Ayelet Gonen

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

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

Low Doses of Lipopolysaccharide and Minimally Oxidized Low-Density Lipoprotein Cooperatively Activate Macrophages via Nuclear Factor κB and Activator Protein-1: Possible Mechanism for Acceleration of Atherosclerosis by Subclinical Endotoxemia

Rationale: Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. Objective: In this study, we examined cooperative macrophage activation by low levels of bacterial lipopolysaccharide (LPS) and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. Methods and Results: We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express proinflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1&agr;), and Ccl4 (MIP-1&bgr;). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an activator protein (AP)-1 binding site were significantly upregulated by costimulation with mmLDL and LPS. In a nuclear factor–DNA binding assay, the cooperative effect of mmLDL and LPS costimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of extracellular signal-regulated kinase (ERK) 1/2. In addition, mmLDL induced c-Jun N-terminal kinase (JNK)-dependent derepression of AP-1 by removing nuclear receptor corepressor (NCoR) from the chemokine promoters. Conclusions: The cooperative engagement of AP-1 and nuclear factor (NF)-&kgr;B by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions.Rationale: Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. Objective: In this study, we examined cooperative macrophage activation by low levels of bacterial lipopolysaccharide (LPS) and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. Methods and Results: We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express proinflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1α), and Ccl4 (MIP-1β). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/mL) at which LPS alone induce...

Tissue-specific gene therapy directed to tumor angiogenesis

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of ‘vascular-directed’ gene therapy will be applicable to both vascular diseases and cancer.

Innate Response Activator B Cells Aggravate Atherosclerosis by Stimulating T Helper-1 Adaptive Immunity

Background— Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose, and complement each other’s function and shape the course of disease can illuminate our understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of granulocyte macrophage colony-stimulating factor–secreting cells of hitherto unknown function in atherosclerosis. Methods and Results— Here, we show that IRA B cells arise during atherosclerosis in mice and humans. In response to a high-cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell–derived granulocyte macrophage colony-stimulating factor develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classic dendritic cells, which then generate interferon &ggr;–producing T helper-1 cells. This IRA B cell–dependent T helper-1 skewing manifests in an IgG1-to-IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins. Conclusions— Granulocyte macrophage colony-stimulating factor–producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a T helper-1–associated milieu that aggravates atherosclerosis.

Interleukin-3/Granulocyte Macrophage Colony–Stimulating Factor Receptor Promotes Stem Cell Expansion, Monocytosis, and Atheroma Macrophage Burden in Mice With Hematopoietic ApoE Deficiency

Objective—Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE−/− mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the common &bgr; subunit of the granulocyte macrophage colony–stimulating factor/interleukin-3 receptor (CBS) on HSPCs. ApoE−/− mice also display increased granulocyte macrophage colony–stimulating factor–dependent monocyte production in the spleen. We investigated the role of the CBS in cholesterol-driven HSPC expansion, monocytosis, and atherosclerosis. Approach and Results—Ldlr−/− mice were transplanted with ApoE−/−Cbs−/− or ApoE−/− BM followed by Western-type diet feeding. Compared with ApoE−/− BM–transplanted controls, ApoE−/−Cbs−/− BM–transplanted mice had reduced BM and splenic HSPC proliferation, fewer blood monocytes and neutrophils, and reduced macrophage content and area of early atherosclerotic lesions. More advanced lesions showed diminished macrophage and collagen content; however, lesion size was unchanged, reflecting an increase in necrotic core area, associated with a marked decrease in Abcg1 expression and increased macrophage apoptosis. Compared with wild-type mice, Western-type diet–fed ApoE−/− mice showed increased CBS expression on granulocyte macrophage colony–stimulating factor–producing innate response activator B cells and expansion of this population. ApoE−/−Cbs−/− BM–transplanted Ldlr−/− mice showed a marked decrease in innate response activator B cells compared with ApoE−/− BM–transplanted Ldlr−/− controls. Conclusions—Increased levels of CBS on HSPCs and splenic innate response activator B cells lead to expansion of these populations in ApoE−/− BM–transplanted Ldlr−/− mice, contributing to monocytosis and increased lesional macrophage content. However, in more advanced lesions, the CBS also has a role in atherosclerotic plaque stabilization.

B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis

RATIONALE B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.

Atheroprotective vaccination with MHC-II restricted peptides from ApoB-100

Background: Subsets of CD4+ T-cells have been proposed to serve differential roles in the development of atherosclerosis. Some T-cell types are atherogenic (T-helper type 1), while others are thought to be protective (regulatory T-cells). Lineage commitment toward one type of helper T-cell versus another is strongly influenced by the inflammatory context in which antigens are recognized. Immunization of atherosclerosis-prone mice with low-density lipoprotein (LDL) or its oxidized derivative (ox-LDL) is known to be atheroprotective. However, the antigen specificity of the T-cells induced by vaccination and the mechanism of protection are not known. Methods: Identification of two peptide fragments (ApoB3501–3516 and ApoB978–993) from murine ApoB-100 was facilitated using I-Ab prediction models, and their binding to I-Ab determined. Utilizing a vaccination scheme based on complete and incomplete Freund’s adjuvant (CFA and IFA) [1 × CFA + 4 × IFA], we immunized Apoe−/−mice with ApoB3501–3516 or ApoB978–993 emulsified in CFA once and subsequently boosted in IFA four times over 15 weeks. Spleens, lymph nodes, and aortas were harvested and evaluated by flow cytometry and real time RT-PCR. Total atherosclerotic plaque burden was determined by aortic pinning and by aortic root histology. Results: Mice immunized with ApoB3501–3516 or ApoB978–993 demonstrated 40% reduction in overall plaque burden when compared to adjuvant-only control mice. Aortic root frozen sections from ApoB3501–3516 immunized mice showed a >60% reduction in aortic sinus plaque development. Aortas from both ApoB3501–3516 and ApoB978–993 immunized mice contained significantly more mRNA for IL-10. Both antigen-specific IgG1 and IgG2c titers were elevated in ApoB3501–3516 or ApoB978–993 immunized mice, suggesting helper T-cell immune activity after immunization. Conclusion: Our data show that MHC Class II restricted ApoB-100 peptides can be atheroprotective, potentially through a mechanism involving elevated IL-10.

Helix-Loop-Helix Factor Inhibitor of Differentiation 3 Regulates Interleukin-5 Expression and B-1a B Cell Proliferation

Objective—Natural immunity is emerging as an important mediator of protection from atherogenesis. Natural IgM antibodies that recognize oxidation-specific epitopes on low-density lipoprotein or phospholipids and the B-1a B cells that produce them attenuate atherosclerosis. We previously demonstrated that Apoe−/− mice globally deficient in the helix-loop-helix protein inhibitor of differentiation 3 (Id3) develop early diet-induced atherosclerosis. Furthermore, B cell–mediated attenuation of atherosclerosis in B cell–deficient mice was dependent on Id3. Here, we sought to determine whether Id3 regulates B-1a B cells and the natural antibodies that they produce and identify mechanisms mediating these effects. Approach and Results—Mice lacking Id3 had significantly fewer B-1a B cells in the spleen and peritoneal cavity and reduced serum levels of the natural antibody E06. B cell–specific deletion of Id3 revealed that this effect was not because of the loss of Id3 in B cells. Interleukin (IL)-33 induced abundant, Id3-dependent IL-5 production in the recently identified innate lymphoid cell, the natural helper (NH) cell, but not Th2 or mast cells. In addition, delivery of IL-5 to Id3-deficient mice restored B-1a B cell proliferation. B-1a B cells were present in aortic samples also containing NH cells. Aortic NH cells produced IL-5, a B-1a B cell mitogen in response to IL-33 stimulation. Conclusions—These studies are the first to identify NH and B-1a B cells in the aorta and provide evidence that Id3 is a key regulator of NH cell IL-5 production and B-1a B cell homeostasis.

Atheroprotective immunization with malondialdehyde-modified LDL is hapten specific and dependent on advanced MDA adducts: implications for development of an atheroprotective vaccine

Immunization with homologous malondialdehyde (MDA)-modified LDL (MDA-LDL) leads to atheroprotection in experimental models supporting the concept that a vaccine to oxidation-specific epitopes (OSEs) of oxidized LDL could limit atherogenesis. However, modification of human LDL with OSE to use as an immunogen would be impractical for generalized use. Furthermore, when MDA is used to modify LDL, a wide variety of related MDA adducts are formed, both simple and more complex. To define the relevant epitopes that would reproduce the atheroprotective effects of immunization with MDA-LDL, we sought to determine the responsible immunodominant and atheroprotective adducts. We now demonstrate that fluorescent adducts of MDA involving the condensation of two or more MDA molecules with lysine to form malondialdehyde-acetaldehyde (MAA)-type adducts generate immunodominant epitopes that lead to atheroprotective responses. We further demonstrate that a T helper (Th) 2-biased hapten-specific humoral and cellular response is sufficient, and thus, MAA-modified homologous albumin is an equally effective immunogen. We further show that such Th2-biased humoral responses per se are not atheroprotective if they do not target relevant antigens. These data demonstrate the feasibility of development of a small-molecule immunogen that could stimulate MAA-specific immune responses, which could be used to develop a vaccine approach to retard or prevent atherogenesis.