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The Role of Immunity in Atherogenesis - Implications for Vaccine Development with Reference to Oxidized LDL
Nandini Venkatesan, Ph.D.1 and Vijay R. Prabhakar, M.D. 2, 1Vaccine Research and Development Center,
National Health Research Institutes, Taiwan, 2Associate Medical Director, Pharmanet Inc. Ltd., Singapore
Please address correspondence to:
E-mail: ndvp@nhri.org.tw
Introduction
There is increasing evidence that both inflammation and immunology regulate atherosclerogenesis and that anti-inflammatory processes (via cells and molecules) play a role in the development of atherosclerosis. Development of atherosclerosis is influenced by innate and adaptive immune responses. Thus, research has focused attention on the immune system as a possible novel target in prevention and treatment of cardiovascular disease.
It is common knowledge that LDLs are the major component of “bad cholesterol” and their accumulation in the arterial wall causes inflammation and is thus a key factor in atherosclerosis. However, it was only a decade ago that the ability of oxidized LDL to trigger an immune response in the body was recognized. Studies revealed that these molecules could trigger an autoimmune response. This is, in fact, the basis of an atherosclerosis vaccine. They also revealed that the immunization with oxLDL particles reduces the development of atherosclerosis hampering the deposition of atherosclerotic plaques .It has recently been shown that active immunization with recombinant human IgG (directed against a specific epitope of oxLDL) triggered the production of antibodies which determined the reduction of the atherosclerotic lesions up to 70% and the stabilization/regression of the plaques. These
Innate and adaptive immune responses play a role in the development of atherosclerosis [2]. Innate immunity is based on detection of pattern recognition receptors on macrophages and dendritic cells [3]; those considered most important in atherosclerosis are the scavenger receptors (SRs) and the toll-like receptors (TLRs) that are involved in uptake of oxLDL, promoting the inflammatory response and most importantly modulating subsequent adaptive immunity [4]. Adaptive immunity, though more specific than innate immunity, may take a longer time to be fully mobilized. Very simply, it involves a stochastic rearrangement process in immunoblasts, leading to the generation of a large number of T and B cell receptors and immunoglobins which can recognize foreign antigens [5]. The immune response pathway is as follows. Oxidized LDL (after its uptake by the SRs) is presented by MHC class 11 proteins for recognition by antigen-specific CD4+ T cells [6]. Activated CD4+ T cells may differentiate into proinflammatory Th1 cells, Th2 cells promoting antibody production, Tr1 cells that suppress antigen-induced activation of other CD4+ T cells, or TGF-β-producing Th3 cells. Presentation of the lipid antigens by the macrophage class 1-like molecule CD1 results in activation of NK1 1+ CD4+cells promoting Th1 and Th2 responses. Exciting new findings in research point to the role of Tregs, which produce inhibitory signals through immunosuppressive cytokines [7]. Disease progression is, in fact, a net result of the balance between pro-inflammatory and anti-inflammatory responses [2].
Immunization and immunosuppressive drugs usually target adaptive immune responses. However, it is possible to target adaptive immunity by initially targeting the TLRs, which are an element of innate immunity [5]. Interruption of TLR-mediated innate immune signaling through genetic removal of key genes has been shown to be atheroprotective in murine models [8].
Another manipulation of innate immunity can be accomplished by stimulation of TGF-β. TGF-β producing Treg and Th3 cells can provide antigen-specific immuno-suppression by acting on other T cells. A recent study has shown that abrogation of TGF-β signaling by T cells resulted in increased severity of atherosclerosis [9]. This suggests that TGF-β is capable of inhibiting proatherogenic T cells under normal conditions.
Manipulation of the T regulatory cells is another method to target the immune system for atheroprotection. The Tr1 (T regulatory 1) cells in particular secrete IL-10 and thus suppress antigen-induced activation of other CD4+ T cells by acting as a “cytokine secretion inhibiting cytokine.” Activation of these Tr1 cells confirms the protective role of IL-10 in mouse models of atherosclerosis [10,11].
Thus, the antigen-specific activation of TGF-β or IL-10 producing Tregs could be an attractive means of protective immunity against atherosclerosis. One of the important issues and challenges now will be to identify strategies to expand the number of active T-regulatory lymphocytes at sites of atherosclerotic lesions [10].
Another strategy brought to light recently is the activation of natural killer T cells (NKT) cells that recognize lipid antigens presented by class 1-like molecules (CD1) that are expressed by macrophages of atherosclerotic lesions [12].
As opposed to inhibition of T cell activation, it is now emerging that atheroprotective immunity could also be conferred by the B cells. Experiments in support of this demonstrate that aggressive atherosclerosis encountered in splenectomized apo E-/- mice is reversed by injection of spleen B cells from atherosclerotic animals [13]. These observations, in fact, support the idea that a vaccine could indeed be developed for treatment or prevention of atherosclerosis.
Immunization Using Intact Oxidized LDL or Oxidized LDL-associated Antigens
LDL when oxidized undergoes several modifications that lead to its recognition by the immune system .Several studies do support the existence of atheroprotective immune responses against epitopes present in oxidized LDL. When intact LDL is oxidized, several modifications take place, such as peroxidation of polyunsaturated fatty acids in phospholipids and cholesterol esters, subsequently forming breakdown products such as malonaldehyde and 4-hydroxynonenal and degradation of apoB-100 into numerous peptide fragments [14]. In humans, autoantibodies against oxidized LDL are primarily of the IgM type whereas immunization shifts them towards the IgG with a marked inhibition of plaque formation [15]. However, since oxidized LDL has numerous epitopes with the potential of inducing atheroprotective immune responses, a detailed characterization of the antigens in oxidized LDL is essential.
Thus far, two major sub-classes of oxidized LDL antigens have been identified: specific MDA-modified peptide sequences in apoB-100, and oxidized phospholipids containing a phosphorylcholine head group, either present as an isolated lipid or covalently bound to an apoB-100 peptide sequence. Immunization with both types of antigens inhibits atherosclerosis; the immune responses to them however, are distinctly different [5]
Some interesting studies in mice immunized with apo B-100 peptides that were identified to induce autoimmune responses in humans resulted in a reduction of atherosclerosis up to 70% [16] but failed to elicit an immune response if the apo-B-100 sequences were not homologous between human and mice.
The atheroprotective mechanism is associated with an adaptive T-cell-dependent synthesis of IgG in mice. These results need to be extended in humans as it remains to be ascertained if indeed anti-apo-B-100 IgG can provide
The presence of oxidized phospholipids antigens in oxidized LDL was demonstrated by Horrko et al. [17]. Antibodies to this antigen recognized epitopes in the lipid moiety of oxidized LDL as well as in delipidated, modified apo-B-100 suggesting that the antigen can exist as a free lipid as well as an adduct to apo B-100 [18]. Oxidized phospholipids antigens differ from apo-B-100 peptide antigens in that they induce an innate B-cell-dependent synthesis of IgM.
Atheroprotective Effect – Site(s) of Protection
It is as yet unclear if immunization with oxLDL antigens is the same in different sites of the arterial system. Most studies demonstrate an inhibition of atherosclerotic plaque development in the aortic root upon immunization with oxLDL [15,19] whereas apo-B-100 peptides reduce plaque size in the descending aorta only [16]. However, it must be stressed that several inconsistencies exist in reported literature about site-specific
Atherosclerosis Vaccines with Other Antigens
HSPs are chaperones that act to protect nascent proteins during their intracellular maturation. Nasal administration of HSP65 induced more IL-10, higher titers of Th2-dependent antibodies, and protection against atherosclerosis by plaque reduction and also by lowering the number of macrophages and CD4+ T cells in LDLr- mice [20]. Significantly, the production of IL-10 was increased by this treatment. In short, a deviation from the Th1 to the Th2 pathway and an adaptive immunity was induced.
Vaccines using some key proteins in the disease process have also been started, in particular cholesterol ester transfer proteins (CETP)-peptide antigens. CETP is an enzyme responsible for transferring cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL) and very low density lipoproteins (VLDL). Immunization of hypercholesterolemic rabbits with this peptide has resulted in inhibition of CETP activity, increased HDL cholesterol, and reduced development of atherosclerosis [21]. Clinical trials with this promising vaccine are now underway [22].
Thus far, all immunization studies have been conducted on young animals with minimal/no pre-existing atherosclerosis and have focused on prevention of early atherosclerosis. This could be a potential limitation from a clinical perspective. It raises two questions – whether early immunization with an atherosclerosis vaccine would prevent atherosclerosis and are repeated immunizations necessary and whether immunization could stabilize or even prompt a regression of existing plaques in adult humans.
The next important concern is regarding oxLDL. This is a complex, poorly characterized particle with both epitopes that could elicit pro- and anti-atherogenic immune responses. Ther
Our understanding of the complex role of immunity in this disease remains incomplete as for example whether atherosclerosis is associated with HLA and other immune genes. Ther
These questions, however, should not lead us to overlook the fact that we currently have very encouraging results in experimental models of immunization against atherosclerosis. This should act as an impetus for researchers and the biotechnology industry to pursue the immunization approach more vigorously.
References