Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity

Abstract

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.

Figure 1. Oxidized lipid moieties induce lipoprotein accumulation in macrophages

Macrophage lipoprotein uptake mechanisms can be separated into (1) macropinocytosis, when actin polymerization and extensive membrane ruffling result in the ruffles closing into large endosomes and capture of large volumes of extracellular material, including all classes of native and oxidized LDL present in the vicinity of the cell, and (2) micropinocytosis, when ligand-receptor binding leads to membrane invagination and nearly stoichiometric internalization of the ligand or the lipoprotein carrying this ligand. mmLDL and polyoxygenated CE hydroperoxides (OxCE) induce Syk recruitment to TLR4, Syk and TLR4 phosphorylation and subsequent ERK1/2-dependent activation of small GTPases Rac, cdc42 and Rho, and phosphorylation of paxillin, leading to actin reorganization and membrane ruffling. Resulting macropinocytosis promotes foam cell formation . Binding of OxLDL or OxPL to CD36 initiates Lyn-dependent phosphorylation of JNK, which is essential for CD36-mediated OxLDL uptake, although the mechanism linking JNK with the membrane dynamics is unclear . The TLR4- and CD36-mediated uptake mechanisms are only examples; there are numerous other PRRs involved in oxidation-specific epitope-stimulated lipoprotein internalization by macrophages.

Figure 2. Oxidized LDL and OxPL induce ROS generation in vascular cells

In macrophages, OxLDL binding to CD36 induces recruitment of Lyn, a Src kinase, its activation and phosphorylation of Vav, which in turn activates Rac . Unlike OxLDL, mmLDL induces recruitment of Syk to TLR4 and Syk-dependent activation of Vav and Rac . In addition, mmLDL induces TLR4- and Syk-dependent activation of PLCγand PKC, which induces recruitment of p47phox, and p67phox (not shown) to the Nox2 enzyme complex, and activated (GTP-bound) Rac completes the complex, leading to ROS production ,. High levels of extracellular ROS may exacerbate the oxidative damage, while intracellular ROS are important signaling molecules, mediating cytokine secretion. One of mmLDL-induced, Nox2-dependent chemokines, RANTES, induces VSMC migration . Nox4 is the predominant NADPH oxidase in endothelial cells. It is activated by OxPAPC via VEGFR2-dependent Rac recruitment . However, the mechanism of the OxPAPC activation is unclear. Although OxPAPC is capable of inducing VEGF production by endothelial cells , anti-VEGF antibodies do not block OxPAPC activation of VEGFR2 . ROS in endothelial cells mediate secretion of MCP-1 and IL-8 and thereby promote monocyte migration.

Figure 3. mmLDL and low-dose LPS induce cooperative TLR4-mediated signaling in macrophages

mmLDL induces rapid, JNK-dependent phosphorylation of Jun, which leads to removal of NCoR and derepression of AP-1. This, together with rapid phosphorylation of ERK1/2, results in the completion of the AP-1 transcription complex, which is later strengthened by delayed ERK1/2 and Jun phosphorylation induced by LPS . However, LPS-induced Jun phosphorylation is mediated by IKKε, which is brought to the promoter region with the NF-κB transcription factors . Cooperative activation of AP-1 and NF-κB result in increased expression of proinflammatory genes induced by co-stimulation with mmLDL and LPS .

Figure 4. Pattern recognition of oxidation-specific DAMPs and microbial PAMPs

Using the example of the phosphocholine (PC) epitope, we illustrate in this cartoon our hypothesis of the emergence and positive selection of multiple PRRs that recognize common epitopes, shared by modified self and microbial pathogens. According to this hypothesis, oxidation of plasma membrane phospholipids in apoptotic cells alters the conformation of the PC headgroup, yielding an exposed epitope, accessible to recognition by macrophage scavenger receptors, NAbs, and pentraxins, such as CRP. These PRRs were selected to clear apoptotic cells from developing or regenerating tissues. Recognition by the same receptors of the PC epitope of capsular polysaccharide in gram-positive bacteria (e.g. S. pneumoniae) strengthened positive selection of these PRRs and probably helped to select additional strong proinflammatory components to PRR-dependent responses. (Note the PC on the bacteria is not part of a phospholipid.) Finally, oxidized lipoproteins, prevalent in humans as a result of dyslipidemia and impact of environmental factors and in experimental animals, bear OxPLs with the PC epitope exposed in an analogous manner to that of apoptotic cells. This leads to OxLDL recognition by PRRs and initiation of innate immune responses. The balance between pro-inflammatory responses of cellular PRRs and atheroprotective roles of NAbs plays an important role in the development of atherosclerosis. There are likely many more oxidation-specific epitopes that represent such DAMPS and corresponding PRRs that represent respective innate responses.