Optimal NAD+ level is a prerequisite for small intestinal secretory cell differentiation 2299891

Abstract Introduction NAD+ is key to cellular metabolism. NAMPT is the rate limiting enzyme in NAD+ synthesis, accounting for 90% of NAD+ in the body. Children harboring loss-of-function mutations of NAMPT had reduced secretory cell (SecC; goblet, Paneth) densities in the small intestine. SecCs play key roles in gut innate immunity. How NAD+ regulates SecC homeostasis is unclear. We hypothesized that NAMPT-dependent NAD+ production supports sirtuin (SIRT)-dependent Notch degradation, a necessary step in ATOH1 activation and gut SecC differentiation. Methods Intestinal epithelial cells (IECs) from wild type (WT) mice were used for snRNAseq. Various Nampt conditional knockout mice were generated: IECs (NamptΔIEC, via VilCre), SecCs (NamptΔAtoh1, via Atoh1CrePR), and Paneth cells (NamptΔPC, via Defa4Cre). Poly(I:C) was used to induce gut injury. NAD+ levels were defined by mass spectrometry. Immunoblot was used to define Notch intracellular domain (NICD), HES1, ATOH1 and acetyl-NFκB (SIRT activity). Results snRNAseq showed Nampt expression in all IEC cell types. NamptΔIEC IECs and organoids showed reduced SecC densities, without SecC apoptosis, suggesting a differentiation defect. WT organoids contained higher NAD+ levels compared to stem cells. NamptΔAtoh1 and NamptΔPC mice showed no reduction in SecCs, suggesting NAD+ is critical in SecC differentiation before ATOH1 activation. There was increased NICD and HES1 and repressed ATOH1 and SIRT activity in NamptΔIEC IEC and organoids. This was phenocopied in mice and organoids with inhibition of SIRT members. NamptΔIEC mice had more severe Poly(I:C) injury. NAD+ precursor nicotinamide ribonucleoside or overexpression of the tryptophan metabolism enzyme IDO1 rescued SecC density in NamptΔIEC mice and organoids and ameliorated Poly(I:C) injury. Conclusion Appropriate levels of NAD+ during the early phase of IEC differentiation is necessary to generate gut SecCs. This study provides a mechanistic link between cellular metabolic state and SecC proportions in the gut. Funding Source n/a Topic Categories Mucosal and Regional Immunology (MUC)