Daniel Engelbertsen

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.

High levels of IgM against methylglyoxal-modified apolipoprotein B100 are associated with less coronary artery calcification in patients with type 2 diabetes

Abstract.  Engelbertsen D, Anand DV, Fredrikson GN, Hopkins D, Corder R, Shah PK, Lahiri A, Nilsson J, Bengtsson E (Skåne University Hospital, Lund University, Malmö, Sweden; Cardiac Imaging and Research Centre, Wellington Hospital, London, UK; Malmö University, Malmö, Sweden; King’s College Hospital, London, UK; William Harvey Research Institute, London, UK; Cedars‐Sinai Medical Center, Los Angeles, CA, USA; David Geffen School of Medicine, UCLA School of Medicine, Los Angeles, CA, USA). High levels of IgM against methylglyoxal‐modified apolipoprotein B100 is associated with less coronary artery calcification in patients with type 2 diabetes. J Intern Med 2012; 271: 82–89.

Immune responses against aldehyde-modified laminin accelerate atherosclerosis in Apoe(-/-) mice.

BACKGROUND LDL oxidation in the vascular wall is associated with aldehyde modification of surrounding extracellular matrix proteins that may target autoimmune responses against vascular tissues. Here we investigated the possible influence of immunity against a malondialdehyde (MDA)-modified form of the basement membrane protein laminin on atherosclerosis. METHODS AND RESULTS IgM and IgG autoantibodies were present in human plasma and a prospective clinical study demonstrated that individuals who later suffered from acute cardiovascular events had lower levels of MDA-laminin antibodies compared to those in the control group. Immunohistochemical analysis of atherosclerotic plaques from Apoe-/- mice demonstrated co-localization between laminin and MDA epitopes, however MDA-laminin IgG was absent in mouse plasma. To determine the effect of MDA-laminin immunity, Apoe-/- mice were immunized with MDA-laminin. Analysis of circulating leukocytes at 12 weeks demonstrated increased T-cell activation, expansion of Th17 cells and a lower fraction of regulatory T cells (Tregs) in mice immunized with MDA-laminin. At 25 weeks, aortic atherosclerosis was increased by more than 60% in mice immunized with MDA-laminin, together with increased levels of MDA-laminin IgG1 and MDA-laminin-specific T-cells expressing IL-2, IL-4 and IL-6 in the spleen. CONCLUSION The clinical observations suggest that immune responses against MDA-laminin may be involved in the development of cardiovascular disease in humans. Furthermore, observations in mice provide evidence for the presence of aldehyde-modified laminin in atherosclerotic lesions and demonstrate that induction of an immune response against these structures is associated with activation of Th17 cells, reduced fraction of Tregs and a more aggressive development of atherosclerosis.

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.

Low Levels of IgM Antibodies against an Advanced Glycation Endproduct–Modified Apolipoprotein B100 Peptide Predict Cardiovascular Events in Nondiabetic Subjects

Increased glucose levels are associated with the generation of advanced glycation endproduct (AGE) modifications. Interaction between AGE-modified plaque components and immune cells is believed to have an important role in the development of vascular complications in diabetes. Methylglyoxal (MGO) is one type of reactive aldehyde that gives rise to AGE modification. The present study analyzed whether autoantibodies against MGO-modified epitopes of the low-density lipoprotein apolipoprotein B (apoB) 100 predict cardiovascular events. A library consisting of 302 peptides comprising the complete apoB100 molecule was screened to identify peptides targeted by MGO-specific autoantibodies. Peptide (p) 220 (apoB amino acids 3286-3305) was identified as a major target. Baseline IgM and IgG against MGO–peptide 220 (p220) were measured in 700 individuals from the Malmö Diet and Cancer Cohort. A total of 139 cardiovascular events were registered during the 15-y follow-up period. Controlling for major cardiovascular risk factors demonstrated that subjects in the lowest tertile of MGO-p220 IgM had an increased risk for cardiovascular events (hazard ratio [95% confidence interval]: 2.07 [1.22–3.50]; ptrend = 0.004). Interestingly, the association between MGO-p220 IgM and cardiovascular events remained and even tended to become stronger when subjects with prevalent diabetes were excluded from the analysis (2.51 [1.37-4.61]; ptrend = 0.002). MGO-p220 IgM was inversely associated with blood glucose, but not with oxidized low-density lipoprotein. Finally, we demonstrate that anti-MGO-p220 IgM is produced by B1 cells. These data show that subjects with low levels of IgM recognizing MGO-modified p220 in apoB have an increased risk to develop cardiovascular events and that this association is present in nondiabetic subjects.

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-1β 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.

Decreased levels of autoantibodies against apolipoprotein B‐100 antigens are associated with cardiovascular disease in systemic lupus erythematosus

Increased production of autoantibodies is a characteristic feature of systemic lupus erythematosus (SLE) and there is evidence that several of these autoantibodies may contribute to increased cardiovascular disease (CVD) in SLE. Autoantibodies against the apolipoprotein (apo) B‐100 peptides p45 and p210 have been associated with a lower CVD risk in non‐SLE cohorts. The aim of the present study was to investigate how SLE affects the occurrence of these potentially protective autoantibodies. The study cohort consisted of 434 SLE patients and 322 age‐ and sex‐matched population controls. Antibodies against native and malondialdehyde (MDA)‐modified p45 and p210 were measured by enzyme‐linked immunosorbent assay (ELISA). SLE patients had significantly lower levels of p210 immunoglobulin (Ig)G and p45 IgM (both the native and malondialdehyde (MDA)‐modified forms). SLE patients with manifest CVD (myocardial infarction, ischaemic cerebrovascular disease or peripheral vascular disease) had lower levels p210 IgG and p45 IgM than SLE patients without CVD. Decreased levels of these autoantibodies were also observed in SLE patients with permanent organ damage, as assessed by the Systemic Lupus International Collaborating Clinics/American College of Rheumatology (ACR) Damage Index (SDI). The present findings show that patients with SLE, a condition generally characterized by abundance of autoantibodies of multiple specificities, have reduced levels of antibodies against the apo B‐100 antigens p45 and p210 and that the levels of these antibodies are reduced further in SLE patients with CVD. These observations suggest the possibility that an impaired antibody‐mediated removal of damaged LDL particles may contribute to the development of vascular complications and organ damage in SLE.

Expansion of CD25+ Innate Lymphoid Cells Reduces Atherosclerosis

Objective—Innate lymphoid cells (ILCs) are a newly discovered subset of immune cells that promote tissue homeostasis and protect against pathogens. ILCs produce cytokines also produced by T lymphocytes that have been shown to affect atherosclerosis, but the influence of ILCs on atherosclerosis has not been explored. Approach and Results—We demonstrate that CD25+ ILCs that produce type 2 cytokines (ILC2s) are present in the aorta of atherosclerotic immunodeficient ldlr−/−rag1−/− mice. To investigate the role of ILCs in atherosclerosis, ldlr−/−rag1−/− mice were concurrently fed an atherogenic diet and treated with either ILC-depleting anti-CD90.2 antibodies or IL-2/anti-IL-2 complexes that expand CD25+ ILCs. Lesion development was not affected by anti-CD90.2 treatment, but was reduced in IL-2/anti–IL-2-treated mice. These IL-2-treated mice had reduced very low–density lipoprotein cholesterol and increased triglycerides compared with controls and reduced apolipoprotein B100 gene expression in the liver. IL-2/anti-IL-2 treatment caused expansion of ILC2s in aorta and other tissues, elevated levels of IL-5, systemic eosinophila, and hepatic eosinophilic inflammation. Blockade of IL-5 reversed the IL-2 complex–induced eosinophilia but did not change lesion size. Conclusions—This study demonstrates that expansion of CD25-expressing ILCs by IL-2/anti-IL-2 complexes leads to a reduction in very low–density lipoprotein cholesterol and atherosclerosis. Global depletion of ILCs by anti-CD90.2 did not significantly affect lesion size indicating that different ILC subsets may have divergent effects on atherosclerosis.

Induction of T helper 2 responses against human Apolipoprotein B100 does not affect atherosclerosis in ApoE−/- mice

AIMS Immune responses against LDL antigens have been found to play an important modulatory role in atherosclerosis. Immunization with homologous oxidized LDL, as well as human apolipoprotein B100 (ApoB)-derived peptides, inhibits atherosclerosis in hypercholesterolaemic animal models of atherosclerosis. However, the role of antigen-specific T helper 2 (Th2) responses in atherosclerosis remains to be fully clarified. METHODS AND RESULTS ApoE(-/-) mice on high-fat diet were immunized with human ApoB using Alum as an adjuvant at 12, 14, and 16 weeks of age. Alum-injected and non-treated mice were used as controls. At 17 weeks of age, a matrigel plug containing ApoB was placed subcutaneously and T-cell infiltration into the plug as well as the development of aortic root atherosclerotic lesions were analysed after an additional 7 days. Immunization with ApoB resulted in four-fold increased accumulation of effector T cells in ApoB-containing matrigel when compared with control groups. The levels of the Th2 cytokines IL-4, IL-5, and IL-10 were also increased in ApoB-containing matrigel plugs. Moreover, the levels of Th2-associated IgG1 against human and also mouse LDL were increased in the plasma of ApoB-immunized mice. In spite of the induction of a Th2 response partially reacting also with the endogenous LDL, there was no difference in atherosclerosis when compared with the Alum group. CONCLUSIONS This study describes a novel model to study antigen-specific T-cell responses in vivo in mouse models of atherosclerosis. The results suggest that activation of Th2 immunity does not mediate the protective effect of immunization with LDL antigens described previously.

Innate lymphoid cells in atherosclerosis

Abstract The family of innate lymphoid cells (ILCs) consisting of NK cells, lymphoid tissue inducer cells and the ‘helper’‐like ILC subsets ILC1, ILC2 and ILC3 have been shown to have important roles in protection against microbes, regulation of inflammatory diseases and involved in allergic reactions. ILC1s produce IFN‐&ggr; upon stimulation with IL‐12 and IL‐18, ILC2s produce IL‐5 and IL‐13 responding to IL‐33 and IL‐25 while ILC3s produce IL‐17 and IL‐22 after stimulation with IL‐23 or IL‐1. Although few studies have directly investigated the role for ILCs in atherosclerosis, several studies have investigated transcription factors and cytokines shared by ILCs and T helper cells. In this review we summarize our current understanding of the role of ILC in atherosclerosis and discuss future directions.