Inhibition of ADAM10, which mediates shedding of the chemokine CX3CL1, has been shown to improve post-infarction cardiac function through reduced neutrophil infiltration and decreased scar size following myocardial infarction (MI). A prerequisite for this ADAM10-mediated mechanism is the presence of the CX3CR1 receptor on neutrophils, which enables their interaction with CX3CL1. Notably, CX3CR1 is predominantly recognized as a monocyte / macrophage marker, particularly in the context of MI, and its expression in post-MI neutrophils has not yet been described. Interestingly, recent findings suggest that CX3CR1 expression is upregulated in neutrophils at days 3 and 7 post-MI in mice, raising questions about their functional role in the infarcted myocardium.
CX3CR1-positive and -negative neutrophils were isolated from bone marrow and infarcted heart tissue of mice 3 days after MI using magnetic cell sorting (MACS) and fluorescence activated cell sorting (FACS). Comprehensive characterization was performed using bulk RNA sequencing as well as histological and immunofluorescence stainings for nuclear morphology, granules and neutrophil extracellular traps (NETs). Transcriptome analysis revealed that CX3CR1-positive neutrophils exhibit a distinct pro-fibrotic rather than inflammatory phenotype. These cells demonstrated reduced NETosis and decreased cytokine production compared to CX3CR1-negative neutrophils, accompanied by a potential metabolic shift towards enhanced itaconate synthesis due to increased Acod1 expression. The pro-fibrotic signature was characterized by elevated TGF-beta signaling and increased expression of extracellular matrix-associated genes. Cell identity was confirmed through visualization of segmented nuclei, expression of neutrophil elastase (NE), and NET formation capability, definitively establishing their neutrophil lineage rather than monocytic or hybrid cell identity.
Our findings demonstrate that CX3CR1-positive neutrophils contribute to post-MI scar formation through a distinct pro-fibrotic rather than inflammatory phenotype. Targeting CX3CR1 on neutrophils may represent a promising strategy to attenuate excessive fibrotic remodeling following MI.
