The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels.

Patients with coronavirus disease 2019 (COVID-19) present increased risk for ischemic cardiovascular complications up to 1 year after infection. Although the systemic inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques remains unknown. Here we report that SARS-CoV-2 viral RNA is detectable and replicates in coronary lesions taken at autopsy from severe COVID-19 cases. SARS-CoV-2 targeted plaque macrophages and exhibited a stronger tropism for arterial lesions than adjacent perivascular fat, correlating with macrophage infiltration levels. SARS-CoV-2 entry was increased in cholesterol-loaded primary macrophages and dependent, in part, on neuropilin-1. SARS-CoV-2 induced a robust inflammatory response in cultured macrophages and human atherosclerotic vascular explants with secretion of cytokines known to trigger cardiovascular events. Our data establish that SARS-CoV-2 infects coronary vessels, inducing plaque inflammation that could trigger acute cardiovascular complications and increase the long-term cardiovascular risk.

Systems immunology-based drug repurposing framework to target inflammation in atherosclerosis.

The development of new immunotherapies to treat the inflammatory mechanisms that sustain atherosclerotic cardiovascular disease (ASCVD) is urgently needed. Herein, we present a path to drug repurposing to identify immunotherapies for ASCVD. The integration of time-of-flight mass cytometry and RNA sequencing identified unique inflammatory signatures in peripheral blood mononuclear cells stimulated with ASCVD plasma. By comparing these inflammatory signatures to large-scale gene expression data from the LINCS L1000 dataset, we identified drugs that could reverse this inflammatory response. Ex vivo screens, using human samples, showed that saracatinib-a phase 2a-ready SRC and ABL inhibitor-reversed the inflammatory responses induced by ASCVD plasma. In Apoe-/- mice, saracatinib reduced atherosclerosis progression by reprogramming reparative macrophages. In a rabbit model of advanced atherosclerosis, saracatinib reduced plaque inflammation measured by [18F] fluorodeoxyglucose positron emission tomography-magnetic resonance imaging. Here we show a systems immunology-driven drug repurposing with a preclinical validation strategy to aid the development of cardiovascular immunotherapies.

SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels.

COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1ß, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.

Netrin-1 blockade inhibits tumor associated Myeloid-derived suppressor cells, cancer stemness and alleviates resistance to chemotherapy and immune checkpoint inhibitor.

Drug resistance and cancer relapse represent significant therapeutic challenges after chemotherapy or immunotherapy, and a major limiting factor for long-term cancer survival. Netrin-1 was initially identified as a neuronal navigation cue but has more recently emerged as an interesting target for cancer therapy, which is currently clinically investigated. We show here that netrin-1 is an independent prognostic marker for clinical progression of breast and ovary cancers. Cancer stem cells (CSCs)/Tumor initiating cells (TICs) are hypothesized to be involved in clinical progression, tumor relapse and resistance. We found a significant correlation between netrin-1 expression and cancer stem cell (CSC) markers levels. We also show in different mice models of resistance to chemotherapies that netrin-1 interference using a therapeutic netrin-1 blocking antibody alleviates resistance to chemotherapy and triggers an efficient delay in tumor relapse and this effect is associated with CSCs loss. We also demonstrate that netrin-1 interference limits tumor resistance to immune checkpoint inhibitor and provide evidence linking this enhanced anti-tumor efficacy to a decreased recruitment of a subtype of myeloid-derived suppressor cells (MDSCs) called polymorphonuclear (PMN)-MDSCs. We have functionally demonstrated that these immune cells promote CSCs features and, consequently, resistance to anti-cancer treatments. Together, these data support the view of both a direct and indirect contribution of netrin-1 to cancer stemness and we propose that this may lead to therapeutic opportunities by combining conventional chemotherapies and immunotherapies with netrin-1 interfering drugs.

Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice.

Hypercholesterolemia and vascular inflammation are key interconnected contributors to the pathogenesis of atherosclerosis. How hypercholesterolemia initiates vascular inflammation is poorly understood. Here we show in male mice that hypercholesterolemia-driven endothelial activation, monocyte recruitment and atherosclerotic lesion formation are promoted by a crosstalk between macrophages and endothelial cells mediated by the cholesterol metabolite 27-hydroxycholesterol (27HC). The pro-atherogenic actions of macrophage-derived 27HC require endothelial estrogen receptor alpha (ERα) and disassociation of the cytoplasmic scaffolding protein septin 11 from ERα, leading to extranuclear ERα- and septin 11-dependent activation of NF-κB. Furthermore, pharmacologic inhibition of cyp27a1, which generates 27HC, affords atheroprotection by reducing endothelial activation and monocyte recruitment. These findings demonstrate cell-to-cell communication by 27HC, and identify a major causal linkage between the hypercholesterolemia and vascular inflammation that partner to promote atherosclerosis. Interventions interrupting this linkage may provide the means to blunt vascular inflammation without impairing host defense to combat the risk of atherosclerotic cardiovascular disease that remains despite lipid-lowering therapies.

Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

Rapid neutrophil mobilization by VCAM-1+ endothelial cell-derived extracellular vesicles.

AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Erratum: Publisher Correction: Systems immunology-based drug repurposing framework to target inflammation in atherosclerosis.

[This corrects the article DOI: 10.1038/s44161-023-00278-y.].

Long noncoding RNA CHROMR regulates antiviral immunity in humans.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their contribution to immune regulation in humans remains poorly understood. Here, we report that the primate-specific lncRNA CHROMR is induced by influenza A virus and SARS-CoV-2 infection and coordinates the expression of interferon-stimulated genes (ISGs) that execute antiviral responses. CHROMR depletion in human macrophages reduces histone acetylation at regulatory regions of ISG loci and attenuates ISG expression in response to microbial stimuli. Mechanistically, we show that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional corepressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Consequently, CHROMR expression is essential to restrict viral infection of macrophages. Our findings identify CHROMR as a key arbitrator of antiviral innate immune signaling in humans.

The Liver X Receptor Is Selectively Modulated to Differentially Alter Female Mammary Metastasis-associated Myeloid Cells.

Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

Reverse cardio-oncology: Exploring the effects of cardiovascular disease on cancer pathogenesis.

The field of cardio-oncology has emerged in response to the increased risk of cardiovascular disease (CVD) in patients with cancer. However, recent studies suggest a more complicated CVD-cancer relationship, wherein development of CVD, either prior to or following a cancer diagnosis, can also lead to increased risk of cancer and worse outcomes for patients. In this review, we describe the current evidence base, across epidemiological as well as preclinical studies, which supports the emerging concept of 'reverse-cardio oncology', or CVD-induced acceleration of cancer pathogenesis.

Chronic stress primes innate immune responses in mice and humans.

Psychological stress (PS) is associated with systemic inflammation and accelerates inflammatory disease progression (e.g., atherosclerosis). The mechanisms underlying stress-mediated inflammation and future health risk are poorly understood. Monocytes are key in sustaining systemic inflammation, and recent studies demonstrate that they maintain the memory of inflammatory insults, leading to a heightened inflammatory response upon rechallenge. We show that PS induces remodeling of the chromatin landscape and transcriptomic reprogramming of monocytes, skewing them to a primed hyperinflammatory phenotype. Monocytes from stressed mice and humans exhibit a characteristic inflammatory transcriptomic signature and are hyperresponsive upon stimulation with Toll-like receptor ligands. RNA and ATAC sequencing reveal that monocytes from stressed mice and humans exhibit activation of metabolic pathways (mTOR and PI3K) and reduced chromatin accessibility at mitochondrial respiration-associated loci. Collectively, our findings suggest that PS primes the reprogramming of myeloid cells to a hyperresponsive inflammatory state, which may explain how PS confers inflammatory disease risk.

Silencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque Regression.

[Figure: see text].

High-Throughput Screening Identifies MicroRNAs Regulating Human PCSK9 and Hepatic Low-Density Lipoprotein Receptor Expression.

Investigations into the regulatory mechanisms controlling cholesterol homeostasis have proven fruitful in identifying low-density lipoprotein (LDL)-lowering therapies to reduce the risk of atherosclerotic cardiovascular disease. A major advance was the discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9), a secreted protein that binds the LDL receptor (LDLR) on the cell surface and internalizes it for degradation, thereby blunting its ability to take up circulating LDL. The discovery that loss-of-function mutations in PCSK9 lead to lower plasma levels of LDL cholesterol and protection from cardiovascular disease led to the therapeutic development of PCSK9 inhibitors at an unprecedented pace. However, there remain many gaps in our understanding of PCSK9 regulation and biology, including its posttranscriptional control by microRNAs. Using a high-throughput region(3'-UTR) of human microRNA library screen, we identified microRNAs targeting the 3' untranslated region of human PCSK9. The top 35 hits were confirmed by large-format PCSK9 3'-UTR luciferase assays, and 10 microRNAs were then selected for further validation in hepatic cells, including effects on PCSK9 secretion and LDLR cell surface expression. These studies identified seven novel microRNAs that reduce PCSK9 expression, including miR-221-5p, miR-342-5p, miR-363-5p, miR-609, miR-765, and miR-3165. Interestingly, several of these microRNAs were also found to target other genes involved in LDLR regulation and potently upregulate LDLR cell surface expression in hepatic cells. Together, these data enhance our understanding of post-transcriptional regulators of PCSK9 and their potential for therapeutic manipulation of hepatic LDLR expression.

MicroRNA-33 Inhibits Adaptive Thermogenesis and Adipose Tissue Beiging.

[Figure: see text].