Role of Natural Antibodies Against Oxidation Specific Epitopes in Bone Homeostasis

脂质过氧化形成高反应性分子，其与蛋白质的氨基产生加合物， 脂质，称为氧化特异性表位（OSE）。OSE是普遍存在的促炎部分， 如果不及时清除，会造成严重的细胞损伤。先天免疫系统的天然抗体， OSE并阻止其不良影响。在巨噬细胞和其他细胞中表达的清道夫受体（SR） 类型识别OSE并处理氧化修饰的内源性分子。持久性和/或 过量的OSE会压倒这些防御机制， 通过清道夫受体/Toll受体激活引起组织损伤和疾病的发展。 含磷胆碱的氧化磷脂（PC-OxPL）在氧化低密度下形成OSE 脂蛋白（OxLDL）和凋亡细胞表面。IgM E06是一种天然抗体， PC-OxPL。表达E06 IgM的抗原结合结构域的单链（scFv）形式的转基因小鼠 （E06-scFv）被保护免于动脉粥样硬化并且具有增加的寿命。这种效果是由 防止OSE与巨噬细胞结合以及随后的炎症途径激活。在 在人类中，低水平的天然抗体与心血管疾病的发病率增加有关。 人类的流行病学证据以及小鼠的机制研究表明，富含OSE的OxLDL 也是骨质疏松症的致病因素。值得注意的是，我们发现E06-scFv转基因阻止了E06-scFv的表达。 HFD通过增加骨内膜成骨细胞数量和骨形成引起的皮质骨丢失。此外，本发明还提供了一种方法， E06-scFv通过以下方式增加喂食正常饮食的雄性和雌性C57 Bl/6 J小鼠的松质骨和皮质骨： 增加骨形成和成骨细胞数量。机制研究表明，E06 IgM阻止了 OxLDL对培养的成骨细胞的增殖、分化和存活的负面影响表明， 抗OSE抗体通过阻止OSE对成骨细胞的负面影响来促进骨愈合。 因此，E06-scFv对巨噬细胞产生抗成骨细胞因子没有影响 转基因小鼠清除剂受体ScrB 1的缺失，ScrB 1是PC最丰富的清除剂受体， 成骨细胞系细胞中的OxPL防止OxLDL对细胞凋亡和分化的不利影响。 此外，抗PC IgM（包括E06）在小鼠中随着年龄的增长而下降，与骨密度下降有关。 马萨诸塞州因此，我们假设抗PC-OxPLs抗体在骨骼稳态中发挥有益作用， 通过保护免受PC-OxPL对骨形成的有害影响，这些有害影响由ScrB 1传递， 成骨细胞由于细胞凋亡加速、氧化应激或慢性 随着年龄的增长，炎症与抗PC-OxPL抗体水平的下降相一致，有助于 骨质疏松症的发病机制在小鼠和人类。本申请中提出的研究将建立 ScrB 1是否通过表征骨在体内介导PC-OxPLs对成骨细胞的负面影响 在成骨细胞谱系细胞中ScrB 1基因缺失的小鼠的表型。我们还将 确定E06-scFv是否可以防止老年引起的骨形成减少。此外，我们将 研究内源性抗PC IgM水平是否与人体骨密度相关， 测量一组符合条件的退伍军人的抗PC IgM水平，这些退伍军人具有骨矿物质密度（BMD） 通过双能X线吸收测定法（DXA）评估。成功完成这项工作可能会建议一部小说 同时治疗骨质疏松症和动脉粥样硬化。

Lipid peroxidation forms highly reactive molecules that generate adducts with amino groups of proteins and lipids, known as oxidation specific epitopes (OSEs). OSEs are ubiquitous pro-inflammatory moieties that cause extensive cell damage if not promptly eliminated. Natural antibodies of the innate immune system, recognize OSEs and block their adverse effects. Scavenger receptors (SRs) expressed in macrophages and other cell types recognize OSEs and dispose of oxidatively-modified endogenous molecules. Persistence and/or excessive amounts of OSEs overwhelm these defense mechanisms, and the inflammatory response initiated by scavenger receptor/toll-receptor activation causes tissue damage and the development of disease. Phosphocholine-containing oxidized phospholipids (PC-OxPLs) form OSEs on oxidized low density lipoproteins (OxLDLs) and on the surface of apoptotic cells. The IgM E06 is a natural antibody that recognizes PC-OxPL. Transgenic mice expressing a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) are protected against atherosclerosis and have increased lifespan. This effect results from the prevention of OSE binding to macrophages and the subsequent activation of inflammatory pathways. In humans, low levels of natural antibodies are associated with increased incidence of cardiovascular disease. Epidemiologic evidence in humans as well as mechanistic studies in mice indicate that the OSE-rich OxLDL is a pathogenic factor for osteoporosis as well. Remarkably, we found that the E06-scFv transgene prevents the cortical bone loss caused by HFD by increasing endosteal osteoblast number and bone formation. In addition, E06-scFv increases cancellous and cortical bone in male and female C57Bl/6J mice fed a normal diet by increasing bone formation and osteoblast number. Mechanistic studies showed that E06 IgM prevents the negative effects of OxLDL on proliferation, differentiation, and survival of cultured osteoblastic cells suggesting that anti-OSE antibodies promote bone anabolism by preventing the negative effects of OSEs on osteoblasts. Consistently, the production of anti-osteogenic cytokines by macrophages was not affected in E06-scFv transgenic mice. Deletion of the scavenger receptor ScrB1, the most abundant scavenger receptor for PC- OxPL in cells of the osteoblast lineage, prevents the adverse effects of OxLDL on apoptosis and differentiation. Moreover, anti-PC IgM (which includes E06) decline with age in mice, in association with a decline in bone mass. Therefore, we hypothesize that anti PC-OxPLs antibodies play a beneficial role in skeletal homeostasis by protecting against deleterious effects of PC-OxPLs on bone formation, that are transmitted by ScrB1 on osteoblasts. Increased production of OSEs due to accelerated apoptosis, oxidative stress, or chronic inflammation, in concert with declining levels of anti PC-OxPLs antibodies with age, contributes to the pathogenesis of osteoporosis in both mice and humans. Studies proposed in this application will establish whether ScrB1 mediates the negative effects of PC-OxPLs on osteoblasts in vivo by characterizing the bone phenotype of mice in which the ScrB1 gene has been deleted in cells of the osteoblast lineage. We will also determine if E06-scFv prevents the reduction in bone formation caused by old age. Additionally, we will investigate whether endogenous anti-PC IgM levels correlate with bone mineral density in humans by measuring the levels of anti-PC IgM in a cohort of eligible Veterans who had a bone mineral density (BMD) assessment by dual X-ray absorptiometry (DXA). Successful completion of this work may may suggest a novel therapeutic approach for the management of osteoporosis and atherosclerosis simultaneously.