Pulmonary Neuroendocrine Cells Regulate Gas Exchange Through Controlling Lung Fluid Balance

Limiting fluid in lung is critical for efficient gas exchange. Here we discovered a mechanism of neuropeptidergic control of lung fluid balance by pulmonary neuroendocrine cells (PNECs), potent sensors of chemical and mechanical cues. We studied the first animal model of neuroendocrine cell hyperplasia of infancy (NEHI), which faithfully recapitulated patient phenotypes including PNEC hyperplasia and impaired gas exchange. Double mutants showed that increased PNECs and excess PNEC products such as CGRP are responsible for poor gas exchange, acting through downregulating endothelial junctions, increasing vessel leakage and fluid accumulation. Endothelium-specific inactivation of CGRP receptor, or treatment with CGRP receptor antagonist reduced fluid and improved gas exchange. In lungs with acute respiratory distress syndrome (ARDS), including those caused by COVID-19, there was a striking increase of CGRP-expressing PNECs. These findings raise the possibility that increased neuropeptides would contribute to excess extravascular lung fluid and antagonizing their function may improve gas exchange. Single-cell suspensions were prepared from 3 whole mouse lungs (gender mixed) for each indicated genotype. Live epithelial and endothelial cells were FACS-sorted and re-proportionated with 2:1 ratio before loading. Single-cell barcoded droplets were produced using 10X Single Cell 3' v3 chemistry following user guide provided by 10X Genomics. Libraries generated were sequenced using Illumina Novaseq instrument. Reads were aligned and gene level unique molecular identifier (UMI) counts were obtained using the Cell Ranger pipeline. Cell clustering and gene expression profile in different cell type and conditions are performed by Seurat (version 3.0) package.维持肺内液体稳态对于高效气体交换至关重要。本研究揭示了肺神经内分泌细胞（pulmonary neuroendocrine cells, PNECs）通过神经肽能途径调控肺液体平衡的机制——这类细胞是化学与机械信号的强效感受器。我们采用首个婴儿神经内分泌细胞增生症（neuroendocrine cell hyperplasia of infancy, NEHI）动物模型展开研究，该模型可精准重现患者的表型特征，包括PNEC增生与气体交换受损。双突变体实验证实，PNEC数量增多及其分泌的CGRP等产物过量是导致气体交换障碍的原因，其作用机制为下调内皮细胞连接、增加血管渗漏与液体蓄积。特异性灭活内皮细胞的CGRP受体，或使用CGRP受体拮抗剂，均可减少肺内液体蓄积并改善气体交换。在急性呼吸窘迫综合征（acute respiratory distress syndrome, ARDS，包括新型冠状病毒肺炎（COVID-19）引发的病例）患者的肺组织中，表达CGRP的PNEC数量显著升高。上述研究结果提示，神经肽水平升高可能参与肺血管外液体过量蓄积的过程，而拮抗其功能或可改善气体交换。针对每种指定的基因型，我们均从3只混合性别小鼠的完整肺组织制备单细胞悬液。通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）获取活上皮细胞与内皮细胞，并按2:1的比例重新混合后用于上样。按照10X Genomics提供的用户手册，采用10X Single Cell 3' v3 chemistry制备带条形码标记的单细胞液滴。构建完成的测序文库采用Illumina NovaSeq测序仪进行测序。利用Cell Ranger分析流程，对测序reads进行比对并获取基因水平的唯一分子标识符（unique molecular identifier, UMI）计数结果。采用Seurat（版本3.0）软件包完成细胞聚类以及不同细胞类型与实验条件下的基因表达谱分析。