Fig 2: Elevated NET levels are associated with intestinal necroptosis and gut barrier dysfunction in II/R patients. (A) Circulating MPO‐DNA and CitH3‐DNA complexes in healthy controls and II/R patients were analysed by ELISA. (B) Serum D‐lactate and I‐FABP were analysed by ELISA. (C) Correlation between circulating NETs biomarker (CitH3‐DNA) and circulating intestinal injury biomarkers (D‐lactate and I‐FABP). (D) Intestinal inflammatory factors (IL‐1β, IL‐6, TNF‐α and MCP‐1) detected by ELISA. (E,F) Comparisons of the histopathological changes using H&E and Masson staining and the Chiu scoring system. Scale bars = 500 μm. (G) Neutrophil infiltration (Ly6G) and NET formation (CitH3) were assessed by immunofluorescent staining. Scale bars = 100 μm. (H,I) The expression levels of TUNEL and p‐MLKL (a necroptotic biomarker) were analysed through immunofluorescence staining, and the numbers of TUNEL+ and p‐MLKL+ cells were counted. Scale bars = 100 μm. (J,K) NET formation (CitH3), necroptosis (p‐MLKL, MLKL) and intestinal tight junction proteins (claudin‐1 and occludin) were analysed by western blotting. Grayscale values were measured and quantified using ImageJ software. CitH3, citrullinated histone H3; H&E, haematoxylin–eosin; H3, histone H3; I‐FABP, intestinal fatty‐acid binding protein; II/R, intestinal ischemia–reperfusion; IL‐1β, interleukin 1β; IL‐6, interleukin 6; MCP‐1, monocyte chemoattractant protein 1; MPO, myeloperoxidase; NET, neutrophil extracellular trap; TNF‐α, tumour necrosis factor alpha. Data are shown as the means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: The differential effects of CM on different polarizing agents affecting the MPO and NE levels in the supernatant. (A) MPO was increased in the presence of CM, while (B) NE showed a different pattern with the most significant change with C-GSF. The statistical significance was calculated using t-test and is denoted by * = p < 0.05.

Fig 4: Elevated expression levels of NETs in BU patients. A) Schematic diagram of patient sample collection and experimental design. B) Expression levels of serum dsDNA and MPO in HC and BU patients (n = 10 per group). C,D) Expression levels of NETs‐related genes and proteins in neutrophils of HC and BU patients detected by qPCR and IF. Scale bars, 50 µm. E) UMAP plot of scRNA‐seq subset analysis of neutrophils in HC and BU patients. F) Tissue preference analysis of neutrophil subsets from scRNA‐seq in HC and BU patients. G) GSVA pathway analysis of neutrophil subsets from scRNA‐seq. H) Functional gene display of neutrophil subsets from scRNA‐seq. I) Volcano plot of DEGs in neutrophils of HC and BU patients. J) GO pathway analysis of DEGs in neutrophils of HC and BU patients. K) Pseudotime analysis ridge plot of neutrophil subsets from scRNA‐seq. Significance in B‐C was determined using unpaired two‐tailed Student's t‐tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: Inhibition of NETs levels alleviates severity of EAU. A,B) Fundus images, clinical scores, H&E staining, and pathological scores of the normal, EAU, and GSK484 groups (n = 5 per group). C,D) Serum levels of dsDNA and MPO in the normal, EAU, and GSK484 groups (n = 5 per group). E,F) IF expression levels of NETs‐related proteins in the CDLNs of normal, EAU, and GSK484 groups (n = 5 per group). Scale bars, 50 µm. G,H) IF expression levels of NETs‐related proteins in the spleen of normal, EAU, and GSK484 groups (n = 5 per group). Scale bars, 50 µm. I) FCM analysis of the proportion of infiltrating CD4+ T cells in the retina (n = 5 per group). J) FCM analysis of IL‐17A in CD4+ T cells derived from PBMC (n = 5 per group). K,L) FCM analysis of IL‐17A, IFN‐γ, and FOXP3 expression in CD4+ T cells derived from CDLNs and spleen of the normal, EAU, and GSK484 groups (n = 5 per group). Significance in A,B was determined using the Kruskal–Wallis test. Significance in C‐L was determined using one‐way ANOVA and Bonferroni multiple comparison test. Results are presented as mean ± SEM, with ns indicating no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.