Biology of Lymphangiogenesis in the Adult Lung

项目摘要/摘要 肺的淋巴系统通过维持液体动态平衡和服务于 作为免疫细胞运输的管道。然而，尽管这个网络很重要，但有许多重要的 在该系统的基础生物学及其关键细胞成分淋巴管内皮细胞方面的知识差距 (LEC)。首先，人们对成人生活中肺LECs的起源和替代以及如何形成LECs知之甚少。 晶状体上皮细胞的不同来源和显微解剖位置可能与不同的作用有关，特别是在 免疫反应。事实上，肺LEC是否由一组异质性和功能不同的细胞组成 亚型或大体上同源的种群是未知的。在小鼠左肺叶内注射流感后， 我们观察到局部和强烈的淋巴管生成反应，表现为早期血管扩张，随后是 淋巴网络本身的扩张。这与7岁以下LEC显著增加1.5-2倍有关 感染后天数(Dpi)和21dpi时显著增加2-3倍，这是病毒被清除的时间。EDU 标记显示，20%的晶状体上皮细胞在7dpi增殖，而对照组小鼠的增殖速度为1%。这些数据伴随着 观察表明，成人晶状体上皮细胞有两种不同的来源，一种是胎儿的，一种是静脉来源的，另一种是后遗症。 Natal和来自髓系来源的LEC强调了LEC在功能和微观上的异质性的可能性 从解剖学上讲。目标1将通过评估这两个LEC来源的作用和本地化来解决这一问题 在成人肺内稳态和病毒诱导的淋巴管生成过程中进行谱系追踪。这些研究将是 在AIM 2中辅以单核RNA测序，以生成LEC特异性遗传信息， 是了解LEC异质性和感染免疫反应的关键，以及LEC的来源 亚集组织定位标记物。我们还发现，流感感染的肺部以LEC减少为特征 河马信号转导中主要转录效应因子YAP和TAZ的核定位 路径。这一高度保守的途径是许多生物系统中细胞表型的中心调节因子 核YAP/TAZ降低，表明信号抑制。综上所述，这些发现表明， 河马信号可能是调控LEC增殖和表型的关键因素。最后，我们观察到， 对侧未感染肺的LEC增殖，提示LEC增殖的可能性很大 是对流感的系统性反应。这些问题将在目标3中通过选择性地删除河马来进一步研究 在我们的流感模型中，通过检测LEC中的信号以及肝脏和心脏中淋巴管的生成。总而言之，我们假设 最先进的技术表明，显然需要基本信息来解决 肺淋巴生物学领域。有了强大的背景数据和稳健的模型，我们相信这些研究 这笔赠款将有助于解决这些基本问题，从而扩大和加深我们的认识 LEC的起源、扩张机制、异质性、功能和表型调节。

PROJECT SUMMARY/ABSTRACT The lung’s lymphatic system plays a critical role in lung health by maintaining fluid homeostasis and by serving as a conduit for immune cell trafficking. Despite the importance of this network however, there are significant knowledge gaps in the basic biology of this system and its key cellular constituent, the lymphatic endothelial cell (LEC). For one, very little is known regarding the origin and replacement of lung LECs in adult life and how different origins and micro-anatomical locations of LECs might be associated with different roles, specifically in immune responses. Indeed, whether lung LECs comprise a heterogeneously and functionally distinct set of cell subtypes or a largely homogenous population is unknown. After instillation of flu into the mouse left lung lobe, we observed a local and vigorous lymphangiogenic reaction manifested by early vessel dilation followed by expansion of the lymphatic network itself. This is associated with a significant 1.5-2-fold increase of LECs at 7 days post-infection (dpi) and a significant 2-3-fold increase at 21 dpi, a time when the virus is cleared. Edu labeling demonstrated that 20% of LECs are proliferating at 7 dpi vs. <1% in control mice. These data along with observations suggesting 2 distinct sources of adult LECS, one fetal and from a venous origin, and one post- natal and from a myeloid origin, underscore the potential for LEC heterogeneity both functionally and micro- anatomically. This will be addressed in Aim 1 by evaluating the role and localization of these 2 sources of LECs during adult lung homeostasis and viral-induced lymphangiogenesis by lineage tracing. These studies will be supplemented by single nuclear RNA sequencing in Aim 2 to generate LEC specific genetic information that will be key to understanding LEC heterogeneity and immune response to infection, along with derivation of LEC subset tissue localization markers. We also found that the flu-infected lung is characterized by decreased LEC nuclear localization of YAP and TAZ, which are the primary transcriptional effectors in the Hippo signaling pathway. This highly conserved pathway is a central regulator of cell phenotype in many biological systems with decreased nuclear YAP/TAZ signifying signal inhibition. Taken together, these findings suggest that the status of Hippo signaling may be a key factor regulating LEC proliferation and phenotype. Lastly, we observed vigorous LEC proliferation in the contralateral uninfected lung, suggesting the interesting possibility that LEC proliferation is a systemic response to flu. These issues will be further studied in Aim 3 by the selective deletion of hippo signaling in LECs and by examining lymphangiogenesis in liver and heart in our flu model. In sum, we posit that the state-of-the art shows a pronounced need for basic information that can resolve fundamental questions in the field of lung lymphatic biology. With strong background data and robust models, we believe that the studies in this grant will help to resolve these basic questions, and as result will expand and deepen our understanding of LEC origin, mechanism of expansion, heterogeneity, function, and phenotype regulation.