Amanda C. Foks

Vaccination against Foxp3(+) regulatory T cells aggravates atherosclerosis.

OBJECTIVE Regulatory T cells are crucial for immune homeostasis and an impaired regulatory T cell function results in many pathological conditions. Regulatory T cells have already been described to be protective in atherosclerosis. However the exact contribution of Foxp3-expressing natural regulatory T cells in atherosclerosis has not been elucidated yet. METHODS AND RESULTS In this study we vaccinated LDL receptor deficient mice with dendritic cells which are transfected with Foxp3 encoding mRNA and studied the effect on initial atherosclerosis. Vaccination against Foxp3 resulted in a reduction of Foxp3(+) regulatory T cells in several organs and in an increase in initial atherosclerotic lesion formation. Furthermore we observed an increase in plaque cellularity and increased T cell proliferation in the Foxp3 vaccinated mice. CONCLUSION We further establish the protective role of Tregs in atherosclerosis. The results illustrate the important role for Foxp3-expressing regulatory T cells in atherosclerosis, thereby providing a potential opportunity for therapeutic intervention against this disease.

Differential effects of regulatory T cells on the initiation and regression of atherosclerosis

OBJECTIVE Regulatory T cells (Tregs) play an important role in the regulation of T cell-mediated immune responses through suppression of T cell proliferation and cytokine production. In atherosclerosis, a chronic autoimmune-like disease, an imbalance between pro-inflammatory cells (Th1/Th2) and anti-inflammatory cells (Tregs) exists. Therefore, increased Treg numbers may be beneficial for patients suffering from atherosclerosis. In the present study, we determined the effect of a vast expansion of Tregs on the initiation and regression of well-established lesions. METHODS AND RESULTS For in vivo Treg expansion, LDL receptor deficient (LDLr(-/-)) mice received repeated intraperitoneal injections of a complex of IL-2 and anti-IL-2 mAb. This resulted in a 10-fold increase in CD4(+)CD25(hi)Foxp3(+) T cells, which potently suppressed effector T cells ex vivo. During initial atherosclerosis, IL-2 complex treatment of LDLr(-/-) mice fed a Western-type diet reduced atherosclerotic lesion formation by 39%. The effect on pre-existing lesions was assessed by combining IL-2 complex treatment with a vigorous lowering of blood lipid levels in LDLr(-/-) mice. This did not induce regression of atherosclerosis, but significantly enhanced lesion stability. CONCLUSION Our data show differential roles for Tregs during atherosclerosis: Tregs suppress inflammatory responses and attenuate initial atherosclerosis development, while during regression Tregs can improve stabilization of the atherosclerotic lesions.

Treating Atherosclerosis With Regulatory T Cells

Regulatory T cells (Tregs) play an important role in the regulation of T-cell-mediated immune responses through suppression of T-cell proliferation and secretion of inhibitory cytokines, such as interleukin-10 and transforming growth factor-β. Impaired Treg numbers and function have been associated with numerous diseases, and an imbalance between proinflammatory/proatherogenic cells and Tregs promotes atherosclerotic disease. Restoration of this balance by inducing Tregs has great therapeutic potential to prevent cardiovascular disease. In addition to suppressing differentiation and function of effector T cells, Tregs have been shown to induce anti-inflammatory macrophages, inhibit foam cell formation and to influence cholesterol metabolism. Furthermore, Tregs suppress immune responses of endothelial cells and innate lymphoid cells. In this review, we focus on the recent knowledge on Treg subsets, their activity and function in atherosclerosis, and discuss promising strategies to use Tregs as a therapeutic tool to prevent cardiovascular disease.

Interruption of the OX40–OX40 Ligand Pathway in LDL Receptor–Deficient Mice Causes Regression of Atherosclerosis

Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)–OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40–OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr−/− mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L–treated mice compared with control mice. Interference of the OX40–OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein–specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40–OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5–producing T cells and oxidized low-density lipoprotein–specific IgM and reductions in Th2 and mast cells.

Blockade of Tim-1 and Tim-4 Enhances Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Objective— T cell immunoglobulin and mucin domain (Tim) proteins are expressed by numerous immune cells, recognize phosphatidylserine on apoptotic cells, and function as costimulators or coinhibitors. Tim-1 is expressed by activated T cells but is also found on dendritic cells and B cells. Tim-4, present on macrophages and dendritic cells, plays a critical role in apoptotic cell clearance, regulates the number of phosphatidylserine-expressing activated T cells, and is genetically associated with low low-density lipoprotein and triglyceride levels. Because these functions of Tim-1 and Tim-4 could affect atherosclerosis, their modulation has potential therapeutic value in cardiovascular disease. Approach and Results— ldlr −/− mice were fed a high-fat diet for 4 weeks while being treated with control (rat immunoglobulin G1) or anti-Tim-1 (3D10) or -Tim-4 (21H12) monoclonal antibodies that block phosphatidylserine recognition and phagocytosis. Both anti-Tim-1 and anti-Tim-4 treatments enhanced atherosclerosis by 45% compared with controls by impairment of efferocytosis and increasing aortic CD4+T cells. Consistently, anti-Tim-4-treated mice showed increased percentages of activated T cells and late apoptotic cells in the circulation. Moreover, in vitro blockade of Tim-4 inhibited efferocytosis of oxidized low-density lipoprotein–induced apoptotic macrophages. Although anti-Tim-4 treatment increased T helper cell (Th)1 and Th2 responses, anti-Tim-1 induced Th2 responses but dramatically reduced the percentage of regulatory T cells. Finally, combined blockade of Tim-1 and Tim-4 increased atherosclerotic lesion size by 59%. Conclusions— Blockade of Tim-4 aggravates atherosclerosis likely by prevention of phagocytosis of phosphatidylserine-expressing apoptotic cells and activated T cells by Tim-4-expressing cells, whereas Tim-1-associated effects on atherosclerosis are related to changes in Th1/Th2 balance and reduced circulating regulatory T cells.

T-Cell Immunoglobulin and Mucin Domain 3 Acts as a Negative Regulator of Atherosclerosis

Objective—Atherosclerosis is a chronic autoimmune-like disease in which lipids and fibrous elements accumulate in the arterial blood vessels. T cells are present within atherosclerotic plaques, and their activation is partially dependent on costimulatory signals, which can either provide positive or negative signals that promote T-cell activation or limit T-cell responses, respectively. T-cell immunoglobulin and mucin domain 3 (Tim-3) is a coinhibitory type 1 transmembrane protein that affects the function of several immune cells involved in atherosclerosis, such as monocytes, macrophages, effector T cells, and regulatory T cells. In the present study, we determined the role of Tim-3 in the development of atherosclerosis. Approach and Results—Western-type diet–fed low-density lipoprotein receptor–deficient (LDLr−/−) mice were treated with an anti–Tim-3 antibody for 3 and 8 weeks. Anti–Tim-3 administration increased fatty streak formation with 66% and increased atherosclerotic plaque formation after 8 weeks with 35% in the aortic root and with 50% in the aortic arch. Furthermore, blockade of Tim-3 signaling increased percentages of circulating monocytes with 33% and lesional macrophages with 20%. In addition, anti–Tim-3 administration increased CD4+ T cells with 17%, enhanced their activation status, and reduced percentages of regulatory T cells with 18% and regulatory B cells with 37%. Conclusions—It is known that Tim-3 acts as a negative regulator of both innate and adaptive immune responses, and in the present study, we show that anti–Tim-3 treatment augments lesion development, accompanied by an increase in the number of monocytes/macrophages and CD4+ T cells and by decreased regulatory T cells and regulatory B cells.

NLRP3 Inflammasome Inhibition by MCC950 Reduces Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice—Brief Report

Objective— Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1&bgr;. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE−/− mice. Approach and Results— First, we determined the efficacy of MCC950 in vitro. Bone marrow–derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1&bgr; release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide–induced interleukin-1&bgr; secretion, without affecting the tumor necrosis factor-&agr; response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE−/− mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin–stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice. Conclusions— These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development.

Complement factor C5a induces atherosclerotic plaque disruptions

Complement factor C5a and its receptor C5aR are expressed in vulnerable atherosclerotic plaques; however, a causal relation between C5a and plaque rupture has not been established yet. Accelerated atherosclerosis was induced by placing vein grafts in male apoE−/− mice. After 24 days, when advanced plaques had developed, C5a or PBS was applied locally at the lesion site in a pluronic gel. Three days later mice were killed to examine the acute effect of C5a on late stage atherosclerosis. A significant increase in C5aR in the plaque was detectable in mice treated with C5a. Lesion size and plaque morphology did not differ between treatment groups, but interestingly, local treatment with C5a resulted in a striking increase in the amount of plaque disruptions with concomitant intraplaque haemorrhage. To identify the potential underlying mechanisms, smooth muscle cells and endothelial cells were treated in vitro with C5a. Both cell types revealed a marked increase in apoptosis after stimulation with C5a, which may contribute to lesion instability in vivo. Indeed, apoptosis within the plaque was seen to be significantly increased after C5a treatment. We here demonstrate a causal role for C5a in atherosclerotic plaque disruptions, probably by inducing apoptosis. Therefore, intervention in complement factor C5a signalling may be a promising target in the prevention of acute atherosclerotic complications.

Adenosine A2B Receptor Agonism Inhibits Neointimal Lesion Development After Arterial Injury in Apolipoprotein E–Deficient Mice

Objective—The A2B adenosine receptor (A2BR) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A2BR deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A2BR agonism protects against injury-induced intimal hyperplasia. Methods and Results—Apolipoprotein E–deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A2B receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A2B receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583–treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583. Conclusion—Our data show that activation of the adenosine A2B receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A2B receptor agonism as a new therapeutic approach in the prevention of restenosis.

Interference of the CD30–CD30L Pathway Reduces Atherosclerosis Development

Objective—Costimulatory molecules tightly control immune responses by providing positive signals that promote T-cell activation or by transducing inhibitory signals that limit T-cell responses. CD30 and CD30L are members of the tumor necrosis factor receptor superfamily and are involved in the activation and proliferation of T and B cells, which have been implicated in the initiation and progression of atherosclerosis. In the present study, we thus aimed to determine the role of the CD30–CD30L pathway in the development of atherosclerosis. Methods and Results—Western-type diet–fed low-density lipoprotein receptor–deficient mice were treated with an anti-CD30L antibody for 8 weeks, which resulted in a reduction of atherosclerotic lesion formation in the aortic root by 35%. Reduced numbers of adventitial CD3+ T cells were found in anti-CD30L–treated mice, whereas no differences were observed in collagen and macrophage content of the atherosclerotic lesions. B-cell and mast cell responses were also not affected on anti-CD30L treatment. Interestingly, splenocyte proliferation was reduced by 53%, whereas T-cell numbers were concomitantly reduced in anti-CD30L–treated mice compared with control mice. These data thus indicate that the CD30–CD30L pathway solely exerts its function via inhibition of T-cell responses. Conclusion—In the present study, we are the first to show that interruption of the CD30–CD30L pathway reduced initial atherosclerosis development by modulating T-cell function.