Elsevier

Immunobiology

Volume 216, Issue 6, June 2011, Pages 663-669
Immunobiology

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

https://doi.org/10.1016/j.imbio.2010.11.003Get rights and content

Abstract

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.

Introduction

Accumulation and oxidation of low density lipoprotein (LDL) in the vascular wall is a key event in the development of atherosclerosis (Hansson et al. 2006). Oxidized LDL particles promote inflammation, leading to influx of mononuclear phagocytes. These cells ingest the oxidized lipoprotein particles and give rise to foam cells and contribute to the atherogenic process by orchestrating inflammatory responses (Galkina and Ley 2009). Oxidation of LDL results in a large variety of oxidized lipid–phospholipid–protein adducts. Modifications of lysines or histidines in these new molecules render them immunogenic (Palinski and Witztum 2000). Activation of Th1 immune responses against these modified self antigens have been shown to contribute to disease progression while immune responses leading to generation of antibodies and Treg activation appear protective (Nilsson and Hansson 2008).

We have recently shown that aluminum salt-containing gel (alum) injected into a hypercholesterolemic environment can absorb antigens from modified LDL and activate immune responses against these in a manner that contributes to protection against early atherosclerosis (Wigren et al. 2009). The alum immunization was shown to activate immune suppressive Treg cells and downregulate activation markers on CD4+ T cells (Wigren et al. 2009). Alum is the most widely used adjuvant in clinical vaccines. Another experimental study has also demonstrated atheroprotective properties of alum immunization (Khallou-Laschet et al. 2006). In addition to a general immune stimulatory mechanism of an adjuvant, alum has been shown to induce cell death and to thereby enhance antigen presentation by dendritic cells (Kono and Rock, 2008, Kool et al., 2008). In the present study we aimed to extend our previous findings by including a carrier in the immunization procedure, to evaluate if the carrier could enhance an atheroprotective immune response in older ApoBec-1/LDL receptor deficient mice with already existing plaques.

The most commonly used carrier proteins are capable of imparting immunogenecity to covalently coupled haptens. Some of the more useful ones include keyhole limpet hemocyanin (KLH) and BSA because of their proven efficacies (Coligan et al. 2001). However, one disadvantage of KLH may be its large size that makes it more likely to precipitate during cross-linking, making the complex difficult to handle. Studies comparing native and cationized BSA (cBSA) showed an alteration of the immunogenic and regulatory properties due to the cationization (Muckerheide et al., 1987a, Muckerheide et al., 1987b). Both in vivo and in vitro, cBSA was highly immunogenic, producing significantly greater responses than the native BSA. C57BL/6 mice produced a significant antibody response to BSA when immunized with cBSA, in contrast to the immunization with native BSA (Muckerheide et al., 1987a, Muckerheide et al., 1987b). Taken together, this indicates that cBSA, because of its positive charges, has greater affinity for the negatively charged cell surface membrane of antigen presenting cells resulting in a more efficient uptake of the antigen.

Here we tested the hypothesis that using an immunization strategy combining alum with a carrier would produce an atheroprotective response against a self antigen in mice with manifest advanced disease. In our previous study, it was implicated that the atheroprotective alum effect was based on activation of Treg cells. Thus, we decided to use several administrations of the carrier, a strategy known to induce immunosuppression.

Section snippets

Animals and immunization protocol

We used female ApoBec-1/LDL receptor deficient mice on C57BL/6 background (Jackson Laboratories, Bar Harbor, Maine), which express full length apoB-100 in their LDL particles. The mice (n = 10–21 per group) were fed a high fat diet (0.15% cholesterol, 21% fat, Lantmännen, Sweden) from 4 weeks of age, provided ad libitum. The diet was changed to chow diet 20 weeks later. Thereafter the mice were immunized at 25, 27, 29, 31, 33, 35 and 36 weeks of age with cBSA. Each injection contained 50 μg cBSA

Increased levels of plasma apoB/IgG and IgG1 immune complexes in mice immunized with cBSA

In a previous study, young apoE deficient mice were immunized with alum alone, resulting in atheroprotection (Wigren et al. 2009). Alum was shown to precipitate oxidized LDL antigens at the injection site, with a consequential increase in plasma IgG1 recognizing an oxidized LDL epitope. However, in mice with manifest advanced disease it is more difficult to induce atheroprotection. Thus, in the present study we aimed to evaluate if the effect could be more pronounced by combining alum with a

Discussion

In the present study we have demonstrated that immunization using the carrier cBSA inhibited lesion progression in ApoBec-1/LDL receptor deficient mice with advanced plaques. The immunization was also associated with a stabilization of the existing plaques in subvalvular lesions, shown by a reduced amount of macrophages and CD3+ T cells as well as a higher content of collagen. This was also associated with an increased regulatory-to-effector T cell ratio as well as induction of an

Conflicts of interest

No conflicts of interest exist.

Acknowledgements

This study was supported by grants from the Swedish Research Council, the Swedish Heart-Lung Foundation, Swedish Foundation for Strategic Research, VINNOVA, the Crafoord Foundation, the Knut and Alice Wallenberg Foundation, the Söderberg Foundation, the Albert Påhlsson Foundation, the Malmö University Hospital Foundation, the Lundström Foundation and the European Community's Sixth Framework Programme contract (“IMMUNATH”) LSHM-CT-2006-037400.

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