Neutrophil extracellular traps aggravate intestinal epithelial necroptosis in ischaemia-reperfusion by regulating TLR4/RIPK3/FUNDC1-required mitophagy

Neutrophil extracellular trap (NET) has been confirmed to be related to gut barrier injury during intestinal ischaemia-reperfusion (II/R). However, the specific molecular regulatory mechanism of NETs in II/R-induced intestinal barrier damage has yet to be fully elucidated. Here, we reported increased NETs infiltration accompanied by elevated inflammatory cytokines, cellular necroptosis and tight junction disruption in the intestine of human II/R patients. Meanwhile, NETs aggravated Caco-2 intestinal epithelial cell necroptosis, impairing the monolayer barrier in vitro. Moreover, Pad4-deficient mice were used further to validate the role of NETs in II/R-induced intestinal injury. In contrast, NET inhibition via Pad4 deficiency alleviated intestinal inflammation, attenuated cellular necroptosis, improved intestinal permeability, and enhanced tight junction protein expression. Notably, NETs prevented FUN14 domain-containing 1 (FUNDC1)-required mitophagy activation in intestinal epithelial cells, and stimulating mitophagy attenuated NET-associated mitochondrial dysfunction, cellular necroptosis, and intestinal damage. Mechanistically, silencing Toll-like receptor 4 (TLR4) or receptor-interacting protein kinase 3 (RIPK3) via shRNA relieved mitophagy limitation, restored mitochondrial function and reduced NET-induced necroptosis in Caco-2 cells, whereas this protective effect was reversed by TLR4 or RIPK3 overexpression. The regulation of TLR4/RIPK3/FUNDC1-required mitophagy by NETs can potentially induce intestinal epithelium necroptosis.