Maria Wigren

Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine

Abstract.  Wigren M, Kolbus D, Dunér P, Ljungcrantz I, Söderberg I, Björkbacka H, Fredrikson GN, Nilsson J. (Malmö University Hospital, Lund University; Malmö University, Malmo; Sweden) Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine. J Intern Med 2011; 269: 546–556.

Low Levels of Circulating CD4+FoxP3+ T Cells Are Associated With an Increased Risk for Development of Myocardial Infarction But Not for Stroke.

Objective—Regulatory T cells (Tregs) protect against atherosclerosis in experimental models, but their association with cardiovascular disease in humans remains to be elucidated. The aim of the present study was to determine whether circulating Tregs predict the development of acute cardiovascular events in humans. Methods and Results—The study cohort consisted of a random sample of participants (n=700), aged 68 to 73 years, from the Malmö Diet and Cancer Study. Mononuclear leukocytes, stored at −140○C at the baseline investigation in 1991–1994, were thawed and Tregs, defined by the expression of FoxP3 in CD4+ T cells, were analyzed by flow cytometry. There was no detectable loss of cells during storage, and the viability of thawed leukocytes was 95%. A low fraction of both CD4+FoxP3+ and CD4+CD25+FoxP3+ T cells was associated with a higher release of proinflammatory cytokines from activated mononuclear leukocytes, and this association was strongest for CD4+FoxP3+ cells. Eighty-four coronary events and 66 strokes were registered during follow-up until December 31, 2008. In a Cox proportional hazard regression model adjusting for major risk factors, low levels of baseline CD4+FoxP3+ T cells were associated with an increased risk for the development of acute coronary events but not stroke. There were no associations between CD4+CD25+FoxP3+ T cells and development of an acute coronary event or stroke. Conclusion—This study provides prospective evidence for the role of Tregs in the development of myocardial infarction. The findings are in accordance with previous experimental studies and provide clinical support for a protective role of Tregs in atherosclerosis. The lack of association between Tregs and stroke may reflect the more heterogeneous cause of this disease.

CD8+ T cell activation predominate early immune responses to hypercholesterolemia in Apoe-/- mice

BackgroundIt is well established that adaptive immune responses induced by hypercholesterolemia play an important role in the development of atherosclerosis, but the pathways involved remain to be fully characterized. In the present study we assessed immune responses to hypercholesterolemia induced by feeding Apoe-/- mice a high-fat diet for 4 or 8 weeks.ResultsThe primary immune response in lymph nodes draining the aortic root was an increased expression of interferon (IFN)-γ in CD8+CD28+ T cells, while an activation of IFN-γ expression in CD4+ T cells was observed only after 8 weeks of high-fat diet. Contrarily, spleen CD4+ T cells responded with a higher expression of IL-10. Spleen CD8+ T cells expressed both IFN-γ and IL-10 and showed enhanced proliferation when exposed to Concanavalin A. Plasma levels of IgG and IgM against oxidized LDL did not change, but the level of apolipoprotein B/IgM immune complexes was increased.ConclusionHypercholesterolemia leads to unopposed activation of Th1 immune responses in lymph nodes draining atherosclerotic lesions, whereas Th1 activation in the spleen is balanced by a concomitant activation of Th2 cells. The activation of CD8+ T cells implies that hypercholesterolemia is associated with formation of cell autoantigens.

Atheroprotective Effects of Alum Are Associated With Capture of Oxidized LDL Antigens and Activation of Regulatory T Cells

The immune system represents a promising novel target for prevention of atherosclerosis. Several pilot vaccines that reduce atherosclerosis in experimental animals have been developed. The aluminum hydroxide adjuvant Alum has been shown to have antiatherogenic properties in itself, suggesting that it may be a suitable adjuvant in possible future atherosclerosis vaccines. To characterize the immune pathways mediating this protection, we treated wild-type C57BL/6 and Apoe−/− mice with Alum or PBS. Analyses of splenocytes isolated from 12-week-old mice demonstrated that Alum increased the presence of CD4+CD25+FoxP3+ regulatory T cells and downregulated the expression of T cell activation markers CD28 and ICOS in Apoe−/− mice but not in C57BL/6 wild-type mice. A similar immunosuppressive phenotype was found also in 25-week-old Apoe−/− mice and was associated with reduced atherosclerosis. Alum precipitates recovered from the injection site of Apoe−/− mice contained antigens derived from oxidized LDL. These findings demonstrate that treatment of Apoe−/− mice with Alum results in an increase of regulatory T cells and suggest that these are activated by tolerogenic antigen-presenting cells presenting oxidized LDL antigens. Our findings provide improved mechanistic understanding of the atheroprotective properties of aluminum hydroxide adjuvants but also point to the importance of determining if hypercholesterolemia may compromise the efficacy of Alum-containing vaccines used clinically today.

T-Helper 2 Immunity Is Associated With Reduced Risk of Myocardial Infarction and Stroke

Objective—Experimental studies in mice have attributed T-helper (Th) 1 and Th2 cells important roles in atherosclerosis, but the clinical importance of these cells in cardiovascular disease (CVD) remains to be clarified. Here, we investigated associations between Th1 and Th2 cells, carotid intima-media thickness, and cardiovascular risk. Methods and Results—Blood drawn at baseline and incident cardiovascular events during 15-year follow-up were assessed in 700 participants. Baseline Th1 (CD3+CD4+interferon-&ggr;+) and Th2 (CD3+CD4+interleukin-4+) cells were analyzed by flow cytometry, and cytokine-release from activated mononuclear leukocytes was measured by multiplex technology. High numbers of Th2 cells were independently associated with decreased mean common carotid intima-media thickness. High numbers of Th2 cells were also independently associated with a reduced risk of acute myocardial infarction in women (hazard ratio, 0.19; 95% confidence interval, 0.06–0.56; P=0.002 for the highest versus the lowest tertile of Th2 cells). Moreover, release of the Th2 cytokine interleukin-4 from activated mononuclear leukocytes was independently associated with a reduced risk of CVD. No independent associations between Th1 cells and carotid intima-media thickness or CVD risk were found. Conclusion—Our observations provide the first clinical evidence for a protective role of Th2 immunity in CVD. They also suggest this protection is more prominent in women than in men. In spite of convincing evidence from experimental studies, we found no support for a role of Th1 immunity in CVD.

Regulatory T cells and the control of modified lipoprotein autoimmunity-driven atherosclerosis.

It has long been recognized that arterial inflammation plays a key role in the development of atherosclerosis. More recent evidence has suggested that this inflammation is modulated by autoimmune responses against modified self-antigens, such as oxidized low-density lipoprotein, in the vascular wall. However, the role of the immune system in atherosclerosis appears to be more complex than in classic autoimmune diseases; and a number of protective immune responses have also been identified. One of the most important of these is carried out by the regulatory T cells. Regulatory T cells inhibit the development of autoimmunity by controlling the activity of autoreactive T cells. If the function of regulatory T cells is compromised in hypercholesterolemic mouse models of atherosclerosis, the development of disease becomes much more aggressive. In this review, we will discuss the possibility that the inflammatory activity in atherosclerotic lesions depends on the balance between plaque antigen-specific proinflammatory Th1-type T cells and anti-inflammatory regulatory T cells specific for the same antigen. We will also discuss the role of hypercholesterolemia in generation of these modified self-antigens as well as ongoing research aiming to develop novel immune-modulating therapy for prevention of cardiovascular disease by targeting these processes.

FcgammaRIIB inhibits the development of atherosclerosis in low-density lipoprotein receptor-deficient mice

The immune processes associated with atherogenesis have received considerable attention during recent years. IgG FcRs (FcγR) are involved in activating the immune system and in maintaining peripheral tolerance. However, the role of the inhibitory IgG receptor FcγRIIB in atherosclerosis has not been defined. Bone marrow cells from FcγRIIB-deficient mice and C57BL/6 control mice were transplanted to low-density lipoprotein receptor-deficient mice. Atherosclerosis was induced by feeding the recipient mice a high-fat diet for 8 wk and evaluated using Oil Red O staining of the descending aorta at sacrifice. The molecular mechanisms triggering atherosclerosis was studied by examining splenic B and T cells, as well as Th1 and Th2 immune responses using flow cytometry and ELISA. The atherosclerotic lesion area in the descending aorta was ~5-fold larger in mice lacking FcγRIIB than in control mice (2.75 ± 2.57 versus 0.44 ± 0.42%; p < 0.01). Moreover, the FcγRIIB deficiency resulted in an amplified splenocyte proliferative response to Con A stimulation (proliferation index 30.26 ± 8.81 versus 2.96 ± 0.81%, p < 0.0001) and an enhanced expression of MHC class II on the B cells (6.65 ± 0.64 versus 2.33 ± 0.25%; p < 0.001). In accordance, an enlarged amount of CD25-positive CD4 T cells was found in the spleen (42.74 ± 4.05 versus 2.45 ± 0.31%; p < 0.0001). The plasma Ab and cytokine pattern suggested increased Th1 and Th2 immune responses, respectively. These results show that FcγRIIB inhibits the development of atherosclerosis in mice. In addition, they indicate that absence of the inhibiting IgG receptor cause disease, depending on an imbalance of activating and inhibiting immune cells.

Lymphocytes in atherosclerosis

It is well established that atherosclerosis is caused by an inflammatory process in the arterial intima. However, it is only in recent years that it has become clear that this inflammation is modulated by immune responses against plaque antigens. These antigens are primarily believed to be modified self-antigens such as oxidized LDL. The immune system is challenged to determine whether these antigens should be regarded self and tolerated or non-self and eliminated. The latter will result in plaque development while the first will be protective. T cells are key effectors of both types of responses. An activation of regulatory T cells inhibits auto-reactive T effector cells and is anti-inflammatory. In contrast, if Th1 cells become activated in the plaque this is associated with increased inflammation and disease progression. The role of B cells in atherosclerosis remains to be clarified but some species of athero-protective antibodies have been identified. The elucidation of role of immune system in atherosclerosis has revealed new targets for intervention and both vaccines and antibody-based therapies are presently in or due to enter clinical testing.

Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice

Objective of the Study Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. Methodology and Principal Findings Streptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. Conclusions Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

Vaccines against atherosclerosis.

Atherosclerosis is the primary cause of acute myocardial infarction and stroke. It is well established that arterial inflammation in response to accumulation and oxidation of lipoproteins in the vascular wall is the major factor responsible for the development of atherosclerosis. During recent years, it has become apparent that this vascular inflammation is modulated by a complex array of autoimmune responses against modified self-antigens in the atherosclerotic plaque and that both protective and pathogenic immune responses become activated as part of the disease process. Studies of hypercholesterolemia-induced immune activation in mouse models of atherosclerosis have demonstrated that Th1 cells contribute to disease progression while regulatory T cells are protective. It has been suggested that antigen presentation of modified self-antigens in the inflammatory environment of atherosclerotic plaques favors generation of antigen-specific Th1 cells over that of regulatory T cells, resulting in a local loss of tolerance. This concept has stimulated the development of plaque-antigen tolerogenic vaccines to dampen plaque inflammation and disease progression. A first generation of atherosclerosis vaccines based on peptides derived from apoB100 and heat shock proteins have demonstrated promising results in animal studies and are now approaching clinical testing.