Project description:We used the high dimensionality of mass cytometry together with the FlowSOM clustering algorithm to accurately identify and define monocyte subsets in blood of healthy human subjects and those with coronary artery disease (CAD). To study the behavior and functionality of the newly defined monocyte subsets, we performed RNA sequencing, transwell migration, and efferocytosis assays. Here, we identify 8 human monocyte subsets based on their surface marker phenotype. We found that 3 of these subsets fall within the CD16+ nonclassical monocyte population and 4 subsets belong to the CD14+ classical monocytes, illustrating significant monocyte heterogeneity in humans. As nonclassical monocytes are important in modulating atherosclerosis in mice, we studied the functions of our 3 newly identified nonclassical monocytes in subjects with CAD. We found a marked expansion of a Slan+CXCR6+ nonclassical monocyte subset in CAD subjects, which was positively correlated with CAD severity. This nonclassical subset can migrate towards CXCL16 and shows an increased efferocytosis capacity, indicating it may play an atheroprotective role.
Project description:Deep phenotyping by mass cytometry and single cell RNA-sequencing reveals LYN regulated signaling profiles underlying monocyte subset heterogeneity and lifespan
Project description:Regulated chromatin states control genome accessibility and thus influence gene expression. Here we report an analysis pipeline termed ATAC-mass that capitalizes on isotopic labeling to detect the accessible genome by multiplexed ion beam imaging (MIBI) and mass cytometry. With MIBI the accessible genome can be visualized at approximately 100-nm resolution simultaneously with metabolic labeling to enable multi-parameter three-dimensional imaging of nuclear features. Extension of this approach to non-spatial mass cytometry enabled the simultaneous measurement of multiple parameters and total genome accessibility in millions of individual cells. We used ATAC-mass to analyze natural killer cells after stimulation with interleukin (IL)-12 or IL-18 -- demonstrating that IL-18 treatment leads to increased total genome accessibility. Analysis of the spatial organization of open chromatin suggest that IL-12 and IL-18 both induce an increase in chromatin accessibility in noncompacted DNA regions. Deep sequencing of the genomic distribution of open chromatin revealed that IL-18 increased the accessibility of quiescent enhancers whereas genomic loci that become more accessible by IL-12 stimulation are mainly localized in the active promoter regions. This integration of epigenomics, proteomics and high-resolution imaging at the single-cell level provides a tool that can enhance our appreciation of the molecular mechanisms underlying gene regulation.
Project description:New techniques for single-cell analysis have led to insights into hematopoiesis and the immune system, but the ability of these techniques to cross-validate and reproducibly identify the biological variation in diverse human samples is currently unproven. We therefore performed a comprehensive assessment of human bone marrow cells using both single-cell RNA sequencing and multiparameter flow cytometry from twenty healthy adult human donors across a broad age range. These data characterize variation between healthy donors as well as age-associated changes in cell population frequencies. Direct comparison of techniques revealed discrepancy in the quantification of T lymphocyte and natural killer cell populations. Orthogonal validation of immunophenotyping using mass cytometry demonstrated good correlation with flow cytometry. Technical replicates using single-cell RNA sequencing matched robustly, while biological replicates showed variation. Given the increasing use of single-cell technologies in translational research, this resource serves as an important reference dataset and highlights opportunities for further refinement. [Funding source] Project Number: 1ZIAHL006163-05 Contact PI / Project Leader: HOURIGAN, CHRISTOPHER Title: DETECTION, PREVENTION AND TREATMENT OF ACUTE MYELOID LEUKEMIA (AML) RELAPSE. Awardee Organization: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
Project description:Rationale: Monocytes are key effectors of the mononuclear phagocyte system (MPS), playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into two major subsets termed conventional (cMo) and patrolling (pMo) monocytes but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology, however LYN’s role in regulating specific monocyte subset homeostasis has not been investigated. Objective: We sought to comprehensively profile monocytes in order to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis. Methods and Results: CyTOF analysis of monocyte subsets and signaling pathway activation patterns in cMos and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN’s role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn-deficient pMos. Furthermore, single cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn-/- monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr-/- mice from high-fat diet induced atherosclerosis. Conclusions: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.
Project description:Kawasaki disease (KD) is characterized by a disorder of immune response, and its etiology remains unknown. Monocyte is an important member of body's innate immune system, however its role in KD is still elusive due to its ambiguous heterogeneity and complex functions. Here, scRNA-seq was performed to reveal monocytes heterogeneity in healthy and KD infants. Circulating monocytes were separated from peripheral blood and scRNA-seq was used to transcriptionally profile the monocytes in both healthy and KD infants. Four monocyte subsets are identified in infants, in which three clusters are mainly CD14+CD16- monocytes and one cluster is mainly CD14-CD16+ monocytes. The four monocyte subsets possess different biological functions and represent a relatively linear differentiation. CD14+ monocyte subsets in KD are distinct from that of healthy infants, including one subset expressing FOLR3, S100A12 and IL1R2 and the other expressing MT-TN specifically. Moreover, the CD14+ monocyte subsets in KD are poorly differentiated, and their functions mainly involve neutrophil activation. In conclusion, a relatively comprehensive map of circulating monocyte subsets was plotted for the first time in healthy infants. CD14+ monocyte subsets that are distinct from healthy infants were revealed in KD, which may serve as a target for KD treatment in the future.