Biology of Lymphangiogenesis in the Adult Lung

项目总结/摘要 肺的淋巴系统在肺健康中起着至关重要的作用，它通过维持体液平衡， 作为免疫细胞运输的管道。尽管这个网络的重要性，但是， 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空白 （LEC）。首先，关于成年后肺LEC的起源和替代以及如何发生的知之甚少。 LEC的不同起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是否包含一组异质的和功能不同的细胞， 亚型或很大程度上同质的群体是未知的。将流感病毒滴入小鼠左肺叶后， 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， 淋巴网络本身的扩张。这与7 ℃时LEC显著增加1.5-2倍有关 感染后10天（dpi），病毒被清除的21 dpi时显著增加2-3倍。Edu 标记证实20%的LEC在7 dpi增殖，而对照小鼠中<1%。这些数据沿着 观察结果表明成人LECS有2个不同来源，一个是胎儿来源，来自静脉，另一个是术后来源。 纳塔尔和骨髓来源，强调LEC异质性的可能性，无论是功能上还是微观上， 从解剖学上讲这将在目标1中通过评估这两个LEC来源的作用和本地化来解决 在成人肺稳态和病毒诱导的淋巴管生成过程中，通过谱系追踪。这些研究报告将 在Aim 2中补充单核RNA测序，以产生LEC特异性遗传信息， 是理解LEC异质性和感染免疫应答的关键，沿着LEC的衍生 亚组组织定位标记物。我们还发现，流感感染肺的特征是LEC减少， 雅普和TAZ的核定位，它们是Hippo信号传导中的主要转录效应子 通路这种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， 细胞核雅普/TAZ比值降低，信号抑制。综上所述，这些发现表明， Hippo信号转导通路可能是调节LEC增殖和表型的关键因素。最后，我们观察到， LEC增殖在对侧未感染的肺，提示有趣的可能性，LEC增殖 是对流感的系统性反应这些问题将在目标3中通过选择性删除hippo来进一步研究 通过在LEC中的信号传导以及在我们的流感模型中检查肝脏和心脏中的淋巴管生成。总之，我们认为， 最先进的技术表明，对能够解决以下基本问题的基本信息的需求是明显的： 肺淋巴生物学领域。有了强大的背景数据和强大的模型，我们相信这些研究 在这个赠款将有助于解决这些基本问题，并因此将扩大和加深我们的理解 LEC的起源，扩展机制，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.