Membrane‐Active Peptide Protects Against Inflammation by Targeting NLRP3 Activation at the Trans‐Golgi Network

ABSTRACT The NLRP3 inflammasome is a multi‐protein complex that plays a crucial role in inflammatory processes mediated by the innate immune system. Dysregulated NLRP3 activation has been implicated in age‐related inflammatory diseases, making it a promising therapeutic target. Here, we report that the synthetic membrane‐active antimicrobial peptide Pep19‐2.5 directly inhibits NLRP3 inflammasome activation. Through cellular, biophysical, and biochemical analyses, we find that Pep19‐2.5 suppresses NLRP3 inflammasome signaling downstream of NLRP3 activation. Pep19‐2.5 interacts with macrophage membranes, supporting a membrane‐targeting mechanism for its anti‐inflammatory effects. Mechanistically, Pep19‐2.5 binds to phosphatidylinositol (PI)‐containing lipid membranes and dispersed trans‐Golgi network (dTGN) structures, which could potentially affect NLRP3 recruitment to the dTGN. We demonstrate a strong and NLRP3‐dependent induction of IL‐1β secretion from human macrophages by house dust mite (HDM) extract, which can be inhibited by Pep19‐2.5. In line with these findings, therapeutic application of Pep19‐2.5 via the nasal aerosol route reduces IL‐1β levels, eosinophil infiltration in bronchoalveolar lavage and significantly improved lung function in an in vivo HDM‐mouse model of allergic airway inflammation. Our findings highlight the therapeutic potential of targeting NLRP3 activation by the small membrane‐active peptide Pep19‐2.5 for the treatment of NLRP3‐driven inflammatory diseases.