Objective: Atherosclerosis is a chronic inflammatory disease that causes leukocyte-rich plaques in the intimal layer of arteries. Its development depends on the interplay of pro- and anti-inflammatory leukocytes in the plaque. However, the phenotypical and transcriptional diversity of arterial leukocytes on a single cell level is unknown. Here, we applied a multi-modal approach including mass cytometry (CyTOF) and single cell RNA-sequencing (scRNA-seq.) to define leukocyte heterogeneity in murine aortas.
Methods and Results: We established and validated a pan-leukocyte CyTOF panel of 35 markers covering all major hematopoeitic lineage markers. Leukocytes were isolated from aortas of Apoe-/- mice after feeding with a standard chow (moderate atherosclerosis) or a western diet (severe atherosclerosis) for 12 weeks. Myeloid, T, and B cells were the predominant leukocyte subset in atherosclerotic aortas, but 26% of leukocytes could not be assigned to classical leukocyte subsets. By applying an unsupervised cluster detection algorithm by tSNE and Phenograph we detected 27 distinct leukocyte clusters covering all CD45+ leukocytes with known and several novel leukocyte sub-populations, including 11 myeloid, 10 T cell, and 4 B cell sub-populations with distinct surface marker expression profiles and regulation in disease. To gain further insights into their transcriptional regulation, we applied scRNA-seq. after FACS-sorting of CD45+ leukocytes from atherosclerotic aortas. Single cell transcriptomes were obtained from 10 pooled aortas. We detected a median of 1.102 genes with an average of >63.000 reads per cells. tSNE clustering of single cell transcriptomes confirmed an unexpected high leukocyte heterogeneity and could identified gene sets in each cluster that were differentially regulated in disease.
Conclusion: Phenotypical and transcriptional profiling by CYTOF and scRNA-seq discovered an unexpected high heterogeneity of aortic leukocytes with several yet unknown sub-populations. These findings have great implication for understanding the differential role of leukocytes in atherosclerosis and propose higher dimensionality cytometry and transcriptomics as novel tool in vascular biology research.