Department of Intensive Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, People’s Republic of China

Purpose: Excessive neutrophil extracellular traps (NETs) formation worsens disease progression in sepsis. Vascular non-inflammatory molecule 1 (VNN1) is a glycosylphosphatidylinositol-anchored protein. The present study investigated the mechanism of the role of VNN1 in NETs formation and sepsis.
Methods: Neutrophils from mouse bone marrow were isolated for experiments and mRNA sequencing. A cecum ligation and puncture-induced sepsis mouse model was established. Histopathological staining, real-time PCR, ELISA, flow cytometry and Western blot were used to detect phenotypic changes.
Results: VNN1 induced ROS-dependent NETs formation in vitro. S100A9 increases VNN1 protein levels and promoted NET formation in a VNN1-dependent manner. VNN1 also induced the NETs formation in vivo. VNN1 inhibition might ameliorate lung injury in sepsis mice via the reduction of NETs formation. VNN1 inhibition alleviated systemic inflammation in sepsis mice. Meanwhile, VNN1 inhibition was associated with reduced neutrophil recruitment to inflammatory sites. RNA sequencing results showed that immune and inflammation-related pathways were significantly altered in sepsis mice treated with the VNN1 inhibitor.
Conclusion: This study demonstrates that S100A9 increases VNN1 protein levels and induces VNN1-dependent NETs formation in vitro. Inhibition of VNN1 by PFI-653 blocks NADPH oxidase-derived ROS and thereby suppresses NETs formation. In vivo VNN1 inhibition alleviates lung injury and systemic inflammation in septic mice, potentially through the suppression of NET formation.

Keywords: neutrophil extracellular traps, vascular non-inflammatory molecule 1, reactive oxygen species, sepsis-induced acute lung injury, inflammation

Introduction

Sepsis is a complex syndrome defined as a dysregulated host response to infection and results in life-threatening organ dysfunction.1 The potential consequences of sepsis include multiple organ dysfunction syndromes (MODS), such as coagulation disorders, acute lung injury (ALI), hepatic injury, and renal failure.2 ALI is a frequent and severe complication in septic patients and serves as a principal cause of early mortality and diminished long‑term quality of life.3 Globally, sepsis remains a significant challenge to public health, contributing to a considerable percentage of annual fatalities.4 The current treatment of sepsis is mainly based on antibiotics. However, the extensive use of antibiotics leads to bacterial resistance, which affects subsequent treatment.5 Consequently, it is crucial to develop new targets to improve the clinical therapy of sepsis.

Neutrophils play a key role in innate immunity as the most abundant population of leukocytes in human body.6 Neutrophils are the first leukocytes to be recruited to inflammatory sites and have been demonstrated to eliminate pathogens through a variety of mechanisms.7 Neutrophils exert functions mainly through three pathways: phagocytosis, degranulation and release of neutrophil extracellular traps (NETs).8 NETs are extracellular DNA webs decorated by histones, myeloperoxidase and elastase.9 NETs have been demonstrated to capture and kill pathogenic microorganisms and inhibit the growth and reproduction of pathogenic bacteria in vivo.10 Nonetheless, the function of NETs is not entirely beneficial. Excessive NETs have been demonstrated to exacerbate inflammation and cause organ damage11 and led to disseminated intravascular coagulation.12 Growing research indicated that NETs may exacerbate inflammatory damage in sepsis.13 It has been documented that NETs formation in sepsis mice was associated with lung injury, and reduction of NETs formation alleviated lung injury.14 The targeting of NETs is regarded as a potential therapeutic modality for the treatment of septic lung injury.15

The human vanin gene family consists of three genes: vanin-1, vanin-2 and vanin-3.16 Vanins participate in the pathogenesis of multiple disorders including obesity, diabetes and cancer, by modulating neutrophil functions and regulating signaling pathways related to oxidative stress and inflammation.17 Vascular non-inflammatory molecule 1 (VNN1) is a glycosylphosphatidylinositol-anchored protein that has the capacity to hydrolyse pantetheine.18 In addition to its participation in coenzyme A metabolic pathways, VNN1 has been demonstrated to contribute to oxidative stress (OS),19 modulate inflammatory response,20 and regulate cell migration.21 VNN1 has been reported to be involved in the pathological processes of inflammatory diseases,22 renal diseases,23 and metabolic diseases.24 A bioinformatic analysis of neutrophil transcriptomes from whole blood samples of sepsis patients revealed that VNN1 was significantly upregulated and identified as a hub gene among neutrophil differentially expressed genes (DEGs).25 VNN1 is a pantetheinase that degrades pantetheine to pantothenate and cysteamine, which inhibits GSH synthesis, leading to ROS accumulation.26,27 Since ROS is a critical trigger for NET formation,28 this positions VNN1 as a key upstream regulator of NETosis in sepsis. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9.29 S100A9 was reported to regulate VNN1 expression in pancreatic duct cells, and VNN1 knockdown inhibited reactive oxygen species (ROS) release and ameliorated intracellular OS.22 Wang et al found that ROS clearance reduced the formation of NETs and intestinal damage caused by sepsis.30 S100A9 triggered the formation of ROS-dependent NETs in abdominal sepsis.31 In light of the aforementioned research, we speculated that the high expression of VNN1 in neutrophils might affect ALI in sepsis by participating in the formation of ROS-dependent NETs.

Materials and MethodsAnimal Model Establishment

Male C57BL/6 mice (6–8 weeks, 20–25 g) were purchased from Huachuang Sino (Taizhou, China). Prior to the commencement of the experiment, the mice were acclimatized for 1 week. Mice had free access to food and water. All groups of mice were housed under a 12-h light/12-h dark cycle, with the ambient temperature maintained at 22 ± 1°C and relative humidity at 45%-55%. At the start of the study, animals were randomly assigned to different cages, and cage changing was performed regularly throughout the experimental period. A total of 72 mice were used in this study. Mice were randomly allocated into 4 groups (n = 18) using a random number table method: Sham, CLP, CLP + DNase I, and CLP + PFI-653. The sample size of n=18 per group was determined based on multiple independent endpoints requiring incompatible tissue processing: n=6 for lung wet/dry ratio (fresh tissue), n=6 for histopathology (fixed tissue), and n=6 for PCR/Western blot (frozen tissue). A cecum ligation and puncture (CLP) model of mice was developed as a previous report32 to investigate the function of VNN1 in mice with sepsis. Mice were anesthetized using 3% isoflurane (RWD Life Science, Shenzhen, China) for induction, followed by maintenance at 1.5% isoflurane. Following anesthesia of the mice, an incision was made in the abdominal wall to expose the cecum, which was subsequently ligated. A needle was punctured between the ligation site and the terminal portion of the cecum, and a little amount of cecal contents was squeezed out. The cecum was placed back into the abdominal cavity, and the abdominal wall was sutured. The mice in sham group underwent cecum exposure without ligation and puncture. Mice were treated with PFI-653 (50 mg/kg)33 via gavage or deoxyribonuclease I (DNase I, 5 mg/kg)34 via intraperitoneal injection. Mice were anesthetized 24 h after surgery and euthanized by cardiac blood collection. The BALF, lung tissues and blood were collected for subsequent experiments. Blood was diluted using Turk’s solution and the white blood cell count was determined using a cell counting plate. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (2024062). In the experiment, the researchers who performed modeling, drug administration, and sample collection were informed of group assignments. The researchers who conducted data collation and statistical analysis were blinded to group information. No pre-established inclusion or exclusion criteria for animals were set in this experiment, and all experimental subjects meeting the basic feeding conditions were included.

Isolation and Identification of Neutrophils

The mouse (Male C57BL/6 mice, 6–8 weeks, 20–25 g) femurs were separated to collect bone marrow cells. Neutrophils were collected according to the instruction of the mouse bone marrow neutrophil isolation kit (Solarbio, Beijing, China). Peripheral blood-derived neutrophils were collected from mouse blood according to the instruction of the mouse peripheral blood neutrophil isolation kit (Solarbio, Beijing, China). The collected neutrophils were resuspended and incubated with Ly6G antibody (Elabscience, Wuhan, China) and CD11b antibody (Elabscience, Wuhan, China) at 4°C for 30 min before identification by flow cytometry.

Bone marrow-derived neutrophils were incubated with 100 nM Phorbol Myristate Acetate (PMA), PFI-653 (6.25 nM, 12.5 nM or 25 nM),33 Diphe-nyleneiodonium chloride (DPI, 1 μM)31 and/or 100 μM TBHP35 for 4 h. The content of DNA-histone complex and the levels of MPO-DNA complex in neutrophil supernatants, the content of extracellular DNA and the levels of ROS in neutrophils were examined. Neutrophil supernatant refers to the cell-free culture medium collected from neutrophils after stimulation, containing secreted inflammatory mediators, soluble proteins, and NET-associated components. Bone marrow-derived neutrophils were incubated with 100 ng/mL mouse recombinant S100A931 and/or 12.5 nM PFI-653 for 4 h. Subsequently, the DNA-histone complex content, the levels of MPO-DNA complex in cell supernatants and extracellular DNA content were assayed. Mouse bone marrow-derived neutrophils were incubated with 100 nM PMA or 100 ng/mL recombinant mouse S100A9 for 4 h. Cells were then harvested and stained with 1 μg of VNN1 antibody (Bioss, Beijing, China) per 106 cells for 30 min at 4°C in the dark. The cells were incubated with 1 μL of FITC-goat anti-rabbit IgG secondary antibody (Proteintech, Wuhan, China) for 40 min at RT in the dark, followed by flow cytometry analysis.

Immunofluorescence (IF) Staining

Cell climbing slices were fixed in 4% paraformaldehyde (PFA) for 15 min. Cells were permeabilized with 0.1% tritonX-100 and then closed with 1% BSA for 15 min. Cells were incubated with primary antibodies CitH3 (1: 100, Abcam, Cambridge, UK) and MPO (1:50, Santa Cruz, Dallas, TX, USA) at 4°C overnight. Secondary antibodies FITC-Goat Anti-Rabbit IgG (1: 200, Proteintech, Wuhan, China) and Cy3-Goat Anti-Mouse IgG (1: 200, Proteintech, Wuhan, China) were incubated with cell climbing slices at room temperature (RT) for 60 min. The nuclei were stained with DAPI. Slices were sealed using an anti-fluorescence quencher and then photographed under a microscope.

Tissues were embedded using paraffin and then cut into slices (5μm). Tissue sections were dewaxed to water and blocked with 1% BSA for 15 min. Sections were incubated with MPO (1:50, Santa Cruz, Dallas, TX, USA) and VNN1 (1: 100, Proteintech, Wuhan, China) or CitH3 (1: 100, Abcam, Cambridge, UK) antibodies at 4°C overnight. Secondary antibodies FITC-Goat Anti-Rabbit IgG (1: 200, Proteintech, Wuhan, China) and Cy3-Goat Anti-Mouse IgG (1: 200, Proteintech, Wuhan, China) were incubated with tissue sections for 90 min at RT. The nuclei were stained with DAPI. Sections were observed and photographed under a microscope.

Enzyme-Linked Immunosorbent Assay (ELISA)

The mouse serum was collected by centrifugation. The levels of C-X-C motif chemokine 15 (CXCL15, mouse homolog of human IL-8,36 FineTest, Wuhan, China), IL-6 (Liankebio, Hangzhou, China), CXCL1 (Liankebio, Hangzhou, China), and CXCL2 (Liankebio, Hangzhou, China) were detected according to the instruction of kits, respectively.

Capture ELISA

A capture ELISA was performed to identify the MPO-DNA complexes in the neutrophil supernatant. A 96-well plate was coated with MPO antibody overnight at 4°C. Each well was blocked with 1% bovine serum albumin (BSA) and incubated with cell supernatant and peroxidase-labelled Anti-DNA (Sigma, St. Louis, MO, USA) for 2 h at RT. The sample was incubated with the peroxidase substrate (ABTS) solution for 20 min. The absorbance value at 405 nm was measured as the relative expression of MPO-DNA complexes.

Measurement of DNA-Histone Complex

Detection of DNA-histone complex in neutrophil supernatants, mouse serum and BALF was performed using a Cell Detection ELISA PLUS kit (Sigma, St. Louis, MO, USA). The samples were incubated with immunoreagent (80 µL, Anti-histone: Anti-DNA-POD = 1:1) for 2 h at RT, followed by incubation with ABTS solution for 20 min. The relative expression of the DNA-histone complex was shown as the absorbance value.

Measurement of Extracellular DNA Content

Neutrophils were collected and incubated with 1 μM Sytox green (Maokang Biotechnology, Shanghai, China) for 15 min. The results were displayed by fluorescence intensity (Excitation/Emission (Ex/Em) = 504/523 nm).

Detection of Reactive Oxygen Species (ROS) and Cell-Free DNA (cfDNA)

Neutrophils were collected and resuspended in serum-free medium, and the level of ROS in the cells was detected with a ROS assay kit (Solarbio, Beijing, China). The cells were incubated with 10 μM DCFH-DA at 37°C for 1 h and the fluorescence intensity was detected (Ex/Em = 488/525 nm).

The serum and BALF of mice were collected, and the fluorescence value (Ex/Em = 488/520 nm) was determined as the level of cfDNA according to the instruction of the PicoGreen dsDNA Quantification Kit (Solarbio, Beijing, China).

Western Blot (WB)

Mouse lung tissue and peripheral blood neutrophils were collected. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. PVDF membranes were incubated with primary antibodies (PAD4 antibody (1: 1000, Affinity, Changzhou, China), CitH3 antibody (1: 1000, Abcam, Cambridge, UK), Histone H3 antibody (1: 500, Proteintech, Wuhan, China), VNN1 antibody (1: 1000, Proteintech, Wuhan, China), and β-actin antibody (1: 20000, Proteintech, Wuhan, China)) overnight at 4°C, followed by incubation with the corresponding secondary antibodies (goat anti rabbit IgG-HRP (1: 10000, Proteintech, Wuhan, China), goat anti mouse IgG-HRP (1: 10000, Proteintech, Wuhan, China)) for 60 min at RT. Protein bands were further visualized with ECL chemiluminescence detection kit (Proteintech, Wuhan, China).

Hematoxylin-Eosin (H&E) and Giemsa Staining

Mouse lung tissues were fixed and cut into 5 μm slices. The slices were stained with hematoxylin and eosin and then photographed to observe the lung lesions. BALF was prepared as cell smears. Cells were stained using Giemsa A solution for 1 min followed by 7 min of staining with Giemsa B solution. The cells were destained in 80% ethanol and subsequently counted under a microscope.

TUNEL Staining

Mouse bone marrow-derived neutrophils were incubated with various concentrations of the VNN1 inhibitor PFI-653 for 4 h. Cells were then permeabilized with 0.1% Triton X-100 for 15 min. TUNEL staining was performed according to the manufacturer’s instructions using the In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). Nuclei were counterstained with DAPI, and images were acquired using a fluorescence microscope.

Real-Time PCR (qPCR)

Total RNA was extracted from mouse lung tissue using Trizol (BioTeke Corporation, Beijing, China) and reverse transcribed into cDNA. The qPCR was performed using SYBR GREE (Solarbio, Beijing, China) and 2×Fast Taq plus PCR Master Mix (biosharp, Hefei, China). The expression levels of target genes were calculated by the 2−ΔΔCT method. The primer sequences are shown in Table 1.

Table 1 Primers Used in Study

Bioinformatics Analysis

Total RNA was extracted from the peripheral blood neutrophils of the sepsis mouse with or without PFI-653 treatment. A library was constructed for RNA sequencing (RNA-seq) analysis after quality testing of the RNA. Differentially expressed genes (DEGs) were identified based on |Log2 fold change| >1 and P-value <0.05. The DEGs screened from the samples were subsequently analyzed for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 9.5 software. Data are presented as mean ± standard deviation. All data were subjected to Normality and Lognormality Tests to determine their distribution. For comparisons involving two independent groups, an unpaired Student’s t‑test was employed. Multiple groups were compared using either one-way analysis of variance (ANOVA) or Brown-Forsythe and Welch ANOVA tests. Statistical significance was defined as p < 0.05.

VNN1 Induced ROS-Dependent Formation of NETs in vitro

Mouse bone marrow-derived neutrophils were isolated to determine the role of VNN1 on the formation of NETs. The purity of the isolated neutrophils achieved 90% (Figure 1A). VNN1 expression was significantly upregulated in PMA-stimulated neutrophils (Figure 1B). PFI-653 is a selective inhibitor of VNN1 enzymatic activity.33 TUNEL staining confirmed that PFI-653 treatment did not induce neutrophil apoptosis (Figure 1C). DNA-histone released from neutrophils was significantly inhibited by 12.5 nM PFI-653, which was determined to be the minimal effective dose (Figure 1D). Therefore, this concentration was selected for subsequent experiments. PFI-653 significantly inhibited the formation of PMA-induced NETs. This was evidenced by the reduction in the extracellular DNA, in the expression of MPO-DNA complex, and in the co-localization of MPO/CitH3 in neutrophils (Figure 1E–G). Diphenyleneiodonium chloride (DPI) is a well-established NADPH oxidase inhibitor that effectively blocks NET formation.31,37 Treatment with PFI-653 showed a similar inhibitory effect on NETs as DPI (Figure 1E–G). ROS exerts an important role in the NETosis.38 Both PFI-653 and DPI significantly reduced PMA-induced ROS levels (Figure 1H). TBHP is an inducer of ROS,35 and it reversed the decrease in ROS levels and the reduction in NETs formation induced by PFI-653 in neutrophils (Figure 1I and J). These results suggested that inhibition of VNN1 in vitro reduced the formation of ROS-dependent NETs.

Figure 1 Vanin 1 (VNN1) induced reactive oxygen species (ROS)-dependent formation of neutrophil extracellular traps (NETs) in vitro.(A) Isolation and identification of mouse bone marrow‑derived neutrophils. (B) Flow cytometric analysis of VNN1 expression in bone marrow‑derived neutrophils stimulated with 100 nM phorbol myristate acetate (PMA). (C) TUNEL staining of neutrophils treated with different concentrations (6.25 nM, 12.5 nM and 25 nM) of PFI‑653 (a VNN1 inhibitor) to assess apoptosis. Scale bar = 50 μm. (D) ELISA detection of DNA-histone complexes in the supernatant of neutrophils treated with different concentrations of PFI-653 (6.25 nM, 12.5 nM, and 25 nM). (E) Extracellular DNA content released by neutrophils treated with 12.5 nM PFI-653 or 1 μM Diphenyleneiodonium chloride (DPI, NADPH oxidase inhibitor). (F) Capture ELISA analysis of MPO–DNA complexes in the supernatant of neutrophils treated with PFI-653 or DPI. (G) Representative images of immunofluorescence staining for MPO/CitH3 in neutrophils treated with PFI-653 or DPI. Scale bar = 50 μm. (H) The ROS levels in neutrophils incubation with PFI-653 or DPI. (I) The ROS levels in neutrophils incubation with PFI-653 and/or TBHP. (J) Representative images of immunofluorescence staining for MPO/CitH3 in neutrophils treated with PFI-653 and/or TBHP (100 μM, ROS inducer). Scale bar = 50 μm. n = 3 independent experiments. ns: no significance, * p<0.05, ** p<0.01, and *** p<0.001.

S100A9 Increases VNN1 Protein Levels and Induces NET Formation Dependent on VNN1

S100A9 was reported to promote the formation of NETs31 and regulate VNN1 expression in pancreatic duct cells.22 Consequently, we speculated that S100A9 might be involved in the VNN1-induced NETs formation. Flow cytometry analysis confirmed that rS100A9 significantly increased VNN1 protein levels in neutrophils (Figure 2A). As shown in Figure 2B, rS100A9 promoted the release of DNA-histone complexes from neutrophils. PFI-653 inhibited the promotion effect of rS100A9, as evidenced by reduced release of DNA-histone complexes, decreased extracellular DNA in neutrophil supernatants and lower MPO-DNA complex levels (Figure 2C–E). PFI-653 also suppressed rS100A9-induced ROS release in neutrophils (Figure 2F). IF staining further confirmed these findings, showing decreased MPO/CitH3 co-localization in PFI-653-treated neutrophils (Figure 2G). These results suggest that S100A9 increases VNN1 protein expression and induces NET formation dependent on VNN1 in vitro.

Figure 2 S100A9 upregulates VNN1 expression and induces NET formation dependent on VNN1.(A) Flow cytometric analysis of VNN1 expression in mouse bone marrow-derived neutrophils stimulated with 100 ng/mL mouse recombinant S100A9 (rS100A9). (B) ELISA detection of DNA-histone complexes in the supernatant of neutrophils treated with rS100A9. (C) ELISA detection of DNA-histone complexes in the supernatant of neutrophils treated with PFI-653 and/or rS100A9. (D) Extracellular DNA content released by neutrophils with PFI-653 and/or rS100A9 treated. (E) Capture ELISA analysis of MPO-DNA complexes in the supernatant of neutrophils treated with PFI-653 and/or rS100A9. (F) The ROS levels in neutrophils incubation with PFI-653 and/or rS100A9. (G) NETs formation was detected by the immunofluorescence of MPO/CitH3, scale bar = 50 μm. n = 3 independent experiments. ** p<0.01, and *** p<0.001.

VNN1 Induced NETs Formation in Sepsis Mice

To further confirm the role of VNN1 in the induction of NETs formation in sepsis mice, we established a CLP mouse model (Figure 3A). DNase I was reported to inhibit the formation of NETs and alleviate lung injury in sepsis mice,14 therefore, it was taken as a positive control. Inhibition of VNN1 in sepsis mice reduced the levels of DNA-histone complexes and cfDNA in serum and BALF (Figures 3B and C), with an inhibitory effect similar to that of DNase I. Neutrophils from peripheral blood of mice were isolated and identified the purity for the further experiments (Figure 3D). VNN1 expression was significantly upregulated in peripheral blood neutrophils of CLP mice (Figure 3E). DNase I acts by degrading the DNA scaffold of NETs at the final stage of NETosis, without affecting ROS generation.14 Our results showed that PFI-653 treatment inhibited ROS release in peripheral blood neutrophils of CLP mice (Figure 3F). PAD4 drives histone H3 citrullination and is essential for NETosis.39 PFI-653 inhibited PAD4 expression and histone citrullination in peripheral blood neutrophils of sepsis mice (Figure 3G). These results suggested that the NETosis was equally induced by VNN1 in sepsis mice.

Figure 3 VNN1 induced NETs formation in sepsis mice (A) Schematic diagram of the animal experiment procedure. (B) The expression of DNA-histone complex in serum and bronchoalveolar lavage fluid (BALF) of CLP mice with DNase I or PFI-653 treatment. (C) Concentration of cell-free DNA in serum and BALF of CLP mice with DNase I or PFI-653 treatment. (D) Flow cytometric identification of neutrophils from mouse peripheral blood. (E) Immune bands for VNN1 in peripheral blood neutrophils from sham-operated or CLP mice. (F) ROS levels in peripheral blood neutrophils from CLP mouse with/without PFI-653 treatment. (G) Immune bands for PAD4, citrullinated histone H3 (CitH3), and histone H3 in peripheral blood neutrophils from sham-operated or CLP mice. n = 6 in each group. ns: no significance, ** p<0.01, and *** p<0.001.

Inhibition of VNN1-Induced NETs Formation Alleviated Lung Injury in Sepsis Mice

Lung injury occurred in sepsis mice, as evidenced by lung oedema, alveolar collapse, increased lung wet-to-dry ratio and increased inflammatory cell infiltration (Figure 4A and B). Inhibition of VNN1 alleviated lung injury in sepsis mice (Figure 4A and B). IF staining showed that sepsis increased VNN1⁺ and MPO⁺ cell infiltration and their co-localization in the lungs, which were attenuated by PFI-653 (Figure 4C). PFI-653 also reduced sepsis-induced NETs formation, as indicated by decreased CitH3-MPO co-localization (Figure 4D). Both total inflammatory cells and neutrophils in BALF were significantly increased in septic mice, and DNase I or PFI-653 treatment reduced neutrophil counts (Figure 4E). Meanwhile, PFI-653 inhibited the upregulated expression of PAD4 and CitH3 in lung tissue (Figure 4F). These results suggested that VNN1 inhibition reduced neutrophil infiltration and NETosis in lung tissues and alleviated lung injury in sepsis mice.

Figure 4 Inhibition of VNN1-induced NETs formation alleviated lung injury in sepsis mice.(A) The images presented are representative of mouse lung tissue, and images of lung tissues treated with HE staining, bar = 50 μm. (B) Wet-to-dry weight ratio in mouse lungs. (C) Representative images of MPO/VNN1 immunofluorescence staining of mouse lungs, scale bar = 50 μm. (D) Representative images of MPO/CitH3 immunofluorescence staining of mouse lungs, scale bar = 50 μm. (E) The upper panel: Total number of inflammatory cells in 1 mL BALF. The lower panel: The number of neutrophils in 1 mL BALF. (F) Immune bands for PAD4, CitH3, and histone H3 in mouse lungs. n = 6 in each group. ns: no significance, * p<0.05, ** p<0.01, and *** p<0.001.

Inhibition of VNN1 Alleviated CLP-Induced Systemic Inflammation in Sepsis Mice

Systemic inflammation occurs in sepsis mice and further promotes disease progression.40 As shown in Figures 5A and C, VNN1 inhibition suppressed the expression of inflammatory factors, as evidenced by decreased levels of TNF-α, IL-8, and IL-6 in lung tissues and diminished IL-8 and IL-6 levels in serum. CXCL1 and CXCL2 are the main chemokines involved in neutrophil migration to inflammation sites.41 The expression of CXCL1 and CXCL2 in mouse lungs and serum was elevated, whereas PFI-653 inhibited this phenomenon and had comparable effects to those of DNase I (Figures 5B and C). Compared with the sham-operated mice, the mRNA levels of TNF-α, IL-1β, IL-6, and MMP9 were significantly elevated in neutrophils from CLP mice, and this upregulation was markedly reduced by DNase I or PFI-653 treatment (Figure 5D). Meanwhile, the number of leukocytes, mononuclear leukocytes, and polymorphonuclear leukocytes in the sepsis mouse blood was significantly inhibited by PFI-653, and the effect was comparable to that of DNase I (Figure 5E). Inhibition of VNN1 alleviated systemic inflammation in sepsis mice and was associated with reduced neutrophil recruitment to inflammatory sites.

Figure 5 Inhibition of VNN1 alleviated CLP-induced systemic inflammation in sepsis mice.(A) The RNA levels of TNF-α, IL-8, and IL-6 in the lung tissues of mice. (B) The RNA levels of CXCL1 and CXCL2 in the lung tissues of mice. (C) The concentrations of IL-8, IL-6, CXCL1, and CXCL2 in the serums of mice. (D) Quantitative real-time PCR analysis of TNF‑α, IL‑1β, IL‑6, and MMP9 mRNA expression in peripheral blood neutrophils from CLP mice treated with DNase I or PFI‑653. (E) Number of monocytes, polymorphonuclear leukocytes, and total leukocyte in mouse blood. n = 6 in each group. ns: no significance, * p<0.05, ** p<0.01, and *** p<0.001.

Identification of Potential Key Genes Associated with VNN1-Induced NETs Formation

To investigate the possible mechanism of VNN1-induced NETs formation, RNA-seq analysis was performed in peripheral blood neutrophils isolated from sepsis mice with or without PFI-653 treatment (Figure 6A). Heatmap and volcano plot displayed DEGs in neutrophils of sepsis mice after VNN1 inhibition (Figures 6B and C). GO analysis showed that the DEGs were predominantly enriched in biological processes associated with stimulus response and immunity after VNN1 inhibition (Figure 6D). GO analysis displayed the top ten neutrophil-associated downregulation pathways (Figure 6E). KEGG analysis exhibited the top ten pathways after inhibition of VNN1, in which typical inflammatory pathways were significantly downregulated (Figure 6F and G). The log2 fold change (Log2FC) values of NET‑associated genes, including MPO, ELANE, and PADI4, were all downregulated after PFI‑653 treatment (Figure 6H).

Figure 6 Identification of potential key genes associated with VNN1-induced NETs formation. (A) Identification of neutrophils. (B) Volcano plot shows differentially expressed genes upregulated and downregulated in neutrophils. (C) Heatmap displayed differentially expressed genes in neutrophils. (D) Bubble plots showing the top ten terms for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) following Gene Ontology (GO) enrichment analysis. (E) The top ten neutrophil-associated downregulation pathways by GO analysis. (F) The top ten terms in KEGG enrichment analysis. (G) Top ten significantly downregulated pathways analyzed by KEGG. (H) The Log2FC value of NET-related genes MPO, PADI4, and ELANE in peripheral blood neutrophils from CLP mice treated with or without PFI‑653.

Discussion

Sepsis is a clinically critical condition, the pathogenesis of which has not yet been fully elucidated. Due to the lack of effective treatment strategies, sepsis morbidity and mortality remain high. Neutrophils are the first line of immunological defense in sepsis. A previous study reported that targeting NETs was a possible treatment modality for sepsis lung injury.15 Meanwhile, plasma VNN1 might be a biomarker in patients with traumatic sepsis.25 Consequently, it is necessary to investigate the potential mechanisms of VNN1 in sepsis and NETs formation.

VNN1 acts as a pantetheinase with intrinsic enzymatic activity, and its catalytic product cysteamine is the key mediator linking VNN1 to ROS production and redox homeostasis.27 VNN1 directly regulated intracellular OS and inflammatory responses through its product cysteamine in mouse intestinal inflammation.42 Deficiency of VNN1 was reported to limit ROS accumulation in the liver.43 In our study, VNN1 inhibition reduced ROS release in neutrophils in vitro. This is likely attributed to its pantetheinase activity. VNN1 exerts its pantetheinase activity to generate cysteamine, which directly inhibits the synthesis of intracellular reduced glutathione (GSH).26 As a critical endogenous antioxidant regulator, decreased GSH content weakens the cellular antioxidant capacity, thereby disrupting redox balance and driving excessive ROS generation.27 The elevated ROS further serves as a central upstream signal to initiate NET formation and amplify inflammatory cascades.28 Our data demonstrate that DPI treatment significantly suppressed both ROS production and NETosis in PMA‑stimulated neutrophils. The findings indicate that the ROS required for VNN1‑mediated NETosis is primarily derived from NADPH oxidase. The neutrophils release large amounts of MPO, DNA and other reactive substances when NETs formation,44 and the NETs formation is associated with ROS, PAD4 and histone citrullination.45 In the present study, the inhibition of VNN1 reduced the formation of NETs, and the ROS inducer reversed this phenomenon. The findings suggested that VNN1 induced the ROS-dependent NETs formation in vitro. Xiang et al reported that S100A9 knockdown alleviated pancreatitis in mice by targeting VNN1-mediated ROS release, and that S100A9 physically interacts with VNN1 through salt bridge formation.22 This direct protein-protein interaction appears to further trigger VNN1 upregulation. It has been reported that S100A9 can function as a scaffold protein to recruit deubiquitinase and stabilize target proteins.46,47 Whether a similar mechanism contributes to VNN1 stabilization needs further investigation in future studies. The present study demonstrated that S100A9 increased VNN1 protein levels and induces NET formation dependent on VNN1 in vitro.

VNN1 has been demonstrated to play a non-negligible role in the development of chronic diseases.27 However, its role in sepsis remains to be fully clarified. The sepsis induced high expression of CXCL1 and CXCL2 in plasma and lungs, which recruited neutrophils to inflammation sites.48 In the present study, the VNN1 inhibition reduced the expression of chemokines in sepsis mice. We propose that VNN1 inhibition may reduce chemokine expression through the following mechanisms. First, as described above, VNN1 exerts pantetheinase activity to generate cysteamine, which suppresses GSH biosynthesis and triggers intracellular ROS accumulation. ROS acts as an upstream activator of NF-κB.49 In sepsis, NF‑κB nuclear translocation and promoter activation induce early response genes, including inflammatory cytokines that trigger chemokine release.50 Thus, PFI-653 likely suppresses chemokines by reducing ROS-mediated NF-κB activation. Second, VNN1 activates the PI3K/Akt/NF‑κB pathway;51 VNN1 inhibition may directly dampen this signaling. Together, these pathways might contribute to reduced chemokine expression in septic mice. Activated neutrophils released MPO following migration and infiltration into the lungs when sepsis occurred.52 MPO is a crucial enzyme contained in neutrophil lysosomes that regulates local inflammation and ROS production.53,54 In the present study, inhibition of VNN1 effectively suppressed MPO expression in vitro and in vivo. VNN1 inhibition alleviated the infiltration of lung neutrophils and reduced the number of blood neutrophils in sepsis mice. During the development of sepsis, neutrophils release NETs to kill pathogens.55 However, NETs components also serve as damage-associated molecular patterns (DAMPs) to induce sustained inflammatory responses and tissue damage.9 Especially in sepsis, excessive NETs formation promotes inflammation and tissue damage.56 Berruyer et al reported that VNN1 deficiency protected mice from colitis.20 In our study, inhibition of VNN1 alleviated systemic inflammation and lung injury in sepsis mice, which might be achieved by the suppression of NETs formation.

To thoroughly investigate the mechanism of NETs induction by VNN1, an RNA-seq experiment was performed. The terms related to neutrophil-associated migration and chemotaxis functions showed significant downregulation. This observation aligns with our in vivo findings, which show that VNN1 inhibition correlates with reduced neutrophil recruitment to the lungs in septic mice. Among the DEGs enriched in neutrophil chemotaxis, the chemokines CXCL1 and CXCL2 have been reported to promote the formation of NETs.57,58 Inhibition of CXCL1 has been reported to attenuate the NETs formation in mouse kidney.59 VNN1 inhibition reduced the expression of CXCL1 in sepsis mice in the present study. Therefore, this might be related to the fact that inhibition of VNN1 reduced the formation of NETs. KEGG analysis showed significant downregulation of typical inflammatory pathways after VNN1 inhibition. It has been demonstrated that the TNF signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway, and NOD-like receptor signaling pathway were all involved in the formation of NETs and have a promoting role.60–62 VNN1 might induce NETs formation via stimulating these pathways. Among the DEGs enriched in the TNF pathway, matrix metalloproteinase-9 (MMP-9) has attracted our attention. MMP-9 is a granzyme secreted by neutrophils,7 and a bioinformatic analysis has reported that MMP-9 played a role in the NETs formation.63 Interestingly, Dammanahalli et al reported that VNN1 promoted MMP-9 activity in smooth muscle cells.26 This suggested us that VNN1 might induce NETs formation via stimulation of MMP-9. However, Further experiments are needed to verify our speculations.

While our findings provide mechanistic insights, this study has limitations. A limitation of this study is the lack of in vitro evidence using neutrophils isolated from VNN1 knockout mice. Future studies employing neutrophil-specific conditional knockout models are required to definitively establish the role of VNN1. This study did not directly assess CXCR2 surface levels on neutrophils, which may limit the interpretation of chemokine-driven neutrophil migration. These limitations should be addressed in future investigations.

Conclusions

In summary, this study demonstrates that S100A9 increases VNN1 protein levels and induces NET formation dependent on VNN1 in vitro. PFI-653 blocks VNN1-mediated NET production driven by NADPH oxidase-derived ROS. In vivo VNN1 inhibition alleviates lung injury and systemic inflammation in septic mice, possibly by suppressing NET formation.

Data Sharing Statement

The data that support the findings of this study are available on request from the corresponding author.

Ethics Approval

The study was designed, conducted and reported in accordance with the ARRIVE guidelines. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (2024062). All animal procedures were conducted in accordance with the National Standards for Laboratory Animal Welfare of China (GB/T 35892-2018).

Author Contributions

Shishuai Meng and Xinyue Ma contributed equally to this work and share first authorship. Shishuai Meng: Writing-original draft, Writing-review and editing, Methodology, Data curation, Validation. Xinyue Ma: Writing-review and editing, Methodology, Investigation, Visualization, Formal analysis. Wei Yang: Writing-review and editing, Supervision, Methodology, Investigation. Mingyan Zhao: Writing-review and editing, Investigation, Supervision, Project administration. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This study was funded by the Hospital Foundation of the First Affiliated Hospital of Harbin Medical University (Grant number 2023B10).

Disclosure

The authors declare that they have no competing interests.

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