DNase1 reverses peribronchial inflammation in murine explants incubated with activated neutrophils.

INTRODUCTION: Neutrophil extracellular traps (NETs) are chromatin-based structures released by activated neutrophils that are associated with microvascular thrombosis and pulmonary inflammation. These are adverse effects that are relevant in ECMO-associated lung injury. Although enzymatic NET degradation by DNase1 represents a potential anti-inflammatory strategy, its anti-inflammatory effects in the lung tissue and on vascular endothelial growth factor (VEGF) signalling have received limited investigation. We used a tissue-architecture-preserving murine lung explant model to characterise inflammation following exposure to activated neutrophils and to determine whether DNase1 attenuates this response without compromising VEGF expression. METHODS: Murine lung tissue in an explant culture was exposed to phorbol 12-myristate 13-acetate-activated human neutrophils. DNase1 was used to prevent NET-mediated effects. Inflammatory and angiogenic responses were assessed using multiplex proximity extension assay and quantitative immunofluorescence of peribronchiolar regions of interest. RESULTS: In whole-explant lysates, the pre-specified primary analyte C-C motif chemokine ligand 5 (Ccl5) was significantly increased following exposure to activated neutrophils (p = 0.015); among exploratory analytes, C-C motif chemokine ligand 3 remained significant after false-discovery-rate correction (Ccl3 p = 0.005, q = 0.014), while C-X-C motif chemokine ligand 9 was nominally elevated (Cxcl9 p = 0.041, q = 0.061). Immunofluorescence confirmed increased Ccl5, in the peribronchiolar regions (p = 0.002, q = 0.004), which was significantly attenuated by DNase1 (p = 0.019, q = 0.038). Vascular endothelial growth factor expression was not significantly altered by neutrophil exposure or DNase1 treatment (p ≥ 0.29). DISCUSSION: NET-associated inflammation in murine lung explants is detected within peribronchiolar regions of interest and is attenuated by DNase1-mediated degradation of extracellular DNA without compromising VEGF expression. These findings provide a rationale for further evaluating DNase1 as a candidate adjunct anti-inflammatory strategy in ECMO-associated lung injury, where the preservation of VEGF expression alongside NET inhibition is potentially attractive given the bleeding risks of conventional anticoagulation. Clinical applicability requires confirmation in flow-based ex vivo systems, in vivo models, and human ECMO studies.