Citations

Introduction: V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA), a co-inhibitory immune checkpoint protein, is known for regulating adaptive immune responses. Here, we investigated the role of VISTA in atherosclerosis. Hypothesis: VISTA is constitutively expressed on hematopoietic cells, particularly T cells. VISTA functions as both a ligand and receptor to maintain T cell quiescence. We hypothesize that VISTA expression is protective against plaque formation by inhibiting CD4 + T cell activation. Methods: Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) was performed on aortic CD45 + cells of HCD fed ApoE-/- mice. Spatial transcriptomics (VISIUM) was performed on carotid plaques from patients with either asymptomatic or symptomatic cerebrovascular disease. Plaque burden, characterization, and flow cytometry of blood and lymphoid organs were performed on VISTA -/- ApoE -/- (VISTA KO) and VISTA +/+ ApoE -/- (WT) mice that were fed a HCD for 10 weeks. ApoE -/- mice treated with VISTA agonist 8G8 or IgG control (200ug, intra-peritoneal, 3 times per week for the last 6/10 weeks of HCD feeding). In vitro proliferation, migration, and functional T cell assays were performed. Results: We found that VISTA is expressed on plaque T cell populations, particularly regulatory T cells, gamma-delta T cells, and naïve T cells. In humans, individuals with symptomatic CVD have significantly reduced VISTA gene expression in carotid plaques. VISTA KO mice have increased total plaque area and necrotic core area compared to littermate WT controls. VISTA KO mice have increased peripheral CD4 + and CD8 + effector memory T (T EM ) cells with a corresponding decrease in CD4 + and CD8 + naïve T (T N ) cells and CD4 + regulatory T (T reg ) cells. Further, VISTA KO mice have a higher frequency of CD4 + T H 1 (T-bet + IFNy + ) cells and lower frequencies of IL-10 + CD4 + T cells and CD4 + T H 2 (GATA-3 + IL-13 + ) cells. In vitro assays showed that VISTA suppresses CD4 + T cell proliferation, IFNγ production, and migration. Finally, VISTA agonist treatment significantly decreased plaque and necrotic core areas in atherosclerotic mice. Conclusion: Our data reveals a novel role for VISTA in reducing plaque development and in suppressing T cell activation, proliferation, and migration. Further characterization of T-cell specific VISTA-deficient mouse models and mechanistic insights into how VISTA modulates T cells are currently being investigated.

Abstract Background T cell activation and polarization drive atherosclerosis. Among the mediators underlying T cell polarization, the epigenetic enzyme EZH2 catalyses the repressive mark H3K27me3. In humans, EZH2 is upregulated in T cells in advanced atherosclerosis, whereas its deficiency in T cells in mice limits plaque formation via an anti-inflammatory response. Emerging evidence indicates that EZH2 can also regulate lncRNAs and miRNAs by depositing H3K27me3 at their loci, repressing transcription. Purpose We hypothesize that EZH2 governs lncRNAs and miRNAs expression in T cells in the context of atherogenesis. Methods CD4+ T cells were isolated from spleens of atherosclerosis-prone T cell-specific Ezh2 (Ezh2cd4)-KO mice and WT littermates. Small RNA-seq (SMARTer® smRNA-Seq) and transcriptomic profiling (Prime-seq) were performed. Differential expression was assessed using edgeR. Selected miRNAs were validated by RT-qPCR. Putative miRNA targets were retrieved from miRTarBase (v. 8) and TargetScan, and gene ontology enrichment was performed using Metascape. Results In CD4+ T cells from Ezh2cd4-KO mice, 41 out of 2,950 mature miRNAs were differentially expressed (log2FC > |0.8|, FDR < 0.05), with 27 upregulated and 14 downregulated. Top upregulated miRNAs included miR-125a-5p, miR-130a-3p, miR-744-5p, miR-181a-5p, miR-181b-5p, miR-374b-5p, miR-99b, miR-3085, and miR-7049-3p. RT-qPCR confirmed that expression of miR-125a-5p, miR-181a-5p, miR-181b-5p, and miR-99b-5p were upregulated in Ezh2-deficient murine CD4+ T cells. GO enrichment analysis of downregulated genes revealed enrichment of IL-4, IL-9, IL-10, and TGF-β pathways, suggesting their relevance in T-cell function and differentiation. In human Jurkat T cells, pharmacological EZH2 inhibition using GSK126 or tazemetostat resulted only in the upregulation of miR-125a-5p and miR-99b-5p, confirming the conservation of the mechanisms in humans. Integration of our Prime-seq transcriptomics and smallRNAseq datasets with human and mouse predicted and experimentally validated targets of miR-125a-5p and miR-99b-5p identified six conserved target genes: STAT1, FAM169B, NDRG3, PSMB9, TRIB1, and RAB3IP. Conclusions Our findings indicate that Ezh2 represses miRNA expression in T cells during atherogenesis, thereby possibly promoting differentiation toward pro-atherogenic subsets while restraining anti-atherogenic transcriptional programs. Further research is needed to elucidate the underlying mechanisms. Ezh2-regulated miRNAs may represent promising targets to direct T cell plasticity to combat atherosclerosis.

Abstract Myocardial infarction (MI) triggers a systemic neutrophil response, yet the roles of distinct neutrophil subsets in cardiac remodeling remain unclear. Studying this requires murine models that accurately mirror human neutrophil dynamics. Here, we show that a minimally invasive intact-chest MI model is more pathophysiologically relevant than the standard open-chest approach for investigating post-MI immune responses. In the open-chest model, surgical trauma disrupts bone marrow homeostasis, releases large numbers of immature neutrophils, and masks MI-specific immune mechanisms. In contrast, the intact-chest model preserves bone marrow integrity and induces only a modest rise in circulating immature neutrophils, closely reflecting MI patient profiles. We further demonstrate that accumulation of immature neutrophils in the infarcted heart exacerbates cardiac dysfunction. Beyond neutrophils, the overall cardiac immune landscape differs markedly between both models. Collectively, our findings establish the intact-chest model as superior for studying post-MI inflammation and reveal immature neutrophils as mediators of adverse cardiac remodeling.

Abstract Metabolic disorders such as obesity, type 2 diabetes, and dyslipidaemia are major drivers of cardiovascular risk and have reached epidemic levels in Western populations. Recent advances in clinical research have uncovered unexpected and often beneficial effects of pharmacological classes of metabolic drugs on cardiovascular outcomes, revealing complex interactions between metabolism and cardiac pathology. Despite their clinical efficacy, the molecular and cellular mechanisms through which many of these agents act remain only partially understood. This gap in knowledge underscores the urgent need for fundamental research that not only dissects the biology of metabolic disorders and cardiovascular disease but also reveals the mechanistic basis of existing and emerging therapies, thereby enabling rational therapeutic development. This scientific statement, developed by researchers from the European Society of Cardiology (ESC) Council on Basic Cardiovascular Science and several ESC Working Groups, provides a state-of-the-art overview of the emerging molecular and cellular pathways linking metabolic abnormalities with vascular and cardiac disease. By illuminating these critical connections and unresolved questions, this article aims to catalyse innovation in the prevention and management of cardiovascular disease in the context of metabolic dysfunction and to suggest new directions for future research.

BACKGROUND: The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme EZH2 (enhancer of zeste homolog 2) mediates the H3K27me3 (trimethylation of histone H3 lysine 27) and is pivotal in controlling T cell responses. METHODS: To detail the role of T cell EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe (apolipoprotein E)–deficient mice with CD (cluster of differentiation) 4 + or CD8 + T cell–specific Ezh2 deletion (Ezh2 cd4 -knockout [KO], Ezh2 cd8 -KO) were analyzed to unravel the role of T cell Ezh2 in atherosclerosis and T cell–associated immune status. RESULTS: EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2 cd4 -KO, but not Ezh2 cd8 -KO, mice showed reduced atherosclerosis with fewer advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4 + T cells drives atherosclerosis. In-depth analysis of CD4 + T cells of Ezh2 cd4 -KO mice revealed that absence of Ezh2 results in a type 2 immune response with increased Il-4 (interleukin 4) gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2 -deficient T cells polarized macrophages toward an anti-inflammatory phenotype. Single-cell RNA-sequencing of splenic T cells revealed that Ezh2 deficiency reduced naive, Ccl5 + (C-C motif chemokine ligand 5) and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift toward Th2 (type 2 T helper) effector CD4 + T cells in Ezh2 cd4 -KO mice and confirmed a profound increase in splenic iNKT cells with increased expression of Plzf (promyelocytic leukemia zinc finger), which is the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 ([zinc finger and BTB domain containing 16], the Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2 cd4 -KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3–chromatin immunoprecipitation followed by quantitative polymerase chain reaction showed that T cell–Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. CONCLUSIONS: Our study uncovers the importance of T cell EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4 + T cells drives type 2 immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis.

The CD40-CD40L co-stimulatory dyad has been identified as critical player in atherosclerosis. Our previous work has shown that CD40 and CD40L have cell divergent effects on atherosclerosis. Deficiency of the CD40-CD40L dendritic cell-T cell axis decreased atherosclerosis via reducing T helper 1 responses, whereas deficiency of macrophages CD40 reduced atherosclerosis by enhancing efferocytosis and reducing necrotic core content. CD40 is highly expressed on B cells and interacts with CD40L on T cells to induce antibody production and T follicular helper cell responses. We therefore hypothesize that deletion of CD40 will reduce atherosclerosis. We aim to unravel the role of B cell CD40 in atherosclerosis. CD40 expression on B cell subsets was determined on peripheral blood mononuclear cells of patients and correlated with severity of coronary artery disease (CAD). Atherosclerosis was analyzed in CD19 Cre CD40 flfl ApoE -/- mice that were fed an atherogenic diet for 14 weeks. Patients with CAD showed a decrease in CD40 on their putative B1 cells, which was associated with increased plaque burden and decreased plaque fibrosis as detected by coronary intravascular ultrasound. Depletion of CD40 on B cells in ApoE -/- mice resulted in a decreased germinal center formation and inadequate IgM production as expected. However, in line with our patient data, absence of B cell-CD40 increased atherosclerosis. This was caused by a reduction in B1 cells and consequently a reduction in atheroprotective anti-oxidation specific epitope (OSE) IgMs. Transfer of CD40-competent B1b cells in B cell-CD40-deficient mice restored anti-OSE IgM levels and prevented the increase in atherosclerosis. CD40-deficient B1b cells have an altered mTOR signaling pathway, impaired mitochondrial function, excessive uptake of lipids, increased cellular stress and accelerated cell death. Data indicate that CD40-deficiency promotes phosphorylation of RAPTOR and protein kinase B (AKT). We have identified a novel function of CD40 on B1b cells. B1b cell-CD40 exerts an anti-atherogenic role by regulating B1b cell metabolic homeostasis via mTOR pathway. Further mechanistic insights into how CD40 mounts adequate atheroprotective anti-OSE IgM production are currently being investigated.

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

Abstract Background The co-stimulatory CD40–CD40 ligand axis is central in atherogenesis. Upon activation, CD40 recruits tumor necrosis factor receptor-associated factors (TRAFs) to induce downstream signaling. Murine studies with mutated CD40-TRAF binding sites in macrophages have shown that the CD40-TRAF6, rather than CD40-TRAF2/3/5, axis is crucial for atherosclerosis. We thus synthesized the small molecule inhibitor TRAF-STOP 6877002, that selectively blocks CD40-TRAF6 signaling. 6877002 successfully reduced the onset and development of atherosclerosis by reducing monocyte recruitment and macrophage activation in ApoE-/- mice without having immunosuppressive side effects. Purpose To assess whether 6877002 is a suitable candidate to pass the translational pipeline towards a clinical application. Methods Elaborate in vitro and in vivo assays evaluating the pharmacological properties and potential toxicity of 6877002. Results Together with Cyprotex, we performed in vitro assays examining the ADME profile of 6877002. In an intestinal permeability assay, 6877002 had low to moderate resorption with no significant efflux, suggesting it is not a substrate for the efflux transporters Pgp/BCRP or MRP2. A plasma protein binding assay showed very low unbound 6877002 fractions, indicating that it is highly bound to plasma proteins. The potential to inhibit cytochrome P450 enzymes was assessed using human liver microsomes in the presence of enzyme-specific probe substrates. While 6877002 inhibited CYP1A2 (IC50=4.01 µM), no potential for inhibiting other CYP isoforms was detected, suggesting low drug-drug interaction potential. To screen for putative cardiovascular risks, hERG ion channel inhibition was assessed and no IC50 could be determined. Hepatic clearance was rapid in mice, rats and dogs, while minipig and human hepatocytes had lower intrinsic clearance. Together with Aptuit, we developed suitable formulations for oral and i.v. administration. In vivo pharmacokinetics studies in rats showed low bioavailability (8.43%) and rapid clearance of orally administered 6877002 compared to i.v. injected 6877002, likely – and in line with our in vitro data – due to fast first-past effect in mouse hepatocytes and uptake by macrophages. To explain the high efficacy of 6877002 despite its rapid metabolism, we assessed whether 6877002 has active metabolites that bind the CD40-TRAF6 pocket and performed an LC-MS/MS-based metabolite profiling. We detected 17 metabolites. Further MD simulations confirmed that Metabolite (M)4, the most abundant metabolite, binds the CD40-TRAF6 pocket, reflected by the highest binding mode (-19.6; defined as Binding Free Energy derived from 40-50 ns of MD run as compared to -16.5 for 6877002). Similar to 6877002, M4 reduced atherosclerosis by 54% in Western diet-fed ApoE-/- mice, indicative of M4 being an active metabolite. Conclusion CD40-TRAF6 inhibitors are an attractive strategy to combat atherosclerosis with high translational potential.

Abstract Background Atherosclerosis is a chronic inflammatory disease, characterized by the formation of luminal plaques in arteries. Atheroprogression is accompanied by strong immune activation, during which T cells play a pivotal role. Genes driving T-cell activation are regulated by the polycomb repressive complex 2(PRC2), whose major component is the Enhancer of Zeste Homolog 2(EZH2), responsible for the methylation of Histone 3 Lysine 27(H3K27me3) epigenetic mark. Purpose We hypothesize that EZH2 mediates T-cell activation and modulates T-helper (Th) cell polarization, thus atherogenesis. Methods To study the effect of EZH2 in atherosclerosis, we backcrossed mice with Ezh2 flanked by loxP-sites sensitive to Cre-mediated inactivation in T cells to apolipoprotein E (Apoe-/-)mice and fed a high cholesterol diet to have hyperlipidemic Ezh2fl/flCd4CreApoe-/-mice and controls. Atherosclerotic plaque progression and immune cell phenotyping were assessed by histology, flow cytometry (FC), gene and protein expression analysis and single cell (sc)RNA-sequencing (Seq) as well as by functional in vitro assays. Results T cell-specific EZH2 deficiency resulted in a significant reduction in H3K27me3 in T-cells. Female Ezh2fl/flCd4Cre mice developed 34% less atherosclerotic plaques in the aortic root compared to wildtype (WT) littermates. FC analysis of spleen and lymph nodes of Ezh2fl/flCd4Cre mice revealed a shift from naive to effector CD4+ T cells, and the majority were Th2 cells (p<0.0001). Accordingly, plasma levels of lL4 and aortic IL4 transcripts were increased in Ezh2fl/flCd4Cre mice(p=0.05 and p=0.0079, respectively). Functional analysis revealed that splenic CD4+ T cells from Ezh2fl/flCd4Cre mice showed decreased migratory capacity towards the chemokines CXCL10, CCL17, CCL22, CCL19. To unravel mechanisms driving the type 2 immune response, bulk RNA-Seq of CD4+ T cells and scRNA-Seq of splenic CD3+ T cells from Ezh2fl/flCd4Cre and WT mice revealed that CD4+ T cell EZH2 deficiency caused upregulation of promyelocytic leukemia zinc finger (Plzf), a transcription factor of NKT cells of the NKT2 subtype, characterized by high IL4 production. Via FC we confirmed the increase in splenic NKT cells in Ezh2fl/flCd4Cre mice. Likewise, Plzf aortic mRNA levels were also significantly increased(p=0.002). To exploit the translational potential of T cell-specific EZH2 targeting, we treated Jurkat T cells with EZH2 inhibitors, the FDA-approved drug Tazemetostat and GSK126. Corroborating our in vivo data, EZH2 inhibitor-treated cells showed decreased H3K27me3 and 70% reduced migration towards the chemokine CXCL12. Conclusions Our study demonstrates that CD4+ T-cell specific EZH2 deficiency impairs T cell-migration and skews the immune response towards a type 2 response, resulting in protection against atherosclerosis. We aim at investigating whether the epigenetic mark H3K27me3 in T cells may be a promising therapeutic target for future cardiovascular immunotherapies.

Abstract Aims Hyperlipidemia and T cell driven inflammation are important drivers of atherosclerosis, the main underlying cause of cardiovascular disease. Here, we detailed the effects of hyperlipidemia on T cells. Methods and results In vitro, exposure of human and murine CD4+ T cells to very low-density lipoprotein (VLDL), but not to low-density lipoprotein (LDL) resulted in upregulation of Th1 associated pathways. VLDL was taken up via a CD36-dependent pathway and resulted in membrane stiffening and a reduction in lipid rafts. To further detail this response in vivo, T cells of mice lacking the LDL receptor (LDLr), which develop a strong increase in VLDL cholesterol and triglyceride levels upon high cholesterol feeding were investigated. CD4+ T cells of hyperlipidemic Ldlr-/- mice exhibited an increased expression of the C-X-C-chemokine receptor 3 (CXCR3) and produced more interferon-γ (IFN-γ). Gene set enrichment analysis identified IFN-γ-mediated signaling as the most upregulated pathway in hyperlipidemic T cells. However, the classical Th1 associated transcription factor profile with strong upregulation of Tbet and Il12rb2 was not observed. Hyperlipidemia did not affect levels of the CD4+ T cell's metabolites involved in glycolysis or other canonical metabolic pathways but enhanced amino acids levels. However, CD4+ T cells of hyperlipidemic mice showed increased cholesterol accumulation and an increased arachidonic acid (AA) to docosahexaenoic acid (DHA) ratio, which was associated with inflammatory T cell activation. Conclusions Hyperlipidemia, and especially its VLDL component induces an atypical Th1 response in CD4+ T cells. Underlying mechanisms include CD36 mediated uptake of VLDL, and an altered AA/DHA ratio.