Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Heart Disease

Monocytes are innate immune cells that are defined by three subsets: classical (cMo), intermediate (iMo) and non-classical (nMo). How these subsets become more heterogeneous in phenotype and function in CVD progression remains elusive. We employed single-cell transcriptome and protein analysis to define how human monocytes differ in subjects with moderate to severe CVD. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects, undergoing coronary angiography as part of the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using Gensini Score (GS). We analyzed 487 immune-related genes and 49 surface proteins at the single cell level in PBMC from each subject using the BD-Rhapsody platform. WNN Seurat 4.0 was used for high dimensional analysis. Data was normalized using GLMM to control for covariates (age, sex, diabetes, and statin use). We identified cMo, iMo, nMo, plasmacytoid DC, and classical DC. We found a significant increase only in the frequencies of intermediate (iMo) monocytes in subjects with high CVD (GS>30) compared to subjects with low CVD (GS<6) ( p =0.004). Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r=0.314, p =0.014) and further showed sex-dependent positive correlation in female patients (r=0.663, p =0.0037). Interestingly, cMo, thought to be potently atherogenic, did not correlate with CVD severity. Further analysis identified 3 iMo subsets, distinguished by expression of HLA-DR, CD86, CXCR3, and PD-L1. We found that frequencies of two of them, iMo_CXCR3 + PDL1 lo ( p =0.0003) and iMo_HLA-DR + CD86 int ( p =0.019), were associated with CVD severity. The iMo_CXCR3 + PDL1 lo immunoregulatory subset was positively correlated with GS in both females (r=0.66, p =0.004) and males (r=0.315, p =0.037). However, the iMo_HLA-DR + CD86 int subset was positively correlated only in females (r=0.55, p =0.022), illustrating substantial heterogeneity among monocyte subsets in a sex-dependent manner. In sum, we identified novel sex- and CVD-dependent iMo subsets in subjects with CVD. Future functional analysis of iMo populations should provide a deeper understanding of long-known sex-differences in CVD progression.