Welcome to the EAS 2023 Interactive Program

Background and Aims

Atherosclerosis is a lipid-driven chronic inflammatory disease, however, whether it can be classified as an autoimmune disease remains unclear. In this study, we aimed to determine whether atherosclerosis has an autoimmune phenotype by studying enrichment of clonally expanded T-cells is in the plaque versus the circulation.

Methods

Here, we applied single-cell TCR sequencing (scTCRseq) on human carotid artery plaques and matched PBMC samples to assess the extent of TCR clonality and antigen specific activation within the various T-cell subsets.

Results

We observed the highest degree of plaque-specific clonal expansion in effector CD4⁺ T-cells, which were characterized expression of CD69, FOS and FOSB, indicative of antigen-specific stimulation. Furthermore, trajectory analyses identified a CCR4⁺ CCR10⁺ migratory CD4⁺ T-cell subset in the PBMC with shared clonotypes with the plaque-residing effector CD4⁺ T-cells. This subset could potentially be a precursor for the antigen-specific CD4⁺ T-cells in the plaque. Next, we examined local interactions of the clonally expanded CD4⁺ T-cells with antigen-presenting cells in the plaque using CellChat, which revealed multiple potential interactions of these effector CD4⁺ T-cells with foam cells, amongst which through the CD40-CD40L pathway. Finally, we integrated a published scTCRseq dataset of the autoimmune disease psoriatic arthritis and report a similar increase of clonally expanded CD4⁺ T-cell enrichment in synovial tissue, with a similar gene expression profile as the plaque-enriched CD4⁺ T-cells, suggesting that both diseases share comparable autoimmune disease patterns.

Conclusions

In conclusion, our data suggest that atherosclerosis has an autoimmune component driven by autoreactive CD4⁺ T-cells.

Background and Aims

T-cells have a prominent role in the pathogenesis of atherosclerosis, although their exact function remains elusive. Here, we pursued a network-driven approach to identify T-cell-associated gene programs driving the transition from low- to high-risk human plaques.

Methods

In this study, 43 human carotid-arterial plaques were collected and stratified based on absence (low-risk) or presence (high-risk) of intraplaque hemorrhage. Lesion RNA was subjected to microarray gene expression analysis and evaluated by Weighted Gene Co-expression Network Analysis (WGCNA) and gene regulatory analysis. The role of a prominent transcription factor, PRDM1, was studied in vivo in Ldlr^-/- mice with T-cell-specific Prdm1-deficiency to study its role in atherosclerotic plaque development.

Results

We identified a co-expressed gene cluster displaying a strong T-cell signaling signature. WGCNA-based Bayesian network inference and single-cell gene expression further confirmed that this gene network was linked to T-cells, underpinning the central role of T-cells in plaque destabilization. Gene regulatory analysis identified T-cell-related transcription factors, most prominently PRDM1, driving this plaque gene network. PRDM1 expression was significantly downregulated in plaque T-cells from symptomatic compared with asymptomatic patients. This suggested protective role of PRDM1, could be validated in vivo in Ldlr^-/- mice with T-cell-specific Prdm1-deficiency, as the lack of Prdm1 in T-cells resulted in more advanced plaques. Moreover, mice lacking T-cell Prdm1 had a significantly reduced amount of circulating regulatory T-cells.

Conclusions

In conclusion, our study reveals a PRDM1-regulated T-cell footprint in high- versus low-risk human lesions and PRDM1 appears to aggravate atherosclerotic plaque development in mice, by modulating T-cell subsets.

Background and Aims

Atherosclerotic cardiovascular disease is a chronic inflammatory process initiated when cholesterol-carrying low-density lipoprotein (LDL) is retained in the arterial wall. CD4+ T cells, some of which recognize peptide components of LDL as antigen, are recruited to the forming lesion, resulting in T-cell activation. We aimed to study the systemic implications of these responses since they are incompletely characterized.

Methods

LDL-reactive T-cell transfers from T-cell receptor-transgenic mice to human APOB100-transgenic Ldlr^-/- mice were used to study how adaptive immune cells interact with lipoprotein metabolism.

Results

Our new data show that Prdm1^fl/flCd19^cre/+ bone marrow-chimeric mice deficient in antibody-producing cells have reduced plasma cholesterol after LDL-reactive T-cell transfer. This effect is not observed when T and B cell interactions are blocked through the administration of inducible T-cell co-stimulator ligand antibodies. RNA-sequencing of livers from mice receiving LDL-reactive T cells indicates that adaptive immune effects are exerted here.

Conclusions

Taken together, these experiments show that adaptive immune responses regulate plasma lipid levels and connect vascular-immune responses with homeostatic control over systemic cholesterol levels. The findings clarify mechanisms of atheroprotective immunity and should aid the development of immunotherapy to prevent cardiovascular disease.

Background and Aims

Highly caloric-induced chronic metabolic alterations perpetuate constitutive hyper-activation of myeloid cells precursors. Neutrophils physiologically age during their short lifetime, after being released “fresh” from bone marrow (BM) and during their canonical activity against external invaders. We here studied how neutrophil plasticity and aging are affected by unhealthy diets, how this impacts neutrophil infiltration in inflamed metabolic tissues and the metabolic outcomes.

Methods

Our experimental models were: wild-type mice (WT) on HFD and chow diet, mice with constitutively aged (CXCR4fl/flCre+) or fresh neutrophils (CXCR2fl/flCre+) (neutrophil-specific deletion of CXCR4/CXCR2 by Cre recombinase on MRP8) HFD-fed for 20 weeks, and we: immunophenotyped neutrophils and their hematopoietic progenitors in tissues, assessed their systemic metabolic response, histologically characterized their metabolic tissues.

Results

HFD increased granulopoiesis in BM (1,3*10⁴±2,6*10³ vs 5,3*10³±2,1*10³ cells/leg, p=0,003), the number of circulating neutrophils (2,7*10²±0,88*10² vs 1,39*10²±0,69*10² cells/μL, p=0,012), and those homed in liver (5,4*10⁵±2,6*10⁵ vs 6,55*10²± 7,29*10² cells/g, p<0,001) and in visceral adipose tissue (VAT) (6,7*10⁴±2,4*10⁴ vs 5,57±8,3 cells/g, p<0,001) in WT mice.

Besides, CXCR2fl/flCre+ neutrophils infiltrated less in liver (2,5*10⁵±3,3*10⁵ vs 1,3*10⁶±6,5*10⁵ cells/g, p=0,006), and in VAT (2,5*10⁴± 1,1*10⁴ vs 1,8*10⁵± 1,0*10⁵ cells/g, p=0,043) versus CXCR4fl/flCre+.

Metabolically, these different profiles resulted into more hepatic steatosis and accumulation of inflammatory macrophages (M1) in VAT of CXCR4fl/flCre+ versus CXCR2fl/flCre+, while CXCR2fl/flCre+ were less obese, displayed less hepatic steatosis and showed greater reduction of glycaemia after insulin administration versus WT (glycaemia decrease after insulin -152,52± 31,35 vs -48,6± 43,76 iAUC, p=0,002).

Conclusions

The aging of neutrophils could contribute to the development of HFD-induced metabolic alterations.

Background and Aims

Lipid peroxidation products like malondialdehyde(MDA) modify plaque components and are recognized by natural IgM antibodies. Titres of these inversely correlate with cardiovascular events, the mechanism of which is still poorly understood. Amongst others, MDA IgMs inhibit extracellular vesicle(EV)-induced clotting and thrombosis. Besides EVs, other plaque components are prothrombotic and MDA modified. We thus hypothesize that MDA IgMs are protective through inhibition of atherothrombosis.

Methods

We assessed MDA IgMs' anti-thrombotic potential. MDA modified plaque protein or plaque homogenates were used to trigger multiplate platelet aggregation assay, thrombin generation and thromboelastometry in the presence of LR04, an MDA targeting natural IgM, or its IgM isotype control. LR04 or isotype was co-injected with MDA plaque protein to induce pulmonary thrombosis in mice(n=30). Finally, MDA plaque protein IgM levels were measured in 704 cardiovascular patients(427 on statins) and correlated to clinical outcomes.

Results

In vitro, MDA plaque protein or plaque homogenates increased platelet and coagulation cascade activation, which was inhibited by LR04 but not isotype. In vivo, LR04 but not isotype protected from pulmonary thrombosis. Finally, higher levels of IgM targeting MDA modified plaque protein were associated with increased survival particularly in patients on statin therapy(p=0.001, multivariate adjusted, HR=0.431(CI95%:0.284-0.654))

Conclusions

Taken together, we describe a novel protective mechanism of MDA IgMs through atherothrombosis inhibition via reduction of platelet and coagulation cascade activation by MDA modified plaque components. Levels of these antibodies can be used to stratify patients in whom dyslipidaemia is addressed with statin therapy, representing a potential marker of residual risk in patients receiving lipid-lowering therapy.