Professor Washington University School of Medicine, St. Louis, Missouri, United States
Disclosure(s):
Ta-Chiang Liu, MD, PhD: No financial relationships to disclose
Introduction/Rationale: 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 (Nampt∆IEC, via VilCre), SecCs (Nampt∆Atoh1, via Atoh1CrePR), and Paneth cells (Nampt∆PC, 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.
