Neutrophil extracellular traps drive intestinal microvascular endothelial ferroptosis by impairing Fundc1-dependent mitophagy

Microvascular endothelial damage caused by intestinal ischemia‒reperfusion (II/R) is a primary catalyst for microcirculation dysfunction and enterogenous infection. Previous studies have mainly focused on how neutrophil extracellular traps (NETs) and ferroptosis cause intestinal epithelial injury, and little attention has been given to how NETs, mainly from circulatory neutrophils, affect intestinal endothelial cells during II/R. This study aimed to unravel the mechanisms through which NETs cause intestinal microvascular dysfunction. We first detected heightened local NET infiltration around the intestinal microvasculature, accompanied by increased endothelial cell ferroptosis, resulting in microcirculation dysfunction in both human and animal II/R models. However, the administration of the ferroptosis inhibitor ferrostatin-1 or the inhibition of NETs via neutrophil-specific peptidylarginine deiminase 4 (Pad4) deficiency led to positive outcomes, with reduced intestinal endothelial ferroptosis and microvascular function recovery. Moreover, RNA-seq analysis revealed a significant enrichment of mitophagy- and ferroptosis-related signaling pathways in HUVECs incubated with NETs. Mechanistically, elevated NET formation induced Fundc1 phosphorylation at Tyr18 in intestinal endothelial cells, which led to mitophagy inhibition, mitochondrial quality control imbalance, and excessive mitochondrial ROS generation and lipid peroxidation, resulting in endothelial ferroptosis and microvascular dysfunction. Nevertheless, using the mitophagy activator urolithin A or AAV-Fundc1 transfection could reverse this process and ameliorate microvascular damage. We first demonstrate that increased NETosis could result in intestinal microcirculatory dysfunction and conclude that suppressed NET formation can mitigate intestinal endothelial ferroptosis by improving Fundc1-dependent mitophagy. Targeting NETs could be a promising approach for treating II/R-induced intestinal microcirculatory dysfunction.