TY - JOUR
T1 - Atlas of the immune cell repertoire in mouse atherosclerosis defined by single-cell RNA-sequencing and mass cytometry
AU - Winkels, Holger
AU - Ehinger, Erik
AU - Vassallo, Melanie
AU - Buscher, Konrad
AU - Dinh, Huy Q.
AU - Kobiyama, Kouji
AU - Hamers, Anouk A.J.
AU - Cochain, Clément
AU - Vafadarnejad, Ehsan
AU - Saliba, Antoine Emmanuel
AU - Zernecke, Alma
AU - Pramod, Akula Bala
AU - Ghosh, Amlan K.
AU - Michel, Nathaly Anto
AU - Hoppe, Natalie
AU - Hilgendorf, Ingo
AU - Zirlik, Andreas
AU - Hedrick, Catherine C.
AU - Ley, Klaus
AU - Wolf, Dennis
N1 - Funding Information:
This work was supported by grants from the Deutsche Forschungsgemeinschaft to D. Wolf (DFG WO1994/1) and H. Winkels (DFG WI4811/1-1). K. Ley was supported by grants HL115232, HL88093, and HL121697 from the National Heart, Lung, and Blood Institute. K. Ley and C. Hedrick were further supported by grant P01 HL136275 from the National Institutes of Health. The CyTOF Helios Mass Cytometer and the fluorescence-activated cell sorting-Aria-3 cell sorter were supported by the Shared Instrumentation Grant Program S10 OD018499-01 and RR027366-01A1, respectively.
Publisher Copyright:
© 2018 American Heart Association, Inc.
PY - 2018
Y1 - 2018
N2 - 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 RNAsequencing 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.
AB - 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 RNAsequencing 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.
KW - atherosclerosis
KW - flow cytometry
KW - immune system
KW - leukocytes
KW - lymphocytes
KW - macrophages
KW - mass cytometry
KW - single-cell RNA-sequencing
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U2 - 10.1161/CIRCRESAHA.117.312513
DO - 10.1161/CIRCRESAHA.117.312513
M3 - Article
C2 - 29545366
AN - SCOPUS:85046991602
SN - 0009-7330
VL - 122
SP - 1675
EP - 1688
JO - Circulation research
JF - Circulation research
IS - 12
ER -