Rationale: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques have not been fully assessed. Objective: Using single-cell transcriptomics and chromatin accessibility we gained a better understanding of the pathophysiology underlying human atherosclerosis. Methods and Results: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4 + and CD8 + T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included two populations of pro-inflammatory macrophages showing IL1B or TNF expression as well as a foam cell-like population expressing TREM2 and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public GWAS data were particularly enriched in lesional macrophages, endothelial and smooth muscle cells. Conclusions: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

Rationale: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cis-regulatory elements (CREs) but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation. Objective: Single nucleus chromatin accessibility profiling of the human atherosclerotic lesions was used to investigate cell type-specific patterns of CREs, to understand transcription factors establishing cell identity and to interpret CAD-relevant, non-coding genetic variation. Methods and Results: We used single nucleus ATAC-seq to generate DNA accessibility maps in > 7,000 cells derived from human atherosclerotic lesions. We identified five major lesional cell types including endothelial cells, smooth muscle cells, monocyte/macrophages, NK/T-cells and B-cells and further investigated subtype characteristics of macrophages and smooth muscle cells transitioning into fibromyocytes. We demonstrated that CAD associated genetic variants are particularly enriched in endothelial and smooth muscle cell-specific open chromatin. Using single cell co-accessibility and cis-eQTL information, we prioritized putative target genes and candidate regulatory elements for ~30% of all known CAD loci. Finally, we performed genome-wide experimental fine-mapping of the CAD GWAS variants using epigenetic QTL analysis in primary human aortic endothelial cells and STARR-Seq massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal SNP(s) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci. Conclusions: These findings highlight the potential of applying snATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by non-coding GWAS variants.

The ability of intracellular pathogens to manipulate host-cell viability is critical to successful infection. Some pathogens promote host-cell survival to protect their replicative niche, whereas others trigger host-cell death to facilitate release and dissemination of the pathogen after intracellular replication has occurred. We previously showed that the intracellular fungal pathogen Histoplasma capsulatum (Hc) uses the secreted protein Cbp1 to actively induce apoptosis in macrophages; interestingly, cbp1 mutant strains are unable to kill macrophages and display severely reduced virulence in the mouse model of Hc infection. To elucidate the mechanism of Cbp1-induced host-cell death, we performed a comprehensive alanine scanning mutagenesis and identified all amino acid residues that are required for Cbp1 to trigger macrophage lysis. Here we demonstrate that Hc strains expressing lytic CBP1 alleles activate the integrated stress response (ISR) in infected macrophages, as indicated by an increase in eIF2α phosphorylation as well as induction of the transcription factor CHOP and the pseudokinase Tribbles 3 (TRIB3). In contrast, strains bearing a non-lytic allele of CBP1 fail to activate the ISR, whereas a partially lytic CBP1 allele triggers intermediate levels of activation. We further show that macrophages deficient for CHOP or TRIB3 are partially resistant to lysis during Hc infection, indicating that the ISR is critical for susceptibility to Hc-mediated cell death. Moreover, we show that CHOP-dependent macrophage lysis is critical for efficient spread of Hc infection to other macrophages. Notably, CHOP knockout mice display reduced macrophage apoptosis and diminished fungal burden and are markedly resistant to Hc infection. Together, these data indicate that Cbp1 is required for Hc to induce the ISR and mediate a CHOP-dependent virulence pathway in the host.

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