Post-natal development of enteric glial cell-epithelial interactions in repair of ischemic-injured intestine

项目概要/摘要 如果上皮覆盖不能迅速恢复，肠粘膜屏障的破坏会导致败血症和死亡， 尤其是新生儿。与更成熟的个体相比，婴儿死亡率较高的原因 肠道损伤尚未得到解释。与缺血相关的疾病导致婴儿死亡率高/ 再灌注（I/R）损伤，包括坏死性小肠结肠炎和肠扭转。在幼猪（6-8周龄）中，我们 广泛研究了以上皮细胞迁移（恢复）为标志的非常快速的肠道修复。 然而，在该提案修订版中最近发表的一项研究中，我们展示了 有趣的是，缺血性损伤后粘膜修复的恢复与年龄相关，几乎完全丧失 新生仔猪缺乏恢复。有趣的是，这种修复缺陷可以通过匀浆来挽救 缺血损伤的幼年匀浆，表明更成熟的细胞群刺激恢复。一个细胞 特别感兴趣的群体是肠胶质细胞（EGC）网络，我们已经证明该网络已经成熟 猪出生后。在修订后的提案中，我们现已证明猪 EGC 条件培养基 刺激受伤新生猪 IPEC-J2 细胞的细胞迁移（恢复）。但仍存在差距 我们对上皮细胞和底层 EGC 之间的信号传导机制的了解，导致 归还。为了解决这一差距，我们对缺血性损伤的新生儿和新生儿进行了 RNAseq 分析。 幼年粘膜，并创建了一个公正的发现管道方法，重点关注显着缺陷 新生儿的信号通路。其中一种途径包括 EGF，它由 EGC 分泌： proEGF 和上皮膜联蛋白 A2，通过 β1-整合素内化信号启动细胞迁移。我们的 中心假设是缺血性损伤粘膜的修复依赖于 EGC 的出生后迁移 进入固有层，以便向受伤的上皮发出有效恢复的信号。我们将测试这个 有两个具体目标的假设：1）确定迁移信号机制中年龄依赖性缺陷 伤口附近的肠上皮细胞。我们的工作假设是，存在与年龄相关的 损伤后上皮恢复的发展至少部分是通过膜联蛋白 A2 信号传导实现的。 2）确定 围产期粘膜 EGC 网络结构、密度和分泌组的变化。我们的工作假设是 上皮附近没有足够的 EGC 分泌屏障促进因子，包括 proEGF 以刺激新生儿的康复。为了检查这些具体目标，我们将使用我们独特的年龄依赖性 猪粘膜修复模型、受伤猪新生IPEC-J2细胞模型、猪原代培养 EGC、通过质谱法进行 EGC 分泌组分析以及神经胶质细胞最先进的成像技术， 包括溶剂清除器官的免疫标记三维成像 (iDISCO)。使用 无偏见的发现管道方法，我们期望能够定义有缺陷的信号机制 在新生儿受伤的肠道中，最终可以提高肠道衰竭患者的生存率

PROJECT SUMMARY/ ABSTRACT Breaches in the intestinal mucosal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants as compared to more mature individuals with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis and volvulus. In juvenile pigs (6-8-weeks of age), we have extensively studied the remarkably rapid intestinal repair marked by epithelial cell migration (restitution). However, in a recently published study featured in the revised version of this proposal, we have shown an intriguing age-dependent deficiency in the recovery of mucosal repair following ischemic injury with a near-total lack of restitution in neonatal piglets. Interestingly, this defect in repair can be rescued by a homogenate of ischemic injured juvenile homogenate, suggesting more mature cell populations stimulate restitution. One cell population of particular interest is the enteric glial cell (EGC) network, which we have shown matures postnatally in pigs. In the revised proposal, we have now shown that porcine EGC-conditioned media stimulates cell migration (restitution) in wounded neonatal porcine IPEC-J2 cells. However, a gap remains in our knowledge regarding the signaling mechanisms between epithelium and underlying EGC that results in restitution. To address this gap, we have performed RNAseq analyses of ischemic-injured neonatal and juvenile mucosa, and created an unbiased discovery pipeline approach to focus on significantly deficient signaling pathways in the neonate. One such pathway includes EGF, which is secreted by the EGC as proEGF, and epithelial annexin A2, which initiates cell migration by signaling internalization of β1-integrin. Our central hypothesis is that repair of ischemic-injured mucosa is reliant on postnatal migration of EGC into the lamina propria in order to signal wounded epithelium to efficiently restitute. We will test this hypothesis with two specific aims: 1) Determine age-dependent defects in migration signaling mechanisms in wound-adjacent intestinal epithelial cells. Our working hypothesis is that there is an age-dependent development of epithelial restitution in response to injury at least in part via annexin A2 signaling. 2) Determine perinatal changes in mucosal EGC network structure, density and secretome. Our working hypothesis is that there are insufficient EGCs secreting barrier-promoting factors, including proEGF, in proximity to the epithelium to stimulate restitution in neonates. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, a wounded porcine neonatal IPEC-J2 cell model, primary culture of porcine EGC, EGC secretome analyses via mass spectrometry, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Using an unbiased discovery pipeline approach, we expect to be able to define signaling mechanisms that are deficient in neonatal injured intestine that can ultimately be targeted to enhance survival of patients with intestinal failure