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Immunoglobulin M (IgM) autoantibodies to oxidation-specific epitopes (OSEs) can be present at birth and protect against atherosclerosis in experimental models. This study sought to determine whether high titers of IgM titers to OSE (IgM OSE) are associated with a lower risk of acute myocardial infarction (AMI) in humans. IgM to malondialdehyde (MDA)-LDL, phosphocholine-modified BSA, IgM apolipoprotein B100-immune complexes, and a peptide mimotope of MDA were measured within 24 h of first AMI in 4,559 patients and 4,617 age- and sex-matched controls in the Pakistan Risk of Myocardial Infarction Study. Multivariate-adjusted logistic regression was used to estimate odds ratio (OR) and 95% confidence interval for AMI. All four IgM OSEs were lower in AMI versus controls (P < 0.001 for all). Males, smokers and individuals with hypertension and diabetes had lower levels of all four IgM OSE than unaffected individuals (P < 0.001 for all). Compared to the lowest quintile, the highest quintiles of IgM MDA-LDL, phosphocholine-modified BSA, IgM apolipoprotein B100-immune complexes, and MDA mimotope P1 had a lower OR of AMI: OR (95% confidence interval) of 0.67 (0.58-0.77), 0.64 (0.56-0.73), 0.70 (0.61-0.80) and 0.72 (0.62-0.82) (P < 0.001 for all), respectively. Upon the addition of IgM OSE to conventional risk factors, the C-statistic improved by 0.0062 (0.0028-0.0095) and net reclassification by 15.5% (11.4-19.6). These findings demonstrate that IgM OSE provides clinically meaningful information and supports the hypothesis that higher levels of IgM OSE may be protective against AMI.

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 (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 OxPL2-4. 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.

Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6C(hi) monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell-selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde-acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

ObjectiveWestern-type high-fat/high-cholesterol diets used to induce atherogenesis in low-density lipoprotein (LDL) receptor-deficient mice also lead to obesity with concomitant metabolic complications, eg, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. Our aim was to design a diet inducing atherosclerosis through moderate hypercholesterolemia without associated parameters of the metabolic syndrome.Methods and resultsMale LDL receptor-deficient mice were fed regular chow (RC; 0.01% cholesterol/4.4% fat), cholesterol-enriched regular chow (HC; 1% cholesterol/4.4% fat), or Western diet (WD 0.06% cholesterol/21% milk fat) for 28 weeks. HC-feeding led to elevated plasma (approximately 20.7 mmol/L [800 mg/dL]) and LDL cholesterol and accelerated atherosclerosis. Plasma triglycerides were unaffected. Compared with RC-fed controls, HC-fed mice had normal body weight gain and normal fasting levels of glucose, free fatty acids, and insulin. In contrast, WD-fed mice were extremely hypercholesterolemic (>41.4 mmol/L), obese, hypertriglyceridemic, hyperinsulinemic, insulin resistant, and showed adverse health such as skin/fur abnormalities and hepatic steatosis. Although atherosclerotic surface areas in the entire aorta were similar in HC-fed and WD-fed mice, lesions in aortic origin cross sections were significantly larger in WD-fed mice. However, morphology was similar in lesions of equal size.ConclusionsThe HC diet induced moderate hypercholesterolemia and extensive atherosclerosis and should be useful to study specific aspects of atherogenesis in the absence of confounding effects of the metabolic syndrome.

PPARalpha, beta/delta, and gamma regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor-ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARalpha, beta, and gamma agonists on foam-cell formation and atherosclerosis in male LDL receptor-deficient (LDLR(-/-)) mice. Like the PPARgamma agonist, a PPARalpha-specific agonist strongly inhibited atherosclerosis, whereas a PPARbeta-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARalpha and PPARgamma agonists, but not the PPARbeta agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARalpha and PPARgamma agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARalpha required LXRs, activation of PPARgamma reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

PPARalpha, beta/delta, and gamma regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor-ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARalpha, beta, and gamma agonists on foam-cell formation and atherosclerosis in male LDL receptor-deficient (LDLR(-/-)) mice. Like the PPARgamma agonist, a PPARalpha-specific agonist strongly inhibited atherosclerosis, whereas a PPARbeta-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARalpha and PPARgamma agonists, but not the PPARbeta agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARalpha and PPARgamma agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARalpha required LXRs, activation of PPARgamma reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

During atherogenesis, LDL is oxidized, generating various oxidation-specific neoepitopes, such as malondialdehyde-modified (MDA-modified) LDL (MDA-LDL) or the phosphorylcholine (PC) headgroup of oxidized phospholipids (OxPLs). These epitopes are recognized by both adaptive T cell-dependent (TD) and innate T cell-independent type 2 (TI-2) immune responses. We previously showed that immunization of mice with MDA-LDL induces a TD response and atheroprotection. In addition, a PC-based immunization strategy that leads to a TI-2 expansion of innate B-1 cells and secretion of T15/EO6 clonotype natural IgM antibodies, which bind the PC of OxPLs within oxidized LDL (OxLDL), also reduces atherogenesis. T15/EO6 antibodies inhibit OxLDL uptake by macrophages. We now report that immunization with MDA-LDL, which does not contain OxPL, unexpectedly led to the expansion of T15/EO6 antibodies. MDA-LDL immunization caused a preferential expansion of MDA-LDL-specific Th2 cells that prominently secreted IL-5. In turn, IL-5 provided noncognate stimulation to innate B-1 cells, leading to increased secretion of T15/EO6 IgM. Using a bone marrow transplant model, we also demonstrated that IL-5 deficiency led to decreased titers of T15/EO6 and accelerated atherosclerosis. Thus, IL-5 links adaptive and natural immunity specific to epitopes of OxLDL and protects from atherosclerosis, in part by stimulating the expansion of atheroprotective natural IgM specific for OxLDL.

During the progression of atherosclerosis, autoantibodies are induced to epitopes of oxidized low-density lipoprotein (oxLDL) and active immunization of hypercholesterolemic mice with oxLDL ameliorates atherogenesis. We unexpectedly found that many autoantibodies to oxLDL derived from 'naive' atherosclerotic mice share complete genetic and structural identity with antibodies from the classic anti-phosphorylcholine B-cell clone, T15, which protect against common infectious pathogens, including pneumococci. To investigate whether in vivo exposure to pneumococci can affect atherogenesis, we immunized Ldlr(-/-) mice with Streptococcus pneumoniae. This induced high circulating levels of oxLDL-specific IgM and a persistent expansion of oxLDL-specific T15 IgM-secreting B cells primarily in the spleen, which were cross-reactive with pneumococcal determinants. Pneumococcal immunization decreased the extent of atherosclerosis, and plasma from these mice had an enhanced capacity to block the binding of oxLDL to macrophages. These studies show molecular mimicry between epitopes of oxLDL and S. pneumoniae and indicate that these immune responses can have beneficial effects.