MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

描述（由申请人提供）：本项目的目的是表征巨噬细胞上趋化因子样受体1（CMKLR 1）的小鼠直系同源物，并确定其在肥胖和动脉粥样硬化中的作用。目的1：研究CMKLR 1在发育中巨噬细胞和组织中巨噬细胞的表达。将测定骨髓源性巨噬细胞（BMDM）随时间推移的受体表达。还将测定各种组织驻留巨噬细胞（TRM）的受体表达。将用促炎和抗炎因子刺激BMDM和TRM，并测定响应于所述因子的CMKLR 1表达（流式细胞术、免疫组织化学[IHC]）。目的2提出在存在或不存在CMKLR 1配体（chemerin）的情况下表征功能性CMKLR 1对基本巨噬细胞效应器功能的影响。CMKLR 1 KO或WT TRM将用或不用chemerin处理，并测定细胞扩散、细胞因子表达（ELISA或细胞内染色）、一氧化氮产生（Greiss测定）、吞噬作用（FITC标记的E. Coli）、精氨酸酶活性（L-精氨酸代谢）、T细胞抗原呈递能力（抗原T细胞增殖测定）和T细胞共刺激能力（共刺激T细胞增殖测定）。目标3将集中于表征CMKLR 1在肥胖和动脉粥样硬化小鼠模型中的作用。对于肥胖症，CMKLR 1 WT和KO小鼠将喂食高脂肪饮食，并随时间记录其体重/食物摄入。在不同的时间点，将测定全身脂肪含量（双能X射线吸收法或定量MRI）。将测定小鼠的葡萄糖耐量（腹膜内葡萄糖耐量试验）、胰岛素耐量（腹膜内胰岛素耐量试验）和血浆脂质（Reflotron试纸）。将测定脂肪组织的巨噬细胞含量（流式细胞术，IHC）、促炎基因表达（定量RT-PCR，组织巨噬细胞胞内细胞因子表达）和趋化蛋白表达（IHC）。对于动脉粥样硬化，将对ApoE缺陷的CMKLR 1 WT和KO小鼠饲喂正常饮食。定期处死小鼠，沿着血液收获其睾丸。测定血浆脂质水平并定量主动脉粥样硬化斑块（油红O或苏丹V染色）。将分析主动脉巨噬细胞含量（IHC）并评估促炎基因表达（qRT-PCR）。相关性：拟议的研究与公共卫生有关，因为巨噬细胞在炎症性疾病的发展和维持中起着重要作用。拟议的研究将奋进进一步阐明巨噬细胞浸润炎症组织的机制，从而提供一个潜在的治疗靶点。

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mouse ortholog of Chemokine-Like Receptor 1 (CMKLR1) on macrophages and determine its role in obesity and atherosclerosis. Aim 1 proposes to characterize the expression of CMKLR1 on developing and tissue resident macrophages. Bone marrow derived macrophages (BMDM) will be assayed for receptor expression over time. Various tissue resident macrophages (TRM) will also be assayed for receptor expression. BMDMs and TRMs will be stimulated with pro-inflammatory and anti-inflammatory factors and assayed for CMKLR1 expression in response to said factors (flow cytometry, immunohistochemistry [IHC]). Aim 2 proposes to characterize the effect of functional CMKLR1 on basic macrophage effector functions either in the presence or absence of CMKLR1 ligand (chemerin). CMKLR1 KO or WT TRMs will be treated with or without chemerin and assayed for cell spreading, cytokine expression (ELISA or intracellular staining), nitric oxide production (Greiss assay), phagocytosis (FITC-labeled E. Coli), arginase activity (L-arginine metabolism), T cell antigen presentation ability (antigen T cell proliferation assay), and T cell costimulation ability (costimulation T cell proliferation assay) in response to various activating stimuli. Aim 3 will focus on characterizing the role of CMKLR1 in mouse models of obesity and athersclerosis. For obesity, CMKLR1 WT and KO mice will be fed a high fat diet and their weights/food intake recorded over time. At various time points, whole body fat content will be assayed (dual-energy-X-ray-absorptiometry or quantitiative MRI). Mice will be assayed for glucose tolerance (intraperitoneal glucose tolerance assay), insulin tolerance (intraperitoneal insulin tolerance assay), and plasma lipid (Reflotron test strips). Adipose tissue will be assayed for macrophage content (flow cytometry, IHC), proinflammatory gene expression (quantitative RT-PCR, tissue macrophage intracellular cytokine expression), and chemerin expression (IHC). For atherosclerosis, CMKLR1 WT and KO mice deficient for ApoE will be fed a normal diet. Periodically, mice will be sacrificed and their aortas harvested along with blood. Plasma levels of lipids with be assayed and aortic atheromatous plaques quantitated (Oil-Red-O or Sudan V staining). Aortic macrophage content will be analyzed (IHC) and pro- inflammatory gene expression (qRT-PCR) will be assessed. RELEVANCE: The proposed research is relevant to public health as macrophages play a large role in the development and maintenance of inflammatory diseases. The proposed research will endeavor to further elucidate the mechanisms by which macrophages infiltrate inflamed tissue, thus offering a potential therapeutic target.