The Role of dNp63 in Maladaptive Regeneration and Repair Following Severe Pulmonary Injury

摘要 与许多哺乳动物的重要器官不同，肺对严重的损伤表现出强大的再生反应，如 如流感感染，主要针对上皮细胞、呼吸道和肺泡。静息驻肺 肺损伤后，上皮祖细胞进入细胞周期、增殖和分化，参与了两个 不同的再生途径：功能有益的再生和不适应的组织重塑。 小叶内呼吸道驻留的远端p63+祖细胞是一种迁移到肺泡中的祖细胞类型， 附着在剥离的肺泡基底膜上，并迅速增殖产生异位细支气管膜- 像组织一样，形成蜂窝状的囊肿，受伤后不能溶解，也不参与气体 交换。虽然最终是一种不适应的损伤反应，但这种异位的细支气管化似乎确实受益。 严重肺泡损伤的患者可通过提供一种“紧急”的上皮屏障。远端p63+祖细胞 是远端肺中唯一表达主要上皮调节因子Trp63的细胞，特别是ΔN亚型 (ΔNP63)。ΔNp63在皮肤等其他上皮组织的增殖性基底干细胞中高度活跃， 乳腺、前列腺和气管，在其中，它赋予基底细胞干细胞样的身份和转录 调节细胞的迁移、黏附和增殖过程。在我自己的初步数据中，我有 发现流感损伤小鼠在呼吸道上皮细胞中存在广泛的ΔNp63缺失，表现出完全的 取消适应不良的肺泡重塑反应。除了这些数据，还没有直接的研究 探讨ΔNp63在不良适应重塑中的作用及其促进机制 再生途径。该提案的目标1将利用ΔNp63的有条件删除和血统追踪 远端p63+祖细胞研究ΔNp63缺失是否会导致远端p63+祖细胞的细胞特性改变 在肺损伤之后。细胞内流式细胞术、免疫组织化学和定量聚合酶链式反应将用于评估 ΔNp63-/-远端p63+祖细胞在流感损伤前后缺失时的命运决定； 此外，还将使用肺功能测试来评估Δnp63的生理后果 淘汰赛。AIM 2将在体外和体外联合应用CRISPRA介导的p63过表达 迁移试验确定远端p63+祖细胞是否受ΔNp63过表达的影响 在其他ΔNp63表达的细胞和组织类型中。最后，在确认/识别已知和/或 在p63+远端祖细胞中以前未确定的ΔNp63迁移靶点，CRISPR介导的这些靶点的敲除 随后进行体外和体外迁移试验的靶标将被用来评估ΔNP63- 损伤激活的p63+远端祖细胞运动中的驱动迁移程序。这些实验将调查 ΔNp63在损伤后远端p63+祖细胞命运选择和激活中的作用，进而深入了解 严重肺损伤后肺再生途径的机制基础。

Abstract Unlike many mammalian vital organs, the lung exhibits a robust regenerative response to severe injuries such as influenza infection, which primarily targets epithelial cells airways and alveoli. Quiescent lung-resident epithelial progenitors enter the cell cycle, proliferate, and differentiate following lung injury, participating in two distinct regenerative pathways: functionally beneficial regeneration and maladaptive tissue remodeling. Intralobular airway-resident distal p63+ progenitors are one such progenitor cell type that migrates into the alveoli, adheres to the denuded alveolar basement membrane, and rapidly proliferates to generate ectopic bronchiolar- like tissue, forming honeycomb-like cysts that fail to resolve after injury and that do not participate in gas exchange. Though ultimately a maladaptive injury response, this ectopic bronchiolization does appear to benefit individuals with severe alveolar injuries by providing an “emergency” epithelial barrier. Distal p63+ progenitors are the only cells in the distal lung that express the master epithelial regulator Trp63, specifically the ΔN isoform (ΔNp63). ΔNp63 is highly active in the proliferative basal stem cells of other epithelial tissues such as the skin, mammary, prostate, and trachea, in which it confers basal cells with their stem-like identity and transcriptionally regulates the cellular processes of migration, adhesion, and proliferation. In my own preliminary data, I have found that influenza-injured mice with broad ΔNp63 deletion in the airway epithelium display a completely abrogated maladaptive alveolar remodeling response. Besides this data, there have been no studies directly investigating the role of ΔNp63 in maladaptive remodeling and the mechanisms by which it promotes this regenerative pathway. Aim 1 of this proposal will utilize conditional deletion of ΔNp63 in and lineage-tracing of distal p63+ progenitors to investigate if loss of ΔNp63 causes a cell identity change in distal p63+ progenitors following pulmonary injury. Intracellular flow cytometry, immunohistochemistry, and qPCR will be used to assess the fate decisions of ΔNp63-/- distal p63+ progenitors upon deletion both prior to and following influenza injury; pulmonary function tests will additionally be used to evaluate the physiological consequences of ΔNp63 knockout. Aim 2 will employ CRISPRa-mediated overexpression of p63 in tandem with in vitro and ex vivo migration assays to determine if cell motility is affected by ΔNp63 overexpression in distal p63+ progenitors as it is in other ΔNp63-expressing cell and tissue types. Finally, upon confirmation/identification of known and/or previously unidentified ΔNp63 migration targets in distal p63+ progenitors, CRISPR-mediated knockout of these targets followed by in vitro and ex vivo migration assays will be utilized to evaluate the importance of the ΔNp63- driven migration program in injury-activated distal p63+ progenitor motility. These experiments will investigate the role of ΔNp63 in distal p63+ progenitor fate choice and activation following injury, in turn yielding insight into the mechanistic underpinnings of lung regeneration pathways following severe pulmonary injury.