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Circ Res. 2018 Jun 8;122(12):1675-1688. doi: 10.1161/CIRCRESAHA.117.312513. Epub 2018 Mar 15.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

Author information

1
From the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, CA (H.W., E.E., M.V., K.B., H.Q.D., K.K., A.A.J.H., A.B.P., A.K.G., C.C.H., K.L., D.W.).
2
Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.).
3
Helmholtz Institute for RNA-based Infection Research, Würzburg, Germany (E.V., A.-E.S.).
4
Department of Cardiology and Angiology I, University Heart Center Freiburg, Germany (N.A.M., N.H., I.H., A.Z., D.W.).
5
the Faculty of Medicine, University of Freiburg, Germany (N.A.M., N.H., I.H., A.Z., D.W.).
6
Department of Bioengineering, University of California, San Diego (K.L.).
7
Institute of Experimental Biomedicine, University Hospital Würzburg, Germany (C.C., A.Z.) dwolf@lji.org.

Abstract

RATIONALE:

Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood.

OBJECTIVE:

We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis.

METHODS AND RESULTS:

Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients.

CONCLUSIONS:

The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

KEYWORDS:

atherosclerosis; flow cytometry; immune system; leukocytes; lymphocytes; macrophages; mass cytometry; single-cell RNA-sequencing

PMID:
29545366
PMCID:
PMC5993603
[Available on 2019-06-08]
DOI:
10.1161/CIRCRESAHA.117.312513

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