Redefining leukocytes in atherosclerosis

Distinct leukocyte populations have been associated with atherosclerosis development and progression or with atheroprotection. However, most of these subsets were defined using preselected markers, and the actual subset heterogeneity in atherosclerotic lesions and the exact contribution of each leukocyte population is not well defined. Two studies now take advantage of novel high-dimensional techniques to provide a more detailed insight into leukocyte phenotypic and transcriptional heterogeneity in atherosclerosis.

Credit: V. Summersby/Macmillan Publishers Limited

Combining mass cytometry (CyTOF) and single-cell RNA sequencing (single-cell RNA-seq) with bioinformatic tools, Dennis Wolf and colleagues found high leukocyte diversity in mouse atherosclerotic aortas, with 11 leukocyte populations with distinct phenotypic and spatial characteristics. By contrast, mouse healthy aortas had a small number of leukocyte subsets. The phenotypic diversity of the clusters was confirmed with a 35-panel CyTOF and conventional flow cytometry, and the subsets showed a high correlation with previously defined transcriptomes. Genetic deconvolution analysis of microdissected mouse aorta and of 126 human plaques showed that monocytes, macrophages, and T cells were predominantly present in plaques and B cells in the adventitia. Finally, absence of one T-cell cluster was associated with a higher cardiovascular risk in patients. “Our data suggest that the traditional classification of leukocytes based on principal haematopoietic lineages and known lineages is too coarse,” says Wolf. “We hope that our cell atlas will open new avenues in atherosclerosis research and inspire others to more precisely look at leukocyte diversity.” The researchers plan to study further some of the newly identified subsets to understand the biological meaning of their findings.

The team led by Alma Zernecke, Clément Cochain, and Antoine-Emmanuel Saliba, used single-cell RNA-seq to analyse the transcriptional profile of CD45⁺ cells from mouse healthy and atherosclerotic aortas. The analysis identified 13 cell clusters. A population of resident-like macrophages was found in both healthy and atherosclerotic aortas, whereas monocytes, monocyte-derived dendritic cells, and two macrophage populations were almost only detectable in atherosclerotic aortas. The two macrophage populations associated with atherosclerotic lesions were inflammatory macrophages enriched in Il1b and TREM2^high macrophages, characterized by a gene expression signature reminiscent of osteoclasts (which suggests a role in lesion calcification) and putative functions in lipid metabolism and catabolism, as revealed by differential gene expression and gene ontology enrichment analyses. TREM2^high macrophages were also detected in advanced atherosclerosis, and TREM2 expression was found in human atherosclerotic lesions. “Our work provides a global overview of the transcriptional landscape of macrophages and other immune cells in atherosclerotic vessels with unprecedented resolution, and may prove a valuable resource for other researchers,” explain the investigators. The research team plans to assess whether similar macrophage populations are present in human atherosclerotic lesions and to investigate some of the newly identified targets that might control macrophage-mediated inflammation in atherosclerosis.