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

Abstract

<div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d401206e294">Rationale</h5> <p id="P1">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 only poorly understood. </p> </div><div class="section"> <a class="named-anchor" id="S2"> <!-- named anchor --> </a> <h5 class="section-title" id="d401206e299">Objective</h5> <p id="P2">We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to define an atlas of the immune cell landscape in atherosclerosis. </p> </div><div class="section"> <a class="named-anchor" id="S3"> <!-- named anchor --> </a> <h5 class="section-title" id="d401206e304">Methods and Results</h5> <p id="P3">Employing scRNAseq of aortic leukocytes from chow (CD) and western diet (WD) fed <i>Apoe</i> ^−/− and <i>Ldlr</i> ^−/− mice, we detected 11 principal leukocyte clusters with distinct phenotypical and spatial characteristics, while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on a 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 <i>Apoe</i> ^−/− and <i>Ldlr</i> ^−/− mice. We confirmed the phenotypic diversity of these clusters with a novel CyTOF 35-marker panel with metal-labelled 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 scRNAseq, CyTOF, and FACS, we detected three principal B-cell subsets with alterations in surface markers, functional pathways, and <i>in vitro</i> cytokine secretion. Finally, we used leukocyte cluster gene signatures to enumerate 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. </p> </div><div class="section"> <a class="named-anchor" id="S4"> <!-- named anchor --> </a> <h5 class="section-title" id="d401206e337">Conclusion</h5> <p id="P4">The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis. </p> </div>