Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. Methods and Results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing (CITE-seq) to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G + neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils (Cd177, Lcn2, Fpr1), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb). At 3 and 5 days two major subsets of Siglecf hi (enriched for e.g. Icam1 and Tnf) and Siglecf low (nSlpi, Ifitm1) neutrophils were found. CITE-seq analysis in blood and heart revealed that while circulating neutrophils undergo a process of ageing characterized by loss of surface CD62L and upregulation of Cxcr4, heart infiltrating neutrophils acquired a unique SiglecF hi signature. SiglecF hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF hi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF hi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF hi signature.

Objective-After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they can promote both tissue healing and damage. Here, we investigated the dynamics of cardiac neutrophil heterogeneity after infarction.Methods and results-We employed single-cell transcriptomics (scRNA-seq) to investigate temporal neutrophil heterogeneity in the heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, neutrophils could be delineated into six distinct clusters with specific time-dependent patterning and proportions. While the majority of neutrophils at day 1 were characterized by high expression of chemokines (e.g. Cxcl3, Ccl6), and putative activity of transcriptional regulators involved in hypoxic response (Hif1a) and emergency granulopoiesis (Cebpb), two major subsets of Siglecf hi (enriched for e.g. Icam1 and Tnf) and Siglecf low (Slpi, Ifitm1) neutrophils were found at 3 and 5 days. Flow cytometry analysis confirmed the presence of LY6G + SIGLECF hi and LY6G + SIGLECF low neutrophils in the heart from 3 days after infarction onwards. LY6G + SIGLECF hi neutrophils were absent from the bone marrow, blood and spleen, suggesting local acquisition of surface SIGLECF. Acquisition of the SIGLECF hi state was paralleled by features of neutrophil ageing and activation (ICAM1 hi CXCR4 hi CD49d hi CD62L low ). scRNA-seq of atherosclerotic aortas revealed two neutrophil subsets with gene expression patterns reminiscent of the major Siglecf hi and Siglecf low cardiac neutrophil subpopulations, revealing that these populations may be present across distinct contexts of cardiovascular inflammation.Conclusion-Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse ischemic heart at the single-cell level, and suggests that temporal neutrophil heterogeneity is in part driven by local transition to a SIGLECF hi state.