Oxidized Low Density Lipoprotein Immune Complexes in Atherosclerosis

 描述(申请人提供)：心血管疾病(CVD)是美国头号死亡原因。心血管疾病最常见的形式是动脉粥样硬化，其特征是慢性炎症，导致动脉中形成斑块。炎症是由低密度脂蛋白(低密度脂蛋白)在血管壁中的隔离引起的 在那里它们被氧化(氧化低密度脂蛋白)，并对局部细胞造成损害。这导致包括树突状细胞(DC)在内的抗原提呈细胞(APC)的招募。虽然oxLDL抗体滴度和由此产生的免疫复合体(oxLDL-ICs)与疾病的严重程度相关，但oxLDL-ICs是否在疾病发病机制中发挥作用尚不清楚。对巨噬细胞系的研究表明，在oxLDL-IC治疗后，细胞活性增加。然而，树突状细胞在这一过程中的作用在很大程度上还没有研究，关于体内相关性的报道也很少。我们实验室的初步数据显示，通过激活标志物的表达和促炎细胞因子的分泌来衡量，oxLDL-IC导致DC激活增加。这些体外研究表明oxLDL-ICs具有致动脉粥样硬化的作用。鉴于DC具有诱导下游免疫应答的强大能力，了解oxLDL-IC介导的DC激活将填补我们在CVD发病机制方面的一个关键空白，并为治疗干预提供新的途径。我们提出了两个目的来确定oxLDL-ICs对DC的影响以及这些影响对动脉粥样硬化结局的影响。在目标1中，我们将确定oxLDL-ICs调节DC激活和细胞因子产生的机制。Ox--IC可能通过Fc受体(FcR)结合和内化DC而激活DC，Fc受体识别ICs的Fc部分，并存在于包括DC在内的多种白细胞上。然而，鉴于oxLDL已知可以与包括TLR4和CD36在内的其他细胞表面受体结合，我们认为oxLDL-ICs可能通过与几种细胞表面受体的共同作用而增加炎症。我们将通过用oxLDL或oxLDL-ICs处理来自野生型(C57BL/6)小鼠和受体特异性敲除小鼠的骨髓来源的DC来验证这一假设。激活将通过流式细胞仪和细胞因子的产生来测量。骨髓间充质干细胞还将与T细胞共同培养，以确定它们诱导免疫反应的能力。在目标2中，我们将 确定树突状细胞特异性FcR在动脉粥样硬化中的作用。鉴于DC激活和分化幼稚T细胞从而控制下游免疫反应的强大能力，DC Fc受体可能在介导动脉粥样硬化结果中发挥重要作用。为了确定DC FcR如何介导动脉粥样硬化，我们将进行野生型或FcR-/-DC过继转移到低密度脂蛋白受体-/-小鼠。此外，我们将对与CD11c Cre+小鼠杂交的FcFLOX/FLOX小鼠的后代进行骨髓移植，以获得DC特异性的FcR基因敲除到LDLR-/-小鼠。总的来说，拟议的研究将确定oxLDL-ICs在动脉粥样硬化中的作用。

 DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the number one cause of mortality in the United States. The most common form of CVD is atherosclerosis which characterized by chronic inflammation resulting in the formation of plaques in the arteries. Inflammation is initiated by the sequestration of low-density lipoproteins (LDL) in the vessel wall where they become oxidized (ox-LDL) and cause damage to local cells. This results in the recruitment of antigen presenting cells (APCs) including dendritic cells (DCs). While oxLDL antibody titers and resulting immune complexes (oxLDL-ICs) correlate with disease severity, it is unknown if oxLDL-ICs play a role in disease pathogenesis. Studies in macrophage cell lines have shown increased cellular activation following oxLDL-IC treatment. However, the role of DCs in this process is largely unstudied, and reports regarding the in vivo relevance are lacking. Preliminary data from our lab show that oxLDL-ICs cause increased DC activation as measured by expression of activation markers and secretion of pro-inflammatory cytokines. These in vitro studies suggest an atherogenic role for oxLDL-ICs. Given the potent ability of DCs to elicit a downstream immune response, understanding oxLDL-IC mediated DC activation will fill a critical gap in our knowledge of CVD pathogenesis and provide new avenues for therapeutic intervention. We propose two aims to determine the effects of oxLDL-ICs on DCs and the implication of these effects on atherosclerotic outcomes. In aim 1, we will determine the mechanism by which oxLDL-ICs modulate DC activation and cytokine production. It is likely that oxLDL-ICs activate DCs through binding and internalization by Fc receptors (FcRs), which recognize the Fc portion of ICs and can be found on several leukocyte populations including DCs. However, given that oxLDL is known to bind other cell surface receptors including TLR4 and CD36, we believe that oxLDL-ICs may increase inflammation by concomitant interaction with several cell surface receptors. We will test this hypothesis by treating bone marrow derived DCs from wild type (C57BL/6) mice and receptor- specific knock-out mice with oxLDL or oxLDL-ICs. Activation will be measured by flow cytometry and cytokine production. BMDC will also be co-cultured with T cells to determine their ability to elicit an immune response. In aim 2, we will determine the effects of dendritic cell specific FcR on atherosclerosis. Given the robust capacity of DCs to activate and differentiate naïve T cells and thus control downstream immune responses, DC FcRs are likely to be important in mediating atherosclerotic outcomes. To identify how DC FcR mediate atherosclerosis, we will perform an adoptive transfer of wild type or FcR-/- DC into LDLr-/- mice. Additionally, we will perform a bone marrow transplant of the progeny from FcRflox/flox mice crossed with CD11c Cre+ mice to obtain DC specific FcR knockouts into LDLr-/- mice. Collectively, the proposed studies will identify a role for oxLDL-ICs in atherosclerosis.