Iron Recycling Gone Wrong Drives Lung Scarring After Mechanical Ventilation in Mice
In mice, ventilator stretch triggers NCOA4-driven ferritinophagy and ferroptosis in lung cells, and ferritin-laden vesicles activate fibroblasts to scar the lung.
Résumé
Mechanical ventilation saves lives but can leave the lungs scarred. This mouse and cell study traces one route to that scarring through iron handling. High tidal volume ventilation activated the ANG II/AGTR1 axis in alveolar epithelial cells. This raised NCOA4, which drives ferritinophagy, the lysosomal breakdown of the iron-storage protein ferritin. Released iron triggered ferroptosis, an iron-dependent form of cell death. Mechanical stretch also made ferritin undergo phase separation, which supported NCOA4-mediated degradation. Injured cells shed ferritin-containing extracellular vesicles that fibroblasts absorbed, becoming iron-loaded and activated and depositing extracellular matrix. Chloroquine or NCOA4 knockdown reduced ferroptosis and fibrosis in mice. The work is preclinical, but it points to iron metabolism and NCOA4 as possible targets for preventing ventilator-associated fibrosis.
Résumé détaillé
Mechanical ventilation is essential for respiratory failure, yet it can injure the lungs and has been linked to later pulmonary fibrosis, including in COVID-19 patients. How ventilation leads to fibrosis is poorly understood. Death of alveolar epithelial cells (AECs) is thought to start fibrosis, and iron-related cell death pathways have recently drawn attention. This study asked whether ferritinophagy, the NCOA4-directed autophagic breakdown of ferritin that frees stored iron, drives ferroptosis in AECs and fibroblast activation after ventilation.
The team ventilated male C57BL/6 mice for 2 hours with high tidal volume and followed them for 7 days. They ran single-cell RNA sequencing on lung tissue and applied mechanical stretch to cultured AECs. They used fluorescence recovery after photobleaching (FRAP) to capture ferritin phase separation in live cells. They inhibited the pathway in several ways: chloroquine for ferritinophagy, ferrostatin-1 for ferroptosis, and AAV-mediated knockdown of AGTR1 and NCOA4 delivered into the airway. EVs were isolated from bronchoalveolar lavage fluid and cell media by ultracentrifugation and characterized by nanoparticle tracking, electron microscopy and immunoblotting. Uptake by fibroblasts was tracked with a dye.
Single-cell data showed disordered iron metabolism after ventilation, with lower expression of the ferroptosis-suppressor genes Gpx4 and Fth in AECs. The ANG II/AGTR1 axis initiated ferritinophagy, causing iron overload and ferroptosis. Under mechanical stretch, NCOA4 was upregulated and promoted ferritin phase separation, which fed ferritinophagy. Blocking ferritinophagy with chloroquine or NCOA4 knockdown reduced ferroptosis and lessened fibrosis in the mouse model. Stressed AECs also released more ferritin-containing EVs. Fibroblasts took these up and accumulated iron, expressed activation markers such as α-SMA, and deposited extracellular matrix. This links epithelial injury to fibroblast activation through iron transfer.
Taken together, the findings describe a chain from mechanical stress to ANG II/AGTR1 signaling, NCOA4-driven ferritinophagy, ferroptosis, and ferritin-EV signaling to fibroblasts. The pathway suggests targets for preventing fibrosis in ventilated patients, including AGTR1, NCOA4, ferritinophagy and iron chelation.
Several caveats apply. The work used young male mice, a short ventilation protocol and in vitro stretch models, so it may not reflect critically ill humans with comorbidities, varied ventilation strategies or both sexes. Pre-treatment before ventilation does not test therapy after injury. Chloroquine has broad effects on lysosomes, and no human tissue or clinical outcome data are reported in the text provided. Clinical translation will need validation in larger animals and patient samples.
Principales conclusions
- Single-cell RNA-seq of ventilated mouse lungs showed iron dysregulation and reduced ferroptosis-suppressor genes Gpx4 and Fth in alveolar epithelial cells.
- The ANG II/AGTR1 axis initiated ferritinophagy, causing iron overload and ferroptosis in alveolar epithelial cells after ventilation.
- Mechanical stretch upregulated NCOA4, which promoted ferritin phase separation and enhanced ferritinophagy in lung epithelial cells.
- Chloroquine or NCOA4 knockdown reduced ferroptosis and alleviated ventilation-induced pulmonary fibrosis in mice.
- Ferritin-containing EVs from injured epithelial cells were taken up by fibroblasts, causing iron overload, activation and extracellular matrix accumulation.
Méthodologie
Preclinical study combining a 2-hour high tidal volume ventilation model in male C57BL/6 mice (7-day follow-up) with in vitro mechanical stretch of alveolar epithelial cells. Methods included scRNA-seq, FRAP for ferritin phase separation, histology, electron microscopy, EV isolation and uptake assays, and pharmacologic (chloroquine, ferrostatin-1) and AAV-shRNA (AGTR1, NCOA4) inhibition.
Limites de l'étude
Findings come from young male mice and cultured cells, so they may not apply to critically ill patients with comorbidities or to female subjects. Inhibitors were given before ventilation, which does not show benefit when treatment starts after injury, and chloroquine has non-specific lysosomal effects. Only part of the full text (through the methods) was available for analysis, and no human validation is evident.
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