Abstract 4350593: TRAM Deletion Reprograms Neutrophils to Resolve Inflammation and Attenuate Atherosclerosis

Atherosclerosis is a leading cause of morbidity and mortality globally, and neutrophils have emerged as key players in the progression and resolution of vascular inflammation. Although neutrophils are associated with atherosclerosis pathogenesis, the molecular mechanisms governing their pro- or anti-inflammatory states remain poorly defined. Here, we identify the adaptor protein TRAM as a critical molecular switch that regulates neutrophil polarization and function in atherosclerosis. Genetic deletion of TRAM in neutrophils reprograms them into a resolving phenotype, characterized by elevated levels of anti-inflammatory mediators such as Resolvin D1 (RvD1), CD200R, and SESN1, and reduced expression of pro-inflammatory mediators including leukotriene B4 (LTB4), elastase, and myeloperoxidase. Adoptive transfer of TRAM-deficient neutrophils into atherosclerotic ApoE −/− mice significantly reduces plaque size, lipid deposition, and vascular leakage, while enhancing plaque stability and improving endothelial integrity. Consistent with in vivo results, TRAM-deficient neutrophils exert protective effects on co-cultured endothelial cells in an RvD1-dependent manner. Mechanistically, TRAM deletion disrupts oxLDL-induced translocation of 5-lipoxygenase to the nuclear membrane by downregulating FLAP, which may shift lipid mediator secretion from pro-inflammatory LTB4 to pro-resolving RvD1. TRAM also functions as a stress sensor of oxLDL and free cholesterol. Furthermore, TRAM-deficient neutrophils reduce ROS generation and SYK/CaMKII signaling activation. Importantly, our scRNA-sequencing data unveil a human CD177 low neutrophil subset with low TRAM expression, mirroring the resolving phenotype of murine TRAM-deficient neutrophils. These CD177 low neutrophils exhibit reduced swarming, elastase release, and LTB4 secretion. Our findings establish TRAM as a central regulator of neutrophil plasticity and inflammation in atherosclerosis, highlighting the therapeutic potential of TRAM-targeted modulation for cardiovascular disease.