Abstract / Summary
Abstract Background Persistent accumulation of neutrophil extracellular traps (NETs) amplifies inflammation and alveolar–capillary barrier injury in acute lung injury (ALI), yet the mechanisms sustaining pathological NET accumulation remain incompletely understood. Here, integrated single-cell transcriptomic and functional analyses uncovered a previously underappreciated formation–disposal imbalance in ALI, in which enhanced NET formation outpaced post-release processing and degradation, disrupting NET homeostasis. Guided by this mechanistic insight, we developed DNase I@mPDA@NM, an inhalable neutrophil-mimetic nanoplatform that coordinates suppression of ongoing NET formation with degradation of extracellular NETs through a “Block-and-Clear” strategy. Results Single-cell RNA sequencing revealed increased neutrophil oxidative-stress/NET-formation programs without a matched increase in monocyte/macrophage NET-processing programs. Consistently, LPS-induced ALI exhibited increased pulmonary NET burden together with slower degradation of preformed NETs in bronchoalveolar lavage fluid, supporting a formation–disposal imbalance in vivo. Mechanistically, DNase I@mPDA@NM addressed both sides of this imbalance: mPDA attenuated intracellular ROS and NETosis-associated citrullinated histone H3 responses, whereas DNase I efficiently degraded preformed extracellular NETs. The mPDA-based formulation preserved approximately 70% of DNA-degrading activity under mildly acidic and ALI-relevant conditions, while neutrophil-membrane functionalization enhanced neutrophil association and prolonged pulmonary retention after inhalation. In an endothelial–epithelial barrier model, DNase I@mPDA@NM reduced NET accumulation and preserved barrier integrity, whereas NET add-back partially reversed this protection, supporting a functional contribution of extracellular NET removal to barrier preservation. In LPS-induced ALI, inhaled DNase I@mPDA@NM reduced pulmonary NET accumulation, neutrophilic inflammation, alveolar–capillary leakage, pulmonary edema, and histopathological injury. Whole-lung transcriptomics further revealed attenuation of neutrophil activation, degranulation, and NF-κB-associated inflammatory programs, together with directional reversal of the ALI-associated reduction in phagocytosis-related programs, providing complementary transcriptomic support for rebalancing NET formation and post-release disposal. Conclusions Persistent pulmonary NET accumulation in ALI is associated with a formation–disposal mismatch that disrupts NET homeostasis. By translating this mechanism into a coordinated therapeutic architecture, inhaled DNase I@mPDA@NM restrains ROS-associated ongoing NET formation while eliminating extracellular NETs after release, thereby restoring NET homeostasis and protecting the alveolar–capillary barrier. This single-cell transcriptomics-guided “Block-and-Clear” strategy provides a mechanism-based framework for NET-directed therapy and a localized nanotherapeutic approach for acute inflammatory lung injury.