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

  • Cell Commun Signal. 2025 Apr 11;23(1):181. doi: 10.1186/s12964-025-02181-4.
Qingyan Tian  #  1 ,  Haiyue Guo  #  1 ,  Mengyao Zhang  1 ,  Kunmao Jiang  1 ,  Fan Hu  1 ,  Yan Xu  2 ,  Li Wan  1 ,  Xiaokai Zhou  3 ,  Yinbing Pan  4 ,  Wentao Liu  5 ,  Chun-Yi Jiang  #  6
Affiliations
  • 1. Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu, 211166, China.
  • 2. Department of Pain, The First People's Hospital of Changzhou, Soochow University, Changzhou, Jiangsu, China.
  • 3. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Jiangning District, Nanjing, Jiangsu, 210029, China.
  • 4. Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Jiangning District, Nanjing, Jiangsu, 210029, China. [email protected].
  • 5. Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu, 211166, China. [email protected].
  • 6. Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu, 211166, China. [email protected].
  • # Contributed equally.
PMID: 40217343 DOI: 10.1186/s12964-025-02181-4
Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords
AXL; GAS6; Morphine tolerance; NETs; NLRP3.
Products