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.