Oxidized phospholipids (OxPL) are generated at sites of oxidative stress and tissue injury, where they function as prototypic danger-associated molecular patterns that trigger inflammation, cell death, and tissue remodeling. The natural IgM antibody E06, and derivative formats such as E06-scFv, recognize the phosphocholine epitope on OxPL but not on native phospholipids, providing powerful tools to probe the biology of OxPL across cardiovascular and noncardiovascular disease. In this review, we summarize experimental and translational evidence implicating OxPL as mediators of atherogenesis, myocardial ischemia-reperfusion injury, nonalcoholic steatohepatitis and hepatocellular carcinoma, bone loss, lung fibrosis and acute lung injury, sepsis, neuroinflammation, and pain. In multiple murine models, genetic or passive approaches that express or deliver E06-based constructs neutralize OxPL, attenuate inflammation, improve tissue function, and reduce lesion burden, establishing OxPL as an actionable driver rather than a passive byproduct of oxidative stress. We further highlight how E06 has been leveraged to develop sensitive immunoassays for OxPL on apoB-containing lipoproteins and on Lp(a) (lipoprotein[a]; OxPL-apo[a]). These biomarkers consistently associate with incident and recurrent cardiovascular events and link OxPL biology to human lipoprotein metabolism, particularly the preferential enrichment of OxPL on Lp(a). We review emerging data on how established and novel therapies, including statins, PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors, lipoprotein apheresis, and Lp(a)-targeted antisense and RNA-based agents, modulate OxPL, and discuss how these interventions may help test the OxPL hypothesis in outcomes trials. Finally, we outline key challenges and opportunities for translating OxPL-directed strategies to the clinic, including issues of specificity, immunogenicity, timing, and patient selection. Collectively, these insights position OxPL and its selective neutralization by E06-like antibodies as a unifying mechanism and promising therapeutic target across diverse inflammatory and cardiometabolic diseases.

Fluctuation in plasma cholesterol due to intermittent statin adherence is common and worsens cardiovascular outcomes. Oxidized phospholipids (oxPL), derived from, or transported by, oxidized low-density lipoprotein (oxLDL) or Lp(a), respectively, play crucial roles in atherosclerotic disease in mice and humans. E06, a naturally produced IgM antibody targeting OxPL, diminishes macrophage (Mφ) uptake of oxLDL in vitro and attenuates atherosclerosis progression in mouse models. Therefore, we hypothesized that OxPL is a causative factor in accelerated atherosclerosis in mice and increased cardiovascular events in people after depressed LDL-C levels are allowed to rise again. To test this, Ldlr -/- and Ldlr -/ - mice expressing a transgene for E06 (“E06 mice”) were subjected to a plasma cholesterol cycling model (high-low-high), inducing plaque progression (P) for 16 weeks, resolution (PR) after ApoB antisense oligonucleotide (ApoB-ASO) treatment for 4 weeks, and re-progression (PRP) after ApoB-ASO withdrawal for an additional 4 weeks (n≥9/group). As expected, LDL-C rebound accelerated plaque inflammation, assessed by a 30% increase in aortic-root Mφ accumulation in Ldlr -/- mice (PRP vs. PR: 68.6±4.0 vs. 42.7±7.8%; P=0.004), whereas E06 prevented Mφ rebound during PRP compared to Ldlr -/- mice (PRP E06 vs. PRP Ldlr -/- : 46.7±11.2 vs. 68.6±4.0%; P=0.003) and showed no significant change between E06 PR and PRP mice (61.2±11.2% vs. 46.7±4.0; P=0.066). Bioinformatic analysis of plaque Mφ based on single-cell RNA-seq revealed reduced inflammatory pathway activation in PRP E06 mice compared to PRP Ldlr -/- mice. Bone marrow monocytes from P Ldlr -/- mice secreted 50% higher IL-6 levels after LPS stimulation compared with P E06 mice (n=3-4, 93.84±12.46 vs. 53.21±13.39 ng/mL, P=0.037), suggesting reduced innate immune priming in E06 mice. Consistent with a higher inflammatory state, P Ldlr -/- mice exhibited higher circulating white blood cells and monocyte counts than E06 mice (WBC: n=10-12; 4.19±0.68 vs. 3.12±0.79 10 3 /uL; P=0.004; monocytes: 0.82±0.3 vs. 0.50±0.19 10 3 /uL; P=0.009). Additionally, patients in the clinical CHORD study at NYU who underwent cholesterol cycling had increased levels of serum OxPL attached to Lp(a) (n=23, P=0.012). In summary, the increased CV events clinically observed in LDL-C cycling may have its mechanistic basis in immune cell priming by OxPL, resulting in exaggerated inflammatory responses in atherosclerotic plaques following LDL-C rebound.

Background: Oxidized phospholipids (OxPL) promote vascular inflammation and atherosclerosis. Although transgenic expression of the E06 single-chain antibody reduces atherosclerosis in mice, the therapeutic potential and mechanism of passive immunization with full-length recombinant E06 IgG remain unknown.Methods: Male Ldlr-/- mice fed a cholesterol-enriched diet received placebo or recombinant E06 IgG (5 or 25 mg/kg subcutaneously twice weekly) for 12 weeks. Plasma lipids, oxidation-specific biomarkers, atherosclerosis, lesional phosphocholine-containing OxPL (PC-OxPL), macrophage content, collagen, and necrotic core were quantified. To assess whether E06 IgG depletes circulating OxPL, apo(a)- and lipoprotein(a)-transgenic mice received a single intravenous injection of E06 IgG followed by serial measurements of oxidation-specific biomarkers and total plasma OxPL by targeted LC–MS/MS.Results: Terminal plasma E06 IgG concentrations confirmed dose-dependent systemic exposure. Compared with placebo, E06 IgG significantly reduced lesional macrophage content (47.6 ± 14.5% vs 34.4 ± 9.2% vs 34.3 ± 9.1% of lesion area; P=0.013) and lesional PC-OxPL (28.5 ± 8.8% vs 18.2 ± 7.5% vs 20.1 ± 7.0%; P=0.010) in placebo, 5 mg/kg, and 25 mg/kg groups, respectively. No significant differences were observed in plasma cholesterol, triglycerides, circulating OxPL-related biomarkers, aortic root lesion area, plaque collagen, or necrotic core. Acute E06 IgG administration demonstrated target engagement in apo(a)- and Lp(a)-transgenic mice but produced only transient, non-significant changes in circulating OxPL measured by ELISA and LC–MS/MS.Conclusions: These findings provide proof-of-concept that passive E06 IgG selectively neutralizes lesional PC-OxPL and reduces plaque macrophage accumulation despite minimal sustained effects on circulating OxPL, supporting further investigation of OxPL-targeted therapies and suggesting that circulating OxPL may not adequately reflect vascular target engagement.

Intermittent statin adherence has been shown to worsen cardiovascular outcomes compared to individuals who are adherent to their statin treatment. Oxidized phospholipids (OxPL) derived from LDL play a crucial role in pre-clinical models in the development and progression of atherosclerosis, and are thought to have similar effects clinically. The natural antibody E06, binding to OxPL, has been shown to diminish macrophage (Mφ) uptake of OxLDL in vitro and decrease atherosclerosis progression in vivo . In prior work using a model of plasma cholesterol cycling (high-low-high), resulting in atherosclerotic plaque Progression (P), Regression (PR), and re-Progression (PRP), we observed accelerated plaque inflammation in the PRP group relative to mice with non-cycling high cholesterol. We hypothesized that OxPL promotes accelerated inflammation after regression in the plaques of cholesterol-cycled mice via trained immunity, and that the E06 antibody will mitigate this in PRP. To test this, we utilized transgenic "E06 mice" that secrete from liver a single-chain variable fragment of E06 on the Ldlr −/− background. Both E06 and Ldlr -/- mice were subjected to an initial high cholesterol diet and divided into the cholesterol cycling groups: P, PR and PRP (n>10/group). Plaque regression was induced by ApoB-ASO injections, which significantly lower cholesterol levels by inhibiting hepatic ApoB production. Ldlr -/- mice in the PRP group exhibited accelerated plaque inflammation post-regression as assessed by Mφ (CD68+ cells) accumulation in aortic roots. While the accumulation of Mφs in Ldlr -/- mice was 50% higher in PRP vs. PR, there was no significant change of Mφs in the E06 mice (P=0.0003). Peripheral blood exhibited a reduction of total monocytes in the PRP group of E06 mice compared to Ldlr -/- mice. Evidence of effects of the E06 antibody on trained innate immunity was obtained by ex vivo stimulation of bone marrow monocytes with LPS, which demonstrated higher IL-6 levels in the supernatant of cells from Ldlr -/- mice vs E06 mice in the PRP group (n=5-6, 79.6±10.5 vs. 50.8±37.4 ng/mL, P=0.03). In progress is single-cell RNA sequencing of plaque cells from E06 and Ldlr -/- mice, which should be available by the time of this meeting. In summary, this work provides insight into the increased risk of cardiovascular disease in individuals with intermittent adherence to statins and highlights the importance of neutralizing OxPL during the resultant cholesterol cycling.

Background: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. Methods: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr(-/-) mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2(V617F)). Results:Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk(-/-) platelets promoted increased NETosis when incubated with Lnk(-/-) neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk(-/-) platelets, as well as increased priming and response of Lnk(-/-) neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk(-/-) mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2(VF) mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk(+/-) and Jak2(VF) clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2(VF)Lnk(+/+) controls. Conclusions: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2(VF) mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

Oxidized phospholipids (OxPLs) are pro-inflammatory molecules that affect bone remodeling under physiological conditions. Transgenic expression of a single-chain variable fragment (scFv) of the antigen-binding domain of E06, an IgM natural antibody that recognizes the phosphocholine (PC) moiety of OxPLs, increases trabecular and cortical bone in adult male and female mice by increasing bone formation. OxPLs increase with age, while natural antibodies decrease. Age-related bone loss is associated with increased oxidative stress and lipid peroxidation and is characterized by a decline in osteoblast number and bone formation, raising the possibility that increased OxPLs, together with the decline of natural antibodies, contribute to age-related bone loss. We show here that transgenic expression of E06-scFv attenuated the age-associated loss of spinal, femoral, and total bone mineral density in both female and male mice aged up to 22 and 24 months, respectively. E06-scFv attenuated the age-associated decline in trabecular bone, but not cortical bone, and this effect was associated with an increase in osteoblasts and a decrease in osteoclasts. Furthermore, RNA-seq analysis showed that E06-scFv increased Wnt10b expression in vertebral bone in aged mice, indicating that blocking OxPLs increases Wnt signaling. Unlike age-related bone loss, E06-scFv did not attenuate the bone loss caused by estrogen deficiency or unloading in adult mice. These results demonstrate that OxPLs contribute to age-associated bone loss. Neutralization of OxPLs, therefore, is a promising therapeutic target for senile osteoporosis, as well as atherosclerosis and non-alcoholic steatohepatitis (NASH), two other conditions shown to be attenuated by E06-scFv in mice.

Oxidized phospholipids containing phosphocholine (OxPL) are pro-inflammatory lipid peroxidation products that bind to scavenger receptors (SRs), such as Scarb1, and toll-like receptors (TLRs). Excessive OxPL, as found in oxidized low-density lipoprotein (OxLDL), overwhelm these defense mechanisms and become pathogenic in atherosclerosis, nonalcoholic steatohepatitis (NASH), and osteoporosis. We previously reported that the innate IgM natural antibody E06 binds to OxPL and neutralizes their deleterious effects; expression of the single-chain (scFv) form of the antigen-binding domain of E06 (E06-scFv) as a transgene increases trabecular bone in male mice. We show herein that E06-scFv increases trabecular and cortical bone in female and male mice by increasing bone formation and decreasing osteoblast apoptosis in vivo. Homozygous E06-scFv mice have higher bone mass than hemizygous, showing a dose effect of the transgene. To investigate how OxPL restrain bone formation under physiologic conditions, we measured the levels of SRs and TLRs that bind OxPL. We found that osteoblastic cells primarily express Scarb1. Moreover, OxLDL-induced apoptosis and reduced differentiation were prevented in bone marrow-derived or calvaria-derived osteoblasts from Scarb1 knockout mice. Because Scarb1-deficient mice are reported to have high bone mass, our results suggest that E06 may promote bone anabolism in healthy young mice, at least in part, by neutralizing OxPL, which in turn promote Scarb1-mediated apoptosis of osteoblasts or osteoblast precursors. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

Oxidized phospholipids (OxPLs), which arise due to oxidative stress, are proinflammatory and proatherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPLs accumulate in human and mouse NASH. Using a transgenic mouse that expresses a functional single-chain variable fragment of E06, a natural antibody that neutralizes OxPLs, we demonstrate the causal role of OxPLs in NASH. Targeting OxPLs in hyperlipidemic Ldlr(-1-) mice improved multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death, and progression to hepatocellular carcinoma. Mechanistically, we found that OxPLs promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPLs in AMLN-diet-fed Ldlr(-1-) mice reduced oxidative stress, improved hepatic and adipose-tissue mitochondrial function, and fatty-acid oxidation. These results suggest targeting OxPLs may be an effective therapeutic strategy for NASH.

In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a – h , instead of a – g . These errors have been corrected online.

BACKGROUND:Oxidation-specific epitopes (OSEs) are proinflammatory, and elevated levels in plasma predict cardiovascular events. OBJECTIVES:The purpose of this study was to develop novel positron emission tomography (PET) probes to noninvasively image OSE-rich lesions. METHODS:An antigen-binding fragment (Fab) antibody library was constructed from human fetal cord blood. After multiple rounds of screening against malondialdehyde-acetaldehyde (MAA) epitopes, the Fab LA25 containing minimal nontemplated insertions in the CDR3 region was identified and characterized. In mice, pharmacokinetics, biodistribution, and plaque specificity studies were performed with Zirconium-89 (89Zr)-labeled LA25. In rabbits, 89Zr-LA25 was used in combination with an integrated clinical PET/magnetic resonance (MR) system. 18F-fluorodeoxyglucose PET and dynamic contrast-enhanced MR imaging were used to evaluate vessel wall inflammation and plaque neovascularization, respectively. Extensive ex vivo validation was carried out through a combination of gamma counting, near infrared fluorescence, autoradiography, immunohistochemistry, and immunofluorescence. RESULTS:LA25 bound specifically to MAA epitopes in advanced and ruptured human atherosclerotic plaques with accompanying thrombi and in debris from distal protection devices. PET/MR imaging 24 h after injection of 89Zr-LA25 showed increased uptake in the abdominal aorta of atherosclerotic rabbits compared with nonatherosclerotic control rabbits, confirmed by ex vivo gamma counting and autoradiography. 18F-fluorodeoxyglucose PET, dynamic contrast-enhanced MR imaging, and near-infrared fluorescence signals were also significantly higher in atherosclerotic rabbit aortas compared with control aortas. Enhanced liver uptake was also noted in atherosclerotic animals, confirmed by the presence of MAA epitopes by immunostaining. CONCLUSIONS:89Zr-LA25 is a novel PET radiotracer that may allow noninvasive phenotyping of high-risk OSE-rich lesions.

Aims:Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results:OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023). Conclusions:OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.

Ischemia reperfusion (I/R) injury accounts for up to 50% of the final myocardial infarction size despite successful percutaneous coronary intervention. No effective pharmacologic therapies exist in humans to reduce I/R injury. Monoclonal antibody (MAb) E06 binds to the phosphocholine (PC) head group

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes(1). Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL(2-4). Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr(-/-) background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr(-/-)mice, Ldlr(-/-)E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Objectives: Aortic stenosis (AS), affecting 7.6 million patients in Europe and North America, initially manifests as aortic sclerosis that progresses to severe fibro-calcific disease requiring aortic valve replacement (AVR). Effective medical therapies do not exist and many patients are ineligible for AVR. OxPL promote calcification and predict the progression of AS and need for AVR. We tested the hypothesis that inactivating OxPL with E06, binding to the phosphocholine head group of OxPL but not normal PL, prevents development of early signs of AS.

Innate immunity utilizes evolutionarily conserved pattern recognition receptors (PRRs) to provide an early and effective response against Pathogen-Associated Molecular Patterns (PAMPs) on microbial pathogens and/or against Danger Associated Molecular Patterns (DAMPs) on endogenous modified-self structures. Atherosclerosis is a chronic inflammatory disease in which lipid peroxidation is greatly increased leading to the generation of OxLDL, which contains a variety of proinflammatory oxidation-specific neoepitopes (OSE), such as phosphocholine (PC) containing oxidized phospholipids (OxPL). Our group has shown that OSEs are DAMPs, to which has evolved a concerted innate immune response mediated by PRRs. For example, CD36, CRP and IgM E06 all recognize the PC of OxPL, but also the PC on apoptotic cells as well as the PC on the cell wall of S. pneumonia (but not as part of a lipid). Accordingly, we postulated that both endogenous DAMPs and exogenous PAMPs provide natural selection for PPR responses to PC. Malondialdehyde (MDA) is another prominent OSE target of three different PRR’s: SR-A, CFH, and the IgM NAb E014. Thus, we hypothesized that EO14 should also recognize an epitope/mimotope on an infectious pathogen. We screened a pathogen library with E014 and discovered it avidly bound to group A streptococcus (GAS). Because it was known that CFH also bound to GAS, and specifically to protein M, the major virulence factor of GAS, we used GAS with and without protein M to show that E014 specifically bound to protein M. Using a series of recombinant protein M fragments, we identified a 125 aa sequence required for binding. Using a synthetic peptide array to generate 15 aa-length overlapping peptides, we identified a 24 aa mimitope that E014 bound. We subsequently showed immunological cross reactivity between GAS, Protein M, the mimotope, and MDA in vitro and in vivo in mice and humans. Further, compared to immunization of mice with protein M, immunization with MDA-LDL provided partial protection against lethal infection with GAS. These data support the hypothesis that OSE are important targets of innate immunity and both oxidative events and pathogens have contributed to the natural selection of potent, shared innate immune responses to oxidation-specific epitopes.