Lack of Ability to Present Antigens on Major Histocompatibility Complex Class II Molecules Aggravates Atherosclerosis in ApoE-/- Mice.

BACKGROUND: Hypercholesterolemic mice lacking factors required for activation of CD4+ T cells are characterized by reduced development of atherosclerosis. Consequently, it has been assumed that atherosclerosis involves loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on major histocompatibility complex class II (MHCII) leads to activation of proatherogenic Th1 cells. In this study, we wanted to determine the role of antigen presentation on MHCII in atherosclerosis development. METHODS: Apolipoprotein E (ApoE-/-) mice deficient for MHCII (ApoE-/-MHCII-/-) were used to study the role of MHCII in atherosclerosis development. RESULTS: Compared with ApoE-/- mice, ApoE-/-MHCII-/- mice had reduced levels of CD4+ T cells, immunoglobulin G and M levels, and Th1 and Th2 cytokines in plasma. CD8+ T cells were increased and regulatory T cells were reduced both in spleen and in lesions of ApoE-/-MHCII-/- mice. Decreased plasma levels of inflammatory cytokines in ApoE-/-MHCII-/- mice indicated reduced systemic inflammation. Despite this, ApoE-/-MHCII-/- mice had significantly more atherosclerosis as assessed by en face Oil Red O staining of the aorta (4.7±2.9% versus 1.9±1.3%; P<0.01) and cross-sectional area of subvalvular lesions (7.7±2.2×105 µm2 versus 4.6±2.8×105 µm2; P<0.05). Cell transfer and blocking antibody studies suggested that loss of regulatory T cells is the most important cause of aggravated atherosclerosis in ApoE-/-MHCII-/- mice. CONCLUSIONS: Our observations demonstrate that antigen presentation on MHCII has important protective functions in atherosclerosis and that this is primarily the result of activation of regulatory T cells. These findings have implications for understanding the possible risks and benefits of immunosuppressive therapy in patients with cardiovascular disease.

IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice.

OBJECTIVE: IL-22 is a recently discovered cytokine that belongs to the family of IL-10 related cytokines. It is produced by activated T-cells and innate lymphoid cells and has been suggested to be involved in tissue repair. As both inflammation and repair play important roles in atherosclerosis we investigated if IL-22 deficiency influences the disease process in Apoe(-/-) mice. METHODS: We generated IL-22(-/-)Apoe(-/-) mice and fed them high-fat-diet for 14 weeks to characterize atherosclerosis development. RESULTS: IL-22(-/-)Apoe(-/-) mice exhibited reduced plaque size both in the aorta (p = 0.0036) and the aortic root compared (p = 0.0012) with Apoe(-/-) controls. Moreover, plaque collagen was reduced in IL-22(-/-)Apoe(-/-) mice (p = 0.02) and this was associated with an increased expression of smooth muscle cell (SMC)-α-actin (p = 0.04) and caldesmon (p = 0.016) in the underlying media. Carotid arteries from IL-22(-/-)Apoe(-/-) mice displayed increased expression of genes associated with a contractile SMC phenotype e.g. α-actin (p = 0.004) and caldesmon (p = 0.03). Arterial SMCs were shown to express the IL-22 receptor and in vitro exposure to IL-22 resulted in a down-regulation of alpha actin and caldesmon gene expression in these cells. CONCLUSION: Our observations demonstrate that IL-22 is involved in plaque formation and suggest that IL-22 released by immune cells is involved in activation of vascular repair by stimulating medial SMC dedifferentiation into a synthetic phenotype. This response contributes to plaque growth by enabling SMC migration into the intima but may also help to stabilize the plaque.

IL-25 inhibits atherosclerosis development in apolipoprotein E deficient mice.

OBJECTIVE: IL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice. METHODS AND RESULTS: Administration of 1 µg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 µg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease. CONCLUSIONS: The present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.

Increased inflammation in atherosclerotic lesions of diabetic Akita-LDLr⁻/⁻ mice compared to nondiabetic LDLr⁻/⁻ mice.

BACKGROUND: Diabetes is associated with increased cardiovascular disease, but the underlying cellular and molecular mechanisms are poorly understood. One proposed mechanism is that diabetes aggravates atherosclerosis by enhancing plaque inflammation. The Akita mouse has recently been adopted as a relevant model for microvascular complications of diabetes. Here we investigate the development of atherosclerosis and inflammation in vessels of Akita mice on LDLr⁻/⁻ background. METHODS AND RESULTS: Akita-LDLr⁻/⁻ and LDLr⁻/⁻ mice were fed high-fat diet from 6 to 24 weeks of age. Blood glucose levels were higher in both male and female Akita-LDLr⁻/⁻ mice (137% and 70%, resp.). Male Akita-LDLr⁻/⁻ mice had markedly increased plasma cholesterol and triglyceride levels, a three-fold increase in atherosclerosis, and enhanced accumulation of macrophages and T-cells in plaques. In contrast, female Akita-LDLr⁻/⁻ mice demonstrated a modest 29% increase in plasma cholesterol and no significant increase in triglycerides, atherosclerosis, or inflammatory cells in lesions. Male Akita-LDLr⁻/⁻ mice had increased levels of plasma IL-1ß compared to nondiabetic mice, whereas no such difference was seen between female diabetic and nondiabetic mice. CONCLUSION: Akita-LDLr⁻/⁻ mice display considerable gender differences in the development of diabetic atherosclerosis. In addition, the increased atherosclerosis in male Akita-LDLr⁻/⁻ mice is associated with an increase in inflammatory cells in lesions.

TAP1-deficiency does not alter atherosclerosis development in Apoe-/- mice.

Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8(+) T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8(+) T cell population. We have recently reported an increased activation of CD8(+) T cells in hypercholesterolemic Apoe(-/-) mice. Therefore, this study included TAP1-deficient Apoe(-/-) mice (Apoe(-/-)Tap1(-/-)) to test the atherogenicity of CD8(+) T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8(+) T cell numbers were low in Apoe(-/-)Tap1(-/-) mice in comparison to Apoe(-/-) mice, constituting ~1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by en face Oil Red O staining of the aorta and cross-sections of the aortic root between Apoe(-/-)Tap1(-/-) and Apoe(-/-) mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3(+) T cells in Apoe(-/-)Tap1(-/-) compared to Apoe(-/-) mice. The CD3(+)CD4(+) T cell fraction was increased in Apoe(-/-)Tap1(-/-) mice, suggesting a compensation for the decreased CD8(+) T cell population. Interestingly, the fraction of CD8(+) effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development.In conclusion, Apoe(-/-)Tap1(-/-) mice develop atherosclerosis equal to Apoe(-/-) mice, indicating a minor role for CD8(+) T cells and TAP1-dependent antigen presentation in the disease process.

Immunization with cationized BSA inhibits progression of disease in ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis.

Immune responses against modified self-antigens generated by hypercholesterolemia play an important role in atherosclerosis identifying the immune system as a possible novel target for prevention and treatment of cardiovascular disease. It has recently been shown that these immune responses can be modulated by subcutaneous injection of adjuvant. In the present study we immunized 25-week old ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis with adjuvant and two different concentrations of the carrier molecule cationized BSA (cBSA). Plasma levels of Th2-induced apolipoprotein B (apoB)/IgG1 immune complexes were increased in the cBSA immunized groups verifying induction of immunity against a self-antigen. Mice were sacrificed at 36 weeks of age and atherosclerosis was monitored by en face Oil red O staining of the aorta. Immunization with 100 µg cBSA inhibited plaque progression, whereas the lower dose (50 µg) did not. In addition, the higher dose induced a more stable plaque phenotype, indicated by a higher content of collagen and less macrophages and T cells in the plaques. Moreover, there was an increased ratio of Foxp3+/Foxp3⁻ T cells in the circulation suggesting activation of a regulatory T cell response. In conclusion, we show that immunization with cBSA induces an immune response against apoB as well as an activation of Treg cells. This was associated with development of a more stable plaque phenotype and reduced atherosclerosis progression.

Effects of simvastatin on circulating autoantibodies to oxidized LDL antigens: relation with immune stimulation markers.

Statins exert a number of anti-inflammatory and immunomodulatory effects in vitro. However, the immunomodulatory effects in vivo are less clarified. In the present study, we investigated whether simvastatin treatment changed the levels of autoantibodies against specific oxidized LDL (oxLDL) antigens as well as their association with leukocyte activation markers. Eighty volunteers with mild-to-moderate hypercholesterolemia were randomized to either simvastatin 40 mg or placebo for 6 weeks. Autoantibodies against apo B peptide antigens, C-reactive protein (CRP) and interleukin (IL)-6 in plasma were determined by ELISA. Subsets of circulating B and T cells were studied by flow cytometry. Simvastatin significantly reduced CRP by 26%, whereas IL-6 remained unchanged. Levels of IgG against the apo B peptide P-240 (amino acids 3586-3605) increased by 16% (p = 0.03) in the simvastatin group whereas autoantibody levels to other apo B peptides did not change. At baseline and after 6 weeks, the P-240 IgG levels were significantly correlated with the number of CD57+CD28 - CD8+T cells but not to other lymphocyte subsets or inflammatory markers. The P-240 IgG levels after 6 weeks simvastatin therapy was strongly correlated to the relative increase in CD57+CD28 - CD8+T cells (p = 0.003). Simvastatin treatment induced an increase in autoantibodies against an oxLDL antigen. The effect was related to an expansion of a CD8+T cell subset and may involve an immunostimulation by simvastatin.

Recombinant antibodies to an oxidized low-density lipoprotein epitope induce rapid regression of atherosclerosis in apobec-1(-/-)/low-density lipoprotein receptor(-/-) mice.

OBJECTIVES: The present study tested the hypothesis that treatment with human recombinant immunoglobulin G1 (IgG1) antibodies against a specific oxidized low-density lipoprotein (oxLDL) epitope will induce regression of existing atherosclerotic lesions in LDL receptor-deficient mice expressing apolipoprotein B-100 (apoB-100) (Apobec-1(-/-)/LDLR(-/-)). BACKGROUND: Oxidized LDL plays an essential role in the pathogenesis of atherosclerosis. We previously showed that an antibody against oxLDL reduces progression of atherosclerosis in mice. METHODS: Apobec-1(-/-)/LDLR(-/-) mice were fed a high-fat diet until they were 24 weeks and were subsequently transferred to chow. Starting at 25 weeks, mice were given 3 weekly injections of either of 2 recombinant human IgG1 antibodies (IEI-E3 or 2D03) against a malondialdehyde-modified apoB-100 peptide sequence. RESULTS: At 25 weeks, atherosclerotic lesions covered 10.3 +/- 3.7% of the descending aorta. Transfer to chow diet resulted in a modest regression of atherosclerosis over a 5-week period (8.28 +/- 4.36%; p = NS). Antibody treatment induced additional regression of atherosclerosis by 50% (2D03; p = 0.001) and 36% (IEI-E3; p = 0.004) compared with control IgG1. The 2D03 treatment also reduced plaque inflammation, enhanced plaque expression of the adenosine triphosphate-binding cassette transporter A1, and inhibited expression of monocyte chemoattractant protein-1 in cultured monocytes. CONCLUSIONS: Human IgG1 against a specific oxLDL epitope can induce rapid and substantial regression of atherosclerotic lesions, possibly by stimulating lipid efflux and inhibiting macrophage recruitment. These recombinant human antibodies could represent a novel strategy for rapid regression/stabilization of atherosclerotic lesions.

Autoimmune responses against the apo B-100 LDL receptor-binding site protect against arterial accumulation of lipids in LDL receptor deficient mice.

BACKGROUND: Oxidation of LDL is associated with generation of autoantibodies against a large number of different aldehyde-modified peptide sequences in apo B-100. Autoantibodies recognizing peptide sequences in the LDL receptor-binding region of apo B-100 could potentially affect both cholesterol metabolism and atherosclerosis. The aim of the present study was to determine physiological effects of induction of immune responses against the apo B-100 LDL receptor-binding site in mice deficient for the LDL receptor. METHODS AND RESULTS: Mice received three immunizations, beginning at 6 weeks of age, with aldehyde-modified or non-modified peptides corresponding to the amino acid sequence of the LDL receptor-binding site. Analysis of antibody response by ELISA unexpectedly revealed high titers of pre-existing IgG against both native and aldehyde-modified binding site sequences in non-immunized mice. Immunization with aldehyde-modified binding site sequences resulted in an almost complete down-regulation of this autoimmune response. It was also associated with a rapid increase in lipid-rich plaques in the aorta and a substantial depletion of the lipid content of the liver, whereas plasma lipid and apo B values were similar in all groups. CONCLUSIONS: These observations demonstrate existence of an endogenous T cell-dependent autoimmune response against the LDL receptor-binding site in LDL receptor(-/-) mice and suggest that this may help to prevent accumulation of lipoprotein lipids in the artery wall, whereas immunization with the corresponding aldehyde modified sequence down-regulates this response and induces substantial atherosclerotic development.

Medial Expression of TNF-α and TNF Receptors Precedes the Development of Atherosclerotic Lesions in Apolipoprotein E/LDL Receptor Double Knockout Mice.

TNF-α is present in atherosclerotic lesions, activates endothelial adhesion molecule expression, stimulates the release of proinflammatory cytokines and matrix metalloproteinases and promotes smooth muscle cell proliferation and migration. Taken together these observations suggest that TNF-α may be functionally involved in early atherosclerosis development. To further evaluate this hypothesis we compared vascular TNF-α and TNF receptor expression in atherosclerosis-susceptible apoE(-/-)/LDL receptor(-/-) mice and control C57BL/6 mice. The aortas of 8 week old apoE(-/-)/LDLreceptor(-/-) mice displayed immunoreactivity for TNF-α as well as TNF p55 and p75 receptors (2.1 ± 1.6%, 5.6 ± 1.5% and 3.6 ± 1.3% of total media area, respectively), but did not have any detectable lesions. A marginal increase in TNF-α and TNF receptor immunoreactivity was observed at 12 weeks and atherosclerotic plaques were detected in 1 out of 5 animals. At 16 weeks TNF-α expression in the media was increased more than four-fold as compared with 8 week old mice, and atherosclerosis was widespread. TNF-α immunoreactivity was also observed in all plaques. In addition, at the same age a tendency towards increased TNF-α mRNA levels was detected in the double knockout mice compared to age-matched controls. A further increase in TNF-α and TNF receptor immunoreactivity as well as plaque size was observed at 20 weeks. With only a few exceptions, no TNF-α or TNF receptor immunoreactivity was detected in C57BL/6 control mice. These findings demonstrate that medial TNF-α and TNF receptor expression precedes lesion formation in apoE(-/-)/LDL receptor(-/-) mice.

Inhibition of injury-induced arterial remodelling and carotid atherosclerosis by recombinant human antibodies against aldehyde-modified apoB-100.

OBJECTIVE: The immune system plays an important regulatory role in the development of atherosclerotic plaques and neointima formation following various types of angioplasty. In the present study we investigated the effect of antibodies against aldehyde-modified apolipoprotein B-100 (apoB-100), a component of oxidized LDL, on atherosclerosis and response to arterial injury in mice. METHODS: The ability of a high affinity human recombinant antibody (2D03), specific for malondialdehyde-modified apoB-100, to influence formation of atherosclerosis as well as remodelling and neointima formation after a collar-induced injury of the carotid artery was studied in LDL receptor(-/-) mice over-expressing human apoB-100. RESULTS: The antibody recognized epitopes present in mouse plasma and reduced the plasma level of oxidized LDL by 34%. Antibody treatment inhibited injury-induced restrictive vascular remodelling but did not influence the size of the neointima. Atherosclerosis in the uninjured contra lateral carotid artery was determined by computerized image analysis and the mean plaque area in animals given control IgG1 was 7608+/-10,336 micro m(2). In contrast, essentially no plaques were present in animals treated with the 2D03 antibody (397+/-235 micro m(2), P<0.01 versus control IgG1). CONCLUSIONS: Treatment with antibodies against aldehyde-modified apoB-100 dramatically reduces atherosclerosis and inhibits restrictive vascular remodelling in mice expressing human apoB-100.

Atheroprotective immunization with MDA-modified apo B-100 peptide sequences is associated with activation of Th2 specific antibody expression.

OBJECTIVE: The objective of this study was to evaluate if immunization with MDA-modified human apo B-100 fragments is associated with a shift in the Th1/Th2 balance. METHODS AND RESULTS: Apo E deficient mice were immunized with one of the peptides (P45; amino acids 688-707, P74; amino acids 1123-1142 or P240; amino acids 3613-3632) at 6, 9 and 11 weeks of age and compared to controls given carrier alone. Immunization with P45 and P74 reduced atherosclerosis in the aorta of 25-week-old mice by 48% (p=0.02) and 31% (p=0.06) and macrophage content in atherosclerotic plaques by 33% (p=0.02) and 39% (p=0.02), respectively. The levels of Th2-specific IgG1 against each peptide increased more than 50-fold in response to immunization, whereas the levels of specific IgM and Th1-associated IgG2a were only marginally affected. However, there was an increase in the plaque expression of both Th1 and Th2 cytokines as assessed by real time PCR. Immunization with P240, a non-homologous peptide used as control, induced a 10-fold increase of specific IgG1 but did not influence atherosclerosis or plaque content. CONCLUSIONS: Immunization with MDA apo B-100 fragments induce a shift from Th1 to a Th2 specific oxidized LDL antibody expression, but without a concomitant downregulation of plaque IFN-gamma expression.

Humoral immune response against defined oxidized low-density lipoprotein antigens reflects structure and disease activity of carotid plaques.

BACKGROUND: Immune responses against oxidized low-density lipoprotein (LDL) play an important role in atherosclerosis. The aim of this study was to investigate if humoral immune response against specific oxidized LDL antigens, such as aldehyde-modified peptide sequences of apolipoprotein B-100, reflects disease activity and structure of atherosclerotic plaques. METHODS AND RESULTS: Plaques were obtained from 114 symptomatic subjects referred to carotid endarterectomy and characterized immunohistochemically and histologically. Plasma levels of IgG and IgM against aldehyde-modified apolipoprotein B-100 amino acid sequences 661 to 680, 3136 to 3155 (peptide 210), and 3661 to 3680 (peptide 240) were determined by enzyme-linked immunosorbent assay. High levels of IgG against peptide 210 were associated with increased plaque content of lipids (r=0.24, P<0.05) and hemorrhage (r=0.27, P=0.005), with decreased content of fibrous tissue (r=-0.25, P=0.01), but also with lower total plaque volume (r=-0.21, P<0.05). In contrast, high levels of IgM against peptide 240 were associated with plaques with more fibrous tissue (r=0.35, P<0.001), less lipids (r=-0.34, P<0.001), and less macrophages (r=-0.24, P<0.05). IgM against peptide 210 were found to be associated with plaque fibrous tissue (r=0.20, P<0.05), less lipids (r=-0.21, P<0.05), and less macrophages (r=-0.27, P=0.01). CONCLUSIONS: These findings support the notion that immune responses against oxidized LDL epitopes are involved in atherosclerosis and that the level of circulating antibodies against these structures may reflect disease activity in the arterial wall.

Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis.

BACKGROUND: Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. METHODS AND RESULTS: Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE-/- mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. CONCLUSIONS: These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.

Inhibition of atherosclerosis in apoE-null mice by immunization with apoB-100 peptide sequences.

OBJECTIVE: LDL oxidation is believed to play an important role in the development of atherosclerosis, and oxidized LDL particles have been shown to become targets for the immune system. Immunization of animals with oxidized LDL results in reduction of atherosclerosis, suggesting an atheroprotective effect of this immune response. METHODS AND RESULTS: Using a polypeptide library covering the complete sequence of apoB-100, a large number of native and malondialdehyde-modified peptide sequences in apoB-100 that are recognized by antibodies in human plasma were identified. We report here that immunization with apoB-100 peptide sequences, against which high levels of IgG and IgM antibodies are present in healthy human controls, reduce atherosclerosis in apoE-null mice by about 60%. Immunizations with these peptides were also found to increase the collagen content of subvalvular lesions. CONCLUSIONS: These studies have identified peptide sequences in apoB-100 that induce immune responses, which inhibits atherosclerosis. This suggests a way of developing an immunization therapy for coronary heart disease.