Inhibition of neutrophil extracellular traps alleviates blood-brain barrier disruption and cognitive dysfunction via Wnt3/β-catenin/TCF4 signaling in sepsis-associated encephalopathy

Background: Neutrophils and neutrophil extracellular traps (NETs) have been identified as crucial contributors in several neuroinflammatory models, such as stroke and traumatic brain injury, but their role in sepsis-associated encephalopathy (SAE) has not been thoroughly investigated.

Methods: In this study, we established an SAE model using cecal ligation puncture (CLP) surgery to examine neutrophil infiltration and NETs formation. A Protein Arginine Deiminase 4 (PAD4) inhibitor, GSK484, was employed to suppress NETs release. To assess changes in hippocampal gene expression induced by GSK484 treatment in CLP mice, we utilized RNA Sequencing (RNA-Seq) combined with bioinformatics analysis. Additionally, the Elisa, cognitive function test, western bolt and immunofluorescence staining were used to measured hippocampal inflammatory cytokine, cognitive function, and the protein levels of tight junctions (TJs) and adherens junctions (AJs) in SAE mice. We also established a Transwell™ co-culture system using bEnd.3 cells and bone marrow-derived neutrophils to examine the effects of GSK484 on endothelial cell function. This comprehensive approach allowed us to evaluate the impact of NETs inhibition on neuroinflammation, cognitive function, and the underlying molecular mechanisms in the CLP-induced SAE model.

Results: Our findings revealed that neutrophils were significantly overactivated, releasing abundant NETs in the hippocampus of CLP-induced SAE mice. Inhibition of NET formation using GSK484 led to reduced neuroinflammatory responses, improved blood-brain barrier (BBB) integrity, and enhanced survival rates and cognitive function in SAE mice. RNA-Seq and bioinformatics analyses identified the Wnt signaling pathway as the most significant pathway affected. Subsequent experiments demonstrated that NETs inhibition alleviated BBB damage primarily by increasing the expression of Occludin, a TJs protein, and promoting the formation of the VCL/β-catenin/VE-cadherin complex at AJs, mediated by the Wnt3/β-catenin/TCF4 signaling pathway.

Conclusions: Our results suggest that inhibition of NETs may protect BBB permeability and cognitive function through the Wnt3/β-catenin/TCF4 signaling pathway in the context of CLP-induced SAE, which provides a promising strategy for SAE therapy.

Adhesion junction; Blood–brain barrier; Neuroinflammation; Neutrophil extracellular traps; Sepsis-associated encephalopathy.