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Response by Kim and Choi to Letter Regarding Article, "Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models".

Response by Kim and Choi to Letter Regarding Article, “Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models” In Response: We appreciate the comments by Drs Cochain, Saliba, and Zernecke on our recent publication. Two highly intriguing articles were published during our first revision that reported the analysis of immune cell heterogeneity by single-cell RNA sequencing (scRNA-seq). Specifically, Cochain et al identified 3 macrophage populations, termed resident-like macrophages, inflammatory macrophages, and Trem2 (triggering receptor expressed on myeloid cells 2) macrophages, in murine atherosclerotic aorta. In their letter, they showed that Trem2 macrophages indeed correspond to the foamy macrophages described by our study. These results prompted us to confirm the corresponding macrophage cell clusters in our scRNA-seq data. As shown in Figure IA in the online-only Data Supplement, the cells in cluster 4 strongly expressed Trem2, suggesting that cluster 4 may correspond to the Trem2 macrophages. We further compared their gene expression signatures with our scRNA-seq data. First, we selected the representative genes (cZ score >3.0) in each macrophage population. As shown in Figure IB in the online-only Data Supplement, the representative genes of inflammatory macrophages were mostly found in clusters 1 and 5 but were not enriched in the foam cell clusters. The representative genes of resident-like macrophages were enriched in clusters 0, 3, 5, and 7, but were not found in the foam cell clusters. Interestingly, Trem2 macrophage genes were mostly found in cluster 4 and were also enriched in the CD45 cell clusters of the total foam cell scRNAseq data. These data clearly suggest that Trem2 macrophages are intimal foamy macrophages, inflammatory macrophages contain intimal and adventitial nonfoamy inflammatory macrophages, and resident-like macrophages are adventitial macrophages. Collectively, the consistency in the scRNA-seq data of aortic macrophages obtained by independent research groups suggests that scRNA-seq analysis is a highly useful method to analyze cell subpopulations and their transcriptomic profiles in experimental animal models of cardiovascular disease. However, there are several issues that need to be considered. The first issue is the cell dissociation method used for scRNA-seq. The immune cell landscape in our scRNA-seq analysis was not consistent with that in the 2 other reports. While we were unable to identify certain immune cell populations, including B cells, natural killer cells, and granulocytes, the 2 previous studies could identify these cells in their scRNA-seq analyses. This discrepancy can be attributed to the different cell dissociation methods used in each study. As discussed in our article, we used a higher concentration of the enzyme mixture, as well as longer digestion times than both these studies. Hence, our cell extraction method is optimized for isolating macrophages that are more firmly attached to the aortic tissue than other immune cells. Therefore, if a specific immune cell population is to be isolated, then the cell dissociation method must be optimized for the isolation of that target cell. The second issue is the anatomic location of target cells. In our study, we removed the adventitial tissue, which contained the majority of aortic macrophages to analyze the transcriptome of intimal foamy and nonfoamy macrophages using bulk-RNA sequencing. scRNA-seq analysis on separately isolated intimal immune and nonimmune cells from atherosclerotic aorta will be useful in further characterizing the cell populations in the primary inflammation site of atherosclerosis. Finally, we fully agree with the comment by Drs Cochain, Saliba, and Zernecke that researchers need to carefully compare and analyze the available transcriptomic datasets to understand the exact characteristics of aortic immune cells in atherosclerosis.