Single-cell transcriptomics predicts colony stimulating factor-1 regulation of age-dependent intestinal epithelial restitution

Abstract

Intestinal diseases associated with ischemic injury, which damages the principal barrier against noxious luminal contents, result in unacceptably poor outcomes in newborns. Ischemia-induced loss of the intestinal 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. While this age-dependence in repair has not been demonstrated in traditional rodent models, we have developed a highly translational pig model of intestinal ischemic injury and repair that does reflect this difference. We have shown that, while juvenile (weaned) pigs recover rapidly after ischemic intestinal injury, barrier repair is markedly underdeveloped in neonatal (nursing) pigs due to complete failure of epithelial restitution. Importantly, we found that restitution in neonates can be rescued by the direct application of homogenized mucosa from ischemia-injured small intestine from juvenile pigs. The mechanisms that allow for this restitution and rescue remain to be defined. 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 collected from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing, unbiased clustering and upstream regulator analysis. A porcine intestinal epithelial cell line (IPEC-J2) and banked tissues from prior rescue experiments were qualitatively and functionally assessed for activity of predicted upstream regulators. Single cell transcriptomics in recovering juvenile epithelium revealed a subcluster of absorptive enterocytes that express several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by many potential molecules, including colony stimulating factor-1 (CSF-1) which is known to induce cell migration in non-intestinal epithelial tissues. To begin validating this prediction, we demonstrated that CSF-1 was enriched in the ischemic juvenile mucosa which rescues neonatal restitution and documented expression of the CSF-1 receptor (CSF1R) in both neonatal and juvenile epithelium, indicating that these cells are equipped to respond to CSF-1. 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. Single cell transcriptomics have the power to inform potential novel therapeutic targets, such as CSF-1, to improve mucosal recovery in neonates with intestinal failure in this unique and powerful pig model. NIH K01 OD028207, NIH R01 HD095876, NIH U01 TR002953, NIH T32 OD011130, NIH P30 DK034987, USDA NIFA 1007263 and 07985. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.