Fig. 3

Massive infiltration of neutrophils and formation of NETs in plasma, BALF, and bronchial casts were observed in MPP associated PB. (A) Chest computed tomography prior to bronchoscopy reveals consolidation and atelectasis in a single lobe of the lung. (B) Bronchoscopy revealed mucosal hyperemia and/or edema, as well as the presence of gelatinous, tree-like casts causing obstruction in the airway. (C) Representative immunofluorescence microscopy images depict the staining for MPO in green, citH3 in red, and DNA in blue within bronchial casts. The co-localization of DNA with both MPO and citH3 is evident, confirming the presence of NETs. n = 10. Scale bar = 20 μm. (D) A representative scanning electron microscopy image reveals the presence NET-like structures in close proximity to neutrophils. Scale bar = 30 μm. (E) Representative images of hematoxylin-eosin staining of the bronchial casts reveal chromatolysis and the presence of cell-free neutrophils, which are indicative of the formation of NETs. n = 10. Scale bar = 100 mm (left),100 μm (middle) and 10 μm (right). (F-G) Levels of dsDNA and MPO-DNA complex, biomarkers for the presence of NETs, were quantified in the plasma across three distinct study cohorts: patients diagnosed with PB (PB, n = 21), patients without PB (non-PB, n = 42), and healthy controls (HC, n = 42). (H-I) Levels of NETs were quantified in the BALF across two distinct study cohorts: patients diagnosed with PB (PB, n = 21), patients without PB (non-PB, n = 42). (J-M) Correlation curves were constructed to delineate the relationship between NETs levels in plasma and the counts of peripheral neutrophils, as well as between NETs levels in BALF and peripheral neutrophil counts

Statistical analysis: (F-I) The graphical representation illustrates the median NETs levels, with each bar accompanied by its corresponding 95% CI. Statistical significance among the groups is indicated by asterisks, using the following convention: *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. (H-I) The Spearman rank correlation test was utilized to evaluate the strength and direction of the association between plasma NETs concentrations and peripheral neutrophil counts. BALF: bronchoalveolar lavage fluid. citH3: citrullinated histone 3; CI: confidence interval; dsDNA: double-stranded DNA; MPO: myeloperoxidase; NETs: neutrophil extracellular traps; PB: plastic bronchitis