NETs activate the GAS6-AXL-NLRP3 axis in macrophages to drive morphine tolerance

The development of morphine tolerance presents a major clinical challenge in the effective management of severe pain. This study aims to explore the mechanisms underlying morphine tolerance from a novel perspective, with the ultimate goal of uncovering new insights and identifying promising therapeutic targets for its treatment. C57BL/6J mice were used in the tail-flick test to evaluate morphine tolerance. Neutrophils derived from mouse bone marrow were employed to investigate the mechanisms underlying morphine-induced NETs formation. Bone marrow-derived macrophages (BMDMs) were harvested from the femur and tibia to study the role of NETs-induced inflammation in analgesic tolerance. Proinflammatory cytokines were measured using Western blotting and real-time PCR. The levels of NETs and the TLR7/9-NLRP3-related signaling pathway were assessed through Western blotting, real-time PCR, and ELISA. Confocal laser scanning microscopy was utilized to visualize NETs in the dorsal root ganglion (DRG) and in cells. Our experiments demonstrated that the levels of NETs in the plasma of patients using morphine for analgesia, as well as in morphine-tolerant animals, were significantly elevated. Genetic elimination of Pad4, neutrophil depletion, and treatment with DNase 1 and RNase A to disrupt NETs formation all effectively alleviated morphine tolerance. These findings indicate that NETs play a critical role in the development of morphine tolerance. Mechanistically, we discovered that morphine-induced NETs can be engulfed by macrophages through the GAS6-AXL axis, which subsequently triggers the activation of the TLR7/TLR9-mediated NLRP3 inflammasome, leading to significantly increased levels of IL-1β and IL-18, and ultimately contributing to tolerance. Deletion of Axl, Gas6, or Nlrp3 each significantly improved morphine tolerance. Furthermore, in the murine model, treatment with the IL-1 receptor antagonist anakinra and the IL-18 decoy receptor IL-18BP prevented the development of morphine tolerance. This study identifies morphine-induced NETs as a key contributor to morphine tolerance, with the GAS6-AXL-TLR7/9 axis emerging as a potential therapeutic target. Strategies focused on disrupting NETs and modulating this axis may offer a promising approach to combat morphine tolerance.