Siglecf(Hi) marks late-stage neutrophils of the infarcted heart: A single-cell transcriptomic analysis of neutrophil diversification

Abstract

BACKGROUND: Neutrophils are thought to be short-lived first responders to tissue injuries such as myocardial infarction (MI), but little is known about their diversification or dynamics. METHODS AND RESULTS: We permanently ligated the left anterior descending coronary arteries of mice and performed single-cell RNA sequencing and analysis of >28 000 neutrophil transcriptomes isolated from the heart, peripheral blood, and bone marrow of mice on days 1 to 4 after MI or at steady-state. Unsupervised clustering of cardiac neutrophils revealed 5 major subsets, 3 of which originated in the bone marrow, including a late-emerging granulocyte expressing SiglecF, a marker clas-sically used to define eosinophils. SiglecFHI neutrophils represented ≈25% of neutrophils on day 1 and grew to account for >50% of neutrophils by day 4 post-MI. Validation studies using quantitative polymerase chain reaction of fluorescent-activated cell sorter sorted Ly6G+SiglecFHI and Ly6G+SiglecFLO neutrophils confirmed the distinct nature of these populations. To con-firm that the cells were neutrophils rather than eosinophils, we infarcted GATA-deficient mice (∆dblGATA) and observed similar quantities of infiltrating Ly6G+SiglecFHI cells despite marked reductions of conventional eosinophils. In contrast to other neutrophil subsets, Ly6G+SiglecFHI neutrophils expressed high levels of Myc-regulated genes, which are associated with longevity and are consistent with the persistence of this population on day 4 after MI. CONCLUSIONS: Overall, our data provide a spatial and temporal atlas of neutrophil specialization in response to MI and re-veal a dynamic proinflammatory cardiac Ly6G+SigF+(Myc+NFκB+) neutrophil that has been overlooked because of negative selection.