Determining the functions of LYVE1 on macrophages during autoantibody-induced valve disease

项目摘要/摘要 风湿性疾病包括类风湿性关节炎、风湿性心脏病和系统性红斑狼疮 与心血管疾病风险增加有关。虽然炎症是 对于风湿性疾病和心血管疾病，还需要继续研究以确定病因机制。K/B.G7 自发性自身抗体诱导的关节炎和二尖瓣病变模型提供了有价值的前期研究 临床工具，以阐明与瓣膜性心脏炎相关的疾病驱动机制，目前很难 在发病前检测到心功能减退。我们小组之前的工作展示了一个角色 巨噬细胞在促进瓣膜病中的作用。发炎MV的单细胞测序数据报告了 巨噬细胞在该组织中的异质性群体促使进一步探讨巨噬细胞亚群 以及驱动疾病的特定子集的角色。我们小组的初步数据表明，淋巴管 内皮细胞促进了MV病的早期阶段，但推动这些新生细胞生长的机制是 淋巴结构尚不清楚。我们鉴定了透明质酸受体LYVE1+巨噬细胞的存在 正常瓣膜和病变瓣膜均靠近内皮细胞和中膜晶状体上皮细胞。基因表达和 流式细胞仪分析显示这些细胞是组织驻留的巨噬细胞。LYVE1+巨噬细胞 在心脏的其他位置以及包括肺、脂肪和腹膜在内的其他组织中被发现。 在这些缝隙中，LYVE1+巨噬细胞与组织天然透明质酸相互作用，调节细胞外基质 动态平衡，促进新生血管生长。这项研究中提出的工作将检验这一假设 A)在疾病早期，LYVE1+巨噬细胞至少部分地通过促进淋巴管生成来促进MV病 和b)LYVE1蛋白不仅描述了这一巨噬细胞群，而且是 巨噬细胞执行这些功能。利用RNA测序，LYVE1+和LYVE1-MV巨噬细胞 子集将相互比较，并跨越疾病时间点，以确定基因表达谱 LYVE1+巨噬细胞独有的，我们预测它将包括淋巴管生成和HA结合相关 成绩单。除了这些细胞的特征外，LYVE1+巨噬细胞耗竭和LYVE1的诱导模型 WT和KO骨髓嵌合体将用于测试该细胞群体和LYVE1表达的必要性 巨噬细胞在推动MV病和病变瓣膜淋巴管生长中的作用。深层组织 将应用成像和分析技术进一步询问LYVE1+巨噬细胞 优先与LECs相互作用，促进血管生长。总之，这些研究将阐明重要的 关于先天免疫反应在驱动瓣膜心脏炎中的作用的信息。这些数据可用于 提高我们对导致人类疾病的早期机制的理解，并有可能发现新的 治疗靶点或诊断标志物。

PROJECT SUMMARY/ABSTRACT Rheumatic diseases including rheumatoid arthritis, rheumatic heart disease, and systemic lupus erythematosus are associated with increased risk of cardiovascular disease. While inflammation is a critical link between rheumatic diseases and CVD, continued research is needed to determine causal mechanisms. The K/B.g7 model of spontaneous autoantibody-induced arthritis and mitral valve (MV) disease provides a valuable pre- clinical tool to elucidate disease-driving mechanisms related to valvular carditis, which is currently difficult to detect prior to the onset of reduced cardiac function. Prior work from our group demonstrated a role for macrophages in promoting valve disease. Single-cell sequencing data from inflamed MV reported a heterogeneous population of macrophages in this tissue prompting further inquiry into the macrophage subsets and subset-specific roles that drive disease. Preliminary data from our group demonstrated that lymphatic endothelial cells promote early stages of MV disease, but the mechanisms driving the growth of these neo- lymphatic structures remains unclear. We identified the presence of hyaluronan receptor LYVE1+ macrophages near the atrial layer of endothelium and MV LECs in both normal and diseased valves. Gene expression and flow cytometry profiling revealed that these cells are tissue resident macrophages. LYVE1+ macrophages have been identified in other locations in the heart as well as other tissues including the lung, adipose, and peritoneum. In these niches, LYVE1+ macrophages interact with tissue native hyaluronan, regulate extracellular matrix homeostasis, and promotes neovascular growth. Work proposed in this fellowship will test the hypothesis that a) early in disease, LYVE1+ macrophages promote MV disease at least in part by promoting lymphangiogenesis and b) the LYVE1 protein does not simply delineate this population of macrophages but is required for the macrophages to perform these functions. Using RNA sequencing, LYVE1+ and LYVE1- MV macrophage subsets will be compared to one another and across disease time points to identify the gene expression profile unique to LYVE1+ macrophages, which we predict will include lymphangiogenesis- and HA-binding-related transcripts. Beyond characterizing these cells, an inducible model of LYVE1+ macrophage depletion and LYVE1 WT and KO bone marrow chimeras will be used to test the necessity of this cell population and LYVE1 expression on macrophages, respectively, in driving both MV disease and lymphatic growth in diseased valves. Deep-tissue imaging and analysis techniques will be applied to further interrogate whether LYVE1+ macrophages preferentially interact with LECs to promote vessel growth. In all, these studies will elucidate important information about the role of innate immune responses in driving valvular carditis. These data can be used to improve our understanding of the early mechanisms driving human disease and potentially identify novel therapeutic targets or diagnostic markers.