Johan Kuiper

Aims: Aging is a major risk factor for cardiovascular disease (CVD) and is also linked to a functional decline of the immune system. B cells contribute to atherosclerosis progression by antibody secretion, antigen presentation and T-cell regulation. During aging, CD11c+CD21low B cells accumulate, but it is unclear whether these age-associated B cells (ABCs) are linked with incident cardiovascular events in humans and if they contribute to the progression of atherosclerosis. Methods and results: To investigate associations with first-time coronary events (CEs), circulating B cells were phenotyped with flow cytometry in a case–control study (N = 604) nested in the Malmö Diet and Cancer study cohort with a median of 14 years follow-up. Splenic B cells in young (5 months) and aged (21 months) female Ldlr−/− mice were characterized with single-cell RNA-sequencing coupled with B-cell receptor (BCR)-sequencing. Atherogenicity of ABCs was determined by adoptive transfer to Ldlr−/− and Ldlr−/−Rag1−/− mice. Compared with other B-cell populations, ABCs showed a significant positive correlation with age (Spearman’s rho 0.24; P < 0.0001; across 46–68 years of age) and accumulated in aged atherosclerotic mice. CE cases had higher counts of ABCs than controls and regression analysis revealed an association with incident CEs, that was independent of cardiovascular risk factors [odds ratio: 1.92 (95% confidence interval 1.13–3.27), P = 0.016, comparing the fourth vs. first quartile]. Adoptive transfer of ABCs to recipient mice promoted atherosclerotic lesion development. In Ldlr−/− Rag1−/− mice, ABCs also increased necrotic cores (P < 0.05). B cells recovered from recipient mice after transfer expressed CD138 and secreted antibodies. In line with these findings, we demonstrated that ABCs expressed plasma cell (PC) differentiation genes and showed the greatest clonal expansion within the B-cell compartment, with extensive clonal overlap with PCs. Conclusion: We show that ABCs predict first-time CEs in humans and contribute to the immunopathology of atherosclerosis in mice, indicating that clonal expansion and accumulation of ABCs contribute to the strong association between age and CVD. ABCs may serve as potential prognostic and therapeutic targets for CVD.

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 β5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit β1i/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.

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 TGFβ -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.

BACKGROUND: Tissue resident memory T (T _RM ) 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, T _RM cells have also been implicated in inflammatory disorders. T _RM 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, T _RM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of T _RM cells in atherosclerosis. METHODS: To identify T _RM 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 T _RM cells. The presence and phenotype of T _RM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing T _RM cells. To explore the function of T _RM cells during atherogenesis, RAG1 ^−/− (recombination activating gene 1 deficient) LDLr ^−/− (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from Hobit ^KO/CRE Blimp-1 ^flox/flox mice, which exhibit abrogated T _RM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a T _RM cell–associated gene signature. Moreover, a fraction of these T cells clustered together with predefined T _RM cells upon integration. The presence of Hobit-expressing T _RM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived T _RM 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: T _RM 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.

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-β sequencing and single-cell T-cell receptor (α and β) 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.

The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E–deficient (Apoe^−/−) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68⁺ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

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.

Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr^−/−) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans. Here, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr^−/− mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leucocytes of young vs. aged Ldlr^−/− mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK⁺CD8⁺ T-cells and previously in atherosclerosis undefined CD11b⁺CD11c⁺T-bet⁺ age-associated B-cells (ABCs). ABCs of Ldlr^−/− mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood. Collectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease.

Epigenetic age estimators (clocks) are known to be predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques can be used for predicting secondary events. Here we estimated an age adjusted measure of epigenetic age, epigenetic age acceleration (EAA), using DNA methylation of human atherosclerotic plaques and of blood. EAA of plaque, but not blood, independently predicted secondary events in a 3-year follow-up (HR=1.3, p= 0.018). Plaque EAA concurred with a high metabolic epigenetic and transcriptional state in plaques. Patients with diabetes and a high body mass index had a higher plaque EAA. EAA was lower in female plaques compared to male plaques by approximately 2 years. Single-cell RNA-seq revealed mesenchymal smooth muscle cells and endothelial cells as main drivers of EAA. Plaque-specific ageing may help identify processes that explain poor health outcomes.

Women presenting with coronary artery disease (CAD) 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. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.