yunhai","middleName":"","lastName":"Luo","suffix":""},{"id":595615928,"identity":"08e8a84b-fe22-4309-93bf-c624dda6c7f1","order_by":2,"name":"Liangxu Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liangxu","middleName":"","lastName":"Wang","suffix":""},{"id":595615929,"identity":"bb8fa1f7-9155-40fe-bbd0-47a5f5057b4c","order_by":3,"name":"Zhengli Tan","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengli","middleName":"","lastName":"Tan","suffix":""},{"id":595615930,"identity":"49abc9b8-776c-4eb3-85a5-73faa78b9575","order_by":4,"name":"Qi Li","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Li","suffix":""},{"id":595615931,"identity":"b93699e9-818a-44f4-a506-09f181217cd3","order_by":5,"name":"Denghui Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Denghui","middleName":"","lastName":"Wang","suffix":""},{"id":595615932,"identity":"36b9cd60-8061-4638-88cc-9817fabc760b","order_by":6,"name":"Zhongjun Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYHCCBCC24eFnbyBeSyJQbZqMZM8B4rUwArUctjG44UCkeoPzB54/+Nl2nofhBgPjh485xGg5cCCxsbftNg/j7AZmyZnbiNBidrAhsZkRqIVZ5gAbMy9RWg4zgLSc42GTSCBWyzGwlgM8PERrsT/DkDiz51wyjwTPwWbi/CLZfybhw48yO3v7480HP3wkRgsDA08CAyMbiAGKH+IA+wEGhj/EKh4Fo2AUjIIRCQC/PDhMzZrvDgAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhongjun","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-02-08 15:39:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8822760/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8822760/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103330606,"identity":"5e905087-0a91-4138-9e9c-4fb2a0af0d1d","added_by":"auto","created_at":"2026-02-24 13:41:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1309636,"visible":true,"origin":"","legend":"\u003cp\u003eExamination of Neutrophil Extracellular Traps (NETs) Infiltration in the Serum of Liver Transplant Recipients (A) Differential gene expression analysis was conducted between the mouse ischemia-reperfusion injury (IRI) group and the control group using the GSE14951 dataset, followed by the construction of a volcano plot. (B) The intersection of differentially expressed genes from both groups with the NETs gene set was identified, resulting in 27 shared genes, which were subsequently illustrated using a Venn diagram. (C) The ssGSEA algorithm was employed to evaluate the scores of samples from the IRI and control groups, and a bar chart was generated to demonstrate a significant increase in NETs content in the IRI group. (D, E) Representative immunofluorescence microscopy images depict the co-localization of NETs components (H3Cit, DAPI, and NE) in the serum of liver transplant recipients (within 24 hours post-surgery), healthy subjects, and a positive control group, along with the average fluorescence intensity (magnification: 100x; scale bar = 75 μm). Data analysis was performed using one-way analysis of variance, and results are expressed as Mean ± SD (n = 5). Statistical significance is indicated as follows: * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001.(F) The levels of circulating free double-stranded DNA (dsDNA) in the serum of liver transplant recipients and healthy individuals were measured. (G) The relationship between the levels of circulating serum neutrophil extracellular traps (NETs) in liver transplant recipients and the severity of liver injury was assessed. The data were analyzed using a t-test and are presented as Mean ± SD (n = 5). Statistical significance was determined at *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/8b5ee87a9d59e3f3b71766f2.png"},{"id":103330368,"identity":"11eea1aa-9c02-466f-a03a-c25a2b44fa44","added_by":"auto","created_at":"2026-02-24 13:40:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1732839,"visible":true,"origin":"","legend":"\u003cp\u003eIn the murine liver ischemia-reperfusion injury (IRI) model, the infiltration of neutrophil extracellular traps (NETs) exacerbates the severity of acute hepatic injury.\u003c/p\u003e\n\u003cp\u003e(A) Hematoxylin and eosin (H\u0026amp;E) staining demonstrated that the most extensive coagulative necrosis occurred in the 70% warm liver ischemia-reperfusion injury model at 12 hours post-reperfusion. (B) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining indicated that the highest level of hepatocyte apoptosis was observed in the 70% warm liver ischemia-reperfusion injury model at 12 hours. (C) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels reached their peak at 12 hours following reperfusion and showed a significant decrease by 24 hours. (D) The measurement of serum double-stranded DNA (dsDNA) levels confirmed a peak at 12 hours, followed by a decline at 24 hours.Immunofluorescence staining of liver sections, along with the quantification of average fluorescence intensity, revealed that the aggregation of neutrophil extracellular traps (NETs) displayed a time-dependent pattern consistent with liver injury. This was evidenced by the co-localization of neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (CitH3). The deposition of NETs peaked at 12 hours post-reperfusion, subsequently declining by 24 hours (magnification: 40x; scale bar = 100 μm). The data were subjected to one-way analysis of variance and are presented as Mean ± SD (n = 4). Statistical significance was denoted as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/461ab196ba570d0362c00c8d.png"},{"id":103330481,"identity":"751c17d6-3628-4554-b09d-3636f983b5d1","added_by":"auto","created_at":"2026-02-24 13:41:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2127643,"visible":true,"origin":"","legend":"\u003cp\u003eIn the murine liver ischemia-reperfusion injury (IRI) model, neutrophil extracellular trap (NET) infiltration promotes an increase in pro-inflammatory M1 macrophage polarization. (A) The heatmap depicting immune cell infiltration, derived from the GSE14951 dataset, indicates an elevated level of M1 macrophage polarization in the IRI model. (B) Pearson correlation analysis demonstrates a positive correlation between NETs and M1 polarization within the IRI model. (C) Western blot analysis confirms the expression of polarization markers for NETs (H3, MPO) and macrophages M1 (iNOS, IL-6), as well as M2 (CD206, IL-10), in the murine liver at various reperfusion time points. (D) Western blot analysis further verifies the impact of the NETs inhibitor DNase I (10 mg/kg) administered to the I1R12 group on macrophage polarization. Data were analyzed using one-way analysis of variance and are presented as mean ± standard deviation (n = 4). Statistical significance is denoted as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.(E) Four distinct in vitro cell models were established: Mø, Mø+PMA, Mø+PMN, and Mø+PMA+PMN. Western blot analysis was employed to assess the presence of neutrophil extracellular traps (NETs) and polarization markers. The data were subjected to one-way analysis of variance and are presented as the mean ± standard deviation (n = 3). Statistical significance was denoted as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. (F) Flow cytometry was utilized to evaluate the expression of M1 (CD86) and M2 (CD11b) markers across the four macrophage in vitro models.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/f087c8086b7dfba87fd5e6a0.png"},{"id":103330563,"identity":"e9b79e25-5a78-4e0d-8412-942a473f4e8a","added_by":"auto","created_at":"2026-02-24 13:41:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1264683,"visible":true,"origin":"","legend":"\u003cp\u003eillustrates the induction of M1-type macrophage polarization by neutrophil extracellular traps (NETs) through direct intercellular interactions. (A) Macrophages (Mø) were co-cultured with purified human NETs under both direct and indirect conditions. (B) Neutrophils isolated from healthy donors were labeled with CMFDA (green) and stimulated with PMA to promote NET formation, followed by co-culture with Mø macrophages. Immunofluorescence staining was employed to visualize citrullinated histone H3 (red), CMFDA (green), and DAPI (blue). Arrows indicate cells that were negative for CMFDA (i.e., not neutrophils) but positive for citrullinated histone H3, suggesting the uptake or internalization of NET components by macrophages (magnification: 200x; scale bar = 25 μm). (C) Alterations in macrophage polarization markers, specifically M1 markers (IL-6, iNOS) and M2 markers (CD206, IL-10), were assessed under both Transwell and direct co-culture conditions. Data were analyzed using one-way ANOVA and are presented as Mean ± SD (n = 3). Statistical significance is indicated as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/a3f260768ed27b8907b85d47.png"},{"id":103330338,"identity":"f88483a7-58c4-4b57-876d-5073b40cdf2a","added_by":"auto","created_at":"2026-02-24 13:40:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1373727,"visible":true,"origin":"","legend":"\u003cp\u003eillustrates the pivotal role of the cGAS-STING pathway in the NETs-mediated M1 polarization of macrophages. (A) An analysis of the GSE14951 dataset reveals significant differences in the NF-κB, MAPK, cGAS-STING, and mitochondrial oxidative stress pathways between the ischemia-reperfusion injury (IRI) group and the control group. (B) Western blot analysis was employed to confirm the expression of NETs markers (H3, MPO) and cGAS-STING pathway markers (cGAS, STING, P-STING, TBK1, P-TBK1, IRF3, P-IRF3, NF-κB) at various reperfusion time points in the mouse liver. The data were analyzed using one-way ANOVA and are presented as Mean ± SD (n = 4). Statistical significance is indicated as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. (C) Western blot analysis was conducted to verify the presence of NETs and markers of the cGAS-STING pathway across four distinct in vitro cell model groups (Mø, Mø+PMA, Mø+PMN, Mø+PMA+PMN). (D) Enzyme-linked immunosorbent assay (ELISA) was utilized to detect M1 polarization indicators, specifically TNF-α, IL-6, and IL-1β, within the same four in vitro cell model groups. The data were subjected to one-way ANOVA for statistical analysis and are presented as Mean ± SD (n = 3). Statistical significance is denoted as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/d0fb086220c46776afdc196d.png"},{"id":103330249,"identity":"d68b6136-224d-40be-9151-f92af7860920","added_by":"auto","created_at":"2026-02-24 13:40:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1951523,"visible":true,"origin":"","legend":"\u003cp\u003eThe pharmacological inhibition of neutrophil extracellular traps (NETs) or cyclic GMP-AMP synthase (cGAS) mitigates M1 macrophage polarization and liver injury in murine models of ischemia-reperfusion injury (IRI). (A) Western blot analysis was conducted to assess the expression levels of NET components, specifically myeloperoxidase (MPO) and Histone H3, along with markers of M1/M2 macrophage polarization within a macrophage-NET co-culture model, with and without the application of RU.521, a cGAS inhibitor. (B) The expression of markers associated with the cGAS-STING signaling pathway was evaluated under identical conditions. Data were subjected to one-way ANOVA and are presented as mean ± standard deviation (SD) with a sample size of n = 3. Statistical significance is denoted as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001. (C) Western blot analysis was performed to verify the expression of NET components and macrophage polarization markers in the liver tissues of hepatic IRI (HIRI) mice following administration of DNase I or RU.521. (D) The expression levels of cGAS-STING pathway markers in hepatic tissues were assessed under varying treatment conditions. (E) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and hematoxylin and eosin (HE) staining were employed to evaluate cellular apoptosis and inflammation in liver tissues under different experimental conditions. (F) Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in mice subjected to different treatment regimens. Data were analyzed using one-way ANOVA and are presented as mean ± SD with a sample size of n = 4. Statistical significance is indicated as follows: * P\u0026lt;0.05, ** P\u0026lt;0.01, *** P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/14c27332e7424b80b99b2003.png"},{"id":103330771,"identity":"510a5451-4c04-4156-8d45-289591eb92f2","added_by":"auto","created_at":"2026-02-24 13:42:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12897133,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/8595a7ea-2eae-49f8-a075-7240a7b5a30d.pdf"},{"id":103330340,"identity":"e7e779ed-3b19-453d-a465-152eb7b2b65f","added_by":"auto","created_at":"2026-02-24 13:40:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8350162,"visible":true,"origin":"","legend":"","description":"","filename":"WesternBlotSupplementaryInfoFile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8822760/v1/ca2db5acd56f04d51f19f118.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neutrophil Extracellular Traps Exacerbate Liver Ischemia-Reperfusion Injury by Promoting Macrophage M1 Polarization via the cGAS-STING Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatic ischemia-reperfusion injury (HIRI) is an unavoidable pathological consequence following liver resection and transplantation, and it represents a primary cause of graft dysfunction and biliary complications. The underlying mechanism of HIRI can be delineated into two distinct phases: during the ischemic phase, there is a disruption of the mitochondrial electron transport chain accompanied by the accumulation of abnormal metabolites. Subsequently, the reperfusion phase, characterized by the restoration of blood flow, precipitates a surge in reactive oxygen species (ROS), which in turn activates sterile inflammatory responses and programmed cell death pathways, including necrosis, apoptosis, and ferroptosis. Current therapeutic strategies emphasize mechanical perfusion techniques as a pivotal advancement. Hypothermic oxygenated perfusion (HOPE) mitigates oxidative damage by restoring mitochondrial metabolism, whereas normothermic mechanical perfusion (NMP) emulates the physiological environment to evaluate organ function. Both techniques have demonstrated superiority over traditional cold preservation methods, significantly enhancing transplant outcomes. Furthermore, adjunctive approaches such as antioxidants and ischemic preconditioning are subjects of ongoing investigation.Nevertheless, ischemia-reperfusion injury (IRI) remains the predominant factor contributing to the failure of transplanted organs, underscoring the urgent necessity for novel molecularly targeted therapies. The involvement of the inflammatory cascade in liver IRI underscores inflammation as the principal factor in this condition.\u003c/p\u003e \u003cp\u003eThe pathogenesis of liver IRI is driven by a robust sterile inflammatory cascade, in which neutrophils play a crucial role. These neutrophils are recruited to the injured liver, where they inflict direct tissue damage by releasing reactive oxygen species and proteases, forming neutrophil extracellular traps (NETs), and obstructing microvessels, thereby exacerbating the injury.Neutrophil extracellular traps (NETs) are a complex network structure consisting of loosely condensed chromatin (double-stranded DNA) and cytotoxic proteins released by neutrophils. They are recognized as an independent pathogenic factor contributing to liver cell damage [8]. NETs are rapidly deployed during reperfusion, facilitating the formation of \"immune thrombosis\" by capturing platelets, which leads to microcirculation obstruction and \"reperfusion failure,\" thereby exacerbating tissue hypoxia [9]. Furthermore, the extracellular histones and proteases released by NETs exhibit direct cytotoxic effects, further damaging the liver sinusoidal endothelium and hepatocytes, thereby amplifying the local sterile inflammatory response [10].\u003c/p\u003e \u003cp\u003eDuring hepatic ischemia-reperfusion injury (HIRI), resident macrophages, known as Kupffer cells, in the liver significantly promote the polarization of necrotic hepatocytes towards a pro-inflammatory M1 phenotype. This process occurs through the phagocytosis of damage-associated molecular patterns (DAMPs) released by the necrotic hepatocytes. The M1 macrophages serve as the primary effectors of tissue destruction, releasing substantial quantities of reactive oxygen species and cytotoxic cytokines [11]. While it is established that neutrophils and macrophages engage in functional interactions, and the HMGB1-TLR4 pathway may contribute partially to this process [12, 13], the precise synergistic mechanism that connects the upstream formation of neutrophil extracellular traps (NETs) with the downstream polarization of M1 macrophages remains understood.\u003c/p\u003e \u003cp\u003eRecent substantial evidence has unequivocally demonstrated that the activation of the cGAS-STING pathway serves as a potent and predominant driving force for M1 macrophage polarization [14]. The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway has emerged as the principal sensor for cytoplasmic double-stranded DNA, initiating an inflammatory response upon detecting the mislocalization of endogenous DNA [15]. Ischemia-reperfusion injury (IRI) leads to the overactivation of the cGAS-STING pathway, and extensive recent evidence has confirmed that this activation is a significant driving factor for M1 macrophage polarization. This reprogramming occurs through the activation of the interferon signaling pathway regulated by IRF3, coupled with a metabolic shift towards glycolysis to sustain the pro-inflammatory state [14].\u003c/p\u003e \u003cp\u003eHerein, we propose a theory: in the context of liver ischemia-reperfusion injury (IRI), double-stranded DNA generated by neutrophil extracellular traps (NETs) is internalized by macrophages and acts as the primary extracellular danger signal. We posit that this DNA from NETs serves as one of the significant contributing factors for the activation of the cGAS-STING pathway within macrophages, functioning as an upstream molecular switch that drives pathogenic M1 polarization. This process, in turn, exacerbates and sustains liver cell damage. The present study seeks to validate this hypothesis through a comprehensive analysis involving clinical transplantation samples, in vitro co-culture systems, and murine models of liver ischemia-reperfusion injury. Furthermore, the study aims to elucidate the role of the NETs-cGAS-STING-M1 axis, with the ultimate goal of identifying potential therapeutic targets for liver ischemia-reperfusion injury.\u003c/p\u003e"},{"header":"Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Human Subjects and Sample Collection\u003c/h2\u003e \u003cp\u003e This study adhered to the principles outlined in the Declaration of Helsinki and received approval from the Institutional Review Board of the Human Experiment Ethics Committee at the First Affiliated Hospital of Chongqing Medical University (IIT: KX2025-KYC0751-01). Written informed consent was obtained from all participants. Peripheral blood samples were collected from patients who had undergone orthotopic liver transplantation (n\u0026thinsp;=\u0026thinsp;5) within 24 hours post-operation. Additionally, samples were obtained from age- and gender-matched healthy volunteers (n\u0026thinsp;=\u0026thinsp;5), serving as the control group. Blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) for the purpose of neutrophil separation, or in serum separation tubes for subsequent enzyme-linked immunosorbent assay (ELISA) analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy Controls (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive Control\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLiver Transplant 24h (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP Value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDemographics\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge,years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48 (42\u0026ndash;54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54 (45\u0026ndash;63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52 (48\u0026ndash;59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.682\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale Sex, n (%)\u003c/p\u003e \u003cp\u003eFemale Sex, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (60%)\u003c/p\u003e \u003cp\u003e2 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (60%)\u003c/p\u003e \u003cp\u003e2 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (80%)\u003c/p\u003e \u003cp\u003e1 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI(kg/m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.5 (22.1\u0026ndash;24.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.1 (22.8\u0026ndash;25.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.2 (25.5\u0026ndash;29.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.038*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepatic Biochemistry\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22 (18\u0026ndash;28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e145 (112\u0026ndash;188)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e703 (679\u0026ndash;780)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 (20\u0026ndash;31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110 (95\u0026ndash;135)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e891 (680\u0026ndash;934)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST/ALT (Ratio)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.1 (0.9\u0026ndash;1.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.75 (0.71\u0026ndash;0.84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.26 (1.00\u0026ndash;1.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT(U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e65 (55\u0026ndash;78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82 (70\u0026ndash;95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e145 (110\u0026ndash;190)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.021*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Bilirubin(mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8 (0.6\u0026ndash;1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1 (0.8\u0026ndash;1.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.8 (3.2\u0026ndash;6.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSynthetic \u0026amp; Renal Function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin(g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.2 (4.0\u0026ndash;4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.1 (3.9\u0026ndash;4.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9 (2.6\u0026ndash;3.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(INR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum Creatinine(mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9 (0.8\u0026ndash;1.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9 (0.8\u0026ndash;1.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4 (1.2\u0026ndash;1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.015*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHematology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC(\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.8 (4.9\u0026ndash;6.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.1 (5.2\u0026ndash;7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.5 (10.1\u0026ndash;15.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelets(\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250 (210\u0026ndash;285)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e245 (220\u0026ndash;270)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95 (75\u0026ndash;120)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Isolation and Culture of Peripheral Blood Mononuclear Cells (PBMCs)\u003c/h2\u003e \u003cp\u003ePeripheral blood was freshly collected and diluted at a 1:1 ratio with phosphate-buffered saline (PBS). The diluted blood was then gently layered onto the upper surface of Ficoll-Paque PLUS (GE Healthcare, USA). The sample underwent centrifugation at 400\u0026times;g for 30 minutes at 20\u0026deg;C, with both acceleration and deceleration settings adjusted to zero. Following centrifugation, the distinct white membrane layer containing the PBMCs was carefully aspirated and subjected to two wash cycles with PBS, each involving centrifugation at 300\u0026times;g for 10 minutes. To achieve a high degree of purity in the mononuclear cell population, CD14\u0026thinsp;+\u0026thinsp;cells were isolated from the total PBMCs using anti-CD14 magnetic beads, in accordance with the manufacturer's protocol (Miltenyi Biotec, Germany)\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.Following flow cytometry verification, the purity of the sorted CD14\u0026thinsp;+\u0026thinsp;monocytes consistently exceeded 95%. The freshly isolated CD14\u0026thinsp;+\u0026thinsp;monocytes were resuspended in RPMI 1640 medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. To promote differentiation into resting macrophages (M0 phenotype), the cells were seeded at a density of 1\u0026times;10^6 cells/mL onto culture plates and incubated with 50 ng/mL recombinant human macrophage colony-stimulating factor (rhM-CSF) (PeproTech, USA) for a duration of 7 days\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. The medium, containing fresh rhM-CSF, was partially replaced on the 3rd and 5th days. On the 7th day, the differentiated mature macrophages (M0 macrophages) were confirmed via phase-contrast microscopy to exhibit adherent growth and characteristic morphology prior to their use in subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Isolation and Quantification of Human Neutrophil Extracellular Traps (NETs)\u003c/h2\u003e \u003cp\u003eHuman neutrophils were isolated from the peripheral blood of healthy donors using the Human Bone Marrow Neutrophil Isolation Kit (Solarbio), in accordance with the manufacturer's protocol. The isolation process involved density gradient centrifugation. The purity of the isolated neutrophils was evaluated via flow cytometry, revealing a purity level exceeding 95%. To induce NETosis, the purified neutrophils were resuspended at a concentration of 3\u0026times;10⁶ cells/mL in RPMI-1640 medium and stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) at 37\u0026deg;C for a duration of 4 hours\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Following incubation, the culture medium was carefully aspirated, and the wells were washed with 500 \u0026micro;L of pre-cooled Ham's Balanced Salt Solution (HBSS) to collect the released NETs. The suspension was subjected to centrifugation at 4\u0026deg;C for 5 minutes at 400 g to remove any residual cells and large fragments. The supernatant, containing the purified NETs, was collected and stored at -80\u0026deg;C. The concentration of NETs was quantified by measuring the double-stranded DNA (dsDNA) content using the Quant-iT\u0026trade; PicoGreen\u0026trade; dsDNA assay kit (Thermo Fisher Scientific), following the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Animals and Ethical Statement\u003c/h2\u003e \u003cp\u003e All animal experimental protocols were evaluated and approved by the Animal Care and Ethics Committee of Chongqing Medical University (IACUCapproval number: IACUC-CQMU-2025-03088). All experiments were performed in accordance with the relevant guidelines and regulations. Furthermore, we confirm that this study is reported in accordance with the ARRIVE guidelines. At the endpoint of the experiments, mice were euthanized by cervical dislocation to minimize animal suffering. The euthanasia procedures were strictly conducted in compliance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Partial Liver Ischemia-Reperfusion Injury Mouse Model\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted following approval from the Animal Care and Use Committee of ChongqingMedical University (IACUCapproval number: IACUC-CQMU-2025-03088). Male C57BL/6 mice, aged 8 to 12 weeks and weighing between 20 and 25 grams, were utilized in this study. A 70% hepatic warm ischemia non-lethal model was established in accordance with previously documented methodologies\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Briefly, anesthesia was induced in the mice via intraperitoneal injection of 1.25% trichloroethanol (125 mg/kg) and intravenous administration of pentobarbital sodium (150 mg/kg). A median laparotomy was performed, and the hepatic portal triad, consisting of the hepatic artery, portal vein, and bile duct supplying the left and middle lobes of the liver, was occluded using non-invasive microvascular clips for a duration of 60 minutes. Following the removal of the clips, reperfusion was initiated. In the sham operation group, mice underwent identical surgical procedures without the application of vascular clamping. The mice were euthanized at intervals of 6, 12, and 24 hours post-reperfusion, and samples of blood and liver tissues were collected for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.6 In Vivo Inhibitory Effect Study\u003c/h2\u003e \u003cp\u003eThe experimental mice were randomly assigned to four groups, each consisting of four mice, with endpoint assessments conducted 12 hours post-reperfusion. The groups were as follows: (1) sham operation group; (2) ischemia-reperfusion with blank drug control; (3) ischemia-reperfusion with cytidine deaminase I (Dnase I) treatment; and (4) ischemia-reperfusion with RU.521 treatment. In the NETs inhibitor group, recombinant human cytidine deaminase I (Solarbio) was administered intraperitoneally at a dose of 10 mg/kg, 15 minutes prior to reperfusion. In the cGAS inhibitor group, RU.521 was administered intraperitoneally at a dose of 5 mg/kg, 30 minutes before the onset of ischemia. Mice in the drug control group received an equivalent volume of sterile normal saline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.7 In Vitro Co-Culture Experiment\u003c/h2\u003e \u003cp\u003eFor the co-culture experiment, differentiated and matured macrophages (M0 macrophages) were utilized. In the direct co-culture setup, highly purified neutrophil extracellular traps (NETs) were introduced directly into the culture medium of M0 macrophages at a final double-stranded DNA (dsDNA) concentration of 5 \u0026micro;g/mL, and the cells were co-cultured for a duration of 12 hours. In the indirect co-culture setup, a Transwell system with a pore size of 0.4 \u0026micro;m (Corning) was employed. M0 macrophages were cultured in the lower chamber of a 6-well plate, while NETs, also at a dsDNA concentration of 5 \u0026micro;g/mL, were placed in the upper chamber. This configuration prevented direct cell-to-cell contact while allowing the diffusion of soluble molecules. The co-culture period was maintained for 12 hours. As a control, M0 macrophages were exposed to an equivalent volume of Hank's Balanced Salt Solution (HBSS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.8 In Vitro Pharmacological Inhibition Experiment\u003c/h2\u003e \u003cp\u003eThe inhibition assay targeting the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and stimulator of interferon genes (STING) signaling pathway was conducted using the specific cGAS inhibitor RU.521 (Corning). Prior to the introduction of neutrophil extracellular traps (NETs) into the direct co-culture system, differentiated M0 macrophages were pre-treated with RU.521 at a concentration of 2.5 \u0026micro;g/mL or with the solvent dimethyl sulfoxide (DMSO) for a duration of 1 hour. Subsequently, the cells were incubated for an additional 12 hours. The experimental groups were as follows: (1) M0 macrophage control group; (2) M0 macrophage\u0026thinsp;+\u0026thinsp;NETs group; (3) M0 macrophage\u0026thinsp;+\u0026thinsp;RU.521 group; and (4) M0 macrophage\u0026thinsp;+\u0026thinsp;NETs\u0026thinsp;+\u0026thinsp;RU.521 group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Biochemical Analysis\u003c/h2\u003e \u003cp\u003eUsing a commercial colorimetric assay kit (Solarbio) in accordance with the manufacturer's instructions, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.10 Histology and Cytology Immunofluorescence (IF)\u003c/h2\u003e \u003cp\u003eThe liver tissue was initially fixed in 10% neutral buffered formalin, subsequently embedded in paraffin, and sectioned into slices of 5 microns in thickness. For histological evaluation, these sections were stained using hematoxylin and eosin (H\u0026amp;E). The severity of hepatic injury was assessed by scoring the degree of sinusoidal vessel congestion, hepatocyte necrosis, and the extent of inflammatory cell infiltration. Apoptotic cells were identified utilizing the Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) kit (Roche). During the immunofluorescence analysis, the sections, following dewaxing, underwent antigen retrieval.The primary antibodies utilized in this study include anti-mastoperoxidase (MPO; Zenbio), anti-neutrophil elastase (NE; Proteintech), and anti-citrullinated histone H3 (CitH3; Zenbio). For the purpose of cell staining, neutrophils or co-cultured macrophages were fixed with 4% formaldehyde and permeabilized using 0.1% Triton X-100. The sections were incubated with the primary antibodies overnight at 4\u0026deg;C, followed by incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen). The cell nuclei were counterstained with DAPI. Images were acquired using either a fluorescence microscope or a confocal fluorescence microscope (Leica).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.11 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe quantitative assessment of double-stranded DNA concentrations in human and mouse serum samples was conducted utilizing the Quant-iT\u0026trade; PicoGreen\u0026trade; dsDNA Assay Kit (Thermo Fisher Scientific). The detection of interleukin-6 (IL-6) in human cell suspensions was performed using the Human IL-6 ELISA Kit (Abcam). Similarly, the presence of tumor necrosis factor-alpha (TNF-α) in human cell suspensions was identified using the Human TNF alpha ELISA Kit (Abcam), and interleukin-1 beta (IL-1β) was detected employing the Human IL-1 beta ELISA Kit (Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.12 Protein Western Blotting\u003c/h2\u003e \u003cp\u003eTotal protein extraction from cultured cells or frozen liver tissues was conducted using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified utilizing the BCA assay. Equivalent protein amounts (30 micrograms) were subjected to separation via SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were then blocked with 5% non-fat milk protein or BSA and incubated overnight at 4\u0026deg;C with the following antibodies: MPO, histone H3cit, cGAS, STING, p-STING, TBK1, p-TBK1 (serine 172), IRF3, p-IRF3, NF-κB p65, iNOS, IL-6, CD206, IL-10 (sourced from Zenbio or Abmart), and GAPDH, which served as a loading control. Following incubation with an HRP-conjugated secondary antibody, protein bands were visualized using the enhanced chemiluminescence (ECL) detection system. Optical density analysis was conducted using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.13 Flow Cytometry\u003c/h2\u003e \u003cp\u003eMacrophages in the M0 state, derived from in vitro inhibition experiments, were subjected to staining with fluorescently labeled antibodies. Specifically, antibodies targeting CD11b were labeled with FITC, those targeting CD86 (a marker for M1 macrophages) were labeled with PE, and those targeting CD206 (a marker for M2 macrophages) were labeled with APC (Elabscience). Data acquisition was performed using a FACSCanto II flow cytometer (BD Biosciences), and subsequent data analysis was conducted with FlowJo software. The macrophages were sorted based on CD11b expression, and the proportions of CD86-positive and CD206-positive cells within this population were quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.14 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), obtained from a minimum of three independent experiments. Statistical analyses were conducted utilizing GraphPad Prism 9 software. For comparisons between two groups, an unpaired two-tailed Student's t-test was employed. In cases involving more than two groups, a one-way analysis of variance (ANOVA) was performed, followed by Tukey's post-hoc test. A p-value of less than 0.05 was deemed to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 After liver transplantation, increased neutrophil infiltration and elevated neutrophil extracellular traps (NETs) worsen liver damage.\u003c/h2\u003e \u003cp\u003eInitially, we conducted an analysis of the transcriptomic dataset (GSE14951) pertaining to mouse liver ischemia-reperfusion to identify differentially expressed genes. The findings indicated a significant upregulation of NETs expression in the ischemia-reperfusion injury (IRI) group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To further substantiate the involvement of NETs in liver ischemia-reperfusion injury, we assessed NETs levels in the peripheral circulation of patients who had recently undergone liver transplantation. Immunofluorescence staining was performed on neutrophils isolated from the peripheral blood of these patients, revealing a marked increase in NETs formation in the transplant group compared to the healthy control group. These structures exhibited the characteristic net-like morphology of NETs, with key components such as myeloperoxidase (MPO), neutrophil elastase (NE), and histone H3 showing pronounced co-localization. The morphology and abundance of NETs in the patient group closely resembled those in the positive control group stimulated by phorbol 12-myristate 13-acetate (PMA), a potent inducer of NET formation.The quantitative analysis of immunofluorescence images demonstrated a statistically significant increase in neutrophil extracellular traps (NETs) in the patient cohort post-transplantation compared to the healthy control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, we assessed the concentration of circulating free double-stranded DNA (dsDNA) in the serum. In alignment with the imaging findings, the serum dsDNA levels in post-transplantation patients were markedly elevated relative to the healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). To further investigate the relationship between circulating serum NETs content in post-liver transplantation patients and the severity of liver injury, we quantified serum dsDNA levels and measured the classic liver injury biomarker, alanine aminotransferase (ALT). The analysis revealed a positive correlation between elevated serum NETs content and increased severity of liver injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In conclusion, these clinical data suggest a significant activation of neutrophils and subsequent release of NETs in the serum following liver transplantation, which is closely associated with post-transplantation liver injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.2 In the mouse liver IRI model, neutrophil extracellular traps (NETs) worsen acute liver injury.\u003c/h2\u003e \u003cp\u003eTo elucidate the role of NETs in liver injury, we developed a model of 70% warm liver ischemia-reperfusion injury in mature mice. Histological analyses, employing hematoxylin and eosin (H\u0026amp;E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, demonstrated that prolonged reperfusion time correlated with increased inflammation and apoptosis in the mouse liver. The most pronounced coagulative necrosis and hepatocyte apoptosis were observed at 12 hours post-reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Concurrently, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicative of liver cell injury, showed significant elevation at the I1R6 time point and peaked at I1R12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, we investigated the temporal dynamics of NETs deposition and its association with the severity of liver injury. Immunofluorescence staining of liver sections revealed a time-dependent aggregation of NETs, confirmed by the co-localization of neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3 (CitH3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).In the sham operation group, neutrophil extracellular traps (NETs) were scarcely detected; however, they began to appear significantly at 6 hours post-reperfusion (I1R6). By 12 hours post-reperfusion (I1R12), there was a marked increase in NET deposition, reaching a peak and forming extensive aggregations within the hepatic sinusoids. At 24 hours post-reperfusion (I1R24), the quantity of NETs began to decline. This pattern was corroborated by serum double-stranded DNA (dsDNA) level measurements, which also peaked at 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Notably, the dynamic progression of liver injury closely paralleled the deposition process of NETs. The strong temporal correlation between the peak of NET infiltration and the peak of liver injury suggests that NETs play a pivotal role in the pathogenic process. The subsequent reduction in NETs and injury indicators after 24 hours may indicate the activation of endogenous clearance and repair mechanisms, underscoring the critical therapeutic intervention window within the first 12 hours post-reperfusion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e4.3 In the mouse liver ischemia-reperfusion injury model, NETs infiltration induces macrophage polarization to the pro-inflammatory M1 phenotype.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the association between NETs infiltration and hepatic injury, we examined the heatmap of immune cell infiltration proportions within the GSE14951 dataset. The findings revealed a significant increase in the polarization level of M1 macrophages in the ischemia-reperfusion (IR) group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Pearson correlation analysis demonstrated a positive correlation between NETs and pro-inflammatory M1 polarization, and a negative correlation with anti-inflammatory M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). By constructing murine IRI models at various reperfusion time points and performing Western blot analysis, it was observed that the expression of pro-inflammatory M1 polarization markers, such as inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6), in macrophages exhibited similar temporal kinetics to NET deposition and liver injury, peaking at 12 hours post-reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).To elucidate the impact of neutrophil extracellular trap (NET) components on macrophage polarization, we employed the NET inhibitor DNase I within the hepatic ischemia-reperfusion injury (HIRI) group. The findings indicated that DNase I markedly suppressed NET formation and reduced M1 polarization levels in macrophages, thereby confirming that NET infiltration is one of the significant contributing factors in promoting M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, an in vitro co-culture experiment involving NETs (comprising polymorphonuclear leukocytes [PMN] and phorbol 12-myristate 13-acetate [PMA]) with macrophages (M\u0026oslash;) revealed that activated neutrophils releasing NETs significantly elevated M1 polarization levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Flow cytometry analysis corroborated these results, demonstrating a substantial increase in the proportion of macrophages expressing the M1 marker CD86 following NET exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these data provide compelling evidence that, within the murine liver ischemia-reperfusion injury model, NET infiltration drives macrophage polarization towards the pro-inflammatory M1 phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Neutrophil extracellular traps (NETs) promote M1-type polarization of macrophages via direct intercellular interactions.\u003c/h2\u003e \u003cp\u003eIt has been established that the accumulation of neutrophil extracellular traps (NETs) following liver ischemia-reperfusion can promote M1 polarization of macrophages. In our study, we sought to elucidate the specific mechanisms by which NETs influence macrophage function in vitro. Utilizing the Transwell system, we co-cultured M\u0026oslash; macrophages with purified human NETs, either directly or indirectly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Immunofluorescence imaging of the direct co-culture revealed that macrophages were in close proximity to the NETs. Double staining with histone H3 and CMFDA demonstrated that the reticular NETs not only adhered to the macrophage surface but also exhibited significant co-localization signals within the cytoplasm, suggesting internalization of the NETs by the macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conversely, such intimate interactions were absent in the indirect Transwell co-culture system.Moreover, Western blot analysis demonstrated that the M1 markers, iNOS and IL-6, were significantly upregulated exclusively in macrophages that had direct contact with NETs, while the M2 markers, CD206 and IL-10, were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Conversely, macrophages isolated from the Transwell membrane, which were physically separated from NETs, exhibited no significant alterations in polarization when compared to the control group. These findings suggest that NETs-induced macrophage polarization is contingent upon direct interaction between NETs and macrophages. The mere presence of soluble factors released by NETs is insufficient to induce M1 polarization. This direct physical contact suggests that macrophages may recognize and internalize the NETs structure, thereby activating intracellular signaling pathways rather than those at the cell surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.5 NETs enhance M1 macrophage polarization by activating the cGAS-STING pathway.\u003c/h2\u003e \u003cp\u003eIn order to investigate how NETs activate the M1 polarization of macrophages, we examined the activation status of classical signaling pathways using the GSE14951 dataset, comparing the ischemia-reperfusion injury (IRI) group with the control group. The analysis revealed significant differences in the activation of four pathways: NF-κB, MAPK, cGAS-STING, and mitochondrial oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Considering that NETs are characterized by double-stranded DNA (dsDNA) and their interaction with macrophages necessitates direct contact, and given that the cGAS-STING pathway is the primary cytoplasmic dsDNA sensor, its activation is recognized as a factor in driving M1 polarization of macrophages. Consequently, we postulated that the intracellular cGAS-STING DNA sensing pathway is the principal mediator of NETs-induced M1 polarization. To test this hypothesis, we assessed the expression of the cGAS-STING pathway in a mouse hepatic ischemia-reperfusion injury (HIRI) model at various reperfusion time points.The Western blot analysis revealed that the expression level of the cGAS-STING pathway peaked at 12 hours post-reperfusion, aligning with the kinetics of NET deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This observation suggests that the activation of the cGAS-STING pathway during mouse liver ischemia-reperfusion injury (IRI) predominantly occurs through post-transcriptional modifications, such as phosphorylation, rather than alterations in protein abundance. In vitro experiments further corroborated these findings, demonstrating that the presence of NETs (composed of PMN and PMA) released by activated neutrophils in conjunction with macrophages resulted in an upregulation of the cGAS-STING pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and a marked enhancement of M1 macrophage polarization. Collectively, these data provide compelling evidence that the activation of the cGAS-STING pathway is one of the significant contributing factors in the NETs-mediated M1 polarization of macrophages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e4.6 Inhibiting NETs or cGAS pharmacologically reduces M1 macrophage polarization and liver damage in mouse models of ischemia-reperfusion injury.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the role of the NETs-cGAS-STING-M1 macrophage axis in liver IRI, we performed an in vitro co-culture experiment involving NETs and macrophages. The findings indicated that NETs markedly activated the cGAS-STING signaling pathway in macrophages, thereby promoting M1 polarization. Furthermore, the administration of the cGAS inhibitor RU.521 significantly attenuated the activation of cGAS-STING pathway proteins and reduced M1 polarization levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In the mouse hepatic ischemia-reperfusion injury (HIRI) model, administration of NETs inhibitors, specifically DNase I, resulted in a reduction in downstream cGAS-STING expression levels and a decrease in M1 macrophage polarization (refer to Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). This intervention significantly mitigated liver injury, restoring it to levels comparable to the control group, thereby suggesting that NETs inhibition can effectively alleviate hepatic damage (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Furthermore, the application of RU.521 in the mouse HIRI model demonstrated that, despite elevated NETs expression, there was a marked inhibition of the cGAS-STING pathway activation (refer to Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings imply that NETs primarily contribute to liver injury through the activation of the macrophage cGAS-STING pathway. Inhibition of this pathway can reduces the impact of NETs on liver damage and M1 polarization, thereby delineating a distinct pathogenic axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHepatic ischemia-reperfusion injury (IRI) represents a significant complication associated with hemorrhagic shock, liver resection, and transplantation \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. This condition is characterized by severe hepatic damage resulting from oxidative stress, inflammation, and mitochondrial dysfunction \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The critical role of IRI is primarily attributed to its nature as a sterile inflammatory response, driven by damage-associated molecular patterns (DAMPs) that activate innate immune cells, such as macrophages \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Previous research has established that neutrophils release neutrophil extracellular traps (NETs), which can independently exacerbate liver IRI \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, while the M1 polarization of macrophages serves as a pivotal factor in the progression of this injury \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In this context, we propose a more intricate scenario: NETs derived from neutrophils may compensate for the insufficiency of other DAMP signals and represent a crucial upstream mechanism for the M1 polarization of macrophages in hepatic ischemia-reperfusion. While other damage-associated molecular patterns (DAMPs), such as mitochondrial DNA (mtDNA) \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e and high mobility group protein B1 (HMGB1) \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, have been extensively studied, their effects appear to be cell type-dependent. In contrast, neutrophil extracellular traps (NETs), as potent amplifiers of inflammation, may exert a more pervasive influence \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThrough direct measurements of the liver immune microenvironment in an in vivo hepatic ischemia-reperfusion injury (HIRI) model, the results demonstrated that, compared to the control group, the levels of neutrophil extracellular traps (NETs), specifically neutrophil elastase (NE), myeloperoxidase (MPO), and histone H3, were elevated in the livers of HIRI mice. This finding indicates that both macrophages and neutrophils are significantly involved in the inflammatory response induced by HIRI. However, in vitro experiments revealed that stimulating macrophages with the supernatant of NETs containing only soluble factors resulted in a markedly reduced effect compared to stimulation with the purified NETs structure. This suggests that the initiation of the M1 polarization signal is likely dependent on the DNA backbone or related components of NETs, rather than on the soluble chemotactic factors.\u003c/p\u003e \u003cp\u003eConsidering the role of the NETs-macrophage axis in both in vivo and in vitro models, our study demonstrates that hepatic function is compromised following reperfusion, as hepatic ischemia-reperfusion injury (HIRI) results in extensive hepatocyte apoptosis and a pronounced inflammatory response. The application of DNase I to degrade neutrophil extracellular traps (NETs) has been shown to markedly enhance liver function and ameliorate the immune microenvironment. Notably, a recent investigation utilizing macrophage-specific cGAS knockout mice in the context of HIRI revealed a similar protective phenotype \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This phenomenon is contingent upon the cytoplasmic DNA sensing mechanism \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, underscoring the critical role of inhibiting the cGAS-STING pathway in macrophages during HIRI \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Concurrently, bioinformatics analyses indicate that, relative to the control group, the cGAS-STING pathway is significantly upregulated in the HIRI group. Consequently, we identified the cGAS-STING signaling pathway as the principal downstream mechanism by which macrophages detect DNA from neutrophil extracellular traps (NETs) and undergo M1 polarization, addressing the research question. It is important to note that previous studies have shown that cGAS-STING-deficient mice do not exhibit injury under basal conditions \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, suggesting that the activation of the STING signal alone is insufficient to induce liver injury. However, pre-treatment could potentially increase cytoplasmic DNA levels (originating from either mitochondrial DNA or NETs) to a certain threshold \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, at which point the cGAS-STING pathway acts merely as an amplifier. Nonetheless, we acknowledge that while cGAS-STING inhibitors can prevent NETs-induced M1 polarization in vitro \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, the precise contribution of DNA from NETs compared to mitochondrial DNA in the hepatic ischemia-reperfusion injury (HIRI) model requires further in vivo validation \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHaving established that the \"NETs-cGAS-STING\" axis is instrumental in the tissue damage associated with ischemia/reperfusion, targeting this specific immune cell interaction may facilitate advancements in mitigating early graft dysfunction (EAD) in liver transplantation. Consequently, it is pertinent to explore the functional relationship between the NETs-cGAS-STING axis and other inflammatory signaling pathways, such as Toll-like receptors (TLRs) and NLRP3 inflammasomes.\u003c/p\u003e \u003cp\u003eWe highlighted that while STING inhibitors have historically been employed to elucidate the role of the cGAS-STING pathway in hepatic ischemia-reperfusion injury (HIRI), their potential to mitigate damage via unidentified targets or upstream ligands suggests that numerous early investigations may have overlooked the NET-driven M1 polarization pathway\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Consequently, the discovery that this signaling cascade can be activated both in vitro and in vivo through pharmacological interventions, such as DNase I or STING inhibition, presents novel opportunities for the development of immunomodulatory therapies. Furthermore, this study establishes a theoretical framework for future research aimed at understanding how the cytoplasmic DNA of neutrophil extracellular traps (NETs) communicates with macrophages, influencing epigenetic and metabolic reprogramming to induce a stable M1 phenotype.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this work\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhongjun Wu conceived the project. Dingheng Hu and Yunhai Luo performed the experiments and collected the data. Dingheng Hu, Liangxu Wang, and Zhengli Tan performed the statistical analysis. Dingheng Hu, Qi Li, and Denghui Wang prepared the figures and drafted the manuscript (Dingheng Hu prepared Figs. 1, 2, 3, and 6; Qi Li prepared Fig. 4; Denghui Wang prepared Fig. 5). Dingheng Hu and Yunhai Luo provided critical feedback and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLIU, Z. et al. Molecular Mechanisms of Ischemia/Reperfusion Injury and Graft Dysfunction in Liver Transplantation: Insights from Multi-Omics Studies in Rodent Animal Models [J]. \u003cem\u003eInt. J. Biol. Sci.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e (5), 2135\u0026ndash;2154 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNEMES, B. \u0026amp; G\u0026aacute;M\u0026aacute;N, G. Biliary complications after liver transplantation [J]. \u003cem\u003eExpert Rev. Gastroenterol. Hepatol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (4), 447\u0026ndash;466 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHOUCHANI E T, PELL V R, G. A. U. D. E. E. et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS [J]. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e515\u003c/b\u003e (7527), 431\u0026ndash;435 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDE OLIVEIRA T H C \u0026amp; GON\u0026ccedil;ALVES G K N. Liver ischemia reperfusion injury: Mechanisms, cellular pathways, and therapeutic approaches [J]. \u003cem\u003eInt. Immunopharmacol.\u003c/em\u003e \u003cb\u003e150\u003c/b\u003e, 114299 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSCHLEGEL, A. \u0026amp; MULLER, X. Hypothermic oxygenated perfusion protects from mitochondrial injury before liver transplantation [J]. \u003cem\u003eEBioMedicine\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e, 103014 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKIM, S. H. Machine perfusion in liver transplantation: A step forward, but still on the runway [J]. \u003cem\u003eWorld J. Gastroenterol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e (40), 112408 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJAESCHKE, H. \u0026amp; WOOLBRIGHT, B. L. Current strategies to minimize hepatic ischemia-reperfusion injury by targeting reactive oxygen species [J]. \u003cem\u003eTranspl. Rev. (Orlando)\u003c/em\u003e. \u003cb\u003e26\u003c/b\u003e (2), 103\u0026ndash;114 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHUANG, H. et al. Damage-associated molecular pattern-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury [J]. \u003cem\u003eHepatology\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e (2), 600\u0026ndash;614 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJORCH S K, K. U. B. E. S. P. An emerging role for neutrophil extracellular traps in noninfectious disease [J]. \u003cem\u003eNat. Med.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (3), 279\u0026ndash;287 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKAWAI, C. et al. Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury [J]. \u003cem\u003eAm. J. Pathol.\u003c/em\u003e \u003cb\u003e186\u003c/b\u003e (4), 829\u0026ndash;843 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHIRAO, H., NAKAMURA, K. \u0026amp; KUPIEC-WEGLINSKI J, W. Liver ischaemia-reperfusion injury: a new understanding of the role of innate immunity [J]. \u003cem\u003eNat. Rev. Gastroenterol. Hepatol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (4), 239\u0026ndash;256 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTADIE, J. M. et al. HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4 [J]. \u003cem\u003eAm. J. Physiol. Lung Cell. Mol. Physiol.\u003c/em\u003e \u003cb\u003e304\u003c/b\u003e (5), L342\u0026ndash;L349 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTSUNG, A. et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion [J]. \u003cem\u003eJ. Exp. Med.\u003c/em\u003e \u003cb\u003e201\u003c/b\u003e (7), 1135\u0026ndash;1143 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLUO, X. \u0026amp; LI, H. MA L, et al. Expression of STING Is Increased in Liver Tissues From Patients With NAFLD and Promotes Macrophage-Mediated Hepatic Inflammation and Fibrosis in Mice [J]. \u003cem\u003eGastroenterology\u003c/em\u003e, \u003cb\u003e155\u003c/b\u003e(6): (2018). 1971-84.e4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSUN, L. et al. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway [J]. \u003cem\u003eScience\u003c/em\u003e \u003cb\u003e339\u003c/b\u003e (6121), 786\u0026ndash;791 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMosher, B. S., Fulkerson, H. L. \u0026amp; Yurochko, A. D. Collection and Isolation of CD14+ Primary Human Monocytes Via Dual Density Gradient Centrifugation as a Model System to Study Human Cytomegalovirus Infection and Pathogenesis. Methods Mol Biol. ;2244:103\u0026ndash;113. (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-1-0716-1111-1_6\u003c/span\u003e\u003cspan address=\"10.1007/978-1-0716-1111-1_6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 33555584.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamidzadeh, K., Belew, A. T., El-Sayed, N. M. \u0026amp; Mosser, D. M. The transition of M-CSF-derived human macrophages to a growth-promoting phenotype. \u003cem\u003eBlood Adv.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (21), 5460\u0026ndash;5472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/bloodadvances.2020002683\u003c/span\u003e\u003cspan address=\"10.1182/bloodadvances.2020002683\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020). PMID: 33166408; PMCID: PMC7656919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNajmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J. \u0026amp; Ferri, L. E. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. \u003cem\u003eJ. Vis. Exp.\u003c/em\u003e (98), 52687. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/52687\u003c/span\u003e\u003cspan address=\"10.3791/52687\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015). PMID: 25938591; PMCID: PMC4541576.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei, D. et al. MAFF alleviates hepatic ischemia-reperfusion injury by regulating the CLCF1/STAT3 signaling pathway. \u003cem\u003eCell. Mol. Biol. Lett.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (1), 39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s11658-025-00721-x\u003c/span\u003e\u003cspan address=\"10.1186/s11658-025-00721-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025). PMID: 40169936; PMCID: PMC11963299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. et al. Neutrophil Extracellular Traps Regulate HMGB1 Translocation and Kupffer Cell M1 Polarization During Acute Liver Transplantation Rejection. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 823511. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2022.823511\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2022.823511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022). PMID: 35603144; PMCID: PMC9120840.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoimenou, M., Tzoros, G., Skendros, P. \u0026amp; Chrysanthopoulou, A. Methods for the Assessment of NET Formation: From Neutrophil Biology to Translational Research. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (24), 15823. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms232415823\u003c/span\u003e\u003cspan address=\"10.3390/ijms232415823\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022). PMID: 36555464; PMCID: PMC9781911.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHAI, Y. \u0026amp; PETROWSKY, H. Ischaemia-reperfusion injury in liver transplantation\u0026ndash;from bench to bedside [J]. \u003cem\u003eNat. Rev. Gastroenterol. Hepatol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (2), 79\u0026ndash;89 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDE OLIVEIRA T H C \u0026amp; GON\u0026ccedil;ALVES G K N. Liver ischemia reperfusion injury: Mechanisms, cellular pathways, and therapeutic approaches [J]. \u003cem\u003eInt. Immunopharmacol.\u003c/em\u003e \u003cb\u003e150\u003c/b\u003e, 114299 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDERY K J, Y. A. O. S. et al. New therapeutic concepts against ischemia-reperfusion injury in organ transplantation [J]. \u003cem\u003eExpert Rev. Clin. Immunol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (10), 1205\u0026ndash;1224 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIU, Y. et al. Neutrophil extracellular traps and complications of liver transplantation [J]. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1054753 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWANG, H. \u0026amp; XI, Z. Macrophage Polarization and Liver Ischemia-Reperfusion Injury [J]. \u003cem\u003eInt. J. Med. Sci.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (5), 1104\u0026ndash;1113 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHANG, F. et al. The role of extracellular traps in ischemia reperfusion injury [J]. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1022380 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZHAO, J. et al. NETs Promote Inflammatory Injury by Activating cGAS-STING Pathway in Acute Lung Injury [J]. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e, \u003cb\u003e24\u003c/b\u003e(6). (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYOKOYAMA A P H et al. Neutrophil extracellular traps (NETs), transfusion requirements and clinical outcomes in orthotopic liver transplantation [J]. \u003cem\u003eJ. Thromb. Thrombolysis\u003c/em\u003e. \u003cb\u003e56\u003c/b\u003e (2), 253\u0026ndash;263 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLI X J, QU J R, ZHANG, Y. H. et al. The dual function of cGAS-STING signaling axis in liver diseases [J]. \u003cem\u003eActa Pharmacol. Sin\u003c/em\u003e. \u003cb\u003e45\u003c/b\u003e (6), 1115\u0026ndash;1129 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXIONG, Y. et al. Dimethyl fumarate alleviate hepatic ischemia-reperfusion injury through suppressing cGAS-STING signaling [J]. MedComm 2025, 6(2): e70077. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXU, D. et al. The cGAS-STING Pathway: Novel Perspectives in Liver Diseases [J]. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 682736 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCHEN, R. et al. The role of cGAS-STING signalling in liver diseases [J]. \u003cem\u003eJHEP Rep.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (5), 100324 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLEI Z, DENG, M. et al. cGAS-mediated autophagy protects the liver from ischemia-reperfusion injury independently of STING [J]. \u003cem\u003eAm. J. Physiol. Gastrointest. Liver Physiol.\u003c/em\u003e \u003cb\u003e314\u003c/b\u003e (6), G655\u0026ndash;g67 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCOUILLIN I, RITEAU N. STING Signaling and Sterile Inflammation [J]. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 753789 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBRYANT, J. D. et al. Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells [J]. \u003cem\u003eCurr. Protoc.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e (2), e372 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8822760/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8822760/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatic ischemia-reperfusion injury (IRI) is a serious complication during liver transplantation, which triggers a strongrepresents a significant complication in the context of liver transplantation, characterized by the induction of a robust non-specific inflammatory response throughmediated by damage-associated molecular patterns (DAMPs) and leads to, ultimately resulting in dysfunction of the transplanted liverorgan. The pathogenic pro-inflammatory (M1) polarization of hepatic macrophages is a driver of this hepatocyte injury, but the exact upstream trigger factor for this remains unclear. We investigated thecritical contributor to this hepatocellular damage; however, the precise upstream trigger remains unidentified. This study explores the potential role of neutrophil extracellular traps (NETs) as an upstream regulator of M1-type macrophage polarization. Utilizing the in vivo H-IRI mouse model alongside an in vitro macrophage co-culture system, we elucidated that the interaction between neutrophil extracellular traps (NETs) and macrophages is facilitated by the cGAS-STING signaling pathway. Our findings indicate that the double-stranded DNA generated by NETs is internalized by macrophages, subsequently activating the cytoplasmic sensor cGAS and the adaptor protein 3wSTING. This activation serves as a driver for the M1 macrophage phenotype. Our study unveils a novel and critical pathway, \"NETs \u0026rarr; cGAS-STING \u0026rarr; M1 polarization,\" which plays a significant role in hepatic ischemia-reperfusion injury (HIRI). This pathway presents a promising therapeutic target for mitigating graft damage following liver transplantation.\u003c/p\u003e","manuscriptTitle":"Neutrophil Extracellular Traps Exacerbate Liver Ischemia-Reperfusion Injury by Promoting Macrophage M1 Polarization via the cGAS-STING Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 13:38:33","doi":"10.21203/rs.3.rs-8822760/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-13T04:52:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-31T11:36:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64270728334109672109888290200261079413","date":"2026-03-31T10:26:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T15:26:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271178980117871526009869692138728296413","date":"2026-02-23T11:49:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-20T10:43:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T10:33:35+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-18T08:11:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T12:30:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-16T12:26:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91404733-5a9b-4a4a-ba0d-ed56f83c4755","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-13T04:52:35+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63375539,"name":"Biological sciences/Cell biology"},{"id":63375540,"name":"Health sciences/Diseases"},{"id":63375541,"name":"Health sciences/Gastroenterology"},{"id":63375542,"name":"Biological sciences/Immunology"},{"id":63375543,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-05-13T05:09:02+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 13:38:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8822760","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8822760","identity":"rs-8822760","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}