Fig 2: The expression of miR-155-5p, CLDN1, and ZO-1 in IBS mice. Murine model of IBS was established by administrating 1.5 mg of TNBS into the proximal colon. The level of nociceptive threshold (A) and MPO (Myeloperoxidase) activity (B) were then evaluated. (C-E) qRT-PCR analysis of the miR-155-5p (C), CLDN1 (D), and ZO-1 (E) expression level in murine colon tissues from controls (n=7) and IBS mice (n=7). IHC analysis of the (F) CLDN1 and (G) ZO-1 protein level in murine colon tissues from controls and IBS mice. Scale bar = 100 µm. *P<0.05, **P<0.01. MPO, myeloperoxidase; CLDN1, claudin-1; ZO-1, zonula occludens-1; IBS, irritable bowel syndrome; TNBS, trinitrobenzenesulfonic acid; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig 3: C57BL/6J mice were intranasally inoculated with Pseudomonas aeruginosa (PA) at a concentration of 2.5 × 106 colony-forming units per mouse). Subsequently, mice received a single dose of hCitH3-mAb (10 mg/kg of body weight), paquinimod (5 mg/kg of body weight), a combination of hCitH3-mAb and paquinimod, or an equivalent dose of DMSO was administered via tail vein injection 0.5 h post-infection. (a) Alveolar cells were then isolated 24 h after PA infection, cells were subsequently subjected to flow cytometry analysis to quantify the distribution of CD45+, CD11b+, Ly6G+ neutrophil populations (n = 3/group). (b) Bronchoalveolar lavage fluid (BALF) was collected from all the treatment groups of mice 24 h after PA infection, and the levels of NETosis (CitH3, ds-DNA, and MPO-DNA) were determined using ELISA (n = 5/group). Data are representative of three independent experiments expressed as means ± SEM.**p < 0.01 versus DMSO; ***p < 0.001 versus DMSO; ****p < 0.0001 versus DMSO; #p < 0.05 versus hCitH3-mAb+Paquinimod; ##p < 0.01 versus hCitH3-mAb+Paquinimod.

Fig 4: Enriched Cd177+ neutrophils are key contributors to lung ischemia-reperfusion injury in the mouse model(A) Nucleic acid staining and watershed-based segmentation for single-cell analysis. A representative image shows nuclei segmented using the watershed algorithm for spatial distribution analysis (n = 2).(B) Spatial expression of the pan-immune marker Cd45 in lung tissue sections reveals regions of local immune cell infiltration.(C) Spatial mapping of Cd177 expression in lung tissues after IRI. Higher-magnification images (right) show colocalization of Cd177 with the inflammatory genes Pglyrp1 and Ltf.(D) Uniform manifold approximation and projection (UMAP) visualization showing Ly6g+Cd177+ neutrophil populations (red) compared with Ly6g+Cd177− neutrophils (blue). The bubble heatmap on the right demonstrates enrichment of inflammatory Gene Ontology terms in Ly6g+Cd177+ cells.(E) Representative immunofluorescence images from the mouse left lung for Ly6G (green), CD177 (red), and citrullinated histone H3 (Cit-H3, white) showing NET formation in CD177+ neutrophils. Scale bar: 20 μm.(F) Reactive oxygen species (ROS) production was higher in CD177+ compared to CD177− neutrophils from human samples (left) and in Cd177flox/flox neutrophils compared to Cd177flox/flox; Ly6gCre neutrophils from mice (right), with or without PMA stimulation. (n = 5 per group). Neut, neutrophils; UT, untreated.(G) Quantification of MPO-DNA complexes showing increased NET formation in CD177+ compared to CD177− human neutrophils (left) and in Cd177flox/flox compared to Cd177flox/flox; Ly6gCre mouse neutrophils (right) under PMA stimulation. Neut, neutrophils; UT, untreated. (n = 5 per group).(H) Representative H&E staining of lung sections from Cd177flox/flox and Cd177flox/flox; Ly6gCre mice under sham and IRI conditions. Quantification of acute lung injury scores (right) demonstrates significant injury in Cd177flox/flox mice and minimal injury in Cd177flox/flox; Ly6gCre mice after lung IRI. Scale bar: 50 μm. (n = 6 per group).(I) Immunofluorescence staining revealing reduced NET infiltration (Ly6G, Cit-H3, and DNA/H1) in lung tissues of Cd177flox/flox; Ly6gCre mice post-lung IRI. Scale bar: 20 μm.Data are shown as mean ± SD. Statistical significance was assessed by a two-sided Wilcoxon test adjusted with the Bonferroni method in (F), (G), and (H) and Fisher’s exact test in (D). ns, not significant; ∗∗∗p < 0.001.