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Macrophages but not skeletal muscle cells respond to extracellular succinate via SUCNR1/GPR91.

Aims:CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice. Method and Results:Cd40flox/floxandLysM-cre Cd40flox/floxmice on anApoe−/−background were generated (CD40wtandCD40mac−/−, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced inCD40mac−/−compared toCD40wtmice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of theCD40mac−/−atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b−macrophages in the atherosclerotic aorta ofCD40mac−/−compared toCD40wtmice. RNA-sequencing of bone marrow-derived macrophages ofCD40mac−/−mice demonstrated upregulation of genes associated with alternatively activated macrophages (includingFolr2,Thbs1,Sdc1, andTns1). Conclusions:We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.

AimsRecent studies have revealed a close connection between cellular metabolism and the chronic inflammatory process of atherosclerosis. While the link between systemic metabolism and atherosclerosis is well established, the implications of altered metabolism in the artery wall are less understood. Pyruvate dehydrogenase kinase (PDK)-dependent inhibition of pyruvate dehydrogenase (PDH) has been identified as a major metabolic step regulating inflammation. Whether the PDK/PDH axis plays a role in vascular inflammation and atherosclerotic cardiovascular disease remains unclear.Methods and resultsGene profiling of human atherosclerotic plaques revealed a strong correlation between PDK1 and PDK4 transcript levels and the expression of pro-inflammatory and destabilizing genes. Remarkably, the PDK1 and PDK4 expression correlated with a more vulnerable plaque phenotype, and PDK1 expression was found to predict future major adverse cardiovascular events. Using the small-molecule PDK inhibitor dichloroacetate (DCA) that restores arterial PDH activity, we demonstrated that the PDK/PDH axis is a major immunometabolic pathway, regulating immune cell polarization, plaque development, and fibrous cap formation in Apoe−/− mice. Surprisingly, we discovered that DCA regulates succinate release and mitigates its GPR91-dependent signals promoting NLRP3 inflammasome activation and IL-1β secretion by macrophages in the plaque.ConclusionsWe have demonstrated for the first time that the PDK/PDH axis is associated with vascular inflammation in humans and particularly that the PDK1 isozyme is associated with more severe disease and could predict secondary cardiovascular events. Moreover, we demonstrate that targeting the PDK/PDH axis with DCA skews the immune system, inhibits vascular inflammation and atherogenesis, and promotes plaque stability features in Apoe−/− mice. These results point toward a promising treatment to combat atherosclerosis.

Contains fulltext : 286832.pdf (Publisher’s version ) (Open Access) BACKGROUND: Direct evaluation of vascular inflammation in patients with COVID-19 would facilitate more efficient trials of new treatments and identify patients at risk of long-term complications who might respond to treatment. We aimed to develop a novel artificial intelligence (AI)-assisted image analysis platform that quantifies cytokine-driven vascular inflammation from routine CT angiograms, and sought to validate its prognostic value in COVID-19. METHODS: For this prospective outcomes validation study, we developed a radiotranscriptomic platform that uses RNA sequencing data from human internal mammary artery biopsies to develop novel radiomic signatures of vascular inflammation from CT angiography images. We then used this platform to train a radiotranscriptomic signature (C19-RS), derived from the perivascular space around the aorta and the internal mammary artery, to best describe cytokine-driven vascular inflammation. The prognostic value of C19-RS was validated externally in 435 patients (331 from study arm 3 and 104 from study arm 4) admitted to hospital with or without COVID-19, undergoing clinically indicated pulmonary CT angiography, in three UK National Health Service (NHS) trusts (Oxford, Leicester, and Bath). We evaluated the diagnostic and prognostic value of C19-RS for death in hospital due to COVID-19, did sensitivity analyses based on dexamethasone treatment, and investigated the correlation of C19-RS with systemic transcriptomic changes. FINDINGS: Patients with COVID-19 had higher C19-RS than those without (adjusted odds ratio [OR] 2·97 [95% CI 1·43-6·27], p=0·0038), and those infected with the B.1.1.7 (alpha) SARS-CoV-2 variant had higher C19-RS values than those infected with the wild-type SARS-CoV-2 variant (adjusted OR 1·89 [95% CI 1·17-3·20] per SD, p=0·012). C19-RS had prognostic value for in-hospital mortality in COVID-19 in two testing cohorts (high [≥6·99] vs low [

Supplemental Digital Content is available in the text., Rationale: Inflammation is a basic component of the pathogenesis of atherosclerosis. CD200 is an immune checkpoint known to control macrophage activation. CD200 recently emerged in the Framingham Heart Study and 2 other cohorts as being potentially relevant in cardiovascular disease. The role of this pathway in cardiovascular disease is unknown. Objective: We sought to examine the role of CD200 in atherosclerosis. Methods and Results: Using hypercholesterolemic apolipoprotein-E deficient mice, we demonstrate that whole-body CD200 deficiency augments atherosclerotic lesion formation and vulnerability. Administration of a CD200-Fusion protein reduces neointima formation. Our data show that the CD200-CD200R pathway restrains activation of CD200R+ lesional macrophages, their production of CCR2 ligands, and monocyte recruitment in vitro and in vivo in an air pouch model. Loss of CD200 leads to an excessive accumulation of classical Ly6Chi monocytes and CCR2+ macrophages within the atherosclerotic aorta, as assessed by mass cytometry. Moreover, we uncover a previously uncharacterized effect of the CD200/CD200R pathway in limiting dysregulated monopoiesis and Ly6Chi monocytosis in hypercholesterolemic mice. Bone marrow chimera experiments demonstrate that the CD200-CD20R pathway enables 2 complementary and tissue-dependent strategies to limit atherogenesis: CD200 expression by bone marrow–derived cells limits systemic monocytosis, while CD200 expression by nonhematopoietic cells, for example, endothelial cells, prevents local plaque growth. We show that CD200R signaling controls monopoiesis and macrophage activation through inhibiting phosphorylation of STAT1 (signal transducer and activator of transcription 1). Finally, CD200R expression on classical monocytes in peripheral blood of patients with coronary artery disease is associated with a lower burden of coronary artery disease and a more favorable Virtual Histology plaque profile. Conclusions: The CD200 checkpoint is a key-limiting factor for monopoiesis, monocyte-macrophage activation, and recruitment in atherosclerosis with conserved features in human and mouse. It thus offers a novel potential therapeutic pathway to treat cardiovascular disease.

Coronavirus disease 2019 (COVID-19) currently has few effective treatments. Given the uncertainty surrounding the effectiveness and uptake of a vaccine, it is important that the search for treatments continue. An exaggerated inflammatory state is likely responsible for much of the morbidity and mortality in COVID-19. Elevated levels of tumor necrosis factor (TNF), a key pro-inflammatory cytokine, have been shown to be associated with increased COVID-19 mortality. In patients with rheumatoid arthritis, TNF blockade reduces not only biologically active TNF but other pro-inflammatory cytokines important in COVID-19 hyperinflammation. Observational data from patients already on anti-TNF therapy show a reduced rate of COVID-19 poor outcomes and death compared with other immune-suppressing therapies. Anti-TNF has a long history of safe use, including in special at-risk populations, and is widely available. The case to adequately assess anti-TNF as a treatment for COVID-19 is compelling., Robinson et al. discuss the potential of TNF blockade in the management of COVID-19. The rationale for the therapeutic benefit of anti-TNF is outlined, including the pathogenic role of TNF in COVID-19, the mechanism of action of anti-TNF, animal data, and supportive observational data from those on anti-TNF who develop COVID-19.

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub- phenotyping. We hypothesized that unbiased clustering of plaques based on gene expression results in an alternative categorization of late-stage atherosclerotic lesions.We analyzed the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These novel plaque phenotypes associated with clinical presentation (pIn conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in- depth transcriptomic based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to differential underlying biology of symptomatic lesions.

Graphical abstract Establishment of an autoreactive B cell memory after myocardial infarction: a working hypothesis. The demise of cardiac cells leads to the release of cryptoantigens that induce a humoral immune response that leads to accumulation of immunoglobulins in plaques and eventually amplifies atherogenesis at remote sites.

Aims: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability. Methods and results: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE−/− mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE−/−Irf5−/− mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts. Conclusion: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture. Key question: The transcription factor interferon regulatory factor-5 (IRF5) is a master regulator of macrophage activation that has been shown to have a role in murine atherogenesis. Its role in human atherosclerosis and its complications is unknown. Key finding: Interferon regulatory factor-5 is linked to plaque vulnerability and symptoms in human carotid endarterectomies. In a murine model of inducible carotid artery plaque rupture, IRF5 drives plaque rupture. Interferon regulatory factor-5 modulates macrophage phenotype and it colocalises with CD11c+ macrophages at the plaque shoulder. Take-home message: We demonstrate a mechanistic link between the IRF5, plaque macrophages, and plaque vulnerability to rupture. Interferon regulatory factor-5 is a potential candidate therapeutic target in human atherosclerosis.

Treatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete description of specific immune biomarkers. We present here a comprehensive multi-omic blood atlas for patients with varying COVID-19 severity in an integrated comparison with influenza and sepsis patients versus healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity involved cells, their inflammatory mediators and networks, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Systems-based integrative analyses including tensor and matrix decomposition of all modalities revealed feature groupings linked with severity and specificity compared to influenza and sepsis. Our approach and blood atlas will support future drug development, clinical trial design and personalized medicine approaches for COVID-19., Graphical Abstract, A multi-omic analysis of patient blood samples reveals both similarities and specific features of COVID-19 when compared with samples obtained from sepsis or influenza patients which could yield better targetted therapies for severe COVID-19.

Aims GITR—a co-stimulatory immune checkpoint protein—is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). Methods and results GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr−/−Apoe−/− mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr−/−Apoe−/− and Apoe−/− monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr−/−Apoe−/− monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. Conclusion Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets—resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages—and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 + foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4 , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/–CD64– band neutrophils and CD66bhiCD15+CD10lo/–CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation., Bona fide immature neutrophil subsets produce unchecked extracellular ROS that contributes to vascular pathologies.

For decades, the pathological definition of the vulnerable plaque led to invaluable insights into the mechanisms that underlie myocardial infarction and stroke. Beyond plaque rupture, other mechanisms, such as erosion, may elicit thrombotic events underlining the complexity and diversity of the atherosclerotic disease. Novel insights, based on single-cell transcriptomics and other "omics" methods, provide tremendous opportunities in the ongoing search for cell-specific determinants that will fine-tune the description of the thrombosis prone lesion. It coincides with an increasing awareness that knowledge on lesion characteristics, cell plasticity and clinical presentation of ischemic cardiovascular events have shifted over the past decades. This shift correlates with an observed changes of cell composition towards phenotypical stabilizing of human plaques. These stabilization features and mechanisms are directly mediated by the cells present in plaques and can be mimicked in vitro via primary plaque cells derived from human atherosclerotic tissues. In addition, the rapidly evolving of sequencing technologies identify many candidate genes and molecular mechanisms that may influence the risk of developing an atherosclerotic thrombotic event - which bring the next challenge in sharp focus: how to translate these cell-specific insights into tangible functional and translational discoveries?

Atherosclerosis is a multifactorial chronic inflammatory disease and is largely responsible for cardiovascular disease, the most common cause of global mortality. The hallmark of atherogenesis is immune activation following lipid accumulation in the arterial wall. In particular, macrophages play a non-redundant role in both the progression and regression of inflammation in the atherosclerotic lesion. Macrophages are remarkably heterogeneous phagocytes that perform versatile functions in health and disease. Their functional diversity in vascular biology is only partially mapped. Targeting macrophages is often highlighted as a therapeutic approach for cancer, metabolic and inflammatory diseases. Future strategies for therapeutic intervention in atherosclerosis may benefit from attempts to reduce local proliferation of pro-inflammatory macrophage subsets or enhance resolution of inflammation. Thus, characterisation of macrophage subsets during atherosclerosis would empower clinical interventions. Therefore, it would be of fundamental importance to understand how pathological factors modulate macrophage activity in order to exploit their use in the treatment of atherosclerosis and other diseases.

Background The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. Methods We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. Results Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. Conclusion The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. Funding UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.

Dysregulated lipid metabolism induces an inflammatory and immune response leading to atherosclerosis. Conversely, inflammation may alter lipid metabolism. Recent treatment strategies in secondary prevention of atherosclerosis support beneficial effects of both anti-inflammatory and lipid-lowering therapies beyond current targets. There is a controversy about the possibility that anti-inflammatory effects of lipid-lowering therapy may be either independent or not of a decrease in low-density lipoprotein cholesterol. In this Position Paper, we critically interpret and integrate the results obtained in both experimental and clinical studies on anti-inflammatory actions of lipid-lowering therapy and the mechanisms involved. We highlight that: (i) besides decreasing cholesterol through different mechanisms, most lipid-lowering therapies share anti-inflammatory and immunomodulatory properties, and the anti-inflammatory response to lipid-lowering may be relevant to predict the effect of treatment, (ii) using surrogates for both lipid metabolism and inflammation as biomarkers or vascular inflammation imaging in future studies may contribute to a better understanding of the relative importance of different mechanisms of action, and (iii) comparative studies of further lipid lowering, anti-inflammation and a combination of both are crucial to identify effects that are specific or shared for each treatment strategy.

SummaryMonocytes as cells of the innate immunity are prominently involved in the development of atherosclerotic lesions. The heterogeneity of blood monocytes has widely been acknowledged by accumulating experimental and clinical data suggesting a differential, subset-specific contribution of the corresponding subpopulations to the pathology of cardiovascular and other diseases. This document re-evaluates current nomenclature and summarises key findings on monocyte subset biology to propose a consensus statement about phenotype, separation and quantification of the individual subsets.Note: The review process for this paper was fully handled by Lina Badimon, Section Editor.

Macrophages, a significant component of atherosclerotic plaques vulnerable to acute complications, can be pro-inflammatory (designated M1), regulatory (M2), lipid- (Mox) or Heme-induced (Mhem). We showed previously that low (LSS) and oscillatory (OSS) shear stress cause thin-cap fibroatheroma and stable smooth muscle cell-rich plaque formation respectively in ApoE-knockout (ApoE(-/-)) mice. Here we investigated whether different shear stress conditions relate to specific changes in macrophage polarization and plaque morphology by applying a shear stress-altering cast to the carotid arteries of high fat-fed ApoE(-/-) mice. The M1 markers iNOS and IRF5 were highly expressed in macrophage-rich areas of LSS lesions compared to OSS lesions 6weeks after cast placement, while the M2 marker Arginase-1, and Mox/Mhem markers HO-1 and CD163 were elevated in OSS lesions. Our data indicates shear stress could be an important determinant of macrophage polarization in atherosclerosis, with low shear promoting M1 programming.

Atherosclerosis is the major cause of cardiovascular disease (CVD), the leading cause of death worldwide. Despite much focus on lipid abnormalities in atherosclerosis, it is clear that the immune system also has important pro- and antiatherogenic functions. The enzyme indoleamine-2,3-dioxygenase (IDO) catalyses degradation of the essential amino acid tryptophan into immunomodulatory metabolites. How IDO deficiency affects immune responses during atherogenesis is unknown and we explored potential mechanisms in models of murine and human atherosclerosis. IDO deficiency in hypercholesterolemic ApoE(-/-) mice caused a significant increase in lesion size and surrogate markers of plaque vulnerability. No significant changes in cholesterol levels were observed but decreases in IL-10 production were found in the peripheral blood, spleen and lymph node B cells of IDO-deficient compared with IDO-competent ApoE(-/-) mice. 3,4,-Dimethoxycinnamoyl anthranilic acid (3,4-DAA), an orally active synthetic derivative of the tryptophan metabolite anthranilic acid, but not l-kynurenine, enhanced production of IL-10 in cultured splenic B cells. Finally, 3,4-DAA treatment reduced lesion formation and inflammation after collar-induced arterial injury in ApoE(-/-) mice, and reduced cytokine and chemokine production in ex vivo human atheroma cell cultures. Our data demonstrate that endogenous production of tryptophan metabolites via IDO is an essential feedback loop that controls atherogenesis and athero-inflammation. We show that the IDO pathway induces production of IL-10 in B cells in vivo and in vitro, suggesting that IDO may induce immunoregulatory functions of B cells in atherosclerosis. The favorable effects of anthranilic acid derivatives in atherosclerosis indicate a novel approach toward therapy of CVD.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by vascular cell proliferation and endothelial cell apoptosis. Toll-like receptor 3 (TLR3) is a receptor for double-stranded RNA and has been recently implicated in vascular protection. Objective: The goal was to study the expression and role of TLR3 in PAH and to determine whether a TLR3 agonist reduces Pulmonary Hypertension in preclinical models. Methods: Lung tissue and endothelial cells from PAH patients were investigated by polymerase chain reaction, immunofluorescence and apoptosis assays. TLR3-/- and TLR3+/+ mice were exposed to chronic hypoxia and SU5416. Chronic hypoxia or chronic hypoxia/SU5416 rats were treated with the TLR3 agonist polyinosinic:polycytidylic acid [Poly(I:C)]. Measurements and Main Results: TLR3 expression was reduced in PAH patient lung tissue and endothelial cells, and TLR3-/- mice exhibited more severe Pulmonary Hypertension following exposure to chronic hypoxia/SU5416. TLR3 knockdown promoted double-stranded RNA signaling via other intracellular RNA receptors in endothelial cells and this was associated with greater susceptibility to apoptosis, a known driver of pulmonary vascular remodeling. Poly(I:C) increased TLR3 expression via interleukin-10 in rat endothelial cells. In vivo, high dose Poly(I:C) reduced Pulmonary Hypertension in both rat models in proof-of-principle experiments. In addition, Poly(I:C) also reduced right ventricular failure in established Pulmonary Hypertension. Conclusions: Our work identifies a novel role for TLR3 in PAH based on the findings that reduced expression of TLR3 contributes to endothelial apoptosis and pulmonary vascular remodeling.

Significance: Great attention has been placed on the link between metabolism and immune function giving rise to the term “immunometabolism.” It is widely accepted that inflammation and oxidative stress are key processes that underlie metabolic complications during obesity, diabetes, and atherosclerosis. Therefore, identifying the mechanisms and mediators that are involved in the regulation of both inflammation and metabolic homeostasis is of high scientific and therapeutic interest. Recent Advances: G protein-coupled receptors (GPCRs) that signal in response to metabolites have emerged as attractive therapeutic targets in inflammatory disease. Critical Issues and Future Directions: In this review, we discuss recent findings about the physiological role of the main metabolite-sensing GPCRs, their implication in immunometabolic disorders, their principal endogenous and synthetic ligands, and their potential as drug targets in inflammation and metabolic disease.

Atherosclerosis, the major risk factor for cardiovascular disease (CVD) and the leading cause of death worldwide, is a multifactorial chronic inflammatory disease, which, clinically manifests from early lipid-rich lesions to plaque rupture and/or thrombosis in the arterial wall. The myeloid cell compartment, including macrophages and dendritic cells (DCs), is long known to contribute to the initiation and progression of atherosclerosis. However their complex phenotypic heterogeneity hampers our full understanding of their role. Here, we review the biological and functional versatility of the myeloid cells in atherosclerosis. Several distinct subsets of macrophages and myeloid cells have been identified in atherosclerotic plaques, including subsets that are specific to atherosclerosis itself. Our ability to target them therapeutically is still limited. The challenge for the future will be the definition of treatments that target specific myeloid subsets to prevent the activation of pro-atherogenic myeloid cell subsets while preserving the anti-atherogenic and repairable function of myeloid cells.

SummaryWhilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (Breg) have been described. In experimental arthritis and lupus-like disease, Breg are contained within the CD21hiCD23hiCD24hi B cell pool. The existence and role of Breg in vascular disease is not known. We sought to investigate the existence, identity and location of Breg in vascular disease. The representation of B2-B cell subsets in the spleens and lymph nodes (LNs) of Apolipoprotein E-/- (ApoE-/-) mice compared to controls was characterised by flow cytometry. Additionally, we utilised a model of neointima formation based on the placement of a perivascular collar around the carotid artery in ApoE-/- mice to ascertain whether B cells and B cell subsets confer protection against lesion development. Adoptive transfer of B cells was performed from wild type or genetically modified mice. We showed that CD21hiCD23hiCD24hi B cells are unexpectedly increased in the draining LNs of ApoE-/- mice. Adoptive transfer of LN-derived B2-B cells or purified CD21hiCD23hiCD24hi B cells to syngeneic mice reduced lesion size and inflammation without changing serum cholesterol levels. Follicular B2-B cells did not confer protection. IL-10 blockade or transfer of IL10-deficient B cells prevented LN-derived B cell-mediated protection. This is the first identification of a specific LN-derived B2-Breg subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.

Objective Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. Methods and results Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. Conclusions These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy., Highlights • The GPR91 binding site for succinate is identified with an adjacent empty pocket. • The binding pocket structure is used to identify novel synthetic GPR91 agonists. • The non-metabolite GPR91 ligands can be used as pharmacological tools and drug leads. • Novel compounds demonstrate GPR91 control of cytokine expression in M2 macrophages.

Background and aim Oxidative modification of lipoproteins is a crucial step in atherosclerosis development. Isotopic-reinforced polyunsaturated fatty acids (D-PUFAs) are more resistant to reactive oxygen species-initiated chain reaction of lipid peroxidation than regular hydrogenated (H-)PUFAs. We aimed to investigate the effect of D-PUFA treatment on lipid peroxidation, hypercholesterolemia and atherosclerosis development. Methods Transgenic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, were pre-treated with D-PUFAs or control H-PUFAs-containing diet (1.2%, w/w) for 4 weeks. Thereafter, mice were fed a Western-type diet (containing 0.15% cholesterol, w/w) for another 12 weeks, while continuing the D-/H-PUFA treatment. Results D-PUFA treatment markedly decreased hepatic and plasma F2-isoprostanes (approx. -80%) and prostaglandin F2α (approx. -40%) as compared to H-PUFA treatment. Moreover, D-PUFAs reduced body weight gain during the study (-54%) by decreasing body fat mass gain (-87%) without altering lean mass. D-PUFAs consistently reduced plasma total cholesterol levels (approx. -25%), as reflected in reduced plasma non-HDL-cholesterol (- 28%). Additional analyses of hepatic cholesterol metabolism indicated that D-PUFAs reduced the hepatic cholesterol content (-21%). Sterol markers of intestinal cholesterol absorption and cholesterol breakdown were decreased. Markers of cholesterol synthesis were increased. Finally, D-PUFAs reduced atherosclerotic lesion area formation throughout the aortic root of the heart (-26%). Conclusions D-PUFAs reduce body weight gain, improve cholesterol handling and reduce atherosclerosis development by reducing lipid peroxidation and plasma cholesterol levels. DPUFAs therefore represent a promising new strategy to broadly reduce rates of lipid peroxidation, and combat hypercholesterolemia and cardiovascular diseases.

The pathogenesis of human atherosclerotic lesions has long been debated and is still evolving nowadays. First conceptualized as chronically evolving degenerative disease initiating in the mother’s womb, then increasingly accepted as a dynamic process causing severe acute complications that jeopardize the blood flow to the heart. Evolution of the hypothesis mirrored the progress of cellular and molecular biology, leading to progressive broadening of the understanding of cell types and molecules involved in atherogenesis. This chapter describes the current histopathological view on the pathogenesis of atherosclerosis, and touches on a historical perspective weaving in the fundamental discoveries that still influence the perception of this disease in humans.

The development of the atherosclerotic plaque relies on specific cognate interactions between ligands and receptors with the ability to regulate cell recruitment, inflammatory signalling, and the production of powerful inflammatory and bioactive lipid mediators. This chapter describes how signalling is engaged by cell-cell surface interactions when the endothelium interacts with platelets and leukocytes enhancing leukocyte recruitment during atherogenesis. It also exemplifies intracellular signalling pathways induced by the activation of innate immune receptors, the most potent activators of inflammation in physiology and disease. Differences are highlighted in innate signalling pathways in metabolic diseases such as atherosclerosis compared to canonical immunological responses. Finally, the key lipid mediators whose production can affect endothelial function, inflammation, and atherosclerosis development are summarized. This Chapter will take you through these fundamental steps in the development of the atherosclerotic plaque by summarizing very recent knowledge in the field and highlighting recent or ongoing clinical trials that may enrich our ability to target cardiovascular disease in the future.

OBJECTIVE: Identification of patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and a prelude to clinical sequelae. There are currently no clinical imaging tools to assess the inflammatory activity within plaques. This study characterized inflammation in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) as a magnetic resonance imaging (MRI) probe. METHODS: DT-MPIO were used to detect and characterize inflammatory markers, vascular cell adhesion molecule 1 (VCAM-1). and P-selectin on (1) tumor necrosis factor-α-treated cells by immunocytochemistry and (2) aortic root plaques of apolipoprotein-E deficient mice by in vivo MRI. Furthermore, apolipoprotein E-deficient mice with focal carotid plaques of different phenotypes were developed by means of periarterial cuff placement to allow in vivo molecular MRI using these probes. The association between biomarkers and the magnetic resonance signal in different contrast groups was assessed longitudinally in these models. RESULTS: Immunocytochemistry confirmed specificity and efficacy of DT-MPIO to VCAM-1 and P-selectin. Using this in vivo molecular MRI strategy, we demonstrated (1) the DT-MPIO-induced magnetic resonance signal tracked with VCAM-1 (r = 0.69; P = .014), P-selectin (r = 0.65; P = .022), and macrophage content (r = 0.59; P = .045) within aortic root plaques and (2) high-risk inflamed plaques were distinguished from noninflamed plaques in the murine carotid artery within a practical clinical imaging time frame. CONCLUSIONS: These molecular MRI probes constitute a novel imaging tool for in vivo characterization of plaque vulnerability and inflammatory activity in atherosclerosis. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid atherosclerotic disease in the future.

Blood vessels are exposed to multiple mechanical forces that are exerted on the vessel wall (radial, circumferential and Longitudinal forces) or on the endothelial surface (shear stress). The stresses and strains experienced by arteries influence the initiation of atherosclerotic Lesions, which develop at regions of arteries that are exposed to complex blood flow. In addition, plaque progression and eventually plaque rupture is influenced by a complex interaction between biological and mechanical factors mechanical forces regulate the cellular and molecular composition of plaques and, conversely, the composition of plaques determines their ability to withstand mechanical load. A deeper understanding of these interactions is essential for designing new therapeutic strategies to prevent lesion development and promote plaque stabilization. Moreover, integrating clinical imaging techniques with finite element modelling techniques allows for detailed examination of local morphological and biomechanical characteristics of atherosclerotic lesions that may be of help in prediction of future events. In this ESC Position Paper on biomechanical factors in atherosclerosis, we summarize the current 'state of the art' on the interface between mechanical forces and atherosclerotic plaque biology and identify potential clinical applications and key questions for future research.

Tumor necrosis factor (TNF) is one of the most important cytokines produced by macrophages. TNF is a very important component of host defense, released very rapidly after all types of injuries and stimuli. The kinetics of TNF release are short, and so it is perhaps not surprising that prolonged TNF production is associated with pathology. This was first elucidated in rheumatoid arthritis but extends to other chronic inflammatory diseases such as Crohn’s disease and psoriasis. In this chapter, the discovery of anti-TNF therapy is reviewed, with its benefit but also its limitations. The potential of anti-TNF therapy in other diseases, e.g., cardiovascular and fibrosis, is discussed, as is the opportunity to define ways of blocking TNF synthesis.