Rationale:: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they have been defined by the expression of a restricted number of markers. Objective:: We have applied single-cell RNA sequencing as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Method and Results:: We performed single-cell RNA sequencing of total aortic CD45 ⁺ cells extracted from the nondiseased (chow fed) and atherosclerotic (11 weeks of high-fat diet) aorta of low-density lipoprotein receptor–deficient ( Ldlr ^−/− ) mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortas, whereas monocytes, monocyte-derived dendritic cells, and 2 populations of macrophages were almost exclusively detectable in atherosclerotic aortas, comprising inflammatory macrophages showing enrichment in Il1b and previously undescribed TREM2 ^hi (triggered receptor expressed on myeloid cells 2) macrophages showing enrichment in Trem2 . Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these 3 macrophage subsets and monocyte-derived dendritic cells and uncovered putative functions of each cell type. Notably, TREM2 ^hi macrophages seemed to be endowed with specialized functions in lipid metabolism and catabolism and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe ^−/− aortas, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions:: These data unprecedentedly uncovered the transcriptional landscape and phenotypic heterogeneity of aortic macrophages and monocyte-derived dendritic cells in atherosclerotic and identified previously unrecognized macrophage populations and their gene expression signature, suggesting specialized functions. Our findings will open up novel opportunities to explore distinct myeloid cell populations and their functions in atherosclerosis.

Rationale: After myocardial infarction, neutrophils rapidly and massively infiltrate the heart, where they promote both tissue healing and damage. Objective: To characterize the dynamics of circulating and cardiac neutrophil diversity after infarction. Methods and results: We employed single-cell transcriptomics combined with cell surface epitope detection by sequencing to investigate temporal neutrophil diversity in the blood and heart after murine myocardial infarction. At day 1, 3, and 5 after infarction, cardiac Ly6G ⁺ (lymphocyte antigen 6G) neutrophils could be delineated into 6 distinct clusters with specific time-dependent patterning and proportions. At day 1, neutrophils were characterized by a gene expression profile proximal to bone marrow neutrophils ( Cd177 , Lcn2 , Fpr1 ), and putative activity of transcriptional regulators involved in hypoxic response ( Hif1a ) and emergency granulopoiesis ( Cebpb ). At 3 and 5 days, 2 major subsets of Siglecf ^hi (enriched for eg, Icam1 and Tnf ) and Siglecf ^low ( Slpi, Ifitm1 ) neutrophils were found. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) analysis in blood and heart revealed that while circulating neutrophils undergo a process of aging characterized by loss of surface CD62L and upregulation of Cxcr4 , heart infiltrating neutrophils acquired a unique SiglecF ^hi signature. SiglecF ^hi neutrophils were absent from the bone marrow and spleen, indicating local acquisition of the SiglecF ^hi signature. Reducing the influx of blood neutrophils by anti-Ly6G treatment increased proportions of cardiac SiglecF ^hi neutrophils, suggesting accumulation of locally aged neutrophils. Computational analysis of ligand/receptor interactions revealed putative pathways mediating neutrophil to macrophage communication in the myocardium. Finally, SiglecF ^hi neutrophils were also found in atherosclerotic vessels, revealing that they arise across distinct contexts of cardiovascular inflammation. Conclusions: Altogether, our data provide a time-resolved census of neutrophil diversity and gene expression dynamics in the mouse blood and ischemic heart at the single-cell level, and reveal a process of local tissue specification of neutrophils in the ischemic heart characterized by the acquisition of a SiglecF ^hi signature.

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.

Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host¹. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility^2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding⁴. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.

Common stemness regulators of embryonic and cancer stem cells