Function of Macrophages in Disease: Current Understanding on Molecular Mechanisms

Tissue-resident macrophages (TRMs) are heterogeneous populations originating either from monocytes or embryonic progenitors, and distribute in lymphoid and non-lymphoid tissues. TRMs play diverse roles in many physiological processes, including metabolic function, clearance of cellular debris, and tissue remodeling and defense. Macrophages can be polarized to different functional phenotypes depending on their origin and tissue microenvironment. Specific macrophage subpopulations are associated with disease progression. In studies of fate-mapping and single-cell RNA sequencing methodologies, several critical molecules have been identified to induce the change of macrophage function. These molecules are potential markers for diagnosis and selective targets for novel macrophage-mediated treatment. In this review, we discuss some of the recent findings regarding less-known molecules and new functions of well-known molecules. Understanding the mechanisms of these molecules in macrophages has the potential to yield new macrophage-mediated treatments or diagnostic approaches to disease.

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Single-Cell RNA Sequencing to Disentangle the Blood System

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314654 ◽

2021 ◽

Vol 41 (3) ◽

pp. 1012-1018

Author(s):

Jean Acosta ◽

Daniel Ssozi ◽

Peter van Galen

Keyword(s):

Blood Cell ◽

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Single Cells ◽

Cell Types ◽

Blood System ◽

Differentiated Cells ◽

Tissue Organization ◽

Single Cell Rna Sequencing

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells. Graphic Abstract: A graphic abstract is available for this article.

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Review of Single-Cell RNA Sequencing in the Heart

International Journal of Molecular Sciences ◽

10.3390/ijms21218345 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8345

Author(s):

Shintaro Yamada ◽

Seitaro Nomura

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Clinical Medicine ◽

Basic Research ◽

Cellular Level ◽

Cardiovascular Development ◽

Biological Mechanisms ◽

Technological Advances ◽

Single Cell Rna Sequencing

Single-cell RNA sequencing (scRNA-seq) technology is a powerful, rapidly developing tool for characterizing individual cells and elucidating biological mechanisms at the cellular level. Cardiovascular disease is one of the major causes of death worldwide and its precise pathology remains unclear. scRNA-seq has provided many novel insights into both healthy and pathological hearts. In this review, we summarize the various scRNA-seq platforms and describe the molecular mechanisms of cardiovascular development and disease revealed by scRNA-seq analysis. We then describe the latest technological advances in scRNA-seq. Finally, we discuss how to translate basic research into clinical medicine using scRNA-seq technology.

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#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

Journal of the Pediatric Infectious Diseases Society ◽

10.1093/jpids/piaa170.042 ◽

2021 ◽

Vol 10 (Supplement_1) ◽

pp. S14-S14

Author(s):

K E Ocwieja ◽

T K Hughes ◽

J M Antonucci ◽

A L Richards ◽

A C Stanton ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Zika Virus ◽

Molecular Mechanisms ◽

Sequencing Analysis ◽

Human Stem Cell ◽

Zikv Infection ◽

Single Cell Sequencing ◽

Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

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Control of neurogenic competence in mammalian hypothalamic tanycytes

Science Advances ◽

10.1126/sciadv.abg3777 ◽

2021 ◽

Vol 7 (22) ◽

pp. eabg3777

Author(s):

Sooyeon Yoo ◽

Juhyun Kim ◽

Pin Lyu ◽

Thanh V. Hoang ◽

Alex Ma ◽

...

Keyword(s):

Single Cell ◽

Molecular Mechanisms ◽

Radial Glial Cells ◽

Neuronal Progenitors ◽

Physiological Processes ◽

Internal States ◽

Nuclear Factor I ◽

Radial Glial ◽

Accessible Chromatin ◽

Deficient Mice

Hypothalamic tanycytes, radial glial cells that share many features with neuronal progenitors, can generate small numbers of neurons in the postnatal hypothalamus, but the identity of these neurons and the molecular mechanisms that control tanycyte-derived neurogenesis are unknown. In this study, we show that tanycyte-specific disruption of the NFI family of transcription factors (Nfia/b/x) robustly stimulates tanycyte proliferation and tanycyte-derived neurogenesis. Single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analysis reveals that NFI (nuclear factor I) factors repress Sonic hedgehog (Shh) and Wnt signaling in tanycytes and modulation of these pathways blocks proliferation and tanycyte-derived neurogenesis in Nfia/b/x-deficient mice. Nfia/b/x-deficient tanycytes give rise to multiple mediobasal hypothalamic neuronal subtypes that can mature, fire action potentials, receive synaptic inputs, and selectively respond to changes in internal states. These findings identify molecular mechanisms that control tanycyte-derived neurogenesis, which can potentially be targeted to selectively remodel the hypothalamic neural circuitry that controls homeostatic physiological processes.

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Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914423117 ◽

2020 ◽

Vol 117 (5) ◽

pp. 2613-2621 ◽

Cited By ~ 5

Author(s):

Aurélien Vigneron ◽

Michelle B. O’Neill ◽

Brian L. Weiss ◽

Amy F. Savage ◽

Olivia C. Campbell ◽

...

Keyword(s):

Single Cell ◽

Salivary Glands ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Developmental Process ◽

Surface Glycoprotein ◽

Mammalian Host ◽

Transmission Blocking ◽

African Trypanosomes ◽

Single Cell Rna Sequencing

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly’s salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

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Single-cell RNA sequencing of murine islets shows high cellular complexity at all stages of autoimmune diabetes

Journal of Experimental Medicine ◽

10.1084/jem.20192362 ◽

2020 ◽

Vol 217 (6) ◽

Cited By ~ 2

Author(s):

Pavel N. Zakharov ◽

Hao Hu ◽

Xiaoxiao Wan ◽

Emil R. Unanue

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Immune Cell ◽

Autoimmune Diabetes ◽

Diabetic Mouse ◽

Cell Populations ◽

Target Organs ◽

Distinct Cell ◽

Single Cell Rna Sequencing ◽

Macrophage Subpopulations

Tissue-specific autoimmune diseases are driven by activation of diverse immune cells in the target organs. However, the molecular signatures of immune cell populations over time in an autoimmune process remain poorly defined. Using single-cell RNA sequencing, we performed an unbiased examination of diverse islet-infiltrating cells during autoimmune diabetes in the nonobese diabetic mouse. The data revealed a landscape of transcriptional heterogeneity across the lymphoid and myeloid compartments. Memory CD4 and cytotoxic CD8 T cells appeared early in islets, accompanied by regulatory cells with distinct phenotypes. Surprisingly, we observed a dramatic remodeling in the islet microenvironment, in which the resident macrophages underwent a stepwise activation program. This process resulted in polarization of the macrophage subpopulations into a terminal proinflammatory state. This study provides a single-cell atlas defining the staging of autoimmune diabetes and reveals that diabetic autoimmunity is driven by transcriptionally distinct cell populations specialized in divergent biological functions.

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Harnessing Single-Cell RNA Sequencing to Better Understand How Diseased Cells Behave the Way They Do in Cardiovascular Disease

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314776 ◽

2020 ◽

Author(s):

Farwah Iqbal ◽

Adrien Lupieri ◽

Masanori Aikawa ◽

Elena Aikawa

Keyword(s):

Cardiovascular Disease ◽

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Cell Types ◽

Specific Cell ◽

Calcific Aortic Valve Disease ◽

Cardiovascular Tissues ◽

Single Cell Rna Sequencing ◽

Cell Phenotypes

The transition of healthy arteries and cardiac valves into dense, cell-rich, calcified, and fibrotic tissues is driven by a complex interplay of both cellular and molecular mechanisms. Specific cell types in these cardiovascular tissues become activated following the exposure to systemic stimuli including circulating lipoproteins or inflammatory mediators. This activation induces multiple cascades of events where changes in cell phenotypes and activation of certain receptors may trigger multiple pathways and specific alterations to the transcriptome. Modifications to the transcriptome and proteome can give rise to pathological cell phenotypes and trigger mechanisms that exacerbate inflammation, proliferation, calcification, and recruitment of resident or distant cells. Accumulating evidence suggests that each cell type involved in vascular and valvular diseases is heterogeneous. Single-cell RNA sequencing is a transforming medical research tool that enables the profiling of the unique fingerprints at single-cell levels. Its applications have allowed the construction of cell atlases including the mammalian heart and tissue vasculature and the discovery of new cell types implicated in cardiovascular disease. Recent advances in single-cell RNA sequencing have facilitated the identification of novel resident cell populations that become activated during disease and has allowed tracing the transition of healthy cells into pathological phenotypes. Furthermore, single-cell RNA sequencing has permitted the characterization of heterogeneous cell subpopulations with unique genetic profiles in healthy and pathological cardiovascular tissues. In this review, we highlight the latest groundbreaking research that has improved our understanding of the pathological mechanisms of atherosclerosis and future directions for calcific aortic valve disease.

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Transcriptional evaluation of the ductus arteriosus at the single-cell level uncovers a requirement for vimentin for complete closure

10.1101/2021.10.30.466605 ◽

2021 ◽

Author(s):

Jocelynda Salvador ◽

Gloria E Hernandez ◽

Feiyang Ma ◽

Cyrus W Abrahamson ◽

Robert D Goldman ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Vascular Remodeling ◽

Molecular Mechanisms ◽

Heart Defects ◽

Ductus Arteriosus ◽

Fibroblast Cell ◽

Management Options ◽

Complete Closure ◽

Single Cell Rna Sequencing

OBJECTIVE: Failure to close the ductus arteriosus immediately post-birth, patent ductus arteriosus (PDA), accounts for up to 10% of all congenital heart defects. Despite significant advances in PDA management options, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. APPROACH AND RESULTS: As anticipated, single-cell RNA sequencing on the ductus arteriosus in mouse embryos at E18.5, P0.5, and P5, revealed broad transcriptional alterations in the endothelial, smooth muscle, and fibroblast cell compartments. Making use of these data sets, vimentin emerged as an interesting candidate for further investigation. Subsequent studies demonstrated that, in fact, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus, as per presence of a functional lumen. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus postnatally in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus potentially through regulation of the Notch signaling pathway.

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Single-cell RNA sequencing reveals a role for reactive oxygen species and peroxiredoxins in fatty-acid-induced rat β-cell proliferation

10.1101/2021.12.22.473904 ◽

2021 ◽

Author(s):

Alexis Vivoli ◽

Julien Ghislain ◽

Ali Filali-Mouhim ◽

Zuraya Elisa Angeles ◽

Anne-Laure Castell ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Proliferation ◽

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Reactive Oxygen ◽

Oxygen Species ◽

Β Cells ◽

Β Cell ◽

Single Cell Rna Sequencing

The functional mass of insulin-secreting pancreatic β cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing β cells. To decipher the underlying molecular mechanisms, we assessed β-cell proliferation in isolated rat islets exposed to glucose and oleate or palmitate for 48 h and analyzed the transcriptional response by single-cell RNA sequencing. Unsupervised clustering of pooled βcells identified subpopulations, including proliferating β cells. β-cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved energy metabolism and mitochondrial activity. Comparison of proliferating vs. non-proliferating β cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, the antioxidant N-acetyl cysteine and the peroxiredoxin inhibitor Conoidin A both blocked oleate-induced β-cell proliferation. Our data reveal a key role for ROS signaling in β-cell proliferation in response to nutrients.

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PseudotimeDE: inference of differential gene expression along cell pseudotime with well-calibrated p-values from single-cell RNA sequencing data

10.1101/2020.11.17.387779 ◽

2020 ◽

Cited By ~ 1

Author(s):

Dongyuan Song ◽

Jingyi Jessica Li

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Real Data ◽

Sequencing Data ◽

Single Cell Rna Sequencing ◽

Ill Posed ◽

Differential Gene ◽

Cell Trajectory ◽

Downstream Analysis

AbstractIn the investigation of molecular mechanisms underlying cell state changes, a crucial analysis is to identify differentially expressed (DE) genes along a continuous cell trajectory, which can be estimated by pseudotime inference from single-cell RNA-sequencing (scRNA-seq) data. However, existing methods that identify DE genes based on inferred pseudotime do not account for the uncertainty in pseudotime inference. Also, they either have ill-posed p-values that hinder the control of false discovery rate (FDR) or have restrictive models that reduce the power of DE gene identification. To overcome these drawbacks, we propose PseudotimeDE, a robust method that accounts for the uncertainty in pseudotime inference and thus identifies DE genes along cell pseudotime with well-calibrated p-values. PseudotimeDE is flexible in allowing users to specify the pseudotime inference method and to choose the appropriate model for scRNA-seq data. Comprehensive simulations and real-data applications verify that PseudotimeDE provides well-calibrated p-values essential for controlling FDR and downstream analysis and that PseudotimeDE is more powerful than existing methods to identify DE genes.

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