Peter X. Shaw

Innate and acquired immunity in atherogenesis.

Traditional risk factors like hypercholesterolemia are important for atherogenesis, but it is now apparent that the immune system also plays an important role. Uncovering the mechanisms by which specific components of the immune system impact atherogenesis will not only provide new insights into the pathogenesis of lesion formation, but could also lead to novel therapeutic approaches that involve immune modulation.

Oxidation-Specific Epitopes Are Danger-Associated Molecular Patterns Recognized by Pattern Recognition Receptors of Innate Immunity

Oxidation reactions are vital parts of metabolism and signal transduction. However, they also produce reactive oxygen species, which damage lipids, proteins and DNA, generating “oxidation-specific” epitopes. In this review, we discuss the hypothesis that such common oxidation-specific epitopes are a major target of innate immunity, recognized by a variety of “pattern recognition receptors” (PRRs). By analogy with microbial “pathogen-associated molecular patterns” (PAMPs), we postulate that host-derived, oxidation-specific epitopes can be considered to represent “danger (or damage)-associated molecular patterns” (DAMPs). We also argue that oxidation-specific epitopes present on apoptotic cells and their cellular debris provided the primary evolutionary pressure for the selection of such PRRs. Furthermore, because many PAMPs on microbes share molecular identity and/or mimicry with oxidation-specific epitopes, such PAMPs provide a strong secondary selecting pressure for the same set of oxidation-specific PRRs as well. Because lipid peroxidation is ubiquitous and a major component of the inflammatory state associated with atherosclerosis, the understanding that oxidation-specific epitopes are DAMPs, and thus the target of multiple arcs of innate immunity, provides novel insights into the pathogenesis of atherosclerosis. As examples, we show that both cellular and soluble PRRs, such as CD36, toll-like receptor-4, natural antibodies, and C-reactive protein recognize common oxidation-specific DAMPs, such as oxidized phospholipids and oxidized cholesteryl esters, and mediate a variety of immune responses, from expression of proinflammatory genes to excessive intracellular lipoprotein accumulation to atheroprotective humoral immunity. These insights may lead to improved understanding of inflammation and atherogenesis and suggest new approaches to diagnosis and therapy.

Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

Scavenger receptors, oxidized LDL, and atherosclerosis.

Abstract: Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR‐B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR‐B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.

Immunological responses to oxidized LDL

Considerable evidence now points to an important role for the immune system in experimental models of atherosclerosis. We have reviewed the growing body of evidence that oxidation of LDL generates a wide variety of neoself determinants that lead to cellular and humoral immune responses. In particular, we have demonstrated that at least some of the oxidation-specific epitopes generated on the oxidized LDL particle, such as oxidized phospholipid epitopes, are also generated on apoptotic cells and are also present on the surface of some bacteria. Many of these same epitopes serve as important ligands mediating the binding and clearance of oxidatively damaged lipoprotein particles and apoptotic cells, and the innate immune response to these epitopes can be seen as a concerted response to effect their removal. In addition, other epitopes of OxLDL also undoubtedly play a role in the immune activation that characterizes the progressive atherosclerotic plaque. It will be of great importance to define the importance of the role of these responses and to understand which are beneficial and which deleterious. Such information could lead one day to novel therapeutic approaches to inhibit atherogenesis that take advantage of the ability to manipulate the immune response.

Targeted Molecular Probes for Imaging Atherosclerotic Lesions With Magnetic Resonance Using Antibodies That Recognize Oxidation-Specific Epitopes

Background— Oxidized low-density lipoprotein plays a key role in the initiation, progression, and destabilization of atherosclerotic plaques and is present in macrophages and the lipid pool. The aim of this study was to assess the feasibility of magnetic resonance imaging of atherosclerotic lesions in mice using micelles containing gadolinium and murine (MDA2 and E06) or human (IK17) antibodies that bind unique oxidation-specific epitopes. Methods and Results— MDA2 micelles, E06 micelles, IK17 micelles, nonspecific IgG micelles, and untargeted micelles (no antibody) were prepared and characterized with respect to pharmacokinetics and biodistribution in wild-type and atherosclerotic apolipoprotein E–deficient (apoE−/−) mice. Magnetic resonance imaging was performed at 9.4 T over a 96-hour time interval after the administration of 0.075–mmol Gd/kg micelles. MDA2, E06, and IK17 micelles exhibited a longer plasma half-life than IgG or untargeted micelles in apoE−/− but not wild-type mice. In apoE−/− mice, MDA2 and IK17 micelles showed maximal arterial wall uptake at 72 hours and E06 micelles at 96 hours, manifested by 125% to 231% enhancement in magnetic resonance signal compared with adjacent muscle. Confocal microscopy revealed that MDA2, IK17, and E06 micelles accumulated within atherosclerotic lesions and specifically within macrophages. Intravenous injection of free MDA2 before imaging with MDA2 micelles resulted in significantly diminished magnetic resonance signal enhancement. IgG micelles and untargeted micelles showed minimal enhancement in apoE−/− mice. There was no significant signal enhancement with all micelles in wild-type mice. Conclusions— Magnetic resonance imaging with micelles containing gadolinium and oxidation-specific antibodies demonstrates specific targeting and excellent image quality of oxidation-rich atherosclerotic lesions.

The role of innate immunity in atherogenesis.

Lipid peroxidation is a common event in health and is greatly accelerated in pro-inflammatory settings such as hypercholesterolemia. Consequently, oxidation-specific epitopes are generated, which are pro-inflammatory and immunogenic, leading to both adaptive and innate responses. Because innate immune mechanisms use conserved germline pattern recognition receptors (PRRs) that are preformed and present at birth, it is not obvious why they should bind to such epitopes. In this review, we put forward the hypothesis that because oxidation-specific epitopes are ubiquitous in both health and disease, and because they in essence represent “danger signals,” they constitute a class of pathogen-associated molecular patterns leading to the natural selection of multiple innate PRRs that target such epitopes. We suggest that apoptotic cells, and the blebs and microparticles released from such cells, which are rich in oxidation-specific epitopes and thus pro-inflammatory, constitute an endogenous set of selecting antigens. In turn, natural antibodies, scavenger receptors, and soluble innate proteins, such as pentraxins, all represent PRRs that target such epitopes. We discuss the evidence for this hypothesis and the consequences of such responses in health and disease, such as atherosclerosis.

Oxidation-specific epitopes are important targets of innate immunity.

During the oxidation of LDL, a central pathophysiological component of atherogenesis, a wide variety of chemical and physical changes occur leading to the generation of oxidation‐specific neoepitopes. These epitopes are not only immunogenic, leading to adaptive humoral responses, but are also a prominent target of multiple arcs of innate immunity. The pattern recognition receptors (PRRs) of innate immunity are germ line encoded, conserved by natural selection, and bind to pathogen‐associated molecular patterns (PAMPs) common on multiple structures. However, it is not intuitive as to why they should recognize oxidation‐specific neoepitopes. Yet it is clear that multiple macrophage scavenger receptors, which are classic PRRs, recognize oxidation‐specific epitopes, such as those found on oxidized LDL (OxLDL). Other innate proteins, such as C‐reactive protein, also bind to OxLDL. Natural antibodies (NAbs), the humoral arc of innate immunity, provide a nonredundant role in the first line of defence against pathogens, but are also believed to provide important homeostatic house‐keeping functions against self‐antigens. Our work demonstrates that oxidation‐specific epitopes, as found on OxLDL, are a major target of NAbs. In this review, we will discuss the specific example of the prototypic NAb T15/E06, which is increased in atherosclerotic mice and mediates atheroprotection, and discuss the potential role of NAbs in atherogenesis, and in inflammation in general. We also review data that oxidation‐specific epitopes are generated whenever cells undergo programmed cell death, forming a common set of PAMPs recognized by oxidation‐specific PRRs on macrophages, NAbs and innate proteins. We present the hypothesis that oxidation‐specific epitopes on apoptotic cells exerted evolutionary pressure for the conservation of these PRRs and also serve to maintain the expansion of a substantial proportion of NAbs directed to these stress‐induced self‐antigens.

The Autoreactivity of Anti-Phosphorylcholine Antibodies for Atherosclerosis-Associated Neo-Antigens and Apoptotic Cells

Abs specific for phosphorylcholine (PC) are known to contribute to the immune defense against a variety of microbial infections. To assess for other types of binding interactions, we performed surveys of anti-PC Abs of diverse biologic origins and structural diversity and demonstrated a common autoreactivity for oxidatively modified low density lipoprotein and other oxidation-specific structures containing PC-Ags. We also found that cells undergoing apoptosis sequentially express a range of oxidation-specific neo-self PC determinants. Whereas natural Abs to PC recognized cells at early stages of apoptosis, by contrast, an IgG anti-PC Ab, representative of a T cell-dependent response, recognized PC determinants primarily associated with late stages of apoptosis. Cumulatively, these results demonstrate a fundamental paradigm in which Abs from both the innate and the T cell-dependent tiers of the B cell compartment recognize a minimal molecular motif arrayed both on microbes and as neo-self Ags linked to atherosclerosis and autoimmune disease.

Complement factor H genotypes impact risk of age-related macular degeneration by interaction with oxidized phospholipids

The rs1061170T/C variant encoding the Y402H change in complement factor H (CFH) has been identified by genome-wide association studies as being significantly associated with age-related macular degeneration (AMD). However, the precise mechanism by which this CFH variant impacts the risk of AMD remains largely unknown. Oxidative stress plays an important role in many aging diseases, including cardiovascular disease and AMD. A large amount of oxidized phospholipids (oxPLs) are generated in the eye because of sunlight exposure and high oxygen content. OxPLs bind to the retinal pigment epithelium and macrophages and strongly activate downstream inflammatory cascades. We hypothesize that CFH may impact the risk of AMD by modulating oxidative stress. Here we demonstrate that CFH binds to oxPLs. The CFH 402Y variant of the protective rs1061170 genotype binds oxPLs with a higher affinity and exhibits a stronger inhibitory effect on the binding of oxPLs to retinal pigment epithelium and macrophages. In addition, plasma from non-AMD subjects with the protective genotype has a lower level of systemic oxidative stress measured by oxPLs per apolipoprotein B (oxPLs/apoB). We also show that oxPL stimulation increases expression of genes involved in macrophage infiltration, inflammation, and neovascularization in the eye. OxPLs colocalize with CFH in drusen in the human AMD eye. Subretinal injection of oxPLs induces choroidal neovascularization in mice. In addition, we show that the CFH risk allele confers higher complement activation and cell lysis activity. Together, these findings suggest that CFH influences AMD risk by modulating oxidative stress, inflammation, and abnormal angiogenesis.