# MicroRNA‐223/NE Signaling Pathway Inhibits Lipopolysaccharide‐Induced Acute Lung Injury by Regulating Neutrophil Extracellular Traps

## TL;DR

This study shows that the miR-223/NE pathway reduces lung damage by controlling neutrophil activity in acute lung injury.

## Contribution

The study identifies the miR-223/NE signaling pathway as a novel regulator of neutrophil extracellular traps in acute lung injury.

## Key findings

- miR-223 deficiency increases NETs formation and worsens lung injury in mice.
- Inhibiting NETs or NE reduces inflammation and lung damage in miR-223−/− mice.
- miR-223/NE signaling modulates neutrophil–epithelial interactions and inflammatory responses.

## Abstract

Acute lung injury (ALI) is characterized by significant neutrophil infiltration in the lungs, representing a life‐threatening condition with diverse etiologies. However, the mechanisms regulating neutrophil–alveolar epithelial interactions and the pathophysiological roles of neutrophil infiltration in ALI remain incompletely understood.

A dose of 20 mg/kg lipopolysaccharide (LPS) was intratracheally instilled to induce ALI models in 10‐week‐old male microRNA‐223 knockout mice (miR‐223−/−) and wild‐type (WT) mice, with control group mice receiving an equal volume of phosphate‐buffered saline (PBS). After 24 h of instillation, lung tissues and peripheral blood were collected from the mice. In vivo, quantitative PCR (qPCR) measured miR‐223 and neutrophil elastase (NE) mRNA levels, while Western blot (WB), enzyme‐linked immunosorbent assay (ELISA), and hematoxylin–eosin (H&E) staining assessed neutrophil extracellular traps (NETs) markers (H3Cit, myeloperoxidase [MPO]), inflammatory cytokines (TNF‐α, IL‐1β, and IL‐6), and lung injury severity. In vitro, HL‐60‐derived neutrophil‐like cells were cocultured with alveolar epithelial cells under LPS stimulation. The roles of the miR‐223/NE/NETs axis were further investigated using the NETs inhibitor GSK484 and the NE inhibitor Sivelestat.

WB experiments showed an increase in NETs‐related proteins MPO and H3Cit in the lungs of WT ALI mice, with significantly enhanced expression in miR‐223−/− mice. The lung injury scores and mortality rates in miR‐223−/− mice were significantly exacerbated, accompanied by increased neutrophil infiltration in the lungs. Levels of inflammatory factors (TNF‐α, IL‐1β, and IL‐6) in the serum of miR‐223−/− mice were significantly elevated. In vitro coculture experiments demonstrated that miR‐223 deficiency in neutrophil‐like cells augmented NETs formation and inflammatory responses, leading to increased damage to alveolar epithelial cells. However, in vivo inhibition of NETs with GSK484 or NE with Sivelestat in miR‐223−/− mice significantly attenuated neutrophil infiltration, inflammation, and lung injury, and improved survival. Similarly, Sivelestat pretreatment reduced NET formation and conferred protection against ALI. Consistent with the in vivo findings, inhibition of NETs with GSK484 or NE with Sivelestat in the coculture system similarly attenuated epithelial damage and inflammatory response.

This study reveals that the miR‐223/NE axis critically regulates NETs formation, modulating neutrophil inflammatory infiltration and neutrophil–epithelial interactions to exacerbate ALI. These findings provide potential therapeutic targets for ALI.