Myeloid cell interactions with T cells in atherosclerosis

描述(由申请人提供)：我们假设不同的髓系细胞亚群在动脉粥样硬化中具有不同的和重叠的功能。在这里，我们建议测试哪些亚群负责炎性细胞因子的产生、抗原呈递和泡沫细胞的形成，以及这些过程是如何由血小板因子4(PF4，也称为CXCL4)调节的。巨噬细胞聚集启动脂肪条纹的形成，最终发展为动脉粥样硬化病变。巨噬细胞也与易损斑块密切相关，斑块破裂会引发重大不良心血管事件(MACE)。巨噬细胞和DC是抗原提呈细胞(APC)，与T细胞相互作用，放大炎症。CD4T细胞与APC的相互作用在多光子显微镜下很明显：T细胞在形成免疫突触时暂停。Pf4基因的产物CXCL4具有致动脉粥样硬化作用，并能显著改变巨噬细胞的表型。为了研究APC的功能、泡沫细胞的形成、抗原递呈以及PF4对这些过程的影响，我们建议将新发展的多光子活细胞成像与流式细胞术、免疫荧光和组织学相结合。具体目标1是利用GFP标记的APOE-/-小鼠的主动脉壁流式细胞术和多光子活细胞成像，检测哪些髓系细胞亚群成为主动脉壁泡沫细胞。我们已经产生了LysMGFP APOE-/-、Cx3cr1GFP APOE-/-和CD11cYFP APOE-/-小鼠。在这些小鼠品系中，不同的髓系细胞亚群是绿色(或黄色)荧光的。特异性目的2是检测T细胞与巨噬细胞和树突状细胞相互作用在动脉粥样硬化中的作用。来自OTII转基因小鼠的T细胞表达一种识别卵白蛋白多肽的单克隆性TCR。多克隆T细胞是从dsRed APOE-/-小鼠中分离出来的，其中一些细胞是针对“动脉粥样硬化抗原”的。这些T细胞被转移到LysMGFP APOE-/-、Cx3cr1GFP APOE-/-和CD11cYFP APOE-/-小鼠的主动脉，以测试它们与APC的相互作用，这有望诱导T细胞增殖(BrdU)、极化(干扰素-�，IL-17A)和泡沫细胞形成(oxLDL摄取)。目的3研究血小板第4因子对泡沫细胞形成和功能的影响。PF4是巨噬细胞表型的强大调节剂，在体外促进M4表型。据报道，PF4-/-APOE-/-小鼠出现较小的动脉粥样硬化病变。为了解决可能的机制，我们建议将PF4-/-APOE-/-小鼠与我们的荧光报告小鼠杂交，以可视化这些小鼠的髓系亚群组成、表型和行为的变化。当这项研究完成后，我们将知道哪个巨噬细胞亚群(S)成为泡沫细胞，它与T细胞在生产性免疫反应中相互作用，触发再次刺激、增殖和细胞因子产生，以及PF4如何影响这两个过程。在此研究的PF4或其他分子可能成为预防或治疗动脉粥样硬化及其并发症的靶点。

DESCRIPTION (provided by applicant): We hypothesize that different subsets of myeloid cells have distinct and overlapping functions in atherosclerosis. Here, we propose to test which subsets are responsible for inflammatory cytokine production, antigen presentation, and foam cell formation, and how these processes are modulated by platelet factor 4 (Pf4, also known as CXCL4). Macrophage accumulation initiates the formation of fatty streaks, which eventually develop into atherosclerotic lesions. Macrophages are also critically involved in vulnerable plaque, whose rupture triggers major adverse cardiovascular events (MACE). Macrophages and DCs are antigen- presenting cells (APCs) that interact with T cells, amplifying inflammation. CD4 T cell interaction with APCs is evident in multiphoton microscopy: the T cells pause when they form an immunological synapse. CXCL4, the product of the Pf4 gene, is pro-atherogenic and drastically alters macrophage phenotype. To investigate APC functions, foam cell formation, antigen presentation and the impact of Pf4 on these processes, we propose to combine newly developed multiphoton live cell imaging of the aortic wall with flow cytometry, immunofluorescence and histology. Specific aim 1 is to test which subsets of myeloid cells become foam cells in the aortic wall, using aortic wall flow cytometry and multiphoton live cell imaging in GFP-tagged Apoe-/- mice. We have generated LysMGFP Apoe-/-, Cx3cr1GFP Apoe-/- and CD11cYFP Apoe-/- mice. In each of these mouse strains, different subsets of myeloid cells are green (or yellow) fluorescent. Specific aim 2 is to test the effects of T cell interactios with macrophages and dendritic cells on atherosclerosis. T cells derived from OTII transgenic mice express a monoclonal TCR that recognizes an ovalbumin peptide. Polyclonal T cells, some of which are specific to "atherosclerosis antigens", are isolated from dsRed Apoe-/- mice. These T cells are transferred to aortas of LysMGFP Apoe-/-, Cx3cr1GFP Apoe-/- and CD11cYFP Apoe-/- mice to test their interactions with APCs, which are expected to induce T cell proliferation (BrdU), polarization (interferon-�, IL-17A) and foam cell formation (oxLDL uptake). Specific aim 3 is to investigate the effect of platelet factor 4 on foam cell formation an function. PF4 is a strong modulator of macrophage phenotype, promoting the M4 phenotype in vitro. Pf4-/-Apoe-/- mice have been reported to develop smaller atherosclerotic lesions. To address possible mechanisms, we propose to cross Pf4-/-Apoe-/- mice with our fluorescent reporter mice to visualize changes in myeloid subset composition, phenotype and behavior in these mice. When this research is complete, we will know which macrophage subset(s) become foam cells, which interact with T cells in a productive immune response to trigger re-stimulation, proliferation and cytokine production, and how PF4 influences both processes. PF4 or other molecules investigated here may emerge as targets for prevention or therapy of atherosclerosis and its complications.