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.

Increased neutrophil extracellular traps (NETs) formation has been found to be associated with intestinal inflammation, and it has been reported that NETs may drive the progression of gut dysregulation in sepsis. However, the biological function and regulation of NETs in sepsis-induced intestinal barrier dysfunction are not yet fully understood. First, we found that both circulating biomarkers of NETs and local NETs infiltration in the intestine were significantly increased and had positive correlations with markers of enterocyte injury in abdominal sepsis patients. Moreover, the levels of local citrullinated histone 3 (Cit H3) expression were associated with the levels of BIP expression. To further confirm the role of NETs in sepsis-induced intestinal injury, we compared peptidylarginine deiminase 4 (PAD4)-deficient mice and wild-type (WT) mice in a lethal septic shock model. In WT mice, the Cit H3-DNA complex was markedly increased, and elevated intestinal inflammation and endoplasmic reticulum (ER) stress activation were also found. Furthermore, PAD4 deficiency alleviated intestinal barrier disruption and decreased ER stress activation. Notably, NETs treatment induced intestinal epithelial monolayer barrier disruption and ER stress activation in a dose-dependent manner in vitro, and ER stress inhibition markedly attenuated intestinal apoptosis and tight junction injury. Finally, TLR9 antagonist administration significantly abrogated NETs-induced intestinal epithelial cell death through ER stress inhibition. Our results indicated that NETs could contribute to sepsis-induced intestinal barrier dysfunction by promoting inflammation and apoptosis. Suppression of the TLR9–ER stress signaling pathway can ameliorate NETs-induced intestinal epithelial cell death.

Background: Hesperetin (HES), one of the major flavonoids that has various biological activities, such as anti-inflammatory and antioxidant activities, may preserve the intestinal barrier during sepsis. However, the detailed mechanism remains unclear. Our previous studies confirmed that neutrophil extracellular traps (NETs) may jeopardize the intestinal barrier via a reactive oxygen species (ROS)-dependent pathway during sepsis. Therefore, we hypothesized that HES may inhibit NET formation and protect the intestinal barrier function during sepsis. Methods: Mice were pretreated with HES (50 mg kg⁻¹) intraperitoneally for one week, and sepsis models were then induced using lipopolysaccharides (LPS) (10 mg kg⁻¹). The mice were randomly divided into three groups: (1) sham group; (2) LPS group; and (3) HES + LPS group. Twenty-four hours after LPS injection, the serum and terminal ileum specimens were collected for subsequent studies. To detect ROS production and NET formation in vitro, human neutrophils were collected and incubated with phorbol-12-myristate-13-acetate (PMA) and various concentrations of HES. The level of autophagy was measured by an immunofluorescence assay and western blot analysis. TUNEL staining was utilized to analyze cell apoptosis. Results: The outcomes demonstrated that HES decreased inflammatory cytokine and myeloperoxidase (MPO) levels in serum and attenuated distant organ dysfunction in LPS-induced septic mice. Meanwhile, HES treatment reversed intestinal histopathological damage in septic mice, improving intestinal permeability and enhancing tight junction expression. Moreover, we found that neutrophil infiltration and NET formation in the intestine were suppressed during sepsis after HES pretreatment. In vitro, HES treatment reduced PMA-induced ROS production and NET formation, which were reversed by hydrogen peroxide (H₂O₂) administration. Notably, HES also inhibited NET formation by reducing the microtubule-associated protein light chain 3 (LC3)-II/LC3-I ratio (an indicator of autophagy) in PMA-induced neutrophils, which was reversed by rapamycin. Moreover, when autophagy was suppressed by chloroquine or induced by rapamycin, apoptosis in cells will be switched with autophagy. Conclusion: Taken together, these findings suggest that HES may inhibit NET formation in a ROS/autophagy-dependent manner and switch neutrophil death from NETosis to apoptosis, which reduced NETs-related intestinal barrier damage, providing a novel protective role in intestinal barrier dysfunction during sepsis.

A growing number of studies have recently revealed a potential role for neutrophil extracellular traps (NETs) in the development of inflammation, coagulation and cell death. Deleterious consequences of NETs have been identified in ischemia-reperfusion (I/R)-induced organ damage, thrombosis and sepsis. And exogenous DNase-I has been suggested as a therapeutic strategy to attenuate ischemia-reperfusion (I/R) injuries in the kidney, brain and myocardium. Herein, we designed a study to investigate whether NETs contribute to the pathogenesis of intestinal I/R injury and evaluated the therapeutic value of DNase-1 in a rat model of intestinal I/R injury. In this rat model of intestinal I/R injury, we found that extracellular DNA was readily detectable in rat serum after 1 h of ischemia and 2 h of reperfusion. Treatment with DNase-1 significantly reduced the inflammatory response, restored intestinal barrier integrity and increased the expression of tight junction proteins. Our results indicate the existence of NETs in I/R-challenged intestinal tissues and firstly provide more evidence that DNase-1 may be an effective treatment for attenuating intestinal I/R injury.

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 viaPad4deficiency 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.