Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas

Abstract

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2⁺ foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Keywords: T lymphocytes; atherosclerosis; interferons; macrophages; monocytes; mouse; neutrophils.

Figure 1.. Overview.

This analysis is based on published studies where atherosclerosis was induced in mouse models using genetic knockouts Ldlr^{−/− ,} , Apoe^−/− , diet (chow, western diet, high fat diet, refs) or AAV-PCSK9 induced lipoprotein changes, resulting in atherosclerotic aortas. In some studies, lineage tracking (Cx3cr1-Cre) or chemical labeling (Bodipy) labeled specific cell types. All studies used enzymatic digestion with the attendant problems of cell death and loss of surface markers. Single cells were phenotyped by RNA-Seq^{, ,} , CyTOF or both. Dimensionality reduction and clustering identified cell types and gene signatures. Matching CyTOF with scRNA-Seq data is challenging. Based on gene signatures, genetic labeling was used to visualize, image and sort some cell types to gain functional insights and deep transcriptomes.

Figure 2.. Integration of scRNA-Seq data from 9 atherosclerosis studies on mouse aortas.

scRNA-Seq data was retrieved from NCBI GEO for Harmony integration and visualized using UMAP. 17 clusters were identified by Louvain clustering. One cluster (lilac) was dominated by non-leukocyte genes including Acta2 and one cluster (dark yellow) was dominated by proliferation genes including Top2a and Tuba1b, leaving 15 bone fide leukocyte clusters: 2 B cell clusters (red and pink), one CD8 T cell cluster (light green), 4 macrophage clusters (inflammatory, blue, IFNIC, light purple, resident, purple and Trem2, light blue), 2 CD4 T cell clusters (Th17, brown and Th2, teal), CD4⁺CD8⁺ cells (turquoise), 2 mixed monocyte/macrophage/DC clusters (orange), Xcr1⁺ DCs (hot pink), neutrophils (light pink), NK cells (dark green). Total n= 15,288 cells

Figure 3.. Different abundance of aortic leukocyte subsets in 9 scRNA-Seq mouse atherosclerosis studies.

The UMAP from figure 2 was separated into the 9 different studies. Studies identified by first author^{, , ,} and abbreviated conditions (see online data set for full conditions). Cell subsets as in figure 2.

Figure 4.. Top gene signatures for 15 aortic leukocyte subsets.

The top 10 up-regulated genes in each subset compared to all other subsets are shown by Dot plot. Expression level indicated by saturation of blue (dark blue is highest expression, log2 scale where 0 is global average). Dot diameter represents percentage of cells in cluster expressing corresponding genes (largest circle is 100%).

Figure 5.. Reclustering of myeloid cells and T cells.

We selected myeloid cells (Macrophages, Mixed monocyte/macrophage/DC clusters except neutrophils, left panel), and T cells (T cell, IL-17⁺, NK and ILC cell clusters, right panel) from the UMAP from figure 2 for reclustering. The following myeloid clusters were retrieved: 5 macrophage clusters (inflammatory, dark green, IFNIC, light green, resident, dark pink, Trem2, light pink, and cavity, red), monocytes (blue), MoDC/cDC2 (yellow), and 3 DC clusters (Mature DC, turquoise, cDC1, yellow green, and pDC, purple). T cells clustered into naïve T cells (turquoise), CD4⁺Cd8⁺ (light pink), IL17⁺Cxcr6⁺ (light green), Treg (dark pink), CD8 (yellow), NK cells (purple) and ILC2s (dark green).

Figure 6.. Relationship between Cx3cr1-GFP⁺ and CD11c-YFP⁺ macrophages and the resident, IFNIC, inflammatory and Trem2 foamy macrophages from the scRNA-Seq studies.

Significantly differentially expressed (DE) genes were determined for Cx3cr1⁻GFP⁺, CD11c⁻YFP⁺, GFP⁺YFP⁺ and unlabeled macrophages against the other 3 subsets. The same was done for resident, IFNIC, inflammatory and Trem2 foamy macrophages. The DE gene lists were intersected. Only the genes that were upregulated in common are shown.

Figure 7.. Summary of cell clusters detectable in the atherosclerotic aorta.

The leukocyte infiltrate in atherosclerotic mouse aortas as analyzed in the comprehensive meta-analysis of 9 single cell RNA-Seq studies identified 19 types of leukocytes: neutrophils, monocytes and DCs, 5 types of macrophages, 5 types of T cells, B1, B2, NK and ILC2 cells. Top expressed and characteristic genes are indicated in italics.