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 (Ezh2cd4-knockout [KO], Ezh2cd8-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. Ezh2cd4-KO, but not Ezh2cd8-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 Ezh2cd4-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 Ezh2cd4-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 Ezh2cd4-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.

Aims Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signalling in atherosclerosis. Methods and results Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1-deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on oestrogen receptor (ER)α-oestradiol signalling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing, and mitochondrial respiration among the key processes affected by CB1 signalling, which was supported by metabolic flux assays. Chronic administration of the peripherally restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism, and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. Conclusion Impaired macrophage CB1 signalling is atheroprotective by limiting their arterial recruitment, proliferation, and inflammatory reprogramming in male mice. The importance of macrophage CB1 signalling appears to be sex-dependent.

Abstract Aim Cardiovascular diseases (CVDs) are the leading cause of death globally, claiming an estimated 17.9 million lives each year. A better understanding of the underlying chronic inflammatory disease mechanisms in atherosclerosis might help finding novel treatments and reduce CVD deaths. In particular, the cannabinoid receptor CB1 has been implicated in atherosclerosis, while its cell-specific effects in this disease are not well understood. Methods Myeloid Cnr1 (CB1 encoding gene) knockout mice were generated on apolipoprotein E deficiency (Apoe-/-) background by crossing CB1flox mice with transgenic mice carrying Cre under control of the lysozyme M (LysM) promoter for selective expression in myeloid cells. Age - and sex-matched groups were analyzed at baseline, 4 and 16 weeks Western diet (0,15% cholesterol). For in vitro experiments, murine bone marrow-derived macrophages (BMDM) were used. Results Male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores compared to controls, while only minor effects in females were observed. Male Cnr1 deficient mice had reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in vitro in BMDMs and blunted by female hormone estradiol treatment. Array-based kinase activity profiling of BMDMs stimulated with a synthetic CB1 agonist revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling via bulk RNA-sequencing of CB1 agonist-stimulated BMDMs further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, again only in male mice. Conclusion Impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The biological effects of macrophage CB1 signaling seem to be more pronounced in male mice. This highlights the need to consider the biological sex as an important variable in preclinical studies.

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1 deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences were reproducible in bone marrow derived macrophages and blunted by estradiol. Kinase activity profiling revealed a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further unveiled chromatin modifications, mRNA processing and mitochondrial respiration among the key processes affected by CB1 signaling, which was supported by metabolic flux assays. Chronic administration of the peripherally-restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism and inflammatory markers, hinting to a possible implication of CB1-dependent regulation in human pathophysiology. In conclusion, impaired CB1 signaling in macrophages is atheroprotective by limiting their arterial recruitment, proliferation and inflammatory reprogramming. The importance of macrophage CB1 signaling seems to be more pronounced in male mice. ![Figure][1] Graphical summary (created with [BioRender.com][2]) ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes [2]: http://BioRender.com

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 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.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-rich plaques within arterial walls. T cells play a pivotal role in the pathogenesis of atherosclerosis in which they help orchestrate immune responses and contribute to plaque development and instability. Here, we discuss the recognition of atherosclerosis-related antigens that may trigger T cell activation together with additional signaling from co-stimulatory molecules and lesional cytokines. Although few studies have indicated candidates for the antigen specificity of T cells in atherosclerosis, further research is needed. Furthermore, we describe the pro-atherogenic and atheroprotective roles of diverse subsets of T cells such as CD4+ helper, CD8+ cytotoxic, invariant natural killer, and γδ T cells. To classify and quantify T cell subsets in atherosclerosis, we summarize current methods to analyze cellular heterogeneity including single cell RNA sequencing and T cell receptor (TCR) sequencing. Further insights into T cell biology will help shed light on the immunopathology of atherosclerosis, inform potential therapeutic interventions, and pave the way for precision medicine approaches in combating cardiovascular disease.

Background and Aims : Atherosclerosis is a chronic inflammatory disease, which drives the progression of luminal plaques in arteries. T cells help orchestrate the underlying inflammation through polarization into pro-atherogenic and atheroprotective subsets. Epigenetic signatures including H3K27me3 can skew T-cell polarization, which may ultimately affect atheroprogression. We hypothesize the H3K27 demethylase, Jumonji domain containing 3 (JMJD3) may contribute to atheroprogression through modulation of T helper (Th) cell polarization.Methods: We generated mice with Jmjd3 flanked by loxP-sites that were sensitive to Cre-mediated inactivation in T cells. Jmdj3 deficiency in T cells was confirmed by qPCR. Mice were backcrossed to apolipoprotein E-deficient (Apoe-/-) mice and set on a high fat diet to have hyperlipidemic Jmjd3fl/fl-Cd4Cre-Apoe-/-, Jmjd3fl/fl-Cd8Cre-Apoe-/- mice and respective controls. Atherosclerotic plaque progression and immune cell phenotypes were assessed by histology, flow cytometry (FC), and gene expression analysis.Results: Female Jmjd3fl/fl-Cd4Cre-Apoe-/- mice developed less atherosclerotic plaques in the aortic root compared to wildtype littermates (42% plaque reduction; p=<0.0001), but no difference was observed between Jmjd3fl/fl-Cd8Cre-Apoe-/- mice and controls. FC analysis of Jmjd3fl/fl-Cd4Cre-Apoe-/- revealed a 21 % increase in splenic T regulatory (Treg) populations (p<0.0001) while CCR6+ Th17 populations were decreased by 35% (p<0.0001). Confirmatory results were observed in the aorta, with elevated lesional Foxp3+ Tregs (p=0.04) and absent IL-17A expression in Jmjd3fl/fl-Cd4Cre-Apoe-/-mice.Conclusions: Deficiency of CD4-, but not CD8-specific JMJD3 significantly reduces atheroprogression through decreased Th17 cells. Targeting T-cell H3K27 trimethylation may be a promising new candidate to address in atherosclerosis. Background and Aims : Atherosclerosis is a chronic inflammatory disease, which drives the progression of luminal plaques in arteries. T cells help orchestrate the underlying inflammation through polarization into pro-atherogenic and atheroprotective subsets. Epigenetic signatures including H3K27me3 can skew T-cell polarization, which may ultimately affect atheroprogression. We hypothesize the H3K27 demethylase, Jumonji domain containing 3 (JMJD3) may contribute to atheroprogression through modulation of T helper (Th) cell polarization. Methods: We generated mice with Jmjd3 flanked by loxP-sites that were sensitive to Cre-mediated inactivation in T cells. Jmdj3 deficiency in T cells was confirmed by qPCR. Mice were backcrossed to apolipoprotein E-deficient (Apoe-/-) mice and set on a high fat diet to have hyperlipidemic Jmjd3fl/fl-Cd4Cre-Apoe-/-, Jmjd3fl/fl-Cd8Cre-Apoe-/- mice and respective controls. Atherosclerotic plaque progression and immune cell phenotypes were assessed by histology, flow cytometry (FC), and gene expression analysis. Results: Female Jmjd3fl/fl-Cd4Cre-Apoe-/- mice developed less atherosclerotic plaques in the aortic root compared to wildtype littermates (42% plaque reduction; p=<0.0001), but no difference was observed between Jmjd3fl/fl-Cd8Cre-Apoe-/- mice and controls. FC analysis of Jmjd3fl/fl-Cd4Cre-Apoe-/- revealed a 21 % increase in splenic T regulatory (Treg) populations (p<0.0001) while CCR6+ Th17 populations were decreased by 35% (p<0.0001). Confirmatory results were observed in the aorta, with elevated lesional Foxp3+ Tregs (p=0.04) and absent IL-17A expression in Jmjd3fl/fl-Cd4Cre-Apoe-/-mice. Conclusions: Deficiency of CD4-, but not CD8-specific JMJD3 significantly reduces atheroprogression through decreased Th17 cells. Targeting T-cell H3K27 trimethylation may be a promising new candidate to address in atherosclerosis.

Background and Aims : Cardiovascular diseases are the leading cause of death globally. Research into the mechanisms and signaling of endocannabinoids in atherosclerosis might help finding novel treatments. We aimed to clarify the myeloid-cell-specific effects of CB1 in atherosclerotic plaque formation and macrophage functions.Methods: Myeloid-CB1 knockout mice were generated on ApoE-/- background. Age- and sex- matched groups were analyzed at baseline, 4 and 16-weeks Western-diet. In vitro, bone-marrow-derived-macrophages(BMDM) were generated from global CB1-/-ApoE-/- and ApoE-/- mice.Results: Male mice with myeloid-specific-CB1 deficiency had smaller lesions after 4weeks Western-diet and less macrophage accumulation in the aortic roots compared to controls. In addition, male with myeloid-CB1 deficiency showed reduced plaque progression after 16 weeks-diet. No impact of myeloid-CB1 on plaque formation was observed in female. In vitro, male CB1-/- BMDM had lower proliferation rates compared to CB1+/+ macrophages. Likewise, plaques of male myeloid-CB1 deficient contained less proliferating macrophages. This proliferation phenotype was again not observed in female, supporting a sex-specific impact of CB1 in regulating myeloid-cell proliferation in atherogenesis. Furthermore, in vitro stimulation of male BMDM with LPS resulted in reduced TNFα secretion in CB1-deficient cells. Finally, Seahorse assays revealed a reduced oxygen consumption rate in male BMDM stimulated with CB1 agonist-ACEA, which supports a proinflammatory role of CB1 signaling in macrophages.Conclusions: Our data suggest that the biological function of CB1 signaling in macrophages seems to be sex-dependent, playing a pro-atherosclerotic role of myeloid-CB1 in male, but not female mice. It further highlights the need to consider the biological sex as an important variable in preclinical studies. Background and Aims : Cardiovascular diseases are the leading cause of death globally. Research into the mechanisms and signaling of endocannabinoids in atherosclerosis might help finding novel treatments. We aimed to clarify the myeloid-cell-specific effects of CB1 in atherosclerotic plaque formation and macrophage functions. Methods: Myeloid-CB1 knockout mice were generated on ApoE-/- background. Age- and sex- matched groups were analyzed at baseline, 4 and 16-weeks Western-diet. In vitro, bone-marrow-derived-macrophages(BMDM) were generated from global CB1-/-ApoE-/- and ApoE-/- mice. Results: Male mice with myeloid-specific-CB1 deficiency had smaller lesions after 4weeks Western-diet and less macrophage accumulation in the aortic roots compared to controls. In addition, male with myeloid-CB1 deficiency showed reduced plaque progression after 16 weeks-diet. No impact of myeloid-CB1 on plaque formation was observed in female. In vitro, male CB1-/- BMDM had lower proliferation rates compared to CB1+/+ macrophages. Likewise, plaques of male myeloid-CB1 deficient contained less proliferating macrophages. This proliferation phenotype was again not observed in female, supporting a sex-specific impact of CB1 in regulating myeloid-cell proliferation in atherogenesis. Furthermore, in vitro stimulation of male BMDM with LPS resulted in reduced TNFα secretion in CB1-deficient cells. Finally, Seahorse assays revealed a reduced oxygen consumption rate in male BMDM stimulated with CB1 agonist-ACEA, which supports a proinflammatory role of CB1 signaling in macrophages. Conclusions: Our data suggest that the biological function of CB1 signaling in macrophages seems to be sex-dependent, playing a pro-atherosclerotic role of myeloid-CB1 in male, but not female mice. It further highlights the need to consider the biological sex as an important variable in preclinical studies.

Background: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. Methods: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry–based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. Results: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3 + T cells in the atherosclerotic lesions suggested a T-cell–related effect of homoarginine supplementation, which was mainly attributed to CD4 + T cells. Macrophages, dendritic cells, and B cells were not affected. CD4 + T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. Conclusions: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Abstract The global coronavirus disease 2019 (COVID-19) pandemic has deranged the recent history of humankind, afflicting more than 27 million individuals to date. While the majority of COVID-19 patients recuperate, a considerable number of patients develop severe complications. Bilateral pneumonia constitutes the hallmark of severe COVID-19 disease but an involvement of other organ systems, namely the cardiovascular system, kidneys, liver, and central nervous system, occurs in at least half of the fatal COVID-19 cases. Besides respiratory failure requiring ventilation, patients with severe COVID-19 often display manifestations of systemic inflammation and thrombosis as well as diffuse microvascular injury observed postmortem. In this review, we survey the mechanisms that may explain how viral entry and activation of endothelial cells by severe acute respiratory syndrome coronavirus 2 can give rise to a series of events including systemic inflammation, thrombosis, and microvascular dysfunction. This pathophysiological scenario may be particularly harmful in patients with overt cardiovascular disease and may drive the fatal aspects of COVID-19. We further shed light on the role of the renin–angiotensin aldosterone system and its inhibitors in the context of COVID-19 and discuss the potential impact of antiviral and anti-inflammatory treatment options. Acknowledging the comorbidities and potential organ injuries throughout the course of severe COVID-19 is crucial in the clinical management of patients affecting treatment approaches and recovery rate.

The trimethylation status of Histone 3 Lysine 27 (H3K27), which is regulated by the methylating enzyme Enchancer of zeste homolog 2 (EZH2) and the demethylating enzyme Jumonji domain containing 3 (JMJD3), is a critical epigenetic signature for the differentiation and polarization of T cells. During atherogenesis, T cells initiate and propagate lesion formation through imbalanced polarization of T helper (Th) cells leading to the accumulation of pro-atherogenic T cell subsets, including Th1, that exacerbate inflammation. We hypothesize that T-cell EZH2 and JMJD3 may contribute to inflammation and atherosclerotic plaque formation through opposing polarization of T helper cells. We generated transgenic mice with either Ezh2 or Jmjd3 flanked by loxP-sites that were sensitive to Cre-mediated inactivation. Mice were backcrossed to apolipoprotein E (ApoE−/−) mice expressing Cre in T cells (Cd4Cretg) to generate hypercholesterolemic Ezh2fl/fl-Cd4Cre-ApoE−/− (Ezh2fl/fl) and Jmjd3fl/fl-Cd4Cre-ApoE−/− (Jmjd3fl/fl) mice. Following a 6 week high fat diet, the immune status and atherosclerotic progression of the mice were assessed by histology, flow cytometry, and RNA sequencing. Mass spectrometry of histones isolated from CD4+ T cells confirmed a decrease in H3K27 trimethylation in Ezh2fl/fl mice and an increase in Jmjd3fl/fl mice (p=0.0002 and p=0.01, respectively). In the aortic root, both Ezh2fl/fl and Jmjd3fl/fl mice developed significantly less atherosclerosis (p=0.001 and p=0.0006, respectively). A shift from naive T cells to effectors was observed in the lymph nodes and spleens in both models (p<0.0001 for all). Flow cytometric analysis revealed a systemic accumulation of Th2 in Ezh2fl/flmice (p<0.0001), which was corroborated by a 2.75 log2 fold change of IL-4 expression (padj<0.0001) (RNA-sequencing) in CD4+ T cells as well as elevated IL-4 plasma concentrations (p=0.04). Ingenuity pathway analysis revealed the canonical Th1 pathway was inhibited in Ezh2fl/fl mice. In Jmjd3fl/fl mice, flow cytometric analysis revealed a 2 fold increase of Foxp3-expressing T regulatory (Treg) cells in the blood (p=0.005), lymph nodes (p<0.0001), and spleen (p<0.0001). Our data demonstrate deficiency of either EZH2 or JMJD3 strongly reduce lesion progression in atherosclerosis through polarization of T cells towards anti-atherogenic Th2 or Treg populations, respectively. Targeting T-cell H3K27 trimethylation may be a promising candidate for further investigation to treat atherosclerosis. Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

The global coronavirus disease 2019 (COVID-19) pandemic has deranged the recent history of humankind, afflicting more than 27 million individuals to date. While the majority of COVID-19 patients recuperate, a considerable number of patients develop severe complications. Bilateral pneumonia constitutes the hallmark of severe COVID-19 disease but an involvement of other organ systems, namely the cardiovascular system, kidneys, liver, and central nervous system, occurs in at least half of the fatal COVID-19 cases. Besides respiratory failure requiring ventilation, patients with severe COVID-19 often display manifestations of systemic inflammation and thrombosis as well as diffuse microvascular injury observed postmortem. In this review, we survey the mechanisms that may explain how viral entry and activation of endothelial cells by severe acute respiratory syndrome coronavirus 2 can give rise to a series of events including systemic inflammation, thrombosis, and microvascular dysfunction. This pathophysiological scenario may be particularly harmful in patients with overt cardiovascular disease and may drive the fatal aspects of COVID-19. We further shed light on the role of the renin-angiotensin aldosterone system and its inhibitors in the context of COVID-19 and discuss the potential impact of antiviral and anti-inflammatory treatment options. Acknowledging the comorbidities and potential organ injuries throughout the course of severe COVID-19 is crucial in the clinical management of patients affecting treatment approaches and recovery rate.

Aims GITR a co-stimulatory immune checkpoint protein is known for both its activating and regulating effects on Tcells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). Methods GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular and results events (n= 100) compared to asymptomatic patients (n= 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and gtycophorin A content, and levels of interteukin (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 I 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 role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1(+) NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1(+) NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.

Background and Aims: The co-stimulatory CD40-CD40L axis is a major driver of atherosclerosis. Both platelets and T-cells express CD40L, but its cell-specific role in atherosclerosis remains unresolved. We hypothesize both a platelet- and T cell-specific CD40L deficiency may attenuate maladaptive inflammatory and thrombotic responses while reducing plaque progression.

Background and Aims: The co-stimulatory CD40-CD40L axis is a major driver of atherosclerosis. T-cell CD40L and dendritic cell (DC) CD40 expression are required for optimal T-cell expansion, but their cell-specific role in atherosclerosis remains elusive. We hypothesize that T-cell CD40L and DC CD40 signaling plays a significant role in atherosclerosis.