Outer membrane vesicles derived from probiotic Escherichia coli Nissle 1917 promote metabolic remodeling and M1 polarization of RAW264.7 macrophages

dongxue ma¹yuxin zhang¹jia zhang¹Jun Shi¹shanhu gao¹fei long¹xin wang¹xinyu pu¹jiayao sun¹shuang liang¹Richard Cannon²Ting-Li Han^3*Silas Granato Villas-Boas⁴

• ¹State Key Laboratory of Ultrasound in Medicine and Engineering, Chongqing Medical University, Chongqing, China
• ²Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin, Otago, New Zealand
• ³Department of Obstetrics and Gynecology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
• ⁴Luxembourg Institute of Science and Technology (LIST), Esch-sur-Alzette, Luxembourg

Escherichia coli Nissle 1917 (EcN) is one of the most extensively studied nonpathogenic Gram-negative probiotic strains worldwide. Recent research has highlighted the ability of EcN outer membrane vesicles (OMVs) to enhance the phagocytosis and proliferation of RAW264.7 macrophages. However, the impact of EcN-OMVs on M1/M2 polarization and metabolic modulation remains unknown. In this study, we evaluated the metabolic effects of EcN-OMVs on RAW264.7 macrophage polarization using metabolomic, transcriptomic, and fluxomic approaches. We found that the RAW264.7 macrophages phagocytosed EcN-OMVs, triggering upregulation of the HIF-1, mTORC1, and NF-κB signaling pathways. This metabolic reprogramming enhanced glycolysis, suppressed the TCA cycle, elevated intracellular reactive oxygen species (ROS), TNF-α, IL-6, IL-1β, ATP, and nitric oxide (NO) production, and promoted macrophage proliferation, migration, invasion, and M1type polarization. In summary, this research establishes a theoretical foundation for utilizing probiotic OMVs in immunomodulatory therapeutic applications.