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bioRxiv · 10.1101/2025.08.06.669018

CCR2+ neutrophils exhibit a proinflammatory phenotype and promote plaque destabilization

Farjia, M.·Pan, C.·Braster, Q.·Lemnitzer, P.·Sachs, N.·Schultz, C.·Maegdefessel, L.·Soehnlein, O.·Silvestre-Roig, C.
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Abstract

BackgroundAtherosclerotic plaque destabilization is promoted by inflammatory cell recruitment, tissue cell death and mechanical weakening. Neutrophils are key instigators of vascular tissue injury and perpetuation of inflammation, and targeting their actions is a viable therapeutic opportunity. We here identify a distinct subset of activated neutrophils within atherosclerotic lesions that express the chemokine receptor CCR2, which directs their migration toward areas enriched with smooth muscle cells (SMCs) and contributes to plaque instability. MethodsFlow cytometry and single cell transcriptomic analysis of CCR2+ neutrophils within murine and human atherosclerotic plaques. Tracking of CCR2+ neutrophil in hypercholesterolemic Ldlr-/- mice and in mice reconstituted with Ccr2GFP/- bone marrow. In vivo reactive oxygen species (ROS) and neutrophil extracellular trap (NET) analysis in lipopolysaccharide-induced peritonitis. In vitro migration assays of neutrophils deficient for or treated with specific antagonist against chemokine receptors. In vivo neutrophil recruitment and atherosclerotic plaque destabilization analysis upon CCR2 and CCL2 blockade in a model of advanced atherosclerosis in Apoe-/- mice. ResultsCCR2+ neutrophils preferentially populate mouse and human atherosclerotic lesions and displayed a proinflammatory phenotype with enhanced capacity for ROS production and NET release. Genetic deletion or pharmacologic inhibition of CCR2 significantly reduces neutrophil migration and infiltration to the atherosclerotic lesion, reducing their presence in SMC-rich areas. Consistently, neutralization of the CCR2 ligand CCL2 decreased lesional neutrophil numbers and preserved fibrous cap integrity by increasing SMC content and decreasing overall plaque instability. ConclusionsOur data suggest that a subset of CCR2-expressing neutrophils senses SMC-derived CCR2 ligands to infiltrate and destabilize atherosclerotic lesions. These results support the existence of neutrophil functional heterogeneity within the atherosclerotic lesions contributing to alterations of lesion stability. Specific targeting thereof may improve plaque stability without impacting host defense.

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Farjia, M., Pan, C., Braster, Q., Lemnitzer, P., Sachs, N., Schultz, C., Maegdefessel, L., Soehnlein, O., Silvestre-Roig, C.. 2025-08-08. CCR2+ neutrophils exhibit a proinflammatory phenotype and promote plaque destabilization. https://doi.org/10.1101/2025.08.06.669018

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