High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

BACKGROUND: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. METHODS: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. RESULTS: We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. CONCLUSIONS: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

Suppression of IL-1ß promotes beneficial accumulation of fibroblast-like cells in atherosclerotic plaques in clonal hematopoiesis.

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1ß in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1ß is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr-/- mice modeling Jak2VF or Tet2 CH treated with IL-1ß antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1ß antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF during plaque regression promoted fibroblast accumulation and fibrous cap thickening. Our studies suggest that suppression of inflammasomes promotes plaque stabilization by recruiting fibroblast-like cells to the fibrous cap.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

Background: Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, we have limited understanding of the comprehensive transcriptional and phenotypical landscape of the cells within these lesions. Methods: To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing (scRNA-seq) to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. Results: We identified 25 distinct cell populations each having a unique multi-omic signature, including macrophages, T cells, NK cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Within the macrophage populations, we identified 2 proinflammatory subsets that were enriched in IL1B or C1Q expression, 2 distinct TREM2 positive foam cell subsets, one of which also expressed inflammatory genes, as well as subpopulations displaying a proliferative gene expression signature and one expressing SMC-specific genes and upregulation of fibrotic pathways. An in-depth characterization uncovered several subsets of SMCs and fibroblasts, including a SMC-derived foam cell. We localized this foamy SMC to the deep intima of coronary atherosclerotic lesions. Using CITE-seq data, we also developed the first flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Last, we found that the proportion of efferocytotic macrophages, classically activated endothelial cells, contractile and modulated SMC-derived cell types were reduced, and inflammatory SMCs were enriched in plaques of clinically symptomatic vs. asymptomatic patients. Conclusions: Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. This facilitates both the mapping of cardiovascular disease susceptibility loci to specific cell types as well as the identification of novel molecular and cellular therapeutic targets for treatment of the disease.

Atherosclerosis is a smooth muscle cell-driven tumor-like disease.

Atherosclerosis, the leading cause of cardiovascular disease, is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), and endothelial cells. Multiple lines of evidence have suggested that SMC "phenotypic switching" plays a central role in atherosclerosis development and complications. Yet, SMC roles and mechanisms underlying the disease pathogenesis are poorly understood. Here, employing SMC lineage tracing mice, comprehensive molecular, cellular, histological, and computational profiling, coupled to genetic and pharmacological studies, we reveal that atherosclerosis, in terms of SMC behaviors, share extensive commonalities with tumors. SMC-derived cells in the disease show multiple characteristics of tumor cell biology, including genomic instability, replicative immortality, malignant proliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. SMC-specific expression of oncogenic KrasG12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we present a proof of concept showing that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models. Our work provides systematic evidence that atherosclerosis is a tumor-like disease, deepening the understanding of its pathogenesis and opening prospects for novel precision molecular strategies to prevent and treat atherosclerotic cardiovascular disease.

Effect of mechanical unloading on genome-wide DNA methylation profile of the failing human heart.

Heart failure (HF) is characterized by global alterations in myocardial DNA methylation, yet little is known about the epigenetic regulation of the noncoding genome and potential reversibility of DNA methylation with left ventricular assist device (LVAD) therapy. Genome-wide mapping of myocardial DNA methylation in 36 patients with HF at LVAD implantation, 8 patients at LVAD explantation, and 7 nonfailing (NF) donors using a high-density bead array platform identified 2,079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in nonischemic cardiomyopathy (NICM). LVAD support resulted in normalization of 3.2% of HF-associated DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FBXO16, EFCAB13, and AKAP13) or hypermethylated and downregulated (TBX3) in HF. A potentially novel cardiac-specific super-enhancer long noncoding RNA (lncRNA) (LINC00881) is hypermethylated and downregulated in human HF. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown reduces peak calcium amplitude in the beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These data suggest that HF-associated changes in myocardial DNA methylation within coding and noncoding genomes are minimally reversible with mechanical unloading. Epigenetic reprogramming strategies may be necessary to achieve sustained clinical recovery from heart failure.

Human Macrophage Long Intergenic Noncoding RNA, SIMALR, Suppresses Inflammatory Macrophage Apoptosis via NTN1 (Netrin-1).

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as novel regulators of macrophage biology and inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human lncRNAs that are not conserved in rodents, are limited. METHODS: Through RNA-sequencing of human monocyte-derived macrophages, we identified suppressor of inflammatory macrophage apoptosis lncRNA (SIMALR). Lipopolysaccharide/IFNγ (interferon γ) stimulated human macrophages were treated with SIMALR antisense oligonucleotides and subjected to RNA-sequencing to investigate the function of SIMALR. Western blots, luciferase assay, and RNA immunoprecipitation were performed to validate function and potential mechanism of SIMALR. RNAscope was performed to identify SIMALR expression in human carotid atherosclerotic plaques. RESULTS: RNA-sequencing of human monocyte-derived macrophages identified SIMALR, a human macrophage-specific long intergenic noncoding RNA that is highly induced in lipopolysaccharide/IFNγ-stimulated macrophages. SIMALR knockdown in lipopolysaccharide/IFNγ stimulated THP1 human macrophages induced apoptosis of inflammatory macrophages, as shown by increased protein expression of cleaved PARP (poly[ADP-ribose] polymerase), caspase 9, caspase 3, and Annexin V+. RNA-sequencing of control versus SIMALR knockdown in lipopolysaccharide/IFNγ-stimulated macrophages showed Netrin-1 (NTN1) to be significantly decreased upon SIMALR knockdown. We confirmed that NTN1 knockdown in lipopolysaccharide/IFNγ-stimulated macrophages induced apoptosis. The SIMALR knockdown-induced apoptotic phenotype was rescued by adding recombinant NTN1. NTN1 promoter-luciferase reporter activity was increased in HEK293T (human embryonic kidney 293) cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α (hypoxia-inducible factor 1 subunit alpha), and our studies suggest that SIMALR may interact with HIF1α to regulate NTN1 transcription, thereby regulating macrophages apoptosis. SIMALR was found to be expressed in macrophages in human carotid atherosclerotic plaques of symptomatic patients. CONCLUSIONS: SIMALR is a nonconserved, human macrophage lncRNA expressed in atherosclerosis that suppresses macrophage apoptosis. SIMALR partners with HIF1α (hypoxia-inducible factor 1 subunit alpha) to regulate NTN1, which is a known macrophage survival factor. This work illustrates the importance of interrogating the functions of human lncRNAs and exploring their translational and therapeutic potential in human atherosclerosis.

A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis.

Phagocytic clearance of dying cells, termed efferocytosis, is essential for maintaining tissue homeostasis, yet our understanding of efferocytosis regulation remains incomplete. Here we perform a FACS-based, genome-wide CRISPR knockout screen in primary mouse macrophages to search for novel regulators of efferocytosis. The results show that Wdfy3 knockout in macrophages specifically impairs uptake, but not binding, of apoptotic cells due to defective actin disassembly. Additionally, WDFY3 interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Mechanistically, while the C-terminus of WDFY3 is sufficient to rescue the impaired degradation induced by Wdfy3 knockout, full-length WDFY3 is required to reconstitute the uptake of apoptotic cells. Finally, WDFY3 is also required for efficient efferocytosis in vivo in mice and in vitro in primary human macrophages. This work thus expands our knowledge of the mechanisms of macrophage efferocytosis, as well as supports genome-wide CRISPR screen as a platform for interrogating complex functional phenotypes in primary macrophages.

Thermal Stability, Mechanical Properties and Ceramization Mechanism of Epoxy Resin/Kaolin/Quartz Fiber Ceramifiable Composites.

The application of epoxy resins in high temperature and thermal protection fields is limited due to their low decomposition temperature and low carbon residual rate. In this paper, epoxy resin (EP)/quartz fiber (QF) ceramifiable composites were prepared using a prepreg-molding process. The thermal stability, phase change and mechanical properties after high-temperature static ablation and ceramization mechanism of EP/QF ceramifiable composites were investigated. The addition of glass frits and kaolinite ceramic filler dramatically increases the thermal stability of the composites, according to thermogravimetric (TG) studies. The composite has a maximum residual weight of 61.08%. The X-ray diffraction (XRD) results show that the mullite ceramic phase is generated, and a strong quartz diffraction peak appears at 1000 °C. The scanning electron microscope (SEM) and element distribution analyses reveal that the ceramic phase generated inside the material, when the temperature reaches 1000 °C, effectively fills the voids in composites. The composites have a bending strength of 175.37 MPa at room temperature and retain a maximum bending strength of 12.89 MPa after 1000 °C treatment.

Cis-regulated expression of non-conserved lincRNAs associates with cardiometabolic related traits.

Many complex disease risk loci map to intergenic regions containing long intergenic noncoding RNAs (lincRNAs). The majority of these is not conserved outside humans, raising the question whether genetically regulated expression of non-conserved and conserved lincRNAs has similar rates of association with complex traits. Here we leveraged data from the Genotype-Tissue Expression (GTEx) project and multiple public genome-wide association study (GWAS) resources. Using an established transcriptome-wide association study (TWAS) tool, FUSION, we interrogated the associations between cis-regulated expression of lincRNAs and multiple cardiometabolic traits. We found that cis-regulated expression of non-conserved lincRNAs had a strikingly similar trend of association with complex cardiometabolic traits as conserved lincRNAs. This finding challenges the conventional notion of conservation that has led to prioritization of conserved loci for functional studies and calls attention to the need to develop comprehensive strategies to study the large number of non-conserved human lincRNAs that may contribute to human disease.

Adipocyte-specific tribbles pseudokinase 1 regulates plasma adiponectin and plasma lipids in mice.

OBJECTIVE: Multiple genome-wide association studies (GWAS) have identified SNPs in the 8q24 locus near TRIB1 that are significantly associated with plasma lipids and other markers of cardiometabolic health, and prior studies have revealed the roles of hepatic and myeloid Trib1 in plasma lipid regulation and atherosclerosis. The same 8q24 SNPs are additionally associated with plasma adiponectin levels in humans, implicating TRIB1 in adipocyte biology. Here, we hypothesize that TRIB1 in adipose tissue regulates plasma adiponectin, lipids, and metabolic health. METHODS: We investigate the metabolic phenotype of adipocyte-specific Trib1 knockout mice (Trib1_ASKO) fed on chow and high-fat diet (HFD). Through secretomics of adipose tissue explants and RNA-seq of adipocytes and livers from these mice, we further investigate the mechanism of TRIB1 in adipose tissue. RESULTS: Trib1_ASKO mice have an improved metabolic phenotype with increased plasma adiponectin levels, improved glucose tolerance, and decreased plasma lipids. Trib1_ASKO adipocytes have increased adiponectin production and secretion independent of the known TRIB1 function of regulating proteasomal degradation. RNA-seq analysis of adipocytes and livers from Trib1_ASKO mice indicates that alterations in adipocyte function underlie the observed plasma lipid changes. Adipose tissue explant secretomics further reveals that Trib1_ASKO adipose tissue has decreased ANGPTL4 production, and we demonstrate an accompanying increase in the lipoprotein lipase (LPL) activity that likely underlies the triglyceride phenotype. CONCLUSIONS: This study shows that adipocyte Trib1 regulates multiple aspects of metabolic health, confirming previously observed genetic associations in humans and shedding light on the further mechanisms by which TRIB1 regulates plasma lipids and metabolic health.

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis.

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1ß reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1ß or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis.

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet-fed Ldlr-/- mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps - all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Nonconserved Long Intergenic Noncoding RNAs Associate With Complex Cardiometabolic Disease Traits.

OBJECTIVE: Transcriptome profiling of human tissues has revealed thousands of long intergenic noncoding RNAs (lincRNAs) at loci identified through large-scale genome-wide studies for complex cardiometabolic traits. This raises the question of whether genetic variation at nonconserved lincRNAs has any systematic association with complex disease, and if so, how different this pattern is from conserved lincRNAs. We evaluated whether the associations between nonconserved lincRNAs and 8 complex cardiometabolic traits resemble or differ from the pattern of association for conserved lincRNAs. Approach and Results: Our investigation of over 7000 lincRNA annotations from GENCODE Release 33-GRCh38.p13 for complex trait genetic associations leveraged several large, established meta-analyses genome-wide association study summary data resources, including GIANT (Genetic Investigation of Anthropometric Traits), UK Biobank, GLGC (Global Lipids Genetics Consortium), Cardiogram (Coronary Artery Disease Genome Wide Replication and Meta-Analysis), and DIAGRAM (Diabetes Genetics Replication and Meta-Analysis)/DIAMANTE (Diabetes Meta-Analysis of Trans-Ethnic Association Studies). These analyses revealed that (1) nonconserved lincRNAs associate with a range of cardiometabolic traits at a rate that is generally consistent with conserved lincRNAs; (2) these findings persist across different definitions of conservation; and (3) overall across all cardiometabolic traits, approximately one-third of genome-wide association study-associated lincRNAs are nonconserved, and this increases to about two-thirds using a more stringent definition of conservation. CONCLUSIONS: These findings suggest that the traditional notion of conservation driving prioritization for functional and translational follow-up of complex cardiometabolic genomic discoveries may need to be revised in the context of the abundance of nonconserved long noncoding RNAs in the human genome and their apparent predilection to associate with complex cardiometabolic traits.

Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human.

BACKGROUND: Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. METHODS: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. RESULTS: We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. CONCLUSIONS: Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

ASEP: Gene-based detection of allele-specific expression across individuals in a population by RNA sequencing.

Allele-specific expression (ASE) analysis, which quantifies the relative expression of two alleles in a diploid individual, is a powerful tool for identifying cis-regulated gene expression variations that underlie phenotypic differences among individuals. Existing methods for gene-level ASE detection analyze one individual at a time, therefore failing to account for shared information across individuals. Failure to accommodate such shared information not only reduces power, but also makes it difficult to interpret results across individuals. However, when only RNA sequencing (RNA-seq) data are available, ASE detection across individuals is challenging because the data often include individuals that are either heterozygous or homozygous for the unobserved cis-regulatory SNP, leading to sample heterogeneity as only those heterozygous individuals are informative for ASE, whereas those homozygous individuals have balanced expression. To simultaneously model multi-individual information and account for such heterogeneity, we developed ASEP, a mixture model with subject-specific random effect to account for multi-SNP correlations within the same gene. ASEP only requires RNA-seq data, and is able to detect gene-level ASE under one condition and differential ASE between two conditions (e.g., pre- versus post-treatment). Extensive simulations demonstrated the convincing performance of ASEP under a wide range of scenarios. We applied ASEP to a human kidney RNA-seq dataset, identified ASE genes and validated our results with two published eQTL studies. We further applied ASEP to a human macrophage RNA-seq dataset, identified genes showing evidence of differential ASE between M0 and M1 macrophages, and confirmed our findings by results from cardiometabolic trait-relevant genome-wide association studies. To the best of our knowledge, ASEP is the first method for gene-level ASE detection at the population level that only requires the use of RNA-seq data. With the growing adoption of RNA-seq, we believe ASEP will be well-suited for various ASE studies for human diseases.

Differentiation of Human-Induced Pluripotent Stem Cells to Macrophages for Disease Modeling and Functional Genomics.

Macrophages play important roles in many diseases. We describe a protocol and the associated resources for the differentiation of human induced pluripotent stem cell-derived macrophages (IPSDM) and their applications in understanding human macrophage physiology and relevant diseases. The protocol uses an embryoid body-based approach with a combination of serum-free condition for hematopoiesis specification, followed by adherent culture with serum and M-CSF for myeloid expansion and macrophage maturation. The protocol produced an almost pure culture of CD45+ /CD18+ macrophages yielding up to 2 × 107 cells per 6-well plate of iPSCs within 24 days, demonstrating high efficiency, purity, and scalability. The IPSDM and monocyte-derived macrophages (HMDM) cultured in the same medium were compared at morphological, functional and transcriptomic levels by RNA-sequencing. IPSDM and HMDM showed broadly similar profiles of coding transcriptome, alternative splicing events, and long noncoding RNAs, with advantages and successful applications in disease modeling using patients-derived and CRISPR-edited iPSC lines. © 2018 by John Wiley & Sons, Inc.

Tissue-Specific Differential Expression of Novel Genes and Long Intergenic Noncoding RNAs in Humans With Extreme Response to Evoked Endotoxemia.

BACKGROUND: Cytokine responses to activation of innate immunity differ between individuals, yet the genomic and tissue-specific transcriptomic determinants of inflammatory responsiveness are not well understood. We hypothesized that tissue-specific mRNA and long intergenic noncoding RNA (lincRNA) induction differs between individuals with divergent evoked inflammatory responses. METHODS: In the GENE Study (Genetics of Evoked Response to Niacin and Endotoxemia), we performed an inpatient endotoxin challenge (1 ng/kg lipopolysaccharide [LPS]) in healthy humans. We selected individuals in the top (high responders) and bottom (low responders) extremes of inflammatory responses and applied RNA sequencing to CD14 monocytes (N=15) and adipose tissue (N=25) before and after LPS administration. RESULTS: Although only a small number of genes were differentially expressed at baseline, there were clear differences in the magnitude of the transcriptional response post-LPS between high and low responders, with a far greater number of genes differentially expressed by endotoxemia in high responders. Furthermore, tissue responses differed during inflammation, and we found a number of tissue-specific differentially expressed lincRNAs post-LPS, which we validated. Relative to nondifferentially expressed lincRNAs, differentially expressed lincRNAs were equally likely to be nonconserved as conserved between human and mouse, indicating that conservation is not a predictor of lincRNAs associated with human inflammatory pathophysiology. Differentially expressed genes also were enriched for signals with inflammatory and cardiometabolic disease in published genome-wide association studies. CTB-41I6.2 ( AC002091.1), a nonconserved human-specific lincRNA, is one of the top lincRNAs regulated by endotoxemia in monocytes, but not in adipose tissue. Knockdown experiments in THP-1 monocytes suggest that this lincRNA enhances LPS-induced interleukin 6 ( IL6) expression in monocytes, and we now refer to this as monocyte LPS-induced lincRNA regulator of IL6 ( MOLRIL6). CONCLUSIONS: We highlight mRNAs and lincRNAs that represent novel candidates for modulation of innate immune and metabolic responses in humans. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT00953667.

Interrogation of nonconserved human adipose lincRNAs identifies a regulatory role of linc-ADAL in adipocyte metabolism.

Long intergenic noncoding RNAs (lincRNAs) have emerged as important modulators of cellular functions. Most lincRNAs are not conserved among mammals, raising the fundamental question of whether nonconserved adipose-expressed lincRNAs are functional. To address this, we performed deep RNA sequencing of gluteal subcutaneous adipose tissue from 25 healthy humans. We identified 1001 putative lincRNAs expressed in all samples through de novo reconstruction of noncoding transcriptomes and integration with existing lincRNA annotations. One hundred twenty lincRNAs had adipose-enriched expression, and 54 of these exhibited peroxisome proliferator-activated receptor γ (PPARγ) or CCAAT/enhancer binding protein α (C/EBPα) binding at their loci. Most of these adipose-enriched lincRNAs (~85%) were not conserved in mice, yet on average, they showed degrees of expression and binding of PPARγ and C/EBPα similar to those displayed by conserved lincRNAs. Most adipose lincRNAs differentially expressed (n = 53) in patients after bariatric surgery were nonconserved. The most abundant adipose-enriched lincRNA in our subcutaneous adipose data set, linc-ADAL, was nonconserved, up-regulated in adipose depots of obese individuals, and markedly induced during in vitro human adipocyte differentiation. We demonstrated that linc-ADAL interacts with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) at distinct subcellular locations to regulate adipocyte differentiation and lipogenesis.

Deep RNA Sequencing Uncovers a Repertoire of Human Macrophage Long Intergenic Noncoding RNAs Modulated by Macrophage Activation and Associated With Cardiometabolic Diseases.

BACKGROUND: Sustained and dysfunctional macrophage activation promotes inflammatory cardiometabolic disorders, but the role of long intergenic noncoding RNA (lincRNA) in human macrophage activation and cardiometabolic disorders is poorly defined. Through transcriptomics, bioinformatics, and selective functional studies, we sought to elucidate the lincRNA landscape of human macrophages. METHODS AND RESULTS: We used deep RNA sequencing to assemble the lincRNA transcriptome of human monocyte-derived macrophages at rest and following stimulation with lipopolysaccharide and IFN-γ (interferon γ) for M1 activation and IL-4 (interleukin 4) for M2 activation. Through de novo assembly, we identified 2766 macrophage lincRNAs, including 861 that were previously unannotated. The majority (≈85%) was nonsyntenic or was syntenic but not annotated as expressed in mouse. Many macrophage lincRNAs demonstrated tissue-enriched transcription patterns (21.5%) and enhancer-like chromatin signatures (60.9%). Macrophage activation, particularly to the M1 phenotype, markedly altered the lincRNA expression profiles, revealing 96 lincRNAs differentially expressed, suggesting potential roles in regulating macrophage inflammatory functions. A subset of lincRNAs overlapped genomewide association study loci for cardiometabolic disorders. MacORIS (macrophage-enriched obesity-associated lincRNA serving as a repressor of IFN-γ signaling), a macrophage-enriched lincRNA not expressed in mouse macrophages, harbors variants associated with central obesity. Knockdown of MacORIS, which is located in the cytoplasm, enhanced IFN-γ-induced JAK2 (Janus kinase 2) and STAT1 (signal transducer and activator of transcription 1) phosphorylation in THP-1 macrophages, suggesting a potential role as a repressor of IFN-γ signaling. Induced pluripotent stem cell-derived macrophages recapitulated the lincRNA transcriptome of human monocyte-derived macrophages and provided a high-fidelity model with which to study lincRNAs in human macrophage biology, particularly those not conserved in mouse. CONCLUSIONS: High-resolution transcriptomics identified lincRNAs that form part of the coordinated response during macrophage activation, including specific macrophage lincRNAs associated with human cardiometabolic disorders that modulate macrophage inflammatory functions.