The Positive Feedback Interaction Between Mast Cell-Derived Tryptase and NETs Formation in the Pathogenesis of Sepsis-Associated Encephalopathy.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a serious neurological complication resulting from sepsis, marked by considerable neuroinflammation and deficits in cognitive function. While neutrophil extracellular traps (NETs) have been implicated in various neuroinflammatory conditions, the specific mechanisms underlying NETs formation in SAE, particularly the role of brain-resident mast cells (MCs) and their interplay with neutrophils, remain poorly understood. METHODS: The cecal ligation and puncture (CLP) procedure was employed to create the SAE model in male C57BL/6 mice. Comprehensive methodological approaches included: 1) Pharmacological interventions using MC stabilizer (cromolyn), MC activator (C48/80), tryptase inhibitor (APC366), and Protease-activated receptor 2(PAR2) antagonist (AZ3451); 2) Establishment of MC-neutrophil co-culture systems with PMA/LPS stimulation; 3) Advanced imaging techniques including multiplex immunofluorescence and NETs visualization through Cit-H3/MPO/DAPI triple staining; 4) Molecular pathway analysis through Western blot, co-immunoprecipitation, and lactate quantification; 5) Functional assessments of MC activation through β-hexosaminidase release and tryptase activity assays. RESULTS: MC activation significantly promoted neutrophil infiltration and NET formation in SAE mice. Investigations into the mechanisms have demonstrated that tryptase, derived from MC, activates the PAR2-MAPK signaling pathway in neutrophils, which results in an increased expression of LDHA and boosts lactate production. Subsequently, lactate-mediated lactylation stabilizes PAD4 protein by inhibiting its degradation, thereby facilitating NETosis. Furthermore, NETs promote a positive feedback mechanism by boosting MC activation and elevating the release and activity of tryptase, thereby creating a self-reinforcing cycle of inflammation. CONCLUSION: This study elucidates a novel bidirectional MC-NETs axis in SAE pathogenesis, wherein tryptase-PAR2-MAPK signaling and lactylation-mediated PAD4 stabilization drive progressive neuroinflammation. These findings not only advance our understanding of SAE mechanisms but also identify multiple potential therapeutic targets within this pathway for clinical intervention.