Rose Elizabeth C, Simon Jeremy M, Gomez-Martinez Ismael, Magness Scott T, Odle Jack, Blikslager Anthony T, Ziegler Amanda L
bioRxiv. 2024 Jun 30:2024.06.28.601271. doi: 10.1101/2024.06.28.601271.
Intestinal ischemic injury damages the epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. We have developed a pig model that demonstrates age-dependent failure of intestinal barrier restitution in neonatal pigs which can be rescued by the direct application of juvenile pig mucosal tissue, but the mechanisms of rescue remain undefined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing analysis, and predicted upstream regulators were assessed in a porcine intestinal epithelial cell line (IPEC-J2) and banked tissues. A subcluster of absorptive enterocytes expressed several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation included colony stimulating factor-1 (CSF-1). We validated age-dependent induction of by ischemia and documented that CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued (but not control) neonates. Further, the CSF1R inhibitor BLZ945 reduced restitution in scratch wounded IPEC-J2 cells. These studies validate an approach to inform potential novel therapeutic targets, such as CSF-1, to improve outcomes in neonates with intestinal injury in a unique pig model.
NEW & NOTEWORTHY: These studies validate an approach to identify and predict upstream regulation of restituting epithelium in a unique pig intestinal ischemic injury model. Identification of potential molecular mediators of restitution, such as CSF-1, will inform the development of targeted therapeutic interventions for medical management of patients with ischemia-mediated intestinal injury.
肠道缺血性损伤会破坏上皮屏障，使患者易患危及生命的败血症，除非该屏障能迅速恢复。肠道恢复存在年龄依赖性，即与年长患者相比，新生儿最易因肠道屏障疾病而死亡。我们建立了一种猪模型，该模型显示新生仔猪肠道屏障修复存在年龄依赖性失败，直接应用幼年猪黏膜组织可挽救这种情况，但挽救机制尚不清楚。我们假设，通过根据细胞迁移转录途径的表达来识别恢复中的肠上皮细胞亚群，我们可以预测年龄依赖性修复反应程序的新上游调节因子。对幼年猪缺血空肠恢复过程中的表层黏膜上皮细胞进行单细胞RNA测序分析，并在猪肠上皮细胞系（IPEC-J2）和储存组织中评估预测的上游调节因子。吸收性肠上皮细胞的一个亚群表达了几个对修复至关重要的细胞迁移途径。该亚群中差异表达的基因预测其上游调节包括集落刺激因子-1（CSF-1）。我们验证了缺血对CSF-1的年龄依赖性诱导，并记录到CSF-1和CSF1R在缺血的幼年（而非新生）猪伤口附近上皮细胞以及幼年猪和获救（而非对照）新生猪的修复上皮中共同定位。此外，CSF1R抑制剂BLZ945降低了划痕损伤的IPEC-J2细胞的修复能力。这些研究验证了一种方法，可用于确定潜在的新治疗靶点，如CSF-1，以改善独特猪模型中肠道损伤新生儿的预后。
这些研究验证了一种在独特的猪肠道缺血损伤模型中识别和预测修复上皮上游调节的方法。确定潜在的修复分子介质，如CSF-1，将为缺血介导的肠道损伤患者的医疗管理中靶向治疗干预措施的开发提供信息。
