Difference of immune cell infiltration between stable and unstable carotid artery atherosclerosis

Abstract Atherosclerotic plaque instability contributes to ischaemic stroke and myocardial infarction. This study is to compare the abundance and difference of immune cell subtypes within unstable atherosclerotic tissues. CIBERSORT was used to speculate the proportions of 22 immune cell types based on a microarray of atherosclerotic carotid artery samples. R software was utilized to illustrate the bar plot, heat map and vioplot. The immune cell landscape in atherosclerosis was diverse, dominated by M2 macrophages, M0 macrophages, resting CD4 memory T cells and CD8 T cells. There was a significant difference in resting CD4 memory T cells (p = 0.032), T cells follicular helper (p = 0.033), M0 (p = 0.047) and M2 macrophages (p = 0.012) between stable and unstable atherosclerotic plaques. Compared with stable atherosclerotic plaques, unstable atherosclerotic plaques had a higher percentage of M2 macrophages. Moreover, correlation analysis indicated that the percentage of naïve CD4 T cells was strongly correlated with that of gamma delta T cells (r = 0.93, p < 0.001), while memory B cells were correlated with plasma cells (r = 0.85, p < 0.001). In summary, our study explored the abundance and difference of specific immune cell subgroups at unstable plaques, which would aid new immunotherapies for atherosclerosis.

identification of vulnerable plaque and prevention of rupture or erosion is of great significance. 7 Immune cell infiltration within the vessel wall is closely associated with the initiation and progression of atherosclerosis. 8 Monocyte/macrophage system participated in engulfing oxidizedlow-density lipoprotein (ox-LDL), secreting cytokines and chemokines and interacting with other immune cells. 9 The polarization of macrophages into M1 and M2 exerts pro-inflammatory and antiinflammatory function respectively. 10 T cells were accounted for ~40% of immune cells in human atherosclerotic lesions. Among them, Th1 cells and NK cells secrete pro-inflammatory factors, which disorganize the collagen fibres and promote plaques transition to a vulnerable phenotype, while Treg cells produce transforming growth factorβ and inhibit Th1 and Th17 cell expansion. 11,12 Th17 cell is another type of T cell that promotes the formation of thick collagen fibres, contributing to the stability of plaque. 13 However, the role of Th2, Th9 and CD8+T cells in atherosclerosis remains unclear. 14 In regard to B cells, B1 cells tend to be pro-atherogenic and B2 exert an anti-atherogenic role. 15 Immune cells participate in the pathogenesis, development and regression of atherosclerosis, so modulating cellular or cytokine-based immune response could be a novel therapeutic target for treatment of atherosclerosis. 16 Immune infiltration analysis is a novel tool to estimate the abundances of immune cell subtypes in a mixed cell population based on gene expression data. Profile of immune cell infiltration in atherosclerosis has been previously elucidated. 8,17 However, it is still unclear about the alteration of immune cell proportion between stable and unstable plaques. In this study, we combined the microarray profiles of stable and unstable atherosclerotic carotid artery tissues from Gene Expression Omnibus (GEO) data sets and applied CIBERSORT method to estimate the abundance of 22 immune cell types within tissues. Further, we discussed their effects on the plaque instability of atherosclerosis.

| Data acquisition
The microarray data were downloaded from GEO database (http:// www.ncbi.nlm.nih.gov/geo/). The GSE28829 data set contained 16 advanced atherosclerotic plaque and 13 early atherosclerotic plaque from human carotid arteries. 18 The data set was based on GPL570 platform ([HG-U133_Plus_2], Affymetrix Human Genome U133 Plus 2.0 Array).

| Evaluation of atherosclerosis-infiltrating immune cells
CIBERSORT was used to calculate the fractions of infiltrating immune cells within plaque. CIBERSORT is an analytical tool from the Alizadeh Lab, which estimated the abundances of member cell types in a mixed cell population, using gene expression data. 19 Briefly, it uses a gene expression signature of 547 marker genes called LM22

| Data normalization and visualization
All analyses were performed using R version 3.6.1. Principal component analysis (PCA) was performed using the ggplot2 package. The

| DISCUSS ION
Atherosclerosis is a chronic inflammatory vascular disease. Multiple types of immune cells participate in the pathological progression across the whole stages. In our study, M0 and M2 macrophages, The correlation of immune cell types between stable and unstable atherosclerotic tissues. Red represented positive correlation, and blue represented negative correlation. The p value of correlation was shown in Datasheet S1  22 In atherosclerotic lesions of Ldlr-deficient mice, M1 and M2 subsets accounted for 40% and 20% respectively. 23 Previous report showed that symptomatic plaques displayed fewer proinflammatory genes and more percentage of macrophages with reparative M2 phenotype, 8 which was in consistent with our results.

Increased infiltration of M2 macrophages was atheroprotective and
drove atherosclerotic inflammation resolution and plaque regression, 24 which could be a protective increasement in unstable atherosclerotic plaque.
A strong correlation was also noteworthy between the per- higher average systolic blood pressure. 35 Gut gamma delta T cells were reported to regulate metabolism and accelerate cardiovascular disease. 36 The role of gamma delta T cells in atherosclerosis was scared and deserved to be further investigations. As atherosclerosis is a complicated, multifactorial pathophysiological process, immune cells in the plaque also interacted and exerted a synergetic role which may explain the correlation between immune cells. 37,38 However, whether changes of these correlations may affect the stability remained unclear.
Single-cell proteomic and transcriptomic analyses have been used for uncovering the immune cell landscape in stable and unstable plaques of carotid artery. 8 Compared with it, we focussed unbiasedly on more immune cell subtypes in a mixed tissue and elucidated the abundance and the correlation between different cells.
Our findings also complemented their results. However, some limitations need caution in our study. Firstly, CIBERSORT is insufficient to uncover the whole subtypes in atherosclerosis, and other software could be used, such as EPIC, TIMER and ImmuCellAI. Secondly, this was a bioinformatic analysis, and the predictions need to be confirmed by functional validation in vitro and in vivo.

| CON CLUS ION
In summary, utilizing CIBERSORT method, we uncovered the abundance and difference of immune cell types within unstable atherosclerotic carotid artery tissues. Our finding may provide some clues for future immunotherapy in atherosclerosis.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The microarray used in our study has been deposited in GEO (GSE28829).