Oxidized Low-Density Lipoprotein Immune Complexes Stimulate Proinflammatory Changes in Innate and Adaptive Immunity

项目总结 心血管疾病(CVD)影响着近三分之一的美国人口，是导致 每年约有100万美国人死亡。动脉粥样硬化是心血管疾病最常见的形式， 是一种以动脉内斑块聚集为特征的无菌炎症疾病。这是 被认为是由低密度脂蛋白(低密度脂蛋白)进入和隔离在血管系统中启动的 在那里它会被氧化(OxLDL)。研究表明，循环中的许多oxLDL必然会 特异性抗体与形成oxLDL免疫复合体(oxLDL-ICs)存在正相关 循环oxLDL-ICs滴度与动脉粥样硬化疾病严重程度之间的关系。我们的团队已经证明 氧化低密度脂蛋白-ICs通过Fc-γ受体(Fc-Rs)、Toll样受体4和CD36协同传递信号 在小鼠骨髓来源的树突状细胞(BMDCs)体外增强产生 致动脉粥样硬化细胞因子IL-1。初步数据显示oxLDL-IC预处理的BMDCs增强Th17 并抑制Th1反应，相对于oxLDL处理的BMDCs。然而，这些差异T细胞 回应似乎是另一种机制的结果。OxLDL-ICs在体内的作用还不完全 了解，但我们的实验室已经表明，消除对CD11c细胞的抑制FcR，FcRIIb是 足以增加雌性LDLR-/-小鼠的动脉粥样硬化。FcRs表达在表面 树突状细胞和巨噬细胞等抗原提呈细胞以及激活和抑制Fc受体 分别诱导相反的促炎表型和耐受表型。因为oxLDL-ICs正在发出信号 部分通过FcRs，这些数据提供了一种机制，通过该机制，oxLDL-IC信号特异性地在 DCs对动脉粥样硬化有促进作用。此外，初步数据显示oxLDL-IC刺激 诱导BMDCs的代谢变化，在游离oxLDL中看不到。由oxLDL-IC激活的细胞更多 糖酵解，并有增加的备用呼吸量。总而言之，这些数据导致 假设oxLDL-IC信号改变DC功能，导致T细胞活化和 导致动脉粥样硬化的分化。该提案的目标1将确定oxLDL-IC刺激如何 通过利用BMDC/T细胞与基因共培养改变下游CD4T细胞应答 敲除和阻断抗体、过继转移和体内oxLDL-IC注射。目标2将 研究oxLDL-IC诱导的代谢改变如何促进DC的激活和功能 利用代谢通量实验、RNA测序和BMDC/T细胞与新陈代谢 抑制剂和基因敲除。总体而言，该提案将定义oxLDL-IC如何直接影响DC 以及随后如何影响CD4T细胞的功能。这些研究的成功将告诉我们 OxLDL-ICs如何促进无菌炎症，并将拓宽我们对动脉粥样硬化的理解 和其他oxLDL-IC相关的自体炎症性疾病。

PROJECT SUMMARY Cardiovascular disease (CVD) affects almost one third of the U.S. population and is responsible for the deaths of approximately 1 million Americans annually. Atherosclerosis, the most common form of CVD, is a disease of sterile inflammation characterized by the accumulation of plaque in the arteries. This is thought to be initiated by the entry and sequestration of low-density lipoprotein (LDL) in the vasculature where it becomes oxidized (oxLDL). Studies show that much of the oxLDL in circulation is bound to specific antibody to form oxLDL immune complexes (oxLDL-ICs) and there is a positive correlation between titers of circulating oxLDL-ICs and atherosclerosis disease severity. Our group has shown that oxLDL-ICs cooperatively signal through Fc gamma receptors (FcRs), Toll-like Receptor 4, and CD36 in murine bone marrow-derived dendritic cells (BMDCs) in vitro to enhance the production of proatherogenic cytokine IL-1. Preliminary data indicate oxLDL-IC pretreated BMDCs enhance Th17 and suppress Th1 responses, relative to oxLDL pretreated BMDCs. However, these differential T cell responses appear to be the result of a separate mechanisms. The role of oxLDL-ICs in vivo is not fully understood, but our lab has shown that elimination of the inhibitory FcR, FcRIIb, on CD11c+ cells is sufficient to increase atherosclerosis in female Ldlr-/- mice. FcRs are expressed on the surface of antigen presenting cells like dendritic cells (DCs) and macrophages, and activating and inhibitory FcRs elicit opposing pro-inflammatory and tolerogenic phenotypes, respectively. As oxLDL-ICs are signaling in part through FcRs, these data provide a mechanism by which oxLDL-IC signaling specifically on DCs could promote atherosclerosis. Furthermore, preliminary data demonstrate oxLDL-IC stimulation induces metabolic changes in BMDCs not seen with free oxLDL. Cells activated by oxLDL-IC are more glycolytic and have an increased spare respiratory capacity. Collectively, these data lead to the hypothesis oxLDL-IC signaling alters DC function resulting in changes in T cell activation and differentiation that are proatherogenic. Aim 1 of this proposal will determine how oxLDL-IC stimulation of DCs alters downstream CD4+ T cell responses by leveraging BMDC/T cell co-cultures with gene knockouts and blocking antibodies, adoptive transfers, and in vivo oxLDL-IC injections. Aim 2 will investigate how oxLDL-IC induced changes in metabolism contribute to DC activation and function using metabolic flux experiments, RNA sequencing, and BMDC/T cell co-cultures with metabolic inhibitors and gene knockouts. Overall, this proposal will define how oxLDL-ICs directly impact DC function and how CD4+ T cells are subsequently influenced. The success of these studies will inform how oxLDL-ICs contribute to sterile inflammation and will broaden our understanding of atherosclerosis and other oxLDL-IC associated autoinflammatory disorders.