Role of natural antibodies against oxidation specific epitopes in bone homeostasis

项目摘要/摘要 脂质过氧化导致氧化特异性表位(OSE)的形成，这些表位是促炎的， 除非它们被移除，否则会造成广泛的细胞损伤。OS存在于氧化型低密度脂蛋白中 (OxLDLS)和凋亡细胞表面，并通过与清道夫受体(SRS)结合清除 巨噬细胞。持久性和/或过多的操作系统会压倒防御机制 他们。此外，由SR激活引发的炎症反应会导致组织损伤， 在几种疾病中致病，包括动脉粥样硬化。猪瘟B1细胞产生的天然抗体(NAB) 先天免疫系统识别OS并阻止其不良影响。人类的流行病学证据，如 在小鼠身上的机制研究表明，富含OSE的OxLDL是这两种疾病的致病因素 动脉粥样硬化和骨质疏松。氧化磷脂中的磷胆碱(PC-OxPL)形成的OS是 被包括ScrB1和NAB E06 IgM在内的几个SRs识别。表达单链的转基因小鼠 (ScFv)形式的E06 IgM抗原结合域(E06-scFv)对动脉粥样硬化的保护作用 通过高脂饮食(HFD)，因为转基因阻止了OS与巨噬细胞的结合和激活 炎症途径。我们发现，表达E06-scFv的转基因小鼠的骨小梁增加 骨量增加，伴随骨形成率增加。E06-scFv转基因还可以预防 HFD通过增加骨内成骨细胞数量和骨形成而导致皮质骨丢失。E06 IgM 防止氧化低密度脂蛋白对培养的成骨细胞增殖、分化和存活的负面影响。 我们的发现表明，抗OSE抗体通过预防骨质疏松症的负面影响来促进骨合成代谢 关于成骨细胞和骨形成的研究进展。此外，抗PC-IgM(包括E06)水平随着 小鼠的年龄，与骨量下降有关。我们假设，由于石油产量的增加， 加速细胞凋亡、氧化应激或慢性炎症，同时抗OSE水平不足 抗体，有助于骨质疏松症的发病机制。在本申请中提出的研究将建立 用DXA、MicroCT、DXA和MicroCT研究转基因E06-scFv对骨合成代谢影响的细胞机制 组织形态计量学和炎性细胞因子的测定。在数量和特征上的变化 破骨细胞和成骨细胞前体细胞将被分析，部分是通过使用一种新的FACS方法来研究 荧光标记的成骨细胞。转E06-scFv基因对HFD的作用机制 将进一步探讨该转基因预防骨丢失所致骨丢失的能力。 晚年将会确定。此外，我们还将研究OS是否通过结合对成骨细胞产生负面影响 ScrB1是成骨细胞中PC-OxPL最丰富的SR。这项工作的成功完成应该说明 OSES和抗OSE抗体在骨内稳态中的作用以前未被认识到，并可能提出一种新的 同时处理骨质疏松和动脉粥样硬化的治疗方法。

PROJECT SUMMARY/ABSTRACT Lipid peroxidation leads to the formation of oxidation specific epitopes (OSEs) that are proinflammatory and, unless they are removed, cause extensive cell damage. OSEs are present in oxidized low density lipoproteins (OxLDLs) and on the surface of apoptotic cells and cleared by binding to scavenger receptors (SRs) on macrophages. Persistence and/or excessive amounts of OSEs overwhelm the defense mechanisms against them. Additionally, the inflammatory response initiated by SR activation leads to tissue damage that becomes pathogenic in several diseases, including atherosclerosis. Natural antibodies (nAbs) produced by B1 cells of the innate immune system recognize OSEs and block their adverse effects. Epidemiologic evidence in humans as well as mechanistic studies in mice indicate that the OSE-rich OxLDLs are a pathogenic factor in both atherosclerosis and osteoporosis. Phosphocholine in oxidized phospholipids (PC-OxPL) forms OSEs that are recognized by several SRs including ScrB1 and by the nAb E06 IgM. Transgenic mice expressing a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) are protected against atherosclerosis induced by high fat diet (HFD) because the transgene prevents the binding of OSEs to macrophages and activation of inflammatory pathways. We have found that transgenic mice expressing E06-scFv have increased trabecular bone mass, accompanied by an increase in bone formation rate. The E06-scFv transgene also prevents the cortical bone loss caused by HFD by increasing endosteal osteoblast number and bone formation. E06 IgM prevents the negative effects of OxLDL on proliferation, differentiation, and survival of cultured osteoblastic cells. Our findings suggest that anti-OSE antibodies promote bone anabolism by preventing the negative effects of OSEs on osteoblasts and bone formation. Moreover, the level of anti-PC IgM (which includes E06) declines with age in mice, in association with a decline in bone mass. We hypothesize that increased production of OSEs due to accelerated apoptosis, oxidative stress, or chronic inflammation, in concert with inadequate levels of anti-OSE antibodies, contributes to the pathogenesis of osteoporosis. Studies proposed in this application will establish the cellular mechanisms of the bone anabolic effect of the E06-scFv transgene using DXA, microCT, histomorphometry, and measurement of inflammatory cytokines. Changes in the number and characteristics of osteoclast and osteoblast progenitors will be analyzed, in part by use of a novel FACS approach to study fluorescently labeled osteoprogenitors. The mechanisms by which the E06-scFv transgene attenuates HFD- induced bone loss will be further explored and the ability of this transgene to prevent the bone loss caused by old age will be determined. In addition, we will investigate whether OSEs negatively affect osteoblasts by binding to ScrB1, the most abundant SR for PC-OxPL in osteoblasts. Successful completion of this work should elucidate a previously unappreciated role of OSEs and anti-OSE antibodies in bone homeostasis and may suggest a novel therapeutic approach for the management of osteoporosis and atherosclerosis simultaneously.