RAGE in Neutrophils: A Sensor for Pathogen-Associated Structures and Beyond

Background/Objectives: Neutrophils express the receptor for advanced glycation end products (RAGE), yet its role in antibacterial responses remains incompletely defined. This study aims to elucidate the dual functionality of RAGE as a membrane-bound signaling sensor and a source of soluble RAGE (sRAGE) in human neutrophils challenged with Salmonella typhimurium, a clinically relevant Gram-negative pathogen. Methods: Human peripheral neutrophils from healthy donors were isolated and stimulated with S. typhimurium, LPS, or fMLP. Calcium flux, ROS/RNS production, and phagocytosis were assessed using fluorescent probes and spectroscopy. RAGE expression and localization were analyzed by immunofluorescence microscopy and flow cytometry. Soluble RAGE in supernatants was quantified by ELISA, and its molecular forms were characterized by Western blotting. Results: Resting neutrophils exhibited minimal surface RAGE but a substantial intracellular pool. RAGE inhibition with FPS-ZM1 attenuated bacteria-induced Ca²⁺ mobilization, oxidative burst, nitrosative output, and phagocytosis, with the most pronounced defect at the pathogen-attachment stage-consistent with impaired cytoskeletal remodeling. Upon activation, neutrophils rapidly released sRAGE (peak at ~10 min) via combined metalloprotease-dependent shedding and regulated secretion of pre-formed intracellular stores. Paradoxically, FPS-ZM1 amplified sRAGE release while suppressing membrane-proximal signaling. Conclusions: Neutrophil RAGE functions as a dynamic, multi-compartmental regulator: membrane-associated RAGE licenses effector responses to Gram-negative bacteria, while concomitant sRAGE release provides a fast negative-feedback loop to limit excessive inflammation. This self-limiting circuit balances antimicrobial defense with tissue protection, and its dysregulation may contribute to pathological outcomes in acute and chronic infections.