key: cord-0774609-zizv1n10
authors: Binder, Christoph J.; Papac-Milicevic, Nikolina; Witztum, Joseph L.
title: Innate sensing of oxidation-specific epitopes in health and disease
date: 2016-06-27
journal: Nat Rev Immunol
DOI: 10.1038/nri.2016.63
sha: 4ebe445ce9d252e8d0935d58b262728704d73133
doc_id: 774609
cord_uid: zizv1n10

Ageing, infections and inflammation result in oxidative stress that can irreversibly damage cellular structures. The oxidative damage of lipids in membranes or lipoproteins is one of these deleterious consequences that not only alters lipid function but also leads to the formation of neo-self epitopes — oxidation-specific epitopes (OSEs) — which are present on dying cells and damaged proteins. OSEs represent endogenous damage-associated molecular patterns that are recognized by pattern recognition receptors and the proteins of the innate immune system, and thereby enable the host to sense and remove dangerous biological waste and to maintain homeostasis. If this system is dysfunctional or overwhelmed, the accumulation of OSEs can trigger chronic inflammation and the development of diseases, such as atherosclerosis and age-related macular degeneration. Understanding the molecular components and mechanisms that are involved in this process will help to identify individuals with an increased risk of developing chronic inflammation, and will also help to indicate novel modes of therapeutic intervention. SUPPLEMENTARY INFORMATION: The online version of this article (doi:10.1038/nri.2016.63) contains supplementary material, which is available to authorized users.

Oxidation-specific epitopes (OSEs) . Novel epitopes that form as a result of lipid peroxidation in response to various physiological and pathological situations. These epitopes are enriched on oxidized low-density lipoprotein and on membranes of microvesicles and apoptotic cells. They are recognized by humoral and cellular immune responses.

Small (0.1-1 μm) membrane vesicles shed from activated or dying cells. They expose phosphatidylserine and carry surface markers of their parental cells. They mediate the transfer of proteins, nucleic acids and lipids between cells. Elevated levels of circulating microvesicles have been reported in acute and chronic inflammatory diseases.

(MDA)modified amino groups, have been documented on the surface of apoptotic cells, microvesicles and dam aged structures such as oxidized low-density lipoproteins (OxLDLs) 14, 17, 20 . They are sensed by humoral and cellu lar components of the innate immune system, which mediate their removal and prevent inflammatory effects. However, if not efficiently cleared, they can also act as damage-associated molecular patterns (DAMPs) and can trigger sterile inflammation 21, 22 

.

In this Review, we discuss the role of OSEs in health and disease, and the innate immune responses that specifically target OSEs as markers of oxidative stress.

Oxidized lipids, and their degradation products, can interfere with the normal function of proteins and lipids. However, a unique aspect of some of these lipid derivatives is their ability to modify proteins and lipids and to form OSEs, which are then recognized by the immune system in a haptenspecific manner; for exam ple, the same OSE can be present on different proteins and can be recognized by a common set of innate immune receptors. Several mechanisms can lead to the oxidation of lipids, particularly of polyunsaturated fatty acids (PUFAs) 8, 23, 24 , and important insights into the generation of OSEs have been obtained from studies on the biological activities of OxLDL that are central to the pathogenesis of atherosclerosis 25, 26 . Lipid peroxida tion involves both enzymatic mechanisms, including lipoxygenases, cyclooxygenases and cytochrome p450, and nonenzymatic mechanisms, which are mediated by free radicals and which require catalysis by transition metals or hemin 23, [26] [27] [28] 

. Moreover, there is now ample evidence that during apoptosis membrane lipids undergo oxidation, which results in the generation of OSEs. The exact mechanisms that lead to the oxidation of cellular membrane lipids are still being elucidated 17, 29 .

PUFAcontaining phospholipids, such as the major membrane phospholipid phosphatidylcholine, are particularly prone to oxidative damage, and their per oxidation results in the generation of a complex mix ture of OxPLs and terminal degradation products 8, 23, 24 

. These, in turn, form adducts with, for example, the amino group of lysine residues or aminophospho lipids to generate OSEs. In particular, the oxidation of the PUFA chain at the sn-2 position of phosphatidyl choline results in its fragmentation and the generation of reactive PUFA breakdown products such as MDA and 4hydroxynonenal (4HNE), which can modify selfmolecules and form OSEs (FIG. 1) . OSEs can also be generated by the modification of proteins with truncated phospholipids, such as oxidized phosphatidylcholine, oxidized cardiolipin (OxCL), oxidized phosphatidyl serine (OxPS) and oxidized phosphatidylethanola mine (OxPE) 8, [30] [31] [32] . Furthermore, 2 (ω carboxyethyl) pyrrole (CEP) represents an adduct between (E)4hydroxy7oxohept5enoic acid -an oxidative fragment of docosahexaenoic acid -and the amino groups of lysines or aminophospholipids 24, 33, 34 . Notably, some of these breakdown products, such as MDA and 4HNE, have been studied as prototypical markers of oxidative stress and can be measured by the frequently used 2-thiobarbituric acid reaction (TBAR) method 8 . As a vast diversity of OxPLs can be generated, many more OSEs derived from these may exist 23, 28 . Moreover, the oxidation of other lipids, such as cholesterol and chol esteryl esters, can lead to structural changes and altered biological activities, but their ability to be recognized by innate immune receptors is still being characterized 10, 25, 35 . It will be important to elucidate the exact structures of the relevant OSEs to better understand their recogni tion by the immune system. Owing to their biological activities, these products of lipid peroxidation have been implicated in a wide variety of physiological and patho logical processes, and immune responses that target these products can modulate many of these effects 14, 30, 36 .

OSEs have important roles in physiological processes, as they mark oxidatively modified endogenous molecules, such as proteins and/or lipoproteins and apoptotic cells, as being damaged by increased oxidative stress. This facilitates their removal by the housekeeping func tions of the immune system to maintain homeostasis. In pathological situations -in which oxidative events are greatly increased and the homeostatic functions of innate immunity are overwhelmed and/or impairedthe accumulation of OSEs leads to sterile inflammation, which is a mechanism that should ultimately restore normal tissue integrity 22 

. If this fails as a result of persistent tissue damage and/or impaired resolution, the

Lipid peroxidation is the oxidative damage of lipids that is initiated by the removal of a hydrogen atom from the CH 2 group within the double bonds of polyunsaturated fatty acids (PUFAs). The subsequent addition of oxygen radicals and the formation of lipid-peroxyl radicals, which are transformed into lipid-hydroperoxide molecules can further propagate this reaction. Both enzymatic and non-enzymatic mechanisms can lead to lipid peroxidation. The enzymatic mechanisms involve lipoxygenases, cyclooxygenases and cytochrome P450, and result in the generation of products with high stereo-specificity 23, 144 . Depending on the PUFA substrate, major products of lipoxygenase oxidation are hydroxyperoxyeicosatetraenoic acids (HpETEs) hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins, hydroxyoctadecadienoic acids (HODEs), hepoxylins and resolvins. Oxygenation by cyclooxygenases results in the generation of prostaglandins, prostacyclin and thromboxanes, whereas cytochrome P450 generates epoxyeicosatrienoic acids (EETs), 20-HETE, thromboxanes and prostacyclins 27, 145 . Non-enzymatic mechanisms are mediated by free radicals that are generated by NADPH oxidases and nitric oxide synthases in the presence of transition metal ions (Fe 2+ and Cu 2+ ) and result in a mixture of nonspecific stereoisomers 23, 27 . Moreover, the enzyme myeloperoxidase can also initiate non-enzymatic lipid peroxidation through the generation of reactive oxygen species (ROS), such as HOCl, HOBr and ·NO 145, 146 . In contrast to products of enzymatic lipid oxidation, the products of non-enzymatic processes are considered to be more toxic and damaging to the host 147 . Continuous degradation of lipid-hydroxyperoxides by cyclization and fragmentation in the presence of reducing metal ions (Fe 2+ and Cu 2+ ) results in the generation of highly reactive terminal degradation products such as reactive aldehydes 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) and 2-(ω carboxyethyl) pyrrole (CEP) 8, 24 . Lipid peroxidation products that are generated by both mechanisms modulate a multitude of physiological processes such as cell signalling, wound healing, immune tolerance, skin barrier function, coagulation, vasodilation, modulation and the resolution of inflammation 12, 13, 16, 23, 145, 148, 149 . They are rapidly removed in healthy tissues but accumulate in many pathological conditions in which they can cause adverse effects. 

(TBAR). The TBAR method is used to measure lipid peroxidation products, such as malondialdehyde (MDA) or other reactive aldehydes, as these products generate thiobarbituric acid-reactive substances that can be detected by colorimetric or fluorometric measurements. accumulation of OSEs can trigger chronic inflammation. Therefore, OSEs have important roles in both the regu lation of tissue homeostasis and inflammatory responses (FIG. 2) . The detailed characterization of OSEs and the immune responses against these OSEs have shown that they are recognized by both cellular and soluble pat tern recognition receptors (PRRs), including natural IgM antibodies 14, 31 (TABLE 1) .

OSEs are recognized by a wide variety of cell surface receptors (TABLE 1) , most of which are expressed by macrophages. Macrophages mediate the uptake and phagocytosis of oxidatively altered molecules and cells, and macrophages also sense the accumulation of OSEs in the local micro environment, which triggers signalling pathways that induce the secretion of inflammatory chemokines and cytokines 14, 17, 31 .

Scavenger receptors represent the prototypical class of innate receptors for OSEs 37 . They are multiligand receptors that bind both modified selfstructures and nonself molecules. Different types of scavenger recep tors have been identified, including CD36, SRA1, SRA2, SRB1, CD68 and lectinlike oxidized LDL receptor 1 (LOX1; also known as OLR1) 37 . Among these, SRA1, SRA2 and CD36 are considered to be responsible for most of the OxLDL uptake by macrophages in in vitro assays, because macrophages that are deficient in CD36, SRA1 and SRA2 show 75-90% decreased binding and degradation of OxLDL 38 . Detailed structural studies have shown that OxPL is a highaffinity ligand that mediates OxLDL binding to CD36 (REF. 39) , and, spe cifically, that this is mediated by the phosphocholine (PC) headgroup of OxPL (PCOxPL) but the PC of unoxidized phospholipids does not serve as a ligand 40 . Binding can also be mediated by the truncated sn2 acyl chain of OxPLs 41, 42 , which indicates that other classes of oxidized phospholipids such as OxPS can bind to CD36 (REFS 43, 44) . Similarly, PCOxPL also mediates the uptake of OxLDL by SRB1 on macrophages 45 . Examples of other OSEs that are recognized by scavenger receptors include CEPmodified proteins, which are recognized by CD36 (REF. 46) and MDAmodified proteins, which are taken up by SRA1 and SRA2 47, 48 . A recent study showed that the endothelial scavenger receptor LOX1 mediates MDAinduced nitric oxide (NO) production 49 and this receptor also binds 4HNE 50 .

Tolllike receptors (TLRs) also recognize and respond to OSEs. Macrophages stimulated with oxidized 1palmitoyl 2arachidonylsnglycero3phosphocholine (OxPAPC) secrete interleukin6 (IL6), whereas IL6 production is absent in macrophages that lack TLR4 or TIRdomaincontaining adaptor protein inducing interferonβ (TRIF; also known as TICAM1) 51 . Sensing of PCOxPL seems to be important for the TLR4mediated effects, as the PCspecific IgM T15/E06 inhibited IL6 secretion induced by OxPAPC but did not inhibit IL6 secretion induced by lipopolysaccharide (LPS) 51 . OxPLs have also been reported to stimulate macrophages via a TLR2 pathway 52 . Moreover, chemokine secretion by macrophages stimulated with OxLDL has been shown to require the cooperation of CD36 with a TLR4-TLR6 heterodimer 53 . Conversely, OxPLs induce apoptosis in macrophages via TLR2-TLR6 in cooperation with a CD36 ligand 54 . As most of these studies have used mix tures of different OxPL products, individual OxPL moi eties might use TLR receptors in varying combinations to generate different signalling pathways. The common role of TLRs as sensors of OSEs is further supported by the findings that CEP adducts are bound by TLR2, but not by TLR4, on both endothelial cells 33 and macrophages 46 . Furthermore, CEP selectively augments TLR2-TLR1 signalling in macrophages 55 and has been shown to acti vate platelets via TLR2-TLR1 (REF. 56). TLRs have not yet been directly implicated in chemokine secretion that is induced by MDA or 4HNE, but this process is probably also mediated by the cooperation of scavenger recep tors with other signalling PRRs 57 . Oxidized cholesteryl esters (OxCEs) trigger proinflammatory macrophage responses via TLR4 and these involve nonclassical sig nalling via spleen tyrosine kinase (SYK) 35, 58 . Thus, some OSEs represent endogenous TLR ligands that have the capacity to trigger inflammatory responses. Notably, in endotoxininduced inflammation, soluble OxPLs impair complex formation of TLR4 with CD14, MD2 and LPS binding protein and thereby dampen LPSinduced responses 23, 59 . Recently, epoxycyclopentenones that are present in OxPAPC were identified as active compo nents responsible for the inhibitory effects of OxPAPC on the activation of specific TLRs by synthetic ligands 60 . An interesting property of the inflammatory responses to OxPL is the tight cooperation with scavenger receptors, such as CD36, which mediate recognition of OxPLs and probably also facilitate sensing and signalling by TLRs. Although OxLDL uptake via CD36 seems to be necessary for some macrophage signalling pathways, it is unknown to what extent scavenger receptormediated uptake of other OSEs is required for their cellular responses.

OSE sensing by soluble PRRs. Soluble PRRs include secreted versions of cellular PRR, pentraxins and pro teins of the complement system 61 . Whereas some of

Sterile inflammation is an inflammatory process that is elicited in response to damage-associated molecular patterns (DAMPs), which are released locally in response to tissue damage 22 . DAMPs are intracellular and extracellular host-derived molecules that are not usually sensed by the immune system but that are released or become modified into altered self-molecules upon tissue damage 21 . In analogy to pathogeninduced inflammation, sterile inflammation is triggered by the activation of the innate immune response through the recognition of DAMPs by pattern recognition receptors (PRRs), resulting in the enhanced secretion of cytokines and chemokines. Membranebound PRRs, such as Toll-like receptors (TLRs), and intracellular PRRs, such as the inflammasome, are key mediators of sterile inflammation. Cytokines belonging to the interleukin-1 (IL-1) family have been proposed to be important drivers of sterile inflammation 150 . Increased cytokine and chemokine secretion at the site of initial damage ultimately results in an enhanced recruitment of immune cells, such as neutrophils and macrophages. The resolution of sterile inflammation should lead to tissue repair and the re-establishment of homeostasis. Unresolved sterile inflammation is implicated in the development of several medical conditions, such as gout, Alzheimer disease, rheumatoid arthritis and atherosclerosis. 

Cell-membrane proteins that take up oxidatively or otherwise modified low-density lipoproteins and mediate their clearance by macrophages and other cell types. They have subsequently been shown to bind a large variety of different modified proteins as well as pathogens.

C-reactive protein (CRP). A highly conserved pentraxin that is induced and rapidly secreted by the liver in response to bacterial infections. Increased plasma CRP levels are also found in a wide variety of other non-infectious chronic inflammatory states.

Complement factor H (CFH). A soluble glycoprotein (155 kDa) that is made up of 20 short consensus repeats (SCRs) organized in a 'bead on the string' manner. It is the major inhibitor of the alternative complement pathway and is highly abundant in plasma at steady-state levels of 400-700 μg/ml. these proteins interact with cellular PRRs to mediate the signals of bound ligands, others participate in the housekeeping functions of the host. Soluble PRRs dis tinguish between healthy and damaged tissues by sens ing metabolic byproducts and OSEs presented on dying cells (TABLE 1) .

C-reactive protein (CRP) was originally identified as the Creactive component of plasma that could bind the PC present on the capsular polysaccharide of Streptococcus pneumoniae 62-64 . However, this PC moiety is not part of a phospholipid and CRP was later found to also bind PCOxPL found on OxLDL and on apoptotic cell mem branes 65 . This molecular mimicry between microbial PC and PCOxPL enables CRP to respond to a com mon OSE present during both microbial infections and oxidative stress. By analogy, Porphyromonas gingivalis and group A Streptococcus have been suggested to carry MDAlike epitopes, but their ability to be recognized by MDAspecific PRRs has not been tested in detail 66 .

The identification of complementregulatory pro tein complement factor H (CFH) as a receptor for MDA epitopes has shed important light on its function in disease 67 . CFH consists of 20 short consensus repeats (SCRs) 68 and two of its domains -SCR7 and SCR1920 -mediate the binding of CFH to MDA 67 . Importantly, these binding sites are hotspots for diseaseassociated single nucleotide polymorphisms (SNPs) 69 ; for example, SNP rs1061170 results in a Y>H exchange of amino acid 402 in SCR7 and reduces the ability of plasma CFH to bind MDA by more than 65% for homozygous carriers compared with controls 67 . In addition, the use of recom binant variants of the SCR1920 domain demonstrated that other SNPs in SCR1920 could alter binding to MDA 70 . Thus, genetic variants of both MDAbinding sites of CFH may determine the ability of CFH to bind MDA. Considering the conserved structure of the regu lators of complement activation, MDAepitopes could also serve as binding sites for other members of this family 68 . Interestingly, the C3 cleavage product C3a, which acts as a proinflammatory anaphylatoxin, was also shown to bind MDAepitopes 71 .

Other soluble PRRs may recognize oxidized rather than native lipid structures on cellular surfaces, but the exact nature of these interactions is less well under stood. For example, milk fat globule-epidermal growth factor 8 (MFGE8) has recently been shown to specifi cally recognize OxPS 72 and OxPE 73 . Moreover, another phosphatidylserinebinding protein, annexin A5, has been suggested to bind to OxCL and to neutralize its biological effects 74 .

Therefore, OSEs represent targets for several soluble PRRs of the innate immune system. As different OSEs are often present on the same surfaces, different solu ble PRRs binding to them may synergize and cooperate in their effector functions. For example, apoptotic cells and microvesicles carry both MDA and PCOxPL epitopes 20,75 , each of which recruits different soluble PRRs to the same surface. Tissue damage, cellular stress and cell death result in increased oxidative stress, which promotes lipid peroxidation. Lipid peroxidation can occur through non-enzymatic mechanisms, such as reactive oxygen species (ROS), and through enzymatic mechanisms, including myeloperoxidases, 12/15-lipoxygenases, cyclooxygenases and cytochrome P450. The oxidation of sn-2 polyunsaturated fatty acids (PUFAs) of membrane phospholipids leads to fragmentation and the generation of highly reactive breakdown products, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) 8, [30] [31] [32] . In addition, different types of oxidized phospholipids (OxPLs) can be generated from different phospholipid backbones, including oxidized phosphatidylcholine, oxidized phosphatidylethanolamine (OxPE), oxidized phosphatidylserine (OxPS) and oxidized cardiolipin (OxCL). The newly generated breakdown products and the oxidized and truncated residual core OxPLs can in turn react with free amino groups of protein side chains or lipids that are localized in their vicinity, forming stable covalent adducts and creating oxidation-specific epitopes (OSEs). Because phosphocholine (PC) as an OSE is only presented as an epitope in the context of OxPL, these epitopes are termed PC-OxPLs for clarity. The PC moiety can also be a component of the capsular polysaccharide of bacteria, where it is not part of a phospholipid and is constitutively presented as an epitope. In addition, an adduct between an oxidative fragment of docosahexaenoic acid, (E)-4-hydroxy-7-oxohept-5-enoic acid and lysines of proteins (or aminophospholipids) can lead to the formation of 2-(ω-carboxyethyl) pyrrole (CEP). OSE-modified proteins or lipids are sensed by innate immune responses and represent a unique class of damage-associated molecular patterns (DAMPs). (TABLE 1) . IgM antibodies in human umbili cal cord blood, which represent naive natural antibodies of fetal origin, have specificity for OSEs 75 . In fact, titres of IgM with specificity for OxLDL and MDALDL were higher in cord blood samples compared with matched maternal blood samples. The specific binding proper ties of natural IgM antibodies have been investigated by characterizing IgM antibodies derived both from in vitro stimulated B1 cells and from the plasma of mice lack ing recombinationactivating gene 1 (RAG1) that were selectively reconstituted with B1 cells 75 . These studies revealed that several OSEs, including PCOxPL, MDA and 4HNE adducts are bound by up to 30% of all natural IgM found in the plasma of both wildtype and gnoto biotic mice, and by a similar percentage of IgMsecreting cells from the spleens of wildtype mice 75 . Furthermore, several monoclonal natural IgM antibodies with specific ity for different OSEs have been characterized. For exam ple, the wellstudied PCbinding T15/E06 IgM -which contains a variable region that is composed of a canoni cal rearrangement of germline genes in both the V L and V H chains -was originally cloned from the spleens of atherosclerosisprone apolipoprotein E (Apoe −/− ) mice as an antibody with specificity for OxLDL, which was later found to be specific for PCOxPL [76] [77] [78] . The same antibody idiotype, as an IgA, was originally described due to its ability to bind PC present in the capsular polysaccharide of S. pneumoniae 76, 79, 80 . In addition, several other IgMs that bind different OSEs have been identified, such as the OxCLspecific LR01 and a number of others with speci ficity for MDAtype adducts, including E014, NA17 and LR04. Similar to T15/E06, all of the OSEspecific natural IgM antibodies that have been described exhibit germline or neargermline usage in their complementary determining region 3 (CDR3), identifying them as natu ral IgM 75, 78, 81, 82 . Thus, natural IgM antibodies prominently participate in the response to OSEs.

Role of OSEs in the maintenance of homeostasis Physiological carriers of OSEs. Tissue homeostasis depends on the efficient removal of dying cells, which is achieved by molecular tags that mark cellular debris at different stages of cellular death. The quiescent removal of apoptotic cells is mainly mediated by phagocytes that sense several 'findme' and 'eatme' signals on apoptotic cells [83] [84] [85] . However, in situations of increased cell death and/or inefficient clearance, apoptotic cells can release DAMPs that trigger sterile inflammation 22 and render these cells immunogenic 17 . OSEs on latestage apop totic cells have been identified as important ligands for the clearance of apoptotic cells under these condi tions 17, 18, 75, 81, 86, 87 . In fact, apoptotic mammalian cells of every cellular origin tested display OSEs on their surface independently of the apoptosis trigger, and these OSEs are identical to the ones on OxLDL 18 (FIG. 2) . Notably, only a small proportion of early apoptotic cells dis play OSEs, which is in contrast to late apoptotic cells as >50% of this cell population carries OSEs 18, 81 . A sub set of apoptotic cells is bound by natural IgM antibod ies with specificity for PCOxPL and different types of MDA epitopes, and some are bound by monoclonal anti bodies with specificity for OxCL 17, 18, 75, 81, 82 . Furthermore, mass spectrometry has demonstrated that lipid extracts of thymocytes, in which apoptosis was induced by Milk fat globule-epidermal growth factor 8 (MFG-E8). A ~53 kDa secreted glycoprotein with versatile functions, including binding to exposed phosphatidylserine to mediate the clearance of apoptotic cells via the αvβ3 and αvβ5 integrin receptors of phagocytic cells.

several triggers, were enriched in different OxPLs 17 . Similarly, micro vesicles generated from monocytes that were loaded with unesterified cholesterol, as well as tBuOOH oxidized microvesicles that were derived from endothelial cells, carried both MDA and PCOxPL epitopes in their membranes 88, 89 .

More recently, we also identified a subset of cir culating microvesicles as carriers of various OSEs 20 . Microvesicles have been implicated in several diseases owing to their proinflammatory and procoagulant activities 90 . Interestingly, MDA adducts consisting of simple MDAlysine adducts, as well as more complex adducts made up of a lysine residue and two or more MDA moieties, were the most prominent OSE found on ~50% of circulating microvesicles, whereas PCOxPLs were found on fewer microvesicles but were found to a greater extent on the surface of the same micro vesicles. The presence of OSEs was generally independent of cellular origin, supporting their universal presence 20 . Moreover, a portion of the microvesicles also carried IgM bound on their surface, which was primarily directed against MDA, consistent with their prominent presence. Interestingly, a subset of circulating micro vesicles also carry CRP on their surface, which is consistent with the presence of PCOxPL 91 .

Thus, different mechanisms of cell death and gener ation of microvesicles result in the surface presentation of various types of OSEs. An interesting area for future investigations includes understanding whether the pres ence of OSEs is associated with other types of cell death such as pyroptosis, necroptosis and ferroptosis 85, 92 .

Physiological role of OSEs. In situations that are associ ated with increased oxidative stress, which favour OSE generation, a large amount of cellular waste is gener ated. For example, exposure to high light intensities in an oxygenrich environment results in enhanced lipid peroxidation. Consequently, the retina is one of the tis sues with the highest cell turnover rate, and accordingly requires high phagocytic activity for the clearance of aged photoreceptors, which are enriched in longchain PUFAs. Both retinal pigment epithelium (RPE) and microvascular endothelial cells express several scavenger receptors that mediate the clearance of apoptotic cells, cellular debris and oxidized lipids 93 . Indeed, rats with a deletion variant of CD36 exhibit impaired phagocytosis of photoreceptor rod outer segments 94 . Whereas anionic phospholipids on apoptotic cells have been suggested to mediate this binding 95 , mass spectrometry studies have shown that the exposure of rat retinal cells to light results in elevated levels of OxPLs 96 . Importantly, OxPL inhibited the uptake of rod outer segments by wildtype cells but not by Cd36 −/− RPE cells. OSEs may also be involved in efferocytosis via MFGE8, which binds OxPS exposed on the surface of apoptotic cells 72 . MFGE8 facilitates engulfment by phagocytes via the αvβ3 and αvβ5 integrin receptors, and has been shown to have an important role in the phagocytosis of photoreceptors of the outer segment 97 . Recent work has identified the 12/15 lipoxygenase product OxPE as a ligand for MFGE8 with unique functional consequences in the context of apoptotic cell clearance during peritonitis 73 . In this study, OxPE that is exposed on the surface of alternatively activated macrophages led to the seques tration of MFGE8, which favours the engulfment of apoptotic cells via phosphatidylserine receptors, such as T cell immunoglobulin and mucin domain 4 (TIM4), but blocks MFGE8dependent uptake of apoptotic cells by proinflammatory LY6C hi monocytes. Thus, in envi ronments with high oxidative stress, the OSEdependent clearance of cellular debris represents a physiological housekeeping function that directs the clearance of dying cells to specific pathways.

Accumulating evidence indicates that the recognition of OSEs may also be crucial for complementmediated clearance mechanisms, particularly of late apoptotic and 

The multi-step process by which dying cells are cleared by phagocytic cells, which is initiated by recognition signals exposed on apoptotic cells recognized by soluble receptors or membrane-bound receptors expressed on phagocytes, resulting in their engulfment and removal.

The deposition of opsonins on the surface of dying cells or invading pathogens to enhance their clearance by specific receptors expressed on the surface of leukocytes. Specific opsonins not only enhance uptake but also determine whether the uptake has proor anti-inflammatory consequences.

(AMD). A condition predominantly found in the elderly that is caused by the accumulation of cellular debris (drusen) between the Bruch's membrane and the retinal pigment epithelium and by the loss of photoreceptors. AMD is divided in to a dry form resulting from atrophy and a wet form resulting from abnormal vascularization of the retina.

necrotic cells, including the activity of several comple ment proteins and receptors, such as CRP and natural IgM, as well as pentraxin 83, 98, 99 . Natural IgM antibodies are important in apoptotic cell clearance, as mice defi cient in secreted IgM are more prone to developing auto immunity when crossed with the lupusprone MRL/lpr background 100 . To date, all characterized OSEspecific natural IgM antibodies have also been found to bind apoptotic cells, and the PCspecific T15/E06 IgM and the MDAspecific NA17 antibodies enhance the uptake of apoptotic cells in mice in vivo 75 104 . Importantly, recruitment of CFH to apoptotic blebs and to necrotic RPE cells can be medi ated by MDA 67 and other previously described ligands 105 . This might allow CFH to provide cofactor activity for factor I on dying cells to inactivate C3b into iC3b. The phagocytosis of dying cells that have been opsonized with iC3b can inhibit nuclear factorκB (NFκB) transcrip tion, and can thereby promote antiinflammatory clear ance by phagocytes 106 . Notably, CFH mediates cofactor activity on MDAdecorated surfaces, and MDA binding does not interfere with this process. Moreover, owing to its reduced MDAbinding capacity, the H402 variant of CFH displayed significantly impaired cofactor activity compared with the Y402 variant, which may result in the less efficient clearance of MDAdecorated structures and increased inflammation 67,107 . Interestingly, CFH was reported to slow down efferocytosis in vitro 104, 108 , which is beneficial in circumstances that require decreased release of ROS, nitrogen intermediates and lysosomal enzymes by phagocytes 108, 109 . During this process, addi tional complementindependent neutralizing functions of CFH and OSEspecific IgM may also be crucial to prevent potential proinflammatory effects of the OSEs that have not yet been cleared. For example, CFH blocks MDAinduced CXCchemokine ligand 8 (CXCL8) and CXCL1 production in vitro and in vivo, respectively 67 . Similarly, the MDAspecific IgM LR04 and the PCspecific IgM T15/E06 inhibited microvesicle induced CXCL8 secretion in human monocytes and the activation of endothelial cells, respectively 20, 88 . These findings provide insights into the important physio logi cal function of OSEspecific immune responses in the recognition and neutralization of cellular debris in the circulation.

In summary, OSEs mark dying cells and their cellular debris and distinguish them from viable and healthy tis sues. This enables specific humoral immune responses such as natural IgM antibodies to coordinate the safe disposal of dying cells with a regulated activation of the early steps of the complement cascade. In addition, scav enger receptors that bind to OSEs mediate efferocyto sis. However, if the enhanced generation of dying cells exceeds the clearance capacity of available phagocytes, the OSEs present on the accumulating apoptotic cells mediate proinflammatory signals and may promote autoimmunity.

During acute inflammatory conditions OSEs can trigger sterile inflammation to ultimately restore tissue integ rity. However, under certain conditions, the balance between the generation and the clearance of OSEs by innate immune functions is lost owing to the increased generation of OSEs and/or dysfunctional innate immune responses, which leads to increased or chronic inflammation.

OSEs have been documented in diseased tissues of patients with, for example, infectious and sterile acute lung injury, atherosclerosis, hepatitis, age-related macular degeneration (AMD), multiple sclerosis and Alzheimer disease 31 . The investigation of these diseases has pro vided important insights into the functional role of OSEspecific immunity in both acute and chronic inflammatory models.

The OSE CEP transiently accumulates during acute inflam mation in a mouse model of wound healing, and induces angio genesis in endothelial cells in a TLR2-MYD88dependent manner to stimulate wound heal ing and to protect against hindlimb ischaemia 33, 46 . In this context, F4/80 hi and alternatively activated macrophages scavenge and degrade accumulated CEP in a process that requires binding of both TLR2 and CD36 (REF. 46 ). Notably, CEP accumulates in ageing tissues, which may lead to adverse consequences.

OSEs also accumulate in a wide variety of acute situations 33, 59, 110 . They accumulate in the lungs of mice following acute lung injury induced by sterile acid aspiration, as well as following infection with the avian influenza virus H5N1 (REF. 51 ). The pulmonary lavage fluid of injured mice contains OxPL that stimulates the production of IL6 in macrophages, which can be ameliorated by the natural IgM antibody T15/E06. Administration of OxPL promoted acute lung injury in mice in a TLR4 and TRIFdependent manner. In addition to PCOxPL, MDA adducts also formed in the lungs of these mice 51 , and intranasal administra tion of MDAmodified bovine serum albumin (BSA) induced CXCL1 secretion and neutrophil recruitment into the lungs 111 . This is consistent with the capacity of MDAadducts to trigger chemokine secretion in macro phages, and further demonstrates the proinflamma tory potential of OSEs in vivo. Notably, both PCOxPL and MDA have been documented in the lung tissues

Cholesterol-laden macrophages with a foamy appearance that typify the early atherosclerotic lesion. The excess cholesterol is believed to occur because of unregulated and excessive uptake of modified low-density lipoprotein (LDL), such as oxidized LDL via scavenger receptors. They also secrete pro-inflammatory cytokines and promote atherosclerotic lesion formation and are hallmark cells of atherosclerotic plaques. of patients who have died of H5N1 and SARS infec tions and in mice infected with lethal pathogens such as H5N1, Bacillus anthracis, SARS coronavirus and Yersinia pestis 51 .

Atherosclerosis is the prototypical chronic inflammatory disease in which the innate immune responses to OSEs are involved (FIG. 3) . It is characterized by the deposition of LDL in the intima of large and mediumsized arteries, and the LDL subse quently undergoes oxidation to generate OxLDL. OSEs on OxLDL not only mediate uptake by macrophages to generate foam cells but also mediate many of the chronic inflammatory events that lead to the development of plaques. If plaques rupture, they can trigger thrombus formation, resulting in severe clinical manifestations such as myocardial infarction and stroke 112 .

PCOxPL, MDA, 4HNE, OxCE and CEP have all been documented in atherosclerotic lesions in humans and in animal models of disease 46, 76, [113] [114] [115] . Endothelial cells and macrophages -which express different sets of scavenger receptors and TLRs -are major cellular sensors of OSEs in atherosclerosis. OxPLs induce the expression of chemoattractants, such as CCchemokine ligand 2 (CCL2; also known as MCP1), fibronectin con taining connecting segment 1, CXCL8 and Pselectin, and also trigger monocyte binding to endothelial cells 23 , which is partly mediated by TLR4 (REF. 116 ). 4HNE also induces ERK1/2 and NFκB activation in endothe lial cells via LOX1 (REF. 50 ). LOX1 deficiency results in decreased lesion formation in Ldlr −/− mice 117 . CEP may also trigger a proatherogenic endothelial cell response via TLR2 (REF. 33 ). The expression of TLR2 is increased in endothelial cells at sites of disturbed blood flow, and TLR2 deficiency in nonhaematopoietic tissues reduces atherosclerosis in Ldlr −/− mice 118 . Thus, OSE sensing by endothelial cells is a key response in the development of atherosclerosis. Moreover, a recent study demon strated that CEP accelerates platelet activation and thrombus formation in hyperlipidaemic Apoe −/− mice in a TLR2dependent manner 56 , indicating a role for OSEs in triggering the clinical events that result from atherosclerosis.

A hallmark of atherosclerotic lesions is the forma tion of lipidladen macrophages, known as foam cells, which occurs because of the enhanced uptake of OxLDL mediated by OSE binding to scavenger receptors, such as CD36 and SRA1 (REF. 119 ). The enhanced uptake of cholesterolrich LDL and subsequent inefficient removal of intracellular cholesterol can lead to a situ ation of supersaturation of cholesterol, leading to the formation of cholesterol crystals that damage lysosomes and that activate the NOD, LRR and pyrin domain containing protein 3 (NLRP3) inflammasome, result ing in IL1β secretion 120 . Indeed, cholesterol loading of macro phages with OxLDL induces IL1β secretion, and Ldlr −/− mice reconstituted with NLRP3deficient bone marrow develop less atherosclerosis 120 . Moreover, deficiency of CD36 and/or SRA1 in Apoe −/− and Ldlr −/− mice, respectively, reduces the size and/or complexity of lesions compared with control mice 119 . In addition, binding of OxLDL to CD36 induces chemokine expres sion and inflammasome priming through NFκB acti vation, which is dependent on hetero trimer formation with TLR4-TLR6 (REFS 53, 121) . TLR4 signalling is important, as Tlr4 −/− Apoe −/− mice exhibit significantly reduced lesion size 122 , and clinical studies have found an SNP in TLR4, which results in impaired signalling, to be associ ated with reduced plaque burden and acute coronary events 123, 124 . These studies demonstrate that sensing of OSEs, and in particular PCOxPL, by CD36 is central to several proatherogenic responses in macro phages. OxLDL, as well as excessive foam cell forma tion, induces macrophage apoptosis, which is observed in advanced atherosclerotic lesions 125 . As a result of inefficient efferocytosis, apoptotic macrophages accu mulate, which results in increased plaque necrosis and longterm atherosclerotic burden. Several studies demonstrate that deficiencies in certain proteins or receptors that mediate efferocytosis enhance lesion for mation in atherosclerosisprone mice 125 . Furthermore, the accumulation of apoptotic macrophages within lesions, and their released microvesicles, which all bear OSEs, may compete with both OxLDL and apoptotic cells for clearance by macrophages and thereby further impair efferocytosis.

Humoral immune responses are increasingly being recognized as important modulators of athero genesis 126 . Although the ability of CRP to bind OSEs and to mediate apoptotic cell clearance suggests a role in atherosclero sis, there is no real support for an active role of CRP itself in the pathogenesis of the disease from animal or human studies 127, 128 . However, CRP is an excellent biomarker of inflammation, and increased levels of CRP independently predict manifestations of athero sclerosis 64 . By contrast, natural IgM antibodies clearly modu late athero sclerotic lesion formation, as deficiency in secreted IgM promotes atherosclerosis in Ldlr −/− mice 126 . As many natural IgM antibodies bind OSEs 75 , their athero protective function may be mostly medi ated by OSEspecific IgM, which includes housekeeping mechanisms and other protective effects. For example, T15/E06 IgM neutralizes the pro inflammatory effects on endothelial cells and macrophages of late apoptotic cells and/or blebs carrying PCOxPL 17, 51, 88 . T15/E06 IgM antibodies have also been shown to block the binding and uptake of OxLDL in macrophages, preventing foam cell formation 77 . Importantly, raising plasma levels of T15/E06 IgM by immunization with PCcontaining pneumococcal extracts or passive infusion decreased atherosclerosis in Ldlr −/− mice and vein graft athero sclerosis in Apoe −/− mice, respectively 129, 130 . In ana logy to PCOxPL, MDAepitopes also trigger tumour necrosis factor (TNF) and CXCL8 in monocytes and macrophages 67 . In addition, the numbers of MDA positive micro vesicles substantially increased at the culprit lesion sites of patients suffering from an acute coronary event compared with the number found in peripheral circulation, which could further propagate inflammation 20 126 .

Our recent identification of CFH as a major MDA binding protein in plasma also suggests its potential involvement in atherosclerosis 67 . CFH is present in human atherosclerotic lesions, where it localizes to areas rich in MDAepitopes 67, 131 . Although several studies have 20, 114 . This results in the expression of adhesion molecules and the secretion of chemoattractants leading to monocyte recruitment to the intima of the artery wall. Macrophages internalize OxLDL via scavenger receptors such as scavenger receptor A1 (SRA1), lectin-like oxidized LDL receptor 1 (LOX1), SRB1 and CD36 (REF. 37) , and in cooperation with Toll-like receptor 2 (TLR2) and TLR4-TLR6 receive pro-inflammatory signals from OSEs. The enhanced uptake of OxLDL via scavenger receptors leads to the excess accumulation of intracellular cholesterol and the formation of lipid-laden foam cells, as well as the secretion of cytokines and chemokines. Excessive free cholesterol accumulation induces cholesterol crystal formation that triggers lysosome rupture and activation of the NOD-, LRR-and pyrin domain-containing protein 3 (NLRP3) inflammasome 120 primed by pro-inflammatory OSE-induced signalling. Free intracellular cholesterol also induces endoplasmic reticulum (ER) stress, leading to macrophage apoptosis. As a consequence of this, and impaired efferocytosis, late-stage apoptotic cells accumulate, contributingtogether with OxLDL and microvesiclesto a growing pool of OSEs inside the plaque. Natural IgM specific for OSEs blocks scavenger receptor-mediated uptake and neutralizes the pro-inflammatory effects of OSEs by promoting their complement-dependent clearance. Complement factor H (CFH) blocks the pro-inflammatory effects of malondialdehyde (MDA) and facilitates the anti-inflammatory clearance of MDA-decorated surfaces through Factor I-dependent iC3b generation 67 . The milk fat globule-epidermal growth factor 8 (MFG-E8) is also involved in the clearance of OSEs by recognizing oxidized phosphatidylserine (OxPS). Impaired functions of these humoral immune responses as a result of low abundance or decreased binding capacities, as well as excessive accumulation of OxLDL, microvesicles and apoptotic cells, favours the recognition of OSEs by macrophage receptors, leading to sustained inflammation. CRP, C-reactive protein; CXCL8, CXC-chemokine ligand 8; HNE, 4-hydroxynonenal; IL-1β, interleukin-1β; NF-κB, nuclear factor-κB; PC-OxPL, phosphocholine (PC) of oxidized phospholipid.

Characterized by lipid accumulation in the liver and inflammation, it represents a critical stage in the progression of non-alcoholic fatty liver disease to more severe potentially life-threatening conditions that are associated with liver cirrhosis and end-stage liver disease.

reported an association of the 402H allele -which decreases binding of CFH to MDA -with an increased risk of heart disease and stroke, a meta analysis of eight different study populations failed to find a significant association of this gene variant with CVD 132 . However, to fully understand this function of CFH in CVD, the net contribution of all SNPs that potentially affect MDA binding needs to be explored.

These data support an important role for immune responses that target OSEs in atherogenesis. OxLDL and cellular debris contribute to an excessive accumu lation of OSEs, which cannot be adequately targeted by beneficial clearance mechanisms and, consequently, pro inflammatory responses in macrophages and endothelial cells are sustained. Similarly, OSEs may pro mote non-alcoholic steatohepatitis 133 , which is increasingly being recognized as a risk factor for CVD. Studies in cholesterolfed Ldlr −/− mice indicate that the expression of CD36 and SRA1 in Kupffer cells mediates hepatic inflammation 134 , whereas inducing plasma levels of the T15/E06 IgM is protective 135 . Consistent with these data, we recently showed that deficiency of sialic acid binding immunoglobulintype lectin G (SiglecG), a negative regulator of B1 cell function, results in increased levels of OSEspecific IgM and reduced atherosclerosis and hepatic inflammation in cholesterolfed Ldlr −/− mice 136 .

OSEs have also been implicated in AMD 137 , which is characterized by the accumulation of cellular waste (drusen) in the retina. Several OSEs, including PCOxPLs, and MDA and CEPmodified proteins, have been identified as drusen components and have been shown to trigger sterile inflammation 138 . For example, CEPmodified proteins induce pro inflammatory gene expression and prime the NLRP3 inflammasome in monocytes via TLR2 (REFS 55, 139) . Drusen extracts isolated from AMD lesions activate the NLRP3 inflammasome, resulting in the secretion of IL1β and IL18 (REF. 139) . Similarly, MDAmodified proteins induce the expression of CXCL8 and CXCL1 in RPE cells in in vitro and in vivo models, respectively 67 . Dysregulation of complement represents another major pathological driver of AMD, and genomewide associ ation studies have identified SNPs in genes encoding complement proteins as genetic risk factors of AMD 69,137 . Among these, the SNP rs1061170 in CFH predisposes to increased risk for AMD 140, 141 . Necrotic RPE cells and blebs thereof display MDAepitopes that are rec ognized by CFH, which in turn facilitates the genera tion of iC3b for opsonization and antiinflammatory removal. Compared with the Y402 CFH variant, the cofactor activity on MDAdecorated surfaces is severely impaired when the 402H CFH variant is used, demon strating a functional consequence. As a result, MDA may accumulate and promote inflammation 67 . Indeed, trans genic mice that express the human 402H variant have increased susceptibility to oxidative stress mediated injury in the retina as a result of MDA accumulation and increased inflammation 142 .

The recognition of OSEs by cellular and humoral immune responses enables the immune system to mediate important housekeeping functions, such as the removal of dying cells, cellular debris and damaged molecules. In situations of increased oxidative stress, the generation and burden of OSEs is greatly increased. As a consequence, OSEs are sensed by cellular signalling receptors, leading to the secretion of chemokines and pro inflammatory cytokines. Thus, OSEs represent a unique class of DAMPs, which both mediate the recog nition of cellular debris for housekeeping functions and, under certain circumstances, act as pro inflammatory danger signals themselves. When this fine balance between the presence of OSEs and the capacities of the immune system to survey and respond to them is lost owing to the excessive generation of OSEs and/or their impaired removal, the development and propagation of chronic inflammatory diseases become manifest.

Although there have been a few attempts to treat patients with antioxidants to reduce the progression of CVD, these have not been successful 143 . These very limited studies have been criticized as premature as we currently lack sufficient knowledge to adequately design an effective therapeutic trial to test the role of oxidation in atherogenesis 26 . As discussed in this Review, oxida tive events and the associated immune responses have beneficial effects as well as adverse consequences and we currently lack insight into the relative importance of these in vivo, which could affect the proper design of antioxidant trials.

A better understanding of the role of OSEs and OSEreactive immune responses in the maintenance of homeo stasis and in controlling inflammation are important areas of future investigations. Moreover, it is important to elucidate how different components of OSE specific immune responses cooperate at a functional level. The identification of the genetic variants that affect some of these responses, and mechanistic studies charac terizing these, may provide exciting insights into differ ent individual predispositions to responding to oxidative stress and to developing chronic inflammation.

ROS function in redox signaling and oxidative stress

This is an excellent review on the physiological and pathological regulation and functions of ROS

NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells

Beyond oxidative stress: an immunologist's guide to reactive oxygen species

oxidative stress and the biology of ageing

Myeloperoxidase: friend and foe

NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury

Regulation of reactive oxygen species generation in cell signaling

Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic

25-Hydroxycholesterols in innate and adaptive immunity

Sterols and oxysterols in immune cell function

Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostanemediated thromboxane A2 receptor activation

Pro-resolving lipid mediators are leads for resolution physiology

12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function

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

Cyclopentenone prostaglandins as potential inducers of phase II detoxification enzymes. 15-deoxy-Δ(12,14)-prostaglandin j2-induced expression of glutathione S-transferases

Characterization of platelet aminophospholipid externalization reveals fatty acids as molecular determinants that regulate coagulation

This study demonstrated for the first time that apoptotic cells carrying OSEs have the potential to be pro-inflammatory and to trigger immune responses specific for OSEs. It also directly documented the presence of OxPLs in the membrane preparations of apoptotic

This is the first demonstration of the binding of apoptotic cells by natural IgM antibodies with specificity for MDA-and PC-epitopes, and a demonstration that these epitopes are important for the recognition of apoptotic cells by macrophages

Oxidation-specific epitopes are important targets of innate immunity

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Danger signals: SOS to the immune system

Sterile inflammation: sensing and reacting to damage

Generation and biological activities of oxidized phospholipids

Structural identification and cardiovascular activities of oxidized phospholipids

Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized low density lipoprotein

Oxidized low-density lipoprotein and atherosclerosis

New families of bioactive oxidized phospholipids generated by immune cells: identification and signaling actions

Role of phospholipid oxidation products in atherosclerosis

Programmed cell clearance: molecular regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation

Discovery of carboxyethylpyrroles (CEPs): critical insights into AMD, autism, cancer, and wound healing from basic research on the chemistry of oxidized phospholipids

The innate immune response to products of phospholipid peroxidation

Phosphorylcholine: friend or foe of the immune system?

First demonstration of CEP as a ligand for TLR2 and a demonstration of CEP in angiogenesis and wound healing involving TLR2 in vivo

4-Hydroxy-7-oxo-5-heptenoic acid (HOHA) lactone is a biologically active precursor for the generation of 2-(ωa-Carboxyethyl)pyrrole (CEP) derivatives of proteins and ethanolamine phospholipids

Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages

The influence of innate and adaptive immune responses on atherosclerosis

The macrophage scavenger receptor at 30 years of age: current knowledge and future challenges

Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages

This paper demonstrates that SRA and CD36 are the principal receptors for the uptake of OxLDL by macrophages in vitro and in vivo

The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein

Phosphocholine as a pattern recognition ligand for CD36

Conformation of an endogenous ligand in a membrane bilayer for the macrophage scavenger receptor CD36

The lipid whisker model of the structure of oxidized cell membranes

CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (αvβ3)

Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein

This study describes the homeostatic property of macrophages in scavenging and controlling the tissue levels of CEP via CD36 and TLR2

Scavenger receptors on sinusoidal liver endothelial cells are involved in the uptake of aldehyde-modified proteins

The metabolism of native and malondialdehyde-altered low density lipoproteins by human monocyte-macrophages

Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a. Free Radic

Milk fat globule epidermal growth factor 8 (MFG-E8) binds to oxidized phosphatidylserine: implications for macrophage clearance of apoptotic cells

This study demonstrates how a 12/15-lipoxygenase-derived OSE regulates tolerogenic clearance of apoptotic cells

Oxidized but not native cardiolipin has pro-inflammatory effects, which are inhibited by Annexin A5

This study identified OSEs as major targets of natural IgM antibodies in mice and humans and demonstrates the capacity of MDA-specific IgM to facilitate the clearance of apoptotic cells in vivo

Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity

Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipidprotein adducts inhibit macrophage uptake of oxidized low-density lipoproteins

Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma

Common individual antigenic determinants in five of eight BALB-c IgA myeloma proteins that bind phosphoryl choline

Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

A natural antibody to oxidized cardiolipin binds to oxidized low-density lipoprotein, apoptotic cells, and atherosclerotic lesions

Phagocytosis of apoptotic cells in homeostasis

Apoptotic cell clearance: basic biology and therapeutic potential

Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways

Regulation of dendritic cells and macrophages by an anti-apoptotic cell natural antibody that suppresses TLR responses and inhibits inflammatory arthritis

IgM antibodies to apoptosis-associated determinants recruit C1q and enhance dendritic cell phagocytosis of apoptotic cells

Oxidized membrane vesicles and blebs from apoptotic cells contain biologically active oxidized phospholipids that induce monocyteendothelial interactions

Cholesterol-induced membrane microvesicles as novel carriers of damage-associated molecular patterns: mechanisms of formation, action, and detoxification

Microparticles, vascular function, and atherothrombosis

Activated complement components and complement activator molecules on the surface of cellderived microparticles in patients with rheumatoid arthritis and healthy individuals

Regulated cell death and inflammation: an auto-amplification loop causes organ failure

Human retinal pigment epithelial cells express scavenger receptors BI and BII

CD36 expression is altered in retinal pigment epithelial cells of the RCS rat

Binding of anionic phospholipids to retinal pigment epithelium may be mediated by the scavenger receptor CD36

Light-induced oxidation of photoreceptor outer segment phospholipids generates ligands for CD36-mediated phagocytosis by retinal pigment epithelium: a potential mechanism for modulating outer segment phagocytosis under oxidant stress conditions

Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis

The role of IgM antibodies in the recognition and clearance of apoptotic cells

A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo

Accelerated development of IgG autoantibodies and autoimmune disease in the absence of secreted IgM

IGM is required for efficient complement mediated phagocytosis of apoptotic cells in vivo

Differential oxidative modification of proteins in MRL +/+ and MRL/lpr mice: Increased formation of lipid peroxidation-derived aldehyde-protein adducts may contribute to accelerated onset of autoimmune response

C-Reactive protein binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an antiinflammatory innate immune response: implications for systemic autoimmunity

C4b-binding protein and factor H compensate for the loss of membrane-bound complement inhibitors to protect apoptotic cells against excessive complement attack

Role of complement and complement regulators in the removal of apoptotic cells

iC3b-opsonized apoptotic cells mediate a distinct anti-inflammatory response and transcriptional NF-κB-dependent blockade

Complement regulation at necrotic cell lesions is impaired by the age-related macular degeneration-associated factor-H His402 risk variant

Human complement factor H modulates C1q-mediated phagocytosis of apoptotic cells

Negative regulation of phagocytosis in murine macrophages by the Src kinase family member, Fgr

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs

Malondialdehyde-acetaldehydeadducted protein inhalation causes lung injury

Immune effector mechanisms implicated in atherosclerosis: from mice to humans

Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a)

Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man

Differential expression of oxidation-specific epitopes and apolipoprotein(a) in progressing and ruptured human coronary and carotid atherosclerotic lesions

Receptors involved in the oxidized 1-palmitoyl-2-arachidonoylsn-glycero-3-phosphorylcholine-mediated synthesis of interleukin-8. A role for

Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet

Increased endothelial expression of Toll-like receptor 2 at sites of disturbed blood flow exacerbates early atherogenic events

Macrophages in atherosclerosis: a dynamic balance

NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals

CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation

Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E

Association of the Toll-like receptor 4 gene Asp299Gly polymorphism with acute coronary events

Toll-like receptor 4 polymorphisms and atherogenesis

Macrophage death and defective inflammation resolution in atherosclerosis

B cells and humoral immunity in atherosclerosis

Fcγ receptors and ligands and cardiovascular disease

Genetically elevated C-reactive protein and ischemic vascular disease

Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL

Passive immunization with monoclonal IgM antibodies against phosphorylcholine reduces accelerated vein graft atherosclerosis in apolipoprotein E-null mice

Association between complement factor H and proteoglycans in early human coronary atherosclerotic lesions: implications for local regulation of complement activation

Genetic variation in complement factor H and risk of coronary heart disease: eight new studies and a meta-analysis of around 48,000 individuals

Non-alcoholic steatohepatitis: the role of oxidized low-density lipoproteins

Internalization of modified lipids by CD36 and SR-A leads to hepatic inflammation and lysosomal cholesterol storage in Kupffer cells

Specific immunization strategies against oxidized low-density lipoprotein: a novel way to reduce nonalcoholic steatohepatitis in mice

Sialic acid-binding immunoglobulinlike lectin G promotes atherosclerosis and liver inflammation by suppressing the protective functions of B-1 cells

Age-related macular degeneration and the role of the complement system

How does the macula protect itself from oxidative stress?

NLRP3 has a protective role in agerelated macular degeneration through the induction of IL-18 by drusen components

Complement factor H variant increases the risk of age-related macular degeneration

Complement factor H polymorphism and age-related macular degeneration

A chimeric Cfh transgene leads to increased retinal oxidative stress, inflammation, and accumulation of activated subretinal microglia in mice

Antioxidant vitamin supplements and cardiovascular disease

Lipid peroxidation: mechanisms, inhibition, and biological effects

Oxidized phospholipid signaling in immune cells

Myeloperoxidase, modified lipoproteins, and atherogenesis

Aloxe3 knockout mice reveal a function of epidermal lipoxygenase-3 as hepoxilin synthase and its pivotal role in barrier formation

Antiinflammatory circuitry: lipoxin, aspirin-triggered lipoxins and their receptor ALX

Activation and regulation of the inflammasomes

C-reactive protein: ligands, receptors and role in inflammation

The work carried out in the authors' laboratories related to this Review is supported by the Austrian Science Fund (SFB-30 and SFB-54 to C.J.B.) and the US National Institutes of Health (grants HL086559, HL119828 and HL055798 to J.L.W.). The authors thank V. Krajina for help with the illustrations.

The authors declare competing interests: see Web version for details.