Manipulating gut endocrine cell fate for the treatment of metabolic, inflammatory and cognitive diseases

Enteroendocrine (EEC) cells are hormone-producing cells that constitute 1% of the intestinal epithelium. Their hormones act locally within the gut epithelium, by signalling to its nervous system, or are released into the blood stream where they can influence distant organs such as the pancreas or brain. These signals have well-defined physiological roles including appetite regulation, pancreatic hormone secretion, intestinal motility and gastric emptying. However, knowledge regarding EEC differentiation in the human intestine is scarce. Understanding EEC specification may unveil targets that allow the manipulation of EEC fate offering control over the density of specific EEC populations. Dependent on the EEC population targeted, this strategy could form the basis of novel treatments for metabolic, inflammatory or cognitive disorders. ISX-9 is a small molecule, notable for inducing neuronal and beta-cell differentiation due to its ability to increase NeuroD1 expression. These cell types share similar developmental transcriptional pathways with EEC cells. The potential of ISX-9 to control EEC identity and specification was explored in mouse and human intestinal organoids derived from intestinal adult stem cells. ISX-9 upregulated NeuroD1 and increased the expression of the EEC-associated transcription factors Ngn3 and Pax4, highlighting a potential early mechanism for enterochromaffin biased fate. Analysis of mature EEC transcripts revealed exclusive upregulation of Cck and enterochromaffin cell markers ChgA, Tac1 and Tph1, but not other mature EEC markers. Double immunostaining for CHGA and serotonin revealed that the double-positive population was significantly increased by ISX-9 exposure. The newly generated enterochromaffin cells were biologically relevant and secreted more serotonin than control cells in response to stimulation. Induction of neuronal differentiation by ISX-9 is dependent on intracellular calcium signalling, suggesting this could be a mechanism by which it induces EEC differentiation. KN93, an inhibitor of CaMKII, an important calcium signalling node, partially inhibited the ISX-9-induced expansion of the enterochromaffin cell population, suggesting calcium increases were in part responsible for ISX-9 effects on EEC differentiation. Inhibition of two key epithelial signalling pathways, Notch and MEK, has been shown to stimulate EEC differentiation. Combining inhibition of these pathways with ISX-9 led to considerable increase in enterochromaffin cells, exposing an extremely powerful method for inducing enterochromaffin cells. The scRNA-seq data identified Pax4 as a potential key factor in biasing EEC fate towards enterochromaffin specification. Pax4 overexpression inhibited EEC differentiation and arrested cells in a progenitor-like state. Contrastingly, levels of Pax4 equivalent to the ones observed following ISX-9 treatment promoted comparable expression of CCK and enterochromaffin markers. These data validate Pax4 as an important factor for enterochromaffin specification and offer a mechanism for ISX-9-biased enterochromaffin differentiation. This work provides proof of concept that the manipulation of the intestine epithelium towards specific EEC populations is possible and that small molecules can be used to this effect. The potential to modulate the population of serotonin-producing cells may present a novel target in the treatment of diseases including depression and gut motility disorders, and as an adjuvant therapy in the modulation of liver regeneration and immune responses, however the physiological relevance and translation into a clinical setting will need to be determined.