Uncovering the Intestinal Mechanosensitive Regeneration Signature: Expect More from Hirschsprung Disease

Introduction: Intestinal growth and hemostasis are influenced by convoluted signals and the impact of mechanical stimuli on human bowel has been remained undiscovered. Herein, we defined the Intestinal Regenerative Mechanosensation Signature (IRMS) gene set using a partial obstruction mouse model that exerts non-dissipative force to the intestinal wall.

We characterized the human bowel’s long term regenerative response to mechanical force by assessing IRMS enrichment in the normal segments of the colon that experienced force in children with Hirschsprung disease. These findings reveal a potential mechanosensitive role for lymphatics in intestinal hemostasis.

Method: Tubes were placed around ileum in mice. Dilated ileum proximal to the tube was harvested at serial time points. RNA sequencing was utilized to identify a set of intestinal regenerative mechanosensation signature (IRMS) genes at maximum regeneration following maximum mechanosensation. The abundance of member cell types was imputed by a deconvolutional machine learning algorithm, following the integration of single-cell RNAseq published data of healthy mouse intestines. Gene Set Enrichment Analysis (GSEA) was performed on colon of 8 children with Hirschsprung disease (GSE98502). Leading-edge genes were analyzed through single-cell RNA sequencing of mouse intestine (GSE190037) and human colon (SCP1884) to identify the mechanosensitive signatures.

Results: The maximal reduction of murine crypt density and muscle thickness was observed on POD3, suggesting maximal mechanosensation. A significant increase in crypt bifurcation and Ki-67 expression was observed on POD5 (maximal regeneration). We detected a significant increase in the fraction of intestinal stem cells (Fig.1). GSEA revealed enrichment of IRMS in the force-experiencing segment of colon. Fifty-two genes were identified as IRMS genes associated with regeneration (Fig.2). Investigation of these genes within scRNAseq datasets revealed the exclusive expression of PARD6G in lymphatic endothelial cells (Fig.3 and Fig.4). PARD6G immunostaining in human colon highlighted its presence beneath the crypts, co-localizing with the niche of intestinal stem cells (Fig.5).

Conclusion: We observed that epithelial growth through mechanical stimulation, may not restrict the stemness of intestinal stem cells. We defined a gene set that has a role in intestinal regeneration due to mechanical force. These findings suggest a new gene associated with lymphatic endothelial cells and a role for lymphatics in intestinal homeostasis.