Anna-Karin L. Robertson

Natural regulatory T cells control the development of atherosclerosis in mice

Atherosclerosis is an immunoinflammatory disease elicited by accumulation of lipids in the artery wall and leads to myocardial infarction and stroke. Here, we show that naturally arising CD4+CD25+ regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are powerful inhibitors of atherosclerosis in several mouse models. These results provide new insights into the immunopathogenesis of atherosclerosis and could lead to new therapeutic approaches that involve immune modulation using regulatory T cells.

Disruption of TGF-β signaling in T cells accelerates atherosclerosis

Increasing evidence suggests that atherosclerosis is an inflammatory disease promoted by hypercholesterolemia. The role of adaptive immunity has been controversial, however. We hypothesized that proatherogenic T cells are controlled by immunoregulatory cytokines. Among them, TGF-β has been implied in atherosclerosis, but its mechanism of action remains unclear. We crossed atherosclerosis-prone ApoE-knockout mice with transgenic mice carrying a dominant negative TGF-β receptor II in T cells. The ApoE-knockout mice with disrupted TGF-β signaling in T cells exhibited a sixfold increase in aortic lesion surface area, a threefold increase in aortic root lesion size, and a 125-fold increase in aortic IFN-γ mRNA when compared with age-matched ApoE-knockout littermates. When comparing size-matched lesions, those of mice with T cell–specific blockade of TGF-β signaling displayed increased T cells, activated macrophages, and reduced collagen, consistent with a more vulnerable phenotype. Ab’s to oxidized LDL, circulating T cell cytokines, and spleen T cell activity were all increased in ApoE-knockout mice with dominant negative TGF-β receptors in T cells. Taken together, these results show that abrogation of TGF-β signaling in T cells increases atherosclerosis and suggest that TGF-β reduces atherosclerosis by dampening T cell activation. Inhibition of T cell activation may therefore represent a strategy for antiatherosclerotic therapy.

T Cells in Atherogenesis

The idea that atherosclerosis is an inflammatory disease is no longer controversial. Instead, much of the current research is now focused on understanding what drives this inflammation and how it is regulated. Adaptive immunity, in particular T cells, is highly involved in atherogenesis. It is well known that different subsets of T cells can drive or dampen inflammatory processes, but we still have much to learn about the regulation of this balance in the context of atherosclerosis. This review summarizes our knowledge of T cells in atherogenesis, their potential antigens, their contact-dependent activities, and their secretion of inflammatory and antiinflammatory mediators, aiming to illustrate how T cells can aggravate or attenuate this disease through cross-talk with other cells within or outside the atherosclerotic plaque.

Enhanced T-cell expression of RANK ligand in acute coronary syndrome: possible role in plaque destabilization.

Objective—Based on its role in inflammation and matrix degradation, we hypothesized a role for osteoprotegerin (OPG), RANK, and RANK ligand (RANKL) in coronary artery disease. Methods and Results—We examined the expression of various members of the OPG/RANKL/RANK axis in patients with stable and unstable angina and in the atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice. Our findings were: (1) Serum levels of OPG were raised in patients with unstable angina (n=40), but not in those with stable angina (n=40), comparing controls (n=20); (2) mRNA levels of RANKL were increased in T-cells in unstable angina patients accompanied by increased expression of RANK in monocytes; (3) strong immunostaining of OPG/RANKL/RANK was seen within thrombus material obtained at the site of plaque rupture during acute myocardial infarction; (4) OPG/RANKL/RANK was expressed in the atherosclerotic plaques of apoE−/− mice, with RANKL located specifically to the plaques; and (5) RANKL enhanced the release of monocyte chemoattractant peptide-1 in mononuclear cells from unstable angina patients, and promoted matrix metalloproteinase (MMP) activity in vascular smooth muscle cells. Conclusions—We show enhanced expression of the OPG/RANKL/RANK system both in clinical and experimental atherosclerosis, with enhanced T-cell expression of RANKL as an important feature of unstable disease.

Lesion Development and Response to Immunization Reveal a Complex Role for CD4 in Atherosclerosis

Atherosclerosis is a complex disease, bearing many of the characteristics of a chronic inflammatory process. Both cellular and humoral immune responses may be involved in the disease development. Oxidized low-density lipoprotein (oxLDL) is suggested to be an autoantigen in atherosclerosis. A protective effect against atherosclerosis has been demonstrated in animals immunized with oxLDL. Such a protection is associated with elevation of T cell–dependent IgG antibodies against oxLDL. In addition, it has been shown that immunization with Freund adjuvant alone also confers protection against development of atherosclerosis. We therefore hypothesized that CD4+ T cells are critical in the development of atherosclerosis and that they are involved in protective immune reactions after immunization. The development of atherosclerosis was studied in apolipoprotein E knockout (apoE KO) mice and CD4/apoE double knockout (dKO) mice that were immunized with either oxLDL in Freund adjuvant or adjuvant alone, or left untreated. Our results show that (1) the absence of CD4+ cells in apoE KO mice leads to reduced atherosclerosis, indicating that CD4+ cells constitute a major proatherogenic cell population, and (2) the atheroprotective effect of LDL immunization does not depend on CD4+ cells, whereas (3) the atheroprotective effect of adjuvant injection is CD4-dependent. These findings demonstrate complex roles of immune cell-cell interactions in the regulation of the atherosclerotic process and point to several possible targets in the treatment and prevention of atherosclerosis.

Enhanced Expression of the Homeostatic Chemokines CCL19 and CCL21 in Clinical and Experimental Atherosclerosis. Possible Pathogenic Role in Plaque Destabilization

Objective—Based on their role in T-cell homing into nonlymphoid tissue, we examined the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in coronary artery disease (CAD). Methods and Results—We performed studies in patients with stable (n=40) and unstable (n=40) angina and healthy controls (n=20), in vitro studies in T-cells and macrophages, and studies in apolipoprotein-E–deficient (ApoE−/−) mice and human atherosclerotic carotid plaques. We found increased levels of CCL19 and CCL21 within the atherosclerotic lesions of the ApoE−/− mice, in human atherosclerotic carotid plaques, and in plasma of CAD patients. Whereas strong CCR7 expression was seen in T-cells from murine and human atherosclerotic plaques, circulating T-cells from angina patients showed decreased CCR7 expression. CCL19 and CCL21 promoted an inflammatory phenotype in T-cells and macrophages and increased matrix metalloproteinase (MMP) and tissue factor levels in the latter cell type. Although aggressive statin therapy increased CCR7 and decreased CCL19/CCL21 levels in peripheral blood from CAD patients, conventional therapy did not. Conclusions—The abnormal regulation of CCL19 and CCL21 and their common receptor in atherosclerosis could contribute to disease progression by recruiting T-cells and macrophages to the atherosclerotic lesions and by promoting inflammatory responses in these cells.

Lack of Complement Factor C3, but Not Factor B, Increases Hyperlipidemia and Atherosclerosis in Apolipoprotein E-/- Low-Density Lipoprotein Receptor-/- Mice

Objective—To investigate the effect of complement deficiency on atherogenesis and lipidemia, we used mice deficient in the third complement component (C3−/−) or factor B (FB−/−). Methods and Results—Complement-deficient mice were crossed with mice deficient in both apolipoprotein E and the low-density lipoprotein receptor (Apoe−/−LDLR−/−). The percent lesion area in the aorta at 16 weeks, determined by en face analysis, was 84% higher in C3−/− mice than in controls (11.8%±0.4% versus 6.4%±0.8%, mean±SEM, P<0.00005). The C3−/− mice also had 58% higher serum triglyceride levels (P<0.05) and a more proatherogenic lipoprotein profile, with significantly more low-density lipoprotein cholesterol and very-low-density lipoprotein triglycerides than control mice. The C3−/− mice weighed 13% less (P<0.01) and had a lower body fat content (3.5%±1.0% versus 13.1%±3.0%, P<0.01). There were no differences between FB−/− mice and controls. Conclusions—Complement activation by the classical or lectin pathway exerts atheroprotective effects, possibly through the regulation of lipid metabolism.

T Cell–Mediated Inflammation in Adipose Tissue Does Not Cause Insulin Resistance in Hyperlipidemic Mice

Obesity is associated with chronic inflammation in adipose tissue. Proinflammatory cytokines including tumor necrosis factor-&agr; and interleukin-6 secreted by adipose tissue during the metabolic syndrome are proposed to cause local and general insulin resistance and promote development of type 2 diabetes. We have used a compound mutant mouse, Apoe−/−×CD4dnTGFbR, with dysregulation of T-cell activation, excessive production of proinflammatory cytokines, hyperlipidemia, and atherosclerosis, to dissect the role of inflammation in adipose tissue metabolism. These mice are lean, which avoids confounding effects of concomitant obesity. Expression and secretion of a set of proinflammatory factors including tumor necrosis factor-&agr;, interferon-&ggr;, and monocyte chemoattractant protein-1 was increased in adipose tissue of Apoe−/−×CD4dnTGFbR mice, as was the enzyme 11&bgr;-hydroxysteroid dehydrogenase type 1, which converts cortisone to bioactive cortisol. Interleukin-6, which has an inhibitory glucocorticoid response element in its promoter, was not upregulated. In spite of intense local inflammation, insulin sensitivity was not impaired in adipose tissue of Apoe−/−×CD4dnTGFbR mice unless exogenous interleukin-6 was administered. In conclusion, T-cell activation causes inflammation in adipose tissue but does not lead to insulin resistance in this tissue in the absence of interleukin-6.

T-Cell Activation Leads to Reduced Collagen Maturation in Atherosclerotic Plaques of Apoe−/− Mice

Rupture of the collagenous, fibrous cap of an atherosclerotic plaque commonly causes thrombosis. Activated immune cells can secrete mediators that jeopardize the integrity of the fibrous cap. This study aimed to determine the relationship between T-cell-mediated inflammation and collagen turnover in a mouse model of experimental atherosclerosis. Both Apoe(-/-) x CD4dnTbetaRII mice with defective transforming growth factor-beta receptors in T cells (and hence released from tonic suppression of T-cell activation) and lesion size-matched Apoe(-/-) mice were used. Picrosirius red staining showed a lower content of thick mature collagen fibers in lesions of Apoe(-/-) x CD4dnTbetaRII mice, although both groups had similar levels of procollagen type I or III mRNA and total collagen content in lesions. Analysis of both gene expression and protein content showed a significant decrease of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, in aortas of Apoe(-/-)--CD4dnTbetaRII mice. T-cell-driven inflammation provoked a selective and limited increase in the expression of proteinases that catabolize the extracellular matrix. Atheromata of Apoe(-/-)--CD4dnTbetaRII mice had increased levels of matrix metalloproteinase-13 and cathepsin S mRNAs and of the active form of cathepsin S protein but no increase was detected in collagen fragmentation. Our results suggest that exaggerated T-cell-driven inflammation limits collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation.

TGF-β in Atherosclerosis

To the Editor: In his excellent review on the role of transforming growth factor (TGF)-β in atherosclerosis in the March 2004 issue of Arteriosclerosis, Thrombosis, and Vascular Biology ,1 David J. Grainger discusses the multiple roles of this growth factor and suggests that direct effects on vascular cells as well as effects exerted primarily on immune cells may account for its antiatherogenic properties. He points out the discrepancy between the experiments applying generalized TGF-β suppression and those using dominant-negative (dn) TGF-β receptors in T cells. Using neutralizing anti–TGF-β antibodies in apoE−/− mice, Mallat et al observed an increased lesion size,2 whereas transplantation of bone marrow containing T cells with dn–TGF-β receptors led to reduced lesions.3 Therefore, Dr. Grainger concludes that TGF-β signaling in the inflammatory cell population cannot be the whole story, and he suggests that the effects of this cytokine on smooth muscle cells (SMCs) determine the formation of matrix-rich, stable plaques. However, new data published after the acceptance of his review article suggests that immunosuppressive effects of TGF-β modulate the growth and stability of the atherosclerotic lesion as well as its inflammatory properties. In an article published in the November 2003 issue of the Journal of Clinical Investigation , we show that apoE−/− mice with abrogated TGF-β signaling in T cells develop dramatically accelerated atherosclerosis with a several-fold increase in lesion size as well as a more vulnerable lesion phenotype with reduced collagen and increased inflammation.4 These proatherogenic effects were …