T-cell specificity and regulation in atherosclerosis

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, a chronic inflammatory disease. Subendothelial retention of lipoproteins triggers monocyte-derived macrophages and T-helper (Th) 1 cells to form lipid-laden atherosclerotic plaques in the artery wall. The Th1 cells react to autoantigens from the ApoB protein in low-density lipoprotein (LDL) perpetuating the inflammation initiated by the innate immune reactions to modified lipoproteins. Other T-helper cells are also active in the lesions with regulatory T cells (Treg) limiting the injurious inflammation, while the effects of Th17 cells are less clear. The slow build-up of atherosclerotic plaques is asymptomatic, but eventually the plaque may cause symptoms. Plaque rupture or endothelial erosion induces thrombus formation that causes a heart attack or ischemic stroke. Advanced plaques usually contain large cholesterol-rich necrotic cores. This determines plaque stability along with a stable cap formation by smooth muscle cells and collagen. Prevention of risk factors has reduced mortality, but there is still a need for novel therapies to stabilize plaques and to treat arterial inflammation. The aim for this thesis is to investigate T-cell responses to LDL and regulation of Th cells during atherogenesis. Genetically modified mouse models were used to study LDL-reactive T cells, mechanisms involved in Th cell differentiation, and the subsequent influence on disease development. Paper I shows how inflammatory signals from the atherosclerotic lesions contribute to Th17 cell differentiation by means of IL-6 and transforming growth factor β (TGF-β). Th17 cells produce IL-17A that promotes collagen synthesis by smooth muscle cells. This paper establishes a plaque-stabilizing role for Th17 cells and IL-17A, which is likely to operate in man and reduce incidence of myocardial infarctions. Paper II establishes that Tregs have a protective role in atherosclerosis by modulating lipid metabolism. Depletion of Foxp3+ Tregs during atherogenesis impairs lipoprotein uptake by unleashing liver inflammation that downregulates the very low-density lipoprotein (VLDL)-regulating protein called sortilin. This leads to increased plasma cholesterol and development of large atherosclerotic plaques with lipid-filled necrotic cores. Paper III shows how LDL-reactive T cells survive clonal selection in the thymus, differentiate into T follicular helper cells (Tfh), and promote a protective B-cell response with anti-LDL antibodies. These antibodies mediate lipoprotein clearance and lower plasma cholesterol, which protects against atherosclerosis. All three papers presented in this thesis illustrate an intricate interplay between the immune system and lipoprotein metabolism, resulting in profound effects on atherosclerosis. These notions may lead to new therapies that stabilize atherosclerotic plaques through specific anti-inflammatory actions that are mirrored by lipid-lowering effects

Platelets enhance CD4+ central memory T cell responses via platelet factor 4-dependent mitochondrial biogenesis and cell proliferation

Platelets regulate multiple aspects of CD4+ T cell immunity, and may exert distinct regulations among different T cell subsets. Our aim was to investigate how platelets regulate CD4+ central memory T cell (Tcm) responses. αCD3/αCD28-stimulated human CD4+ Tcm cells were cultured without or with platelets or platelet-derived mediators. Polyclonal stimulation induced cell proliferation and Th1 and Treg cell activation of Tcm cells. Platelet factor 4/PF4 neutralization abolished platelet-enhanced Tcm effector responses, whilst TGFβ neutralization only partially inhibited platelet-enhanced Treg cell activation. PF4 supplementation mimicked the effects of platelet co-cultures, while PF4 receptor CXCR3 blockade and CXCR3 knockdown with siRNAs inhibited or abolished PF4-enhanced Th1 and Treg cell responses. Platelet co-cultures or PF4-treatment increased Tcm cell proliferation, whilst CXCR3 blockade counteracted. PF4-enhanced Tcm proliferation and effector cell responses were associated with mitochondrial biogenesis. Overexpression of mitochondrial transcription factor A (TFAM) mimicked PF4 effects, and PF4 treatment attenuated Akt phosphorylation of activated Tcm cells, leading to mitochondrial biogenesis. Impacts of platelets and PF4 on Tcm proliferation were further confirmed by that CXCR3 knockdown/blockade counteracted PF4-enhanced Tcm cell proliferation. In conclusion, platelets enhance Th1 and Treg cell responses of CD4+ Tcm cells, via PF4-dependent mitochondrial biogenesis and cell proliferation of Tcm cells

Increased carotid artery lesion inflammation upon treatment with the CD137 agonistic antibody 2A

Background: Increased inflammatory activity destabilizes the atherosclerotic lesion and may lead to atherothrombosis and symptomatic cardiovascular disease. Co-stimulatory molecules, such as CD137, are key regulators of inflammation, and CD137 activity regulates inflammation in experimental atherosclerosis. Here, we hypothesized that CD137 activation promotes carotid artery inflammation and atherothrombosis. Methods and Results: In a model of inducible atherothrombosis with surgical ligation of the right carotid artery and a subsequent placement of a polyethene cuff, elevated levels of CD137 and CD137 ligand mRNA in atherothrombotic vs. non-atherothrombotic murine carotid lesions was observed. Mice treated with the CD137 agonistic antibody 2A showed signs of increased inflammation in the aorta and a higher proportion of CD8+ T cells in spleen and blood. In carotid lesions of 2A-treated mice, significantly higher counts of CD8+ and major histocompatibility (MHC)-class II molecule I-Ab+ cells were observed. Treatment with the CD137 agonistic antibody 2A did not significantly affect the atherothrombosis frequency in 16-week-old mice in this model. Conclusions: Levels of CD137 and CD137 ligand mRNA were higher in advanced atherosclerotic disease compared to control vessels, and treatment with the CD137 agonistic antibody 2A, in a murine model for inducible atherothrombosis promoted vascular inflammation, but had no significant effect on atherothrombosis frequency at this early disease stage

Transforming growth factor-β2 is associated with atherosclerotic plaque stability and lower risk for cardiovascular events

AIMS: Transforming growth factor-beta (TGF-β) exists in three isoforms TGF-β1, -β2 and -β3. TGF-β1 has been suggested to be important for maintaining plaque stability, yet the role of TGF-β2 and -β3 in atherosclerosis remains to be investigated. OBJECTIVE: This study explores the association of these three isoforms of TGF-β with plaque stability in the human atherosclerotic disease. METHODS AND RESULTS: TGF-β1, -β2 and -β3 proteins were quantified in 223 human carotid plaques by immunoassays. Indications for the endarterectomy were: symptomatic carotid plaque with stenosis >70% or without symptoms and >80% stenosis. Plaque mRNA levels were assessed by RNA sequencing. Plaque components and extracellular matrix were measured histologically and biochemically. Matrix metalloproteinases were measured with ELISA. Monocyte chemoattractant protein-1 (MCP-1) was measured with immunoassays. The effect of TGF-β2 on inflammation and protease activity was investigated in vitro using THP-1 and RAW264.7 macrophages. Patients were followed longitudinally for cardiovascular events.TGF-β2 was the most abundant isoform and was increased at both protein and mRNA levels in asymptomatic plaques. TGF-β2 was the main determinant separating asymptomatic plaques in an Orthogonal Projections to Latent Structures Discriminant Analysis. TGF-β2 correlated positively to features of plaque stability and inversely to markers of plaque vulnerability. TGF-β2 was the only isoform inversely correlated to the matrix-degrading matrix metalloproteinase-9 and inflammation in the plaque tissue. In vitro, TGF-β2 pre-treatment reduced MCP-1 gene and protein levels as well as matrix metalloproteinase-9 gene levels and activity. Patients with plaques with high TGF-β2 levels had a lower risk to suffer from future cardiovascular events. CONCLUSIONS: TGF-β2 is the most abundant TGF-β isoform in human plaques and may maintain plaque stability by decreasing inflammation and matrix degradation

Genetic deficiency of indoleamine 2,3-dioxygenase aggravates vascular but not liver disease in a nonalcoholic steatohepatitis and atherosclerosis comorbidity model

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe−/− and Apoe−/−Ido1−/− mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe−/−Ido1−/− mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe−/−Ido1−/− mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe−/− and Apoe−/−Ido1−/mice−. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues

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 metabolis