Fig. 4

NETs are present in the bronchoalveolar lavage fluid of βENaC-Tg mice. a ELISA was used to detect MPO-DNA complexes, indicative of NETs, in BAL supernatants of mice at 6 and 8 weeks. There were significantly more MPO-DNA complexes detected in the BAL supernatant of βENaC-Tg mice than WT at both 6 and 8 weeks of age. One-way ANOVA was used to determine statistical significance (n = 6, **, p < 0.01). b Immunofluorescence imaging of 1.5 × 10⁵ cells from 1 to 2 mice per group was performed to detect NETs in the BAL of 8 week-old mice. Images are representative of 1 slide/ group. The larger image represents a closer view of the cells within the red square. The white arrows indicate NETs and the white arrow heads mark resting neutrophils characterized by distinct MPO and DNA staining patterns and lack of citrullinated histone staining. Blue=DAPI, orange=MPO, green=CitH3. Flow cytometry was used to quantify the number of cells undergoing histone citrullination in the BAL of (c) 6 and (d) 8-week-old mice. Pooled samples of 1 × 10⁶ cells from the BAL of 2–3 mice per group were assayed for citrullinated histone (CitH3) positive neutrophils. A Student’s t-test was used to determine statistical significance (n = 5–6/group, *, p < 0.05, **, p < 0.01). Citrullinated histone-positive neutrophils were considered as CD11b⁺/CD115⁻/Ly6G⁺/CitH3⁺. e Representative scatter plots showing CitH3⁺ neutrophils (Ly6G⁺)