The role of the immune system in atherosclerosis and the potential for exploiting the immune system to influence the atherosclerotic disease process are new concepts in atherogenesis and atherosclerosis management.

The primary goals of the Atherosclerosis Research Center at Cedars-Sinai are: 1) to gain an understanding of the molecular mechanisms of atherothrombosis, focusing on novel genes, 2) to use this improved understanding to develop and test novel therapies that can prevent, reverse, or stabilize atherosclerosis. Some of the novel genes identified at this research center through transcriptional profiling of various arteries include PTN, LPP, TNF-, and M-CSF.

HDL-based therapeutics, the development and introduction of the recombinant apo A-1 milano, and oral apo A-1 limited peptide therapy are some of the novel therapies for atherosclerosis studied at the research center. Promising results with recombinant apo A-1 milano are being seen, which is already in clinical trials; much of the pre-clinical work was done at Cedars-Sinai. Gene transfer, using novel vectors to introduce therapeutic genes, is another program at the research center. The benefits of a number of atheroprotective genes, including Apo A-1 wild-type and Apo A-1 milano, is being explored in animals, with the hope of subsequent study in humans. The role of immune mechanisms and developing novel ways of modulating atherosclerosis by influencing the immune system constitutes another large program at the research center.

Inflammatory gene induction and immune cell activation plays a critical role in every step of the evolution from a normal artery to an artery with an atherosclerotic lesion and subsequent plaque rupture and thrombosis. The retention and modification of atherogenic lipoproteins at atherosclerosis-prone sites activate the inflammatory and immune mechanisms that lead to the earliest stages and then progression of atherosclerosis and eventually to de-stabilization of atherosclerosis.

The evidence for the involvement of the immune system in atherosclerosis comes from animal and human models of the disease: 1) In animal models, the presence of activated T-cells, macrophages, mast cells, and dendritic cells, all components of the innate and adaptive immune response, can be found at the earlier stages of lesion evolution. 2) T-cells reactive to various antigens can be demonstrated in atherosclerotic lesions and in circulating blood. 3) Antibodies to a variety of auto-antigens are found in circulating blood and atherosclerotic lesions. 4) T-cell-dependent cytokines can influence and modulate atherosclerosis. 5) Immunization and tolerization experiments clearly can modulate atherosclerosis in a favorable or unfavorable direction. 6) Pro-atherogenic and anti-atherogenic immune responses can be demonstrated to be transferable using adaptive cell transfer techniques. All of these data suggest that the immune system plays an important role in modulating experimental atherosclerosis.

Similarly, in human models of atherosclerosis, there is evidence for a role of the immune system. The presence of all of the cells involved in the immune response has been shown in the earliest to most advanced stages of atherosclerotic lesions.  As in animals, antibodies and reactive T-cells can be demonstrated. Pro-atherogenic effects of immune suppression have been documented in transplant medicine in humans. Accelerated atherosclerosis in autoimmune disease, such as lupus, is further evidence.  Atheroprotective effects of non-specific vaccines, such as influenza vaccine, have recently been demonstrated in cross-sectional and pilot clinical trials in humans.

The macrophage is the major factor in innate immunity. It is a blank response of macrophages, with the receptors CD-36, toll-like receptor (TLR), and SR-AB. When these receptors are engaged by a variety of pathogen-associated molecular patterns (PAMPS) and oxidized lipids, it leads to either foam cell formation in the case of the scavenger receptor or CD-36, or to activation of inflammatory and immune active genes with the engagement of TLR, and then the release of inflammatory mediators.

TLR-4 is a transmembrane receptor, which, along with other co-receptors such as CD-14, is able to signal the effects of bacterial lipopolysaccharide, endotoxin, or heat shock proteins (HSP). Through adaptive molecules, such as MD-88, it is able to activate and translocate NF-ɻB to the nucleus, leading to the induction of inflammatory and immune genes and mediators.

TLR-4 immunoreactivity can be detected in lipid-rich atherosclerotic plaques and co-localizes with macrophages, as recently demonstrated by Shah and colleagues in collaboration with another laboratory. TLR-4 is primarily expressed by macrophages and by the endothelial cells in human and murine atherosclerotic lesions. Interestingly, TLR-4 mRNA is upregulated in a dose-dependent fashion by oxidized LDL, but not by native LDL—indicating a link between lipoproteins and the innate immune system.

The innate immune system seems to play a pro-atherogenic role. TLR signaling is responsible for linking some of the pro-inflammatory effects of hyperlipidemia in the murine model and thus plays an important role in atherogenesis.

In experiments with double-knockout mice that lacked the ApoE gene and either one or both alleles of the MyD88 gene (myeloid differentiation factor), a critical adapter protein in the signal transduction cascade for TLR, Shah and colleagues showed that the introduction of the MyD88 null genotype, in a dose-dependent fashion, is associated with a reduction in atherosclerosis.

The double-knockout mice homozygous for the MyD88 knockout had about a 55% reduction in atherosclerosis. In addition, a dose-dependent decrease in plaque inflammation was measured by macrophage immunoreactivity, with about a 60% reduction when both alleles of the MyD88 gene had been knocked out. Clearly, the innate immune system is playing a pro-atherogenic role. Disrupting the innate immune signaling through disruption of the MyD88 gene results in a major reduction in atherosclerosis and plaque inflammation.

Notably, the effects on atherosclerosis are not mediated through a reduction in circulating lipid concentration. No significant change in total cholesterol or triglyceride levels was seen in concert with the reduction in atherosclerosis, and the lipoprotein profile of all 3 groups of mice was very comparable.

In contrast, knocking out the MyD88 gene and disrupting immune signaling was associated with a significant decrease in circulating levels of chemokines and pro-atherogenic cytokines (MCP-1, IL-1, p40), which were significantly reduced in the double-knockout mice, perhaps playing a role in the atheroprotective effects of disrupting innate immune signaling. TLR-4 and Apo E double-knockout mice also demonstrate about a 25%-30% reduction in atherosclerosis, parallel to that seen with the MyD88 knockout mice, as shown by Shah and colleagues.

In contrast to innate immunity, the adaptive immune response (AIR) in atherosclerosis is more complicated. AIR has the potential to be pro-atherogenic and anti-atherogenic.

AIR is generated when antigen-presenting cells, such as dendritic cells, carry in antigen and expose them to T-cells and B-cells, leading to activation of the T and B cells. The T cells can then become polarized, along the pro-inflammatory TH-1 phenotype with release of interferon-gamma and pro-inflammatory cytokines, or along the TH-2 phenotype with endo-inflammatory cytokine IL-10 release and potential anti-atherogenic effects. Similarly, B-cell engagement and activation can lead to plasma cellformation and formation of antibody to the presented antigen.

The auto-antigens that have elicited an AIR in experimental animal studies and in humans include HSP-60, ß2GP1, and ox-LDL, which is the most important and most studied. The immune response to HSP-60 and HSP-65 is atherogenic, as shown by immunization experiments in mice and rabbits resulting in increased atherosclerosis. Conversely, tolerization to HSP-65 using nasal or gastrointestinal mucosal antigen exposure induces tolerance to HSP-65 and reduces atherosclerosis.

The immune response to ß2GP-1 is also atherogenic. Immunization of experimental animals with ß2GP-1 is associated with accelerated atherosclerosis. Notably, this effect can be transferred to non-immunized mice through the transfer of lymphocytes from immunized mice—indicating that this adaptive transfer conveys the pro-atherogenic effects of the immune response to ß2GP-1.

The evidence for the humoral immune response to ox-LDL on atherosclerosis in humans had been conflicting. Several studies suggested that in the absence of coronary artery disease (CAD) there was less immune response than in the presence of CAD. However, other studies that examined different markers of atherosclerosis showed no significant difference between controls and cases for the magnitude of anti-oxidized LDL antibody levels. Uncertainty about the immune response to oxidized LDL wascreated.

The athero-protective effect of immunization with oxidized LDL or native LDL was first shown by experiments by Shah and colleagues and by Palinski and colleagues. Confirmation came from other investigators in experiments using LDL-R null mice and in another using apo-E null mice.

Shah and colleagues studied the immune response to ox-LDL in animal models. The rationale for the experiments was that oxidative modification of LDL leads to changes in the phospholipid and protein components of LDL and thereby exposes neoantigens, which are then recognized by the immune system and then can react in the form of antibody production or T-cell polarization and cytokine secretion.

Hypercholesterolemic rabbits were immunized with LDL from rabbits as an antigen against homologous unmodified LDL or homologous oxidative modified LDL. After 16 weeks, in which the primary immunization and a booster was given, no significant effect was seen on circulating cholesterol levels. However, contrary to expectations, both native and oxidized LDL as an antigen dramatically reduced atherosclerosis, by nearly 60% in this rabbit model (p<0.05). About the same time, nearly identical results were reported by Palinski and colleagues, with about a 30%-40% reduction in atherosclerosis in the aorta of the Watanabe rabbit model, using malondialdehyde-modified LDL as an antigen.

Apo B100-related peptide vaccines were developed based on 101 peptide sequences that could have antibodies in human serum, which were identified in a collaborative program between Shah and colleagues and Nilsson and colleagues in Sweden to develop a testable strategy in humans.

In one experiment, vaccines were created using 2 peptide sequences with significant homology to mouse Apo B100. Primary immunization then was given to Apo E null mice at 6-7 weeks and a booster at 8-9 weeks. At 25 weeks, the animals were sacrificed to examine the extent of atherosclerosis. No statistically significant difference was seen in serum cholesterol levels with Peptide 1 or Peptide 2 compared to control, although there was a trend for lower cholesterol levels in the Peptide 1 group and a little higher in the Peptide 2 group compared to control (700 mg/dl, 1400 mg/dl, and 1100 mg/dl, respectively). In contrast, about a 50% reduction in atherosclerosis in the aorta was found in the Peptide 2 group compared to the Peptide 1 group or control (p<0.01). Substantially less atheroma production was found in the Peptide 2 group, accompanied by a significant decrease in plaque inflammation as measured by macrophage immunoreactivity, which was reduced significantly in the Peptide 2 group. In parallel, a significant increase in collagen staining in the plaques was seen in the Peptide 2 group, as measured by trichrome stain. Peptide 2 immunization reduced atherosclerosis substantially, although the cholesterol levels were the highest in this group, reduced plaque inflammation, and increased plaque collagen content, indicating a shift in the plaque morphology to a more stable phenotype.

In terms of potential mechanisms, in splenic cytokine expression, both peptide antigens caused an increase in the TH-1 cytokine interferon-gamma, and TH-2 cytokines IL-10 and IL-4. This suggests that the specific athero-protective effects of Peptide 2 immunization are unlikely to be mediated by a shift from a TH-1 to TH-2 cytokine profile. In contrast, there was a significant difference in the antibody levels produced in immunized groups. Data from another set of experiments conducted with 2 additional athero-protective antigens (antigen 35 and antigen 74) showed that compared to control the peptide-immunized animals had an increase IgG1 subtype compared to IgG1 subtype, indicating a significant humoral immune response to the antigen.

Intriguingly, adaptively transferred splenocytes conveyed the atheroprotective effects of Peptide 2 immunization to non-immunized mice—highlighting the important role of the spleen and splenocytes in this process. In an experiment with 3 groups of non-immunized mice, the mice that received splenocytes from Peptide 2 immunized mice had virtually the same degree of reduction of atherosclerosis as the group that had received the actual Peptide 2 immunization.

A passive immunization approach was used in recent experiments, and confirmed that the effects of active immunization could be reproduced by a passive immunization using the pre-formed antibody against one of the protective antigenic epitopes identified during the active immunization experiments. In these experiments, compared to an irrelevant antibody, the specific antibody in a dose-dependent fashion reduced atherosclerosis in Apo E null mice, while the irrelevant antibody had no significant effect.

Shah and colleagues have demonstrated the feasibility and proof of concept that specific Apo B100-related peptide sequences can promote athero-protective and anti-inflammatory response in hypercholesterolemic Apo E null mice. This response is associated with increased antibodies, and can be adaptively transferred through splenocytes. They believe that the potential mechanism by which this immunization approach may work is by creating antibodies that can lead to increased clearance of LDL from the circulation and reduced deposition of LDL within the atherosclerotic lesion. Yet, the possibility that T-cell polarization with TH-2 dominance may also be playing a role in reducing atherosclerotic lesions cannot be ruled out.

The immune system plays a complex role in atherosclerosis with pro-atherogenic and athero-protective effects. The innate immune system appears to be primarily pro-atherogenic, while the adaptive immune response appears to have both pro-atherogenic and athero-protective components. Immunization using LDL or ox-LDL and specific Apo B-100 related peptide sequences reduces atherosclerosis and favorably modifies plaque composition, without altering circulating levels of cholesterol, raising the idea that immunotherapy is feasible. Immunotherapy of atherosclerosis warrants further investigation.