Resident Memory T Cells in the Atherosclerotic Lesion Associate With Reduced Macrophage Content and Increased Lesion Stability.

BACKGROUND: Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. METHODS: To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (recombination activating gene 1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

Virus-Associated CD8+ T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

INTRODUCTION: Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. METHODS: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-ß sequencing and single-cell T-cell receptor (α and ß) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. RESULTS: Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. CONCLUSIONS: This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.

Immunoproteasomal Inhibition With ONX-0914 Attenuates Atherosclerosis and Reduces White Adipose Tissue Mass and Metabolic Syndrome in Mice.

BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.

Atherosclerotic Plaque Epigenetic Age Acceleration Predicts a Poor Prognosis and Is Associated With Endothelial-to-Mesenchymal Transition in Humans.

BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.

Female Gene Networks Are Expressed in Myofibroblast-Like Smooth Muscle Cells in Vulnerable Atherosclerotic Plaques.

BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.

Sexual dimorphism in atherosclerotic plaques of aged Ldlr-/- mice.

BACKGROUND: Atherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall, resulting in plaque formation. It is well-known that atherosclerosis prevalence and manifestation vary by sex. However, sexual dimorphism in the immune landscape of atherosclerotic plaques has up to date not been studied at high-resolution. In this study, we investigated sex-specific differences in atherosclerosis development and the immunological landscape of aortas at single-cell level in aged Ldlr-/- mice. METHODS: We compared plaque morphology between aged male and female chow diet-fed Ldlr-/- mice (22 months old) with histological analysis. Using single-cell RNA-sequencing and flow cytometry on CD45+ immune cells from aortas of aged Ldlr-/- mice, we explored the immune landscape in the atherosclerotic environment in males and females. RESULTS: We show that plaque volume is comparable in aged male and female mice, and that plaques in aged female mice contain more collagen and cholesterol crystals, but less necrotic core and macrophage content compared to males. We reveal increased immune cell infiltration in female aortas and found that expression of pro-atherogenic markers and inflammatory signaling pathways was enriched in plaque immune cells of female mice. Particularly, female aortas show enhanced activation of B cells (Egr1, Cd83, Cd180), including age-associated B cells, in addition to an increased M1/M2 macrophage ratio, where Il1b+ M1-like macrophages display a more pro-inflammatory phenotype (Nlrp3, Cxcl2, Mmp9) compared to males. In contrast, increased numbers of age-associated Gzmk+CD8+ T cells, dendritic cells, and Trem2+ macrophages were observed in male aortas. CONCLUSIONS: Altogether, our findings highlight that sex is a variable that contributes to immunological differences in the atherosclerotic plaque environment in mice and provide valuable insights for further preclinical studies into the impact of sex on the pathophysiology of atherosclerosis.

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics.

RATIONALE: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. OBJECTIVE: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. METHODS AND RESULTS: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. CONCLUSIONS: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.

Blocking Sodium-Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice.

Active secretion of bile salts into the canalicular lumen drives bile formation and promotes biliary cholesterol and phospholipid output. Disrupting hepatic bile salt uptake, by inhibition of sodium-taurocholate cotransporting polypetide (NTCP; Slc10a1) with Myrcludex B, is expected to limit bile salt flux through the liver and thereby to decrease biliary lipid excretion. Here, we show that Myrcludex B-mediated NTCP inhibition actually causes an increase in biliary cholesterol and phospholipid excretion whereas biliary bile salt output and bile salt composition remains unchanged. Increased lysosomal discharge into bile was excluded as a potential contributor to increased biliary lipid secretion. Induction of cholesterol secretion was not a consequence of increased ATP-binding cassette subfamily G member 5/8 activity given that NTCP inhibition still promoted cholesterol excretion in Abcg8-/- mice. Stimulatory effects of NTCP inhibition were maintained in Sr-b1-/- mice, eliminating the possibility that the increase in biliary lipids was derived from enhanced uptake of high-density lipoprotein-derived lipids. NTCP inhibition shifts bile salt uptake, which is generally more periportally restricted, toward pericentral hepatocytes, as was visualized using a fluorescently labeled conjugated bile salt. As a consequence, exposure of the canalicular membrane to bile salts was increased, allowing for more cholesterol and phospholipid molecules to be excreted per bile salt. Conclusion: NTCP inhibition increases biliary lipid secretion, which is independent of alterations in bile salt output, biliary bile salt hydrophobicity, or increased activity of dedicated cholesterol and phospholipid transporters. Instead, NTCP inhibition shifts hepatic bile salt uptake from mainly periportal hepatocytes toward pericentral hepatocytes, thereby increasing exposure of the canalicular membrane to bile salts linking to increased biliary cholesterol secretion. This process provides an additional level of control to biliary cholesterol and phospholipid secretion.

Mast Cell Distribution in Human Carotid Atherosclerotic Plaque Differs Significantly by Histological Segment.

OBJECTIVE: Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated. METHODS: Twenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied. RESULTS: A statistically significant difference in MCs/mm2 between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm2 between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm2 in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion. CONCLUSION: MC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.

Hypercholesterolemia Induces a Mast Cell-CD4+ T Cell Interaction in Atherosclerosis.

Mast cells (MCs) are potent innate immune cells that aggravate atherosclerosis through the release of proinflammatory mediators inside atherosclerotic plaques. Similarly, CD4+ T cells are constituents of the adaptive immune response and accumulate within the plaques following lipid-specific activation by APCs. Recently it has been proposed that these two cell types can interact in a direct manner. However, no indication of such an interaction has been investigated in the context of atherosclerosis. In our study, we aimed to examine whether MCs can act as APCs in atherosclerosis, thereby modulating CD4+ T cell responses. We observed that MCs increased their MHC class II expression under hyperlipidemic conditions both in vivo and in vitro. Furthermore, we showed that MCs can present Ags in vivo via MHC class II molecules. Serum from high-fat diet-fed mice also enhanced the expression of the costimulatory molecule CD86 on cultured MCs, whereas OVA peptide-loaded MCs increased OT-II CD4+ T cell proliferation in vitro. The aortic CD4+ and TH1 cell content of atherosclerotic mice that lack MCs was reduced as compared with their wild-type counterparts. Importantly, we identified MCs that express HLA-DR in advanced human atheromata, indicating that these cells are capable of Ag presentation within human atherosclerotic plaques. Therefore, in this artice, we show that MCs may directly modulate adaptive immunity by acting as APCs in atherosclerosis.

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis. Methods and results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells. Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.

Circulating CD14+CD16- classical monocytes do not associate with a vulnerable plaque phenotype, and do not predict secondary events in severe atherosclerotic patients.

AIMS: Mouse studies have established distinct monocyte subtypes that participate in the process of atherosclerotic lesion formation. The pro-inflammatory Ly6Chigh monocyte subtype actively contributes to murine plaque progression and destabilization. Also in humans, different peripheral monocyte subtypes have been identified, of which the CD14+CD16- classical monocyte is suggested to display similar pro-atherosclerotic properties as the murine Ly6Chigh subtype. We aimed to investigate if circulating CD14+CD16- classical monocytes associate with characteristics of a vulnerable carotid atherosclerotic plaque and if they associate with the risk of secondary adverse manifestations of atherosclerotic disease. METHODS AND RESULTS: We enrolled 175 carotid endarterectomy patients of the Athero-Express biobank in our study. Just prior to surgical procedure, blood was collected and peripheral blood mononuclear cells were isolated. Characterization of monocyte subsets was performed by flow cytometry. Plaque characteristics were semi-quantitatively scored for the presence of fat, collagen, intraplaque hemorrhage and calcification. Vessel density, smooth muscle cells and macrophages were assessed quantitatively on a continuous scale. All features of a vulnerable plaque phenotype, including low amounts of collagen and smooth muscle cells, and increased fat content, vessel density, intraplaque hemorrhage and plaque macrophages were not significantly associated with differential levels of peripheral classical CD14+CD16- monocytes or other monocyte subsets. Using Cox regression models to evaluate the prognostic value of circulating monocyte subtypes, we found that total counts of peripheral monocytes, as well as CD14+CD16- classical and other monocyte subtypes were not associated with the risk of secondary cardiovascular events during 3 years follow-up. CONCLUSION: Circulating classical CD14+CD16- monocytes do not associate with specific vulnerable plaque characteristics. In addition, they do not predict secondary adverse manifestations. This suggests that in patients with established carotid artery disease, the circulating monocytes do not reflect plaque characteristics and have no value in identifying patients at risk for future cardiovascular events.

Increased Plasma IgE Accelerate Atherosclerosis in Secreted IgM Deficiency.

RATIONALE: Deficiency of secreted IgM (sIgM-/-) accelerates atherosclerosis in Ldlr-/-mice. Several atheroprotective effects of increased levels of IgM antibodies have been suggested, including preventing inflammation induced by oxidized low-density lipoprotein and promoting apoptotic cell clearance. However, the mechanisms by which the lack of sIgM promotes lesion formation remain unknown. OBJECTIVE: To identify the mechanisms by which sIgM deficiency accelerates atherosclerosis in mice. METHODS AND RESULTS: We here show that both sIgM-/- and Ldlr-/-sIgM-/- mice develop increased plasma IgE titers because of impaired generation of B cells expressing the low-affinity IgE receptor CD23, which mediates the clearance of IgE antibodies. We further report that Ldlr-/-sIgM-/- mice exhibit increased numbers of activated mast cells and neutrophils in the perivascular area of atherosclerotic plaques. Treatment with an anti-IgE-neutralizing antibody fully reversed vascular inflammation and accelerated atherosclerotic lesion formation in cholesterol-fed Ldlr-/-sIgM-/- mice. CONCLUSIONS: Thus, our data identify a previously unsuspected mechanism by which sIgM deficiency aggravates atherosclerosis.

The many faces of CD8+ T cells in atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis and the clinical consequence of cardiovascular disease remain the leading cause of death worldwide. Both an increase in cholesterol levels, as well as immune responses drive the pathogenesis of this disease. Although much is known about the role of many immune cell subsets in atherogenesis, research into the role of CD8 T cells is limited. RECENT FINDINGS: Both atheroprotective and atherogenic functions of CD8 T cells have been reported. On the one hand, the inflammatory cytokines produced by CD8 T cells exacerbate inflammatory responses, and the cytotoxic activity of these cells toward lesion-stabilizing cells such as endothelial cells drives the progression and instability of atherosclerotic lesions. On the other hand, cytotoxic activity toward antigen presenting cells and the presence of regulatory CD8 T-cell subsets dampen immunity and can limit atherosclerosis. SUMMARY: Here we review the different roles of CD8 T cells in atherosclerosis and discuss possible treatment strategies targeting these cells to reduce atherosclerotic lesion burden.

NLRP3 Inflammasome Inhibition by MCC950 Reduces Atherosclerotic Lesion Development in Apolipoprotein E-Deficient Mice-Brief Report.

OBJECTIVE: Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1ß. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE-/- mice. APPROACH AND RESULTS: First, we determined the efficacy of MCC950 in vitro. Bone marrow-derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1ß release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide-induced interleukin-1ß secretion, without affecting the tumor necrosis factor-α response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE-/- mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin-stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice. CONCLUSIONS: These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development.

Oxidized low-density lipoprotein-induced apoptotic dendritic cells as a novel therapy for atherosclerosis.

Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This could result, in part, from decreased emigration of DCs from atherosclerotic lesions because of the high-cholesterol environment. Nonetheless, local induction of anti-inflammatory responses by apoptotic cell clearance seems to dampen atherosclerosis, because inhibition of apoptotic cell clearance worsens atherosclerosis. In this study, we assessed whether i.v. administration of oxLDL-induced apoptotic DCs (apop(ox)-DCs) and, as a control, unpulsed apoptotic DCs could modulate atherosclerosis by inducing tolerance. Adoptive transfer of apop(ox)-DCs into low-density lipoprotein receptor knockout mice either before or during feeding of a Western-type diet resulted in increased numbers of CD103(+) tolerogenic splenic DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic DCs induced an immediate 40% decrease in Ly-6C(hi) monocyte numbers and a 50% decrease in circulating CCL2 levels, but only apop(ox)-DC treatment resulted in long-term effects on monocytes and CCL2 levels. Although initial lesion development was reduced by 40% in both treatment groups, only apop(ox)-DC treatment prevented lesion progression by 28%. Moreover, progressed lesions of apop(ox)-DC-treated mice showed a robust 45% increase in collagen content, indicating an enhanced stability of lesions. Our findings clearly show that apoptotic DC treatment significantly decreases lesion development, but only apop(ox)-DCs can positively modulate lesion progression and stability. These findings may translate into a safe treatment for patients with established cardiovascular diseases using patient-derived apop(ox)-DCs.

Novel B-cell subsets in atherosclerosis.

PURPOSE OF REVIEW: The number of deaths associated with cardiovascular disease remains high, despite great advances in treating the associated high levels of cholesterol. The main underlying pathology of cardiovascular disease is atherosclerosis, which is recognized as a chronic autoimmune-like inflammatory disease. Hence, there is a pressing need to shed light on the immune pathways associated with atherosclerosis. B cells have long been thought to have a general protective effect in atherosclerosis. However, findings in the last decade have challenged this paradigm, showing that it is crucial to differentiate between the various B-cell subsets when assessing their role/effect on atherosclerosis. RECENT FINDINGS: It has become increasingly recognized lately that B cells can have significant effects on the immune system independent of antibody production. The understanding that B cells form a major source of cytokines and can directly influence T-cell responses via surface markers, have led to the identification of novel B-cell subsets. These subsets are important modulators of autoimmune disorders but have not yet been fully investigated in atherosclerosis. SUMMARY: Here we review the current known roles of B-cell subsets and the putative effects of recently identified B cells on atherosclerosis.

Atherosclerosis: the interplay between lipids and immune cells.

PURPOSE OF REVIEW: Cardiovascular disease is the leading cause of mortality worldwide. The underlying cause of the majority of cardiovascular disease is atherosclerosis. In the past, atherosclerosis was considered to be the result of passive lipid accumulation in the vessel wall. However, today's picture of the pathogenesis of atherosclerosis is much more complex, with a key role for immune cells and inflammation in conjunction with hyperlipidemia, especially elevated (modified) LDL levels. Knowledge on immune cells and immune responses in atherosclerosis has progressed tremendously over the past decades, and the same is true for the role of lipid metabolism and the different lipid components. However, it is largely unknown how lipids and the immune system interact. In this review, we will describe the effect of lipids on immune cell development and function, and the effects of immune cells on lipid metabolism. RECENT FINDINGS: Recently, novel data have emerged that show that immune cells are affected, and behave differently in a hyperlipidemic environment. Moreover, immune cells have reported to be able to affect lipid metabolism. SUMMARY: In this review, we will summarize the latest findings on the interactions between lipids and the immune system, and we will discuss the potential consequences of these novel insights for future therapies for atherosclerosis.

Treating atherosclerosis with regulatory T cells.

Regulatory T cells (Tregs) play an important role in the regulation of T-cell-mediated immune responses through suppression of T-cell proliferation and secretion of inhibitory cytokines, such as interleukin-10 and transforming growth factor-ß. Impaired Treg numbers and function have been associated with numerous diseases, and an imbalance between proinflammatory/proatherogenic cells and Tregs promotes atherosclerotic disease. Restoration of this balance by inducing Tregs has great therapeutic potential to prevent cardiovascular disease. In addition to suppressing differentiation and function of effector T cells, Tregs have been shown to induce anti-inflammatory macrophages, inhibit foam cell formation and to influence cholesterol metabolism. Furthermore, Tregs suppress immune responses of endothelial cells and innate lymphoid cells. In this review, we focus on the recent knowledge on Treg subsets, their activity and function in atherosclerosis, and discuss promising strategies to use Tregs as a therapeutic tool to prevent cardiovascular disease.