Anton Gisterå

Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis

Atherosclerosis is a chronic inflammatory disease promoted by hyperlipidemia. Several studies support FOXP3-positive regulatory T cells (Tregs) as inhibitors of atherosclerosis; however, the mechanism underlying this protection remains elusive. To define the role of FOXP3-expressing Tregs in atherosclerosis, we used the DEREG mouse, which expresses the diphtheria toxin (DT) receptor under control of the Treg-specific Foxp3 promoter, allowing for specific ablation of FOXP3+ Tregs. Lethally irradiated, atherosclerosis-prone, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice received DEREG bone marrow and were injected with DT to eliminate FOXP3(+) Tregs. Depletion of Tregs caused a 2.1-fold increase in atherosclerosis without a concomitant increase in vascular inflammation. These mice also exhibited a 1.7-fold increase in plasma cholesterol and an atherogenic lipoprotein profile with increased levels of VLDL. Clearance of VLDL and chylomicron remnants was hampered, leading to accumulation of cholesterol-rich particles in the circulation. Functional and protein analyses complemented by gene expression array identified reduced protein expression of sortilin-1 in liver and increased plasma enzyme activity of lipoprotein lipase, hepatic lipase, and phospholipid transfer protein as mediators of the altered lipid phenotype. These results demonstrate that FOXP3(+) Tregs inhibit atherosclerosis by modulating lipoprotein metabolism.

Transforming Growth Factor–β Signaling in T Cells Promotes Stabilization of Atherosclerotic Plaques Through an Interleukin-17–Dependent Pathway

Enhanced TGF-β signaling may promote plaque stability and prevent clinical manifestations of atherosclerosis. IL-17 Helps Plaques Lie Dormant Like a dormant volcano, stable atherosclerotic plaques can lull you into a false sense of security. The accumulation of lipids and inflammatory mediators results in arterial hardening and lack of flexibility, but individuals with these plaques may be asymptomatic for decades. However, when an unstable plaque ruptures, thrombi forming on the exposed tissue can block blood flow, resulting in heart attack or stroke. Gisterå et al. now report that transforming growth factor–β (TGF-β) promotes plaque stabilization through the effects of interleukin-17 (IL-17). The authors looked at T cells with enhanced expression of TGF-β in a mouse model of atherosclerosis. They found that these animals had larger atherosclerotic lesions, but these lesions were more stable. Inhibiting IL-17 through neutralizing antibodies decreased the stability of these plaques, whereas IL-17 expression correlated to expression of components of the fibrous cap in human atherosclerotic plaques. These data suggest that patients treated with IL-17 receptor blockers should be closely monitored for cardiovascular events and provide IL-17 as a therapeutic option to prevent plaque eruption. Adaptive immunity has a major impact on atherosclerosis, with pro- and anti-atherosclerotic effects exerted by different subpopulations of T cells. Transforming growth factor–β (TGF-β) may promote development either of anti-atherosclerotic regulatory T cells or of T helper 17 (TH17) cells, depending on factors in the local milieu. We have addressed the effect on atherosclerosis of enhanced TGF-β signaling in T cells. Bone marrow from mice with a T cell–specific deletion of Smad7, a potent inhibitor of TGF-β signaling, was transplanted into hypercholesterolemic Ldlr−/− mice. Smad7-deficient mice had significantly larger atherosclerotic lesions that contained large collagen-rich caps, consistent with a more stable phenotype. The inflammatory cytokine interleukin-6 (IL-6) was expressed in the atherosclerotic aorta, and increased mRNA for IL-17A and the TH17-specific transcription factor RORγt were detected in draining lymph nodes. Treating Smad7-deficient chimeras with neutralizing IL-17A antibodies reversed stable cap formation. IL-17A stimulated collagen production by human vascular smooth muscle cells, and RORγt mRNA correlated positively with collagen type I and α-smooth muscle actin mRNA in a biobank of human atherosclerotic plaques. These data link IL-17A to induction of a stable plaque phenotype, could lead to new plaque-stabilizing therapies, and should prompt an evaluation of cardiovascular events in patients treated with IL-17 receptor blockade.

The immunology of atherosclerosis

Cardiovascular disease is the leading cause of death worldwide, both in the general population and among patients with chronic kidney disease (CKD). In most cases, the underlying cause of the cardiovascular event is atherosclerosis — a chronic inflammatory disease. CKD accelerates atherosclerosis via augmentation of inflammation, perturbation of lipid metabolism, and other mechanisms. In the artery wall, subendothelial retention of plasma lipoproteins triggers monocyte-derived macrophages and T helper type 1 (TH1) cells to form atherosclerotic plaques. Inflammation is initiated by innate immune reactions to modified lipoproteins and is perpetuated by TH1 cells that react to autoantigens from the apolipoprotein B100 protein of LDL. Other T cells are also active in atherosclerotic lesions; regulatory T cells inhibit pathological inflammation, whereas TH17 cells can promote plaque fibrosis. The slow build-up of atherosclerotic plaques is asymptomatic, but plaque rupture or endothelial erosion can induce thrombus formation, leading to myocardial infarction or ischaemic stroke. Targeting risk factors for atherosclerosis has reduced mortality, but a need exists for novel therapies to stabilize plaques and to treat arterial inflammation. Patients with CKD would likely benefit from such preventive measures.

Inhibition of indoleamine 2,3-dioxygenase promotes vascular inflammation and increases atherosclerosis in Apoe-/- mice.

AIMS Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of low-density lipoprotein in the artery, leading to maladaptive response of cells from the immune system and vessel wall. Strong evidence implicates indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway of tryptophan (Trp) degradation, with immune regulation and anti-inflammatory mechanisms in different diseases. However, the role of IDO and the endogenous degradation of Trp have never been directly examined in atherosclerosis development. We used the IDO inhibitor 1-methyl-Trp (1-MT) to determine the role of IDO-mediated Trp metabolism in vascular inflammation and atherosclerosis. METHODS AND RESULTS Apoe(-/-) mice were treated with 1-MT in drinking water for 8 weeks. Systemic IDO inhibition led to a significant increase in atherosclerotic lesions that were ∼58 and 54% larger in the aortic arch and root, respectively. 1-MT treatment enhanced vascular inflammation, up-regulated VCAM-1 and CCL2, and increased CD68 macrophage accumulation into the plaque. Notably, the rise in VCAM-1 expression was not limited to the plaque but also found in smooth muscle cells (SMCs) of the tunica media. Furthermore, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis and vascular inflammation can be reversed by exogenous administration of the Trp metabolite 3-hydroxyanthranilic acid (3-HAA). CONCLUSION IDO-mediated Trp metabolism regulates vascular inflammation and plaque formation in hypercholesterolaemic Apoe(-/-) mice. Our data establish that this pathway plays a major role in the pathological process of atherogenesis.

T Cell-based Therapies for Atherosclerosis

Cardiovascular diseases (CVDs), largely due to atherosclerosis, are the major causes of death in todays world. Atherosclerosis is a chronic inflammatory condition initiated by retention and accumulation of cholesterol-containing lipoproteins, in particular low-density lipoprotein (LDL), in the artery wall. This initiates pathological responses of immune cells that lead to atherosclerotic plaque formation. T cells are present during all stages of the disease, and play an essential role in the initiation and progression of plaques. Whereas most T effector cell responses have been suggested to aggravate atherosclerosis, regulatory T cells (Tregs) have been shown to limit inflammation and inhibit the formation of lesions. In addition to their effects on the local pathological process, T cells and their released mediators modulate systemic lipid metabolism and can increase risk of CVDs. Such knowledge on the pathological and protective function of these cells has led to significant advances in the field. This review examines experimental and pre-clinical studies approaching the manipulation of cellular immunity in atherosclerosis. Modulation of T cells responses by vaccination, antibody therapies, dendritic cell based-therapies, and using amino acid-derived metabolites have shown benefits against atherosclerotic plaque progression in animal models. The clinical benefit of T cell-based therapies in humans still requires further investigation.

Vaccination against T-cell epitopes of native ApoB100 reduces vascular inflammation and disease in a humanized mouse model of atherosclerosis

The T‐cell response to low‐density lipoprotein (LDL) in the vessel wall plays a critical role in atherosclerotic plaque formation and stability. In this study, we used a new translational approach to investigate epitopes from human apolipoprotein B100 (ApoB100), the protein component of LDL, which triggers T‐cell activation. We also evaluated the potential of two selected native ApoB100 epitopes to modulate atherosclerosis in human ApoB100‐transgenic Ldlr−/− (HuBL) mice.

ERV1/ChemR23 Signaling Protects from Atherosclerosis by Modifying oxLDL Uptake and Phagocytosis in Macrophages

Background: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein–coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. Methods: Lipidomic plasma analysis were performed after EPA supplementation in Apoe−/− mice. Erv1/Chemr23−/−xApoe−/− mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. Results: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe−/− mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1–mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. Conclusions: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver

Rationale: The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. Objective: To investigate hepatic T-cell subsets upon hypercholesterolemia. Methods and Results: We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-&bgr;1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr−/−) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr−/− mice led to intrahepatic Th1 cell differentiation and CD11b+CD11c+ leukocyte accumulation. Conclusions: Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4+ T-cell pool in the atherosclerotic aorta.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Abstract Aims Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Hypercholesterolemia Enhances T Cell Receptor Signaling and Increases the Regulatory T Cell Population

Hypercholesterolemia promotes the inflammation against lipoproteins in atherosclerosis. Development of atherosclerosis is affected by the balance between pro-inflammatory effector T cells and anti-inflammatory regulatory T (Treg) cells. However, phenotype and function of T cell subpopulations in hypercholesterolemia remain to be investigated. Here, we found that cholesterol-containing diet increased the expression of the Treg cell lineage-defining transcription factor FoxP3 among thymocytes and splenocytes. Hypercholesterolemia elevated the FoxP3 expression level and population size of peripheral Treg cells, but did not prevent enhanced proliferation of stimulated T cells. Moreover, cholesterol supplementation in diet as well as in cell culture medium promoted T cell antigen receptor (TCR) signaling in CD4+ T cells. Our results demonstrate that hypercholesterolemia enhances TCR stimulation, Treg cell development as well as T cell proliferation. Thus, our findings may help to understand why hypercholesterolemia correlates with altered CD4+ T cell responses.