Interleukin-1 receptor accessory protein blockade limits the development of atherosclerosis and reduces plaque inflammation.

AIMS: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. METHODS AND RESULTS: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. CONCLUSION: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.

Therapeutic S100A8/A9 blockade inhibits myocardial and systemic inflammation and mitigates sepsis-induced myocardial dysfunction.

BACKGROUND AND AIMS: The triggering factors of sepsis-induced myocardial dysfunction (SIMD) are poorly understood and are not addressed by current treatments. S100A8/A9 is a pro-inflammatory alarmin abundantly secreted by activated neutrophils during infection and inflammation. We investigated the efficacy of S100A8/A9 blockade as a potential new treatment in SIMD. METHODS: The relationship between plasma S100A8/A9 and cardiac dysfunction was assessed in a cohort of 62 patients with severe sepsis admitted to the intensive care unit of Linköping University Hospital, Sweden. We used S100A8/A9 blockade with the small-molecule inhibitor ABR-238901 and S100A9-/- mice for therapeutic and mechanistic studies on endotoxemia-induced cardiac dysfunction in mice. RESULTS: In sepsis patients, elevated plasma S100A8/A9 was associated with left-ventricular (LV) systolic dysfunction and increased SOFA score. In wild-type mice, 5 mg/kg of bacterial lipopolysaccharide (LPS) induced rapid plasma S100A8/A9 increase and acute LV dysfunction. Two ABR-238901 doses (30 mg/kg) administered intraperitoneally with a 6 h interval, starting directly after LPS or at a later time-point when LV dysfunction is fully established, efficiently prevented and reversed the phenotype, respectively. In contrast, dexamethasone did not improve cardiac function compared to PBS-treated endotoxemic controls. S100A8/A9 inhibition potently reduced systemic levels of inflammatory mediators, prevented upregulation of inflammatory genes and restored mitochondrial function in the myocardium. The S100A9-/- mice were protected against LPS-induced LV dysfunction to an extent comparable with pharmacologic S100A8/A9 blockade. The ABR-238901 treatment did not induce an additional improvement of LV function in the S100A9-/- mice, confirming target specificity. CONCLUSION: Elevated S100A8/A9 is associated with the development of LV dysfunction in severe sepsis patients and in a mouse model of endotoxemia. Pharmacological blockade of S100A8/A9 with ABR-238901 has potent anti-inflammatory effects, mitigates myocardial dysfunction and might represent a novel therapeutic strategy for patients with severe sepsis.

IL-2Rßγ signalling in lymphocytes promotes systemic inflammation and reduces plasma cholesterol in atherosclerotic mice.

BACKGROUND AND AIMS: The relationship between inflammation and lipid metabolism is complex and bidirectional. Lymphocyte-driven inflammation has been shown to modulate both atherosclerotic plaque development and cholesterol levels, but the mechanisms are incompletely understood. METHODS: The cardiometabolic effects of IL-2Rßγ signalling in atherosclerotic Apoe-/- mice were investigated by treatment with an agonistic IL-2Rßγ-targeting IL-2/anti-IL-2 complex or a monoclonal anti-CD122 (IL-2Rß) blocking antibody. RESULTS: Administration of IL-2Rßγ agonistic IL-2/anti-IL-2 complexes to Apoe-/- mice augmented opposing arms of the adaptive immune system. Expansion of effector/memory T cells and increased levels of circulating pro-inflammatory cytokines were observed along with elevated levels of regulatory T cells and IL-10. Notably, IL-2/anti-IL-2 treatment did not affect plaque size but decreased levels of plasma cholesterol. The cholesterol lowering effect of IL-2Rßγ agonism was not affected by anti-CD8 or anti-NK1.1 depleting antibody treatment but was contingent on the presence of adaptive immunity. Expression of multiple liver X receptor (LXR)-related genes, including Pltp and Srebp1c in the liver, was decreased by IL-2/anti-IL-2 treatment. Although IL-2Rßγ agonism lowered cholesterol levels, blocking IL-2Rßγ signalling using an anti-CD122 monoclonal antibody did not impact cholesterol levels or plaque burden in Apoe-/- mice. CONCLUSIONS: Elevated IL-2Rßγ signalling results in activation of both inflammatory and regulatory lymphocytes with a net zero effect on atherosclerosis and decreased plasma cholesterol levels. Changes in cholesterol levels were associated with reductions in hepatic LXR-related gene expression. Further studies are needed to investigate the clinical significance of IL-2 mediated modulation of hepatic LXR signalling in inflammatory disorders.

Immune responses against oxidized LDL as possible targets for prevention of atherosclerosis in systemic lupus erythematosus.

Patients suffering from systemic lupus erythematosus (SLE) are at increased risk of developing cardiovascular disease (CVD) and traditional therapies including statins provide insufficient protection. Impaired removal of apoptotic material is a common pathogenic mechanism in both SLE and atherosclerosis and is considered to be a key factor in the development of autoimmunity. Since oxidized LDL and apoptotic material bind to the same receptors, we aimed to investigate if targeting the oxidized LDL autoimmunity can affect atherosclerosis in SLE. To investigate the possible role of oxidized LDL autoimmunity in the accelerated atherosclerosis associated with SLE we used a hypercholesterolemic SLE mouse model (B6.lpr.ApoE-/- mice). Promoting LDL tolerance through mucosal immunization with an apolipoprotein B-100 peptide p45 (amino acids 661-680) and cholera toxin B-subunit fusion protein increased regulatory T cells and B cells in mesenteric lymph nodes and reduced plaque development in the aorta by 33%. Treatment with the oxidized LDL-specific antibody Orticumab reduced aortic atherosclerosis by 43%, subvalvular plaque area by 50% and the macrophage content by 31%. The present study provides support for oxLDL as a possible target for prevention of cardiovascular complications in SLE.

ILC2 transfers to apolipoprotein E deficient mice reduce the lipid content of atherosclerotic lesions.

BACKGROUND: Expansion of type 2 innate lymphoid cells (ILC2s) in hypercholesterolaemic mice protects against atherosclerosis while different ILC2 subsets have been described (natural, inflammatory) based on their suppression of tumorigenicity 2 (ST2) and killer-cell lectin like receptor G1 (KLRG1) expression. The aim of the current study is to characterize the interleukin 25 (IL25)-induced splenic ILC2 population (Lin-CD45+IL17RB+ICOS+IL7raintermediate) and address its direct role in experimental atherosclerosis by its adoptive transfer to hypercholesterolaemic apolipoprotein E deficient (apoE-/-) mice. RESULTS: Immunomagnetically enriched, FACS-sorted ILC2s from the spleens of IL-25 treated apoE-/- mice were stained for KLRG1 and ST2 directly upon cell obtainment or in vitro cell expansion for flow cytometric analysis. IL25-induced splenic ILC2s express high levels of both KLRG1 and ST2. However, both markers are downregulated upon in vitro cell expansion. In vitro expanded splenic ILC2s were intraperitoneally transferred to apoE-/- recipients on high fat diet. ApoE-/- mice that received in vitro expanded splenic ILC2s had decreased lipid content in subvalvular heart and brachiocephalic artery (BCA) plaques accompanied by increased peritoneal B1 cells, activated eosinophils and alternatively activated macrophages (AAMs) as well as anti-phosphorylcholine (PC) immunoglobulin (Ig) M in plasma. CONCLUSIONS: With the current data we designate the IL25-induced ILC2 population to decrease the lipid content of atherosclerotic lesions in apoE-/- mice and we directly link the induction of B1 cells and the atheroprotective anti-PC IgM antibodies with ILC2s.

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.

Elevated circulating effector memory T cells but similar levels of regulatory T cells in patients with type 2 diabetes mellitus and cardiovascular disease.

Type 2 diabetes mellitus is associated with an elevated risk of cardiovascular disease, but the mechanism through which diabetes contributes to cardiovascular disease development remains incompletely understood. In this study, we compared the association of circulating regulatory T cells, naïve T cells, effector memory T cells or central memory T cells with cardiovascular disease in patients with and without type 2 diabetes mellitus. Percentage of circulating T cell subsets was analysed by flow cytometry in type 2 diabetes mellitus subjects with and without prevalent cardiovascular disease as well as in non-diabetic subjects with and without prevalent cardiovascular disease from the Malmö SUMMIT cohort. Subjects with type 2 diabetes mellitus had elevated percentages of effector memory T cells (CD4+CD45RO+CD62L-; 21.8% ± 11.2% vs 17.0% ± 9.2% in non-type 2 diabetes mellitus, p < 0.01) and central memory T cells (CD4+CD45RO+CD62L+; 38.0% ± 10.7% vs 36.0% ± 9.5% in non-type 2 diabetes mellitus, p < 0.01). In contrast, the frequency of naïve T cells was reduced (CD4+CD45RO-CD62L+, 35.0% ± 16.5% vs 42.9% ± 14.4% in non-type 2 diabetes mellitus, p < 0.001). The proportion of effector memory T cells was increased in type 2 diabetes mellitus subjects with cardiovascular disease as compared to those without (26.4% ± 11.5% vs 18.4% ± 10.2%, p < 0.05), while no difference in regulatory T cells was observed between these two patient groups. This study identifies effector memory T cells as a potential cellular biomarker for cardiovascular disease among subjects with type 2 diabetes mellitus, suggesting a state of exacerbated immune activation in type 2 diabetes mellitus patients with cardiovascular disease.

B cells treated with CTB-p210 acquire a regulatory phenotype in vitro and reduce atherosclerosis in apolipoprotein E deficient mice.

OBJECTIVE: Intranasal immunization with a fusion protein of the ApoB100-derived peptide p210 and the cholera toxin B subunit (CTB-p210) has previously been shown to induce mucosal tolerance and reduce atherosclerosis development, but the exact mode of action remains to be elucidated. Recent studies have indicated an important role for B cells in mucosal tolerance, in particular by induction of regulatory B (Bregs) and T cells (Tregs). In this study, we aimed to investigate if transfer of B cells pulsed with CTB-p210 can protect against atherosclerosis. METHOD AND RESULTS: First, we studied if CTB-p210 can induce Bregs and Tregs in vitro. After pulsing B cells from Apobtm2Sgyldlr-/- or Apoe-/- mice with CTB-p210 for 1 h and co-culturing them with naïve T cells for 48 h, we observed increased expression of membrane bound TGFß/latency-associated peptide (mTGFß/LAP) on B cells and an increased proportion of CD25hiFoxP3+ Tregs. Adoptive transfer of B cells pulsed with CTB-p210 into high-fat diet-fed Apoe-/- mice at 8, 10 and 12 weeks of age, reduced the plaque area in the aorta at 20 weeks of age as compared with control-treated (CTB-pOVA treated B cells or PBS) mice. Moreover, mice receiving p210-CTB treated B cells had increased levels of anti-p210 IgG antibodies. CONCLUSION: Our observations suggest that CTB-p210 pulsed B cells acquire a regulatory phenotype and induce Tregs in vitro. Adoptive transfer of CTB-p210, but not control-treated, B cells into Apoe-/- mice decreased atherosclerosis development.

Interleukin-25 reduces Th17 cells and inflammatory responses in human peripheral blood mononuclear cells.

BACKGROUND: The absence of interleukin-25 (IL-25) favors the induction of Th1 and Th17 immune responses in mice. Th1 immune responses have been associated with the pathology of atherosclerosis, a lipid and inflammation driven disease of the arterial wall. PURPOSE OF RESEARCH: To evaluate the effect of IL-25 on human peripheral blood mononuclear cells (hPBMCs) in the presence and absence of oxidized low density lipoprotein (oxLDL), a key player in atherosclerosis development. PRINCIPAL RESULTS: Human PBMCs were incubated with recombinant human IL-25 (rhIL-25) in the presence and absence of oxLDL and analyzed with flow cytometry while cytokine secretion was measured in cell culture supernatants. The IL-25 receptor, IL-17RB, was mostly expressed on T cells. Incubation of hPBMCs with IL-25 reduced the frequency of Th17 cells. Furthermore, IL-25 inhibited the release of the Th17-inducing cytokine IL-6 from dendritic cells isolated from hPBMCs indicating that the IL-25 mediated Th17 suppression may be indirect. Moreover, IL-25 reduced the secretion of the proinflammatory cytokine IFNγ from hPBMCs. OxLDL decreased IFNγ release from hPBMCs regardless of the presence or absence of IL-25. CONCLUSIONS: IL-25 reduces Th1 and Th17 immune responses in hPBMCs raising the interesting possibility that IL-25 could have a protective role in human atherosclerosis.

Interleukin-25 (IL-25) has a protective role in atherosclerosis development in the aortic arch in mice.

Atherosclerosis is a chronic inflammatory disease characterized by the entrapment of apolipoprotein B-containing lipoproteins in the arterial intima, leading to local inflammation. T helper (Th) cell 1-mediated immune responses have been associated with atherosclerosis, and the cytokine interleukin-25 (IL-25 or IL-17E) has been reported to potentially regulate Th1 cell- and Th17 cell-related immune responses. In this study, we evaluated the effects of complete IL-25 deficiency or of a temporal IL-25 blockade on atherosclerosis development in apolipoprotein E-deficient (Apoe-/-) mice. Mice deficient in both apolipoprotein E and IL-25 (Apoe-/-/IL-25-/-) had more Th1 cells in the spleen, along with elevated plasma levels of IL-17 and an increased release of splenic interferon-γ (INF-γ). In support of this observation, a 4-week-long treatment of young Apoe-/- mice (at 10-14 weeks of age) with an IL-25-blocking antibody increased the release of Th1/Th17-associated cytokines in the spleen. In both mouse models, these findings were associated with increased atherosclerotic plaque formation in the aortic arch. We conclude that complete IL-25 deficiency and a temporal IL-25 blockade during early plaque development aggravate atherosclerosis development in the aortic arch of Apoe-/- mice, accompanied by an increase in Th1/Th17-mediated immune responses. Our finding that endogenous IL-25 has an atheroprotective role in the murine aortic arch has potential implications for atherosclerosis development and management in humans.

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.

Circulating CD40+ and CD86+ B cell subsets demonstrate opposing associations with risk of stroke.

OBJECTIVE: Accumulating evidence shows that immune cells play an important role in atherosclerosis. Most attention has focused on the role of different T cell subsets, whereas the possible involvement of B cells has been less studied. In this study, we assessed the association of 2 different B cell subsets, CD19(+)CD40(+) and CD19(+)CD86(+) B cells, with risk for development of acute cardiovascular events. APPROACH AND RESULTS: The prospective study included 700 subjects randomly selected from the cardiovascular cohort of the Malmö Diet and Cancer study. Mononuclear leukocytes, stored at -140(○)C at the baseline investigation in 1991-1994, were thawed and B cell subsets analyzed by flow cytometry. Cytokine release from CD3/CD28-stimulated mononuclear leukocytes was measured with multiplex ELISA. Baseline carotid intima-media thickness and stenosis were assessed by ultrasonography, and clinical events were monitored through validated national registers during a median/mean follow-up time of 15 years. The subjects in the highest tertile of CD19(+)CD40(+) B cells had a significantly lower risk of incident stroke after adjustment for other risk factors. In contrast, CD19(+)CD86(+) B cells were associated with higher risk for development of a stroke event and increased release of proinflammatory cytokines from mononuclear leukocytes. CONCLUSIONS: These observations provide evidence for an involvement of B cells in the incidence of stroke and suggest that both pathogenic and protective B cell subsets exist.

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-γ(+)) 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. CONCLUSIONS: 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.

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 degrees 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. CONCLUSIONS: 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.

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.

Elevated CD14++CD16- monocytes predict cardiovascular events.

BACKGROUND: Although monocytes in peripheral blood are no longer considered to be a homogeneous population, associations between distinct monocyte subsets and cardiovascular disease have not been highlighted in large epidemiological studies. METHODS AND RESULTS: The study included 700 randomly selected subjects from the cardiovascular arm of the Malmö Diet and Cancer study. Among these, 123 subjects experienced ischemic cardiovascular events during the follow-up until December 2008. Mononuclear leukocytes frozen at the baseline investigation in 1991 to 1994 were thawed and analyzed with flow cytometry to enumerate monocyte subsets, based on CD14 and CD16 expression. The percentage and number of classical CD14(++)CD16(-) monocytes were increased in the cardiovascular-event group compared with the event-free subjects (median, 69% [interquartile range, 62% to 76%] versus 67% [59% to 72%], P=0.017; 344 [251 to 419] cells/µL versus 297 [212 to 384] cells/µL, P=0.003). The hazard ratio was 1.66 for suffering a cardiovascular event in the highest tertile of the number of CD14(++)CD16(-) monocytes compared with the lowest tertile, even after adjustment for common risk factors (HR, 1.66; 95% CI: 1.02 to 2.72). CD14(++)CD16(-) monocytes did not, however, associate with the extent of atherosclerosis at baseline. In contrast, the percentage of monocytes expressing CD16 was negatively associated to the extent of carotid atherosclerosis measured as intima-media thickness at baseline. The chemokine receptors CCR2, CX3CR1, and CCR5 were not differentially expressed between cases and controls on any of the monocyte subsets, but CCR5 expression on CD14(+)CD16(++) monocytes was negatively associated to carotid intima-media thickness. CONCLUSIONS: This study shows that classical CD14(++)CD16(-) monocytes can predict future cardiovascular risk independently of other risk factors in a randomly selected population.

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.

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.