Welcome to the EAS 2022 Interactive Program

Background and Aims

Viperin (encoded by the Rsad2 gene) is a highly conserved radical SAM-enzyme with anti-viral properties. Viperin is expressed in advanced atherosclerotic plaques in humans, but its role in atherosclerosis development is unknown.

Methods

To investigate the role of Viperin in atherosclerosis, we injected bone marrow from Rsad2^+/+ or Rsad2^-/- donor mice into lethally irradiated low density lipoprotein receptor deficient mice (Ldlr^-/-), which were fed an atherogenic diet (0.2% cholesterol, 21% fat) for either 10 (early atherosclerosis) or 16 weeks (advanced atherosclerosis).

Results

Lesion size in the aortic root did not differ between the groups after 10 weeks of atherogenic diet feeding, but lesion size was markedly increased in Ldlr^-/- mice lacking Viperinin the hematopoietic system after 16 weeks of diet feeding (Rsad2^+/+: 21.4 ± 4.8 vs Rsad2^-/-: 39.6 ± 4.0 x10⁴ µm², P = 0.0083; mean ± SEM.). Because Viperin regulates Toll-like receptor 7 and 9 signaling in innate cells, we examined the activation state of myeloid cells in the spleen and peripheral blood by flow cytometry. Notably, we did not find differences in numbers or activation state of monocytes or neutrophils. Consistent with the role of Viperin in modulating T cell responses, Viperin deletion in the hematopoietic system diminished activated and effector memory T cells in the spleen of atherosclerotic mice. Furthermore, plasma levels of IgG1 and IgG2b antibodies were decreased upon Viperin deletion in the hematopoietic system in mice with advanced atherosclerosis.

Conclusions

Our data suggest that Viperin confers an atheroprotective effect via modulation of adaptive and humoral immunity.

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 Jmjd3^fl/fl-Cd4^Cre-Apoe^-/-, Jmjd3^fl/fl-Cd8^Cre-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 Jmjd3^fl/fl-Cd4^Cre-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 Jmjd3^fl/fl-Cd8^Cre-Apoe^-/- mice and controls. FC analysis of Jmjd3^fl/fl-Cd4^Cre-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 Jmjd3^fl/fl-Cd4^Cre-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

Cellular metabolism defines T cell polarization and activation; Tregulatory (Treg) cells rely on fatty acids oxidation (FAO) for their suppressive function while glycolysis is preferred for cell migration. We aimed at studying how SREBP1c, a key protein regulating intracellular fatty acid (FA) metabolism, impacts Treg cell metabolism and function.

Methods

A detailed immunophenotyping through flow cytometry and metabolic profiling of isolated Tregulatory (CD4+CD25+) and in vitro induced Treg (iTreg) cells were performed together with in vitro and in vivo assays of Treg function from SREBP1c KO and WT littermates.

Results

Srebp1c KO mice presented reduced circulating and tissues’ level of Treg compared to WT mice (-66%,p<0,01). Functionally, Srebp1c deficiency was associated with a reduced suppressive (-21%,p<0,01) and increased migratory function (+40%,p<0,05). In Experimental Autoimmune Encephalomyelitis, a model of immune challenge, Treg from KO mice were less effective compared to WT in limiting disease progression. Taking advantage from iTreg we confirmed that the less suppressive and more migratory phenotype was the consequence of Srebp1c deficiency in Treg rather than an effect of reduced cholesterol and triglycerides plasma levels in KO vs WT (-56%,-61%,p<0.01). Metabolically, KO iTreg showed an increased glycolytic potential with preserved mitochondrial function. Accumulation of lactate (+20%,p<0,01) and increased mTORC1 activation by pS6 phosphorylation (+45%,p<0,01) further confirmed a switch to anaerobic glycolysis in KO Treg.

Conclusions

SREBP1c represents a key player of Treg immunometabolism by controlling glycolysis and cell migration. Metabolic fluxes are ongoing to depict the molecular mechanism/s of this phenotype.

Background and Aims

T cells have a prominent role in the pathogenesis of atherosclerosis, although their function in atherosclerotic plaques is only partly understood. In this study, we utilize the advantages of high-throughput techniques and data analytic strategies to compare the inherent biological changes of T cells during plaque transition from a stable, non-haemorrhaged (low-risk) to a rupture-prone, haemorrhaged (high-risk) phenotype.

Methods

We classified 43 human carotid arterial lesions into high- and low-risk plaques based on the presence/absence of intraplaque hemorrhages. RNA from these lesions was isolated and microarray gene expression data was obtained and analyzed by Weighted Gene Co-expression Network Analysis.

Results

A strong T cell signalling signature was identified in high- versus low-risk plaques, influencing angiogenesis and interferon-related processes. Bayesian network inference, cell type deconvolution and single-cell RNA sequencing analysis revealed that the T cell-associated gene program was linked to effector-memory cytotoxic, CD8⁺ T cells. This gene program appeared driven by CD8⁺ T cell-related transcription factors, including RUNX3, IRF7 and most importantly PRDM1. To validate these findings, we demonstrated in a murine model that T cell PRDM1 plays a key role in plaque formation, as atherosclerotic mice with a T cell specific Prdm1 deficiency developed larger and more advanced atherosclerotic plaques compared to control mice.

Conclusions

In conclusion, our study unveils a clear PRDM1-regulated effector-memory cytotoxic CD8⁺ T cell footprint in plaque development and the shift from low- to high-risk plaques, thereby revealing CD8⁺ T cells and PRMD1 as potential targets for intervention in adverse T cell responses in human atherosclerotic lesions.

Background and Aims

Cytotoxic CD4⁺ T cells have previously been found in peripheral blood of patients with coronary artery disease¹, however their occurrence in atherosclerotic plaques and their association with the pathophysiology of atherosclerosis has not been established.

Methods

Single-cell RNA sequencing was performed on human carotid atherosclerotic plaques of 18 patients to identify specific T cell populations.² Next, human femoral and carotid atherosclerotic plaques (n=95) and matched blood samples (n=49) were analyzed by flow cytometry for the presence of CD4⁺GZMB⁺ T cells. Plaque morphology was assessed by Movat’s Pentachrome staining.

Results

A distinct cytotoxic GZMB⁺PRF1⁺CD28^- CD4⁺ T cell cluster was identified using single-cell RNA sequencing. Furthermore, flow cytometry analysis showed that the percentage of GZMB⁺CD4⁺ T cells was significantly elevated in plaque compared to blood (Blood: 12.17±2.0 vs. Plaque: 17.40±1.0; P=0.0002). Moreover, a significant positive correlation was observed between the percentage of GZMB⁺CD4⁺ T cells in blood versus plaque (P=0.031). In line with the proinflammatory character of these cells, we found a positive association of GZMB⁺CD4⁺ T cells (P=0.036) with necrotic core size, whereas no correlation was found with this subtype in the circulation.

Conclusions

In this study we have shown an enrichment of cytotoxic CD4⁺ T cells in atherosclerotic lesions, which positively correlate with necrotic core size. Future studies are aimed at elucidating the role of these cells in advanced atherosclerosis.

¹Liuzzo et al., J Am Coll Cardiol. 2007

²Depuydt et al., Circ. Res. 2020