METABOLITE MIMICRY REVEALS BUTYRATE ANALOG WITH SELECTIVE PROTECTIVE FUNCTIONS IN THE INTESTINAL MUCOSA

Abstract The intestinal mucosa is a semipermeable barrier that facilitates interactions between the host and the microbiota. A well-orchestrated balance exists in healthy mucosa whereby microbes and microbial metabolites encounter a barrier of specialized intestinal epithelial cells. This homeostasis exists at a harsh interface between the well-oxygenated crypt floor and the anaerobic intestinal lumen. This steep oxygen gradient establishes “physiologic hypoxia” as a key metabolic characteristic of the mucosa. Emerging interest in understanding the dynamic host-microbe interplay has identified microbial metabolites that support host functions at different levels. Of relevance are short-chain fatty acids (SCFA), particularly butyrate. Butyrate is the preferred energy source for mature colonocytes. It also contributes to homeostasis through gene transcription regulation by stabilizing the transcription factor hypoxia-inducible-factors (HIF) and functioning as a histone deacetylase (HDAC) inhibitor. Interestingly, a metabolic gradient exists within the intestinal crypt as butyrate is constantly used as fuel. One limitation of butyrate as a therapeutic is its rapid metabolism in differentiated colonocytes. Furthermore, intestinal stem cells (ISCs) respond differently to butyrate, preferentially using glucose for energy procurement. When the metabolic gradient is disrupted (e.g. disease), butyrate accumulates in ISCs leading to reduced cell proliferation and differentiation, thereby hampering intestinal barrier recovery. Recent studies revealed that HIF stabilization contributes to the inhibition of ISCs activity. To address these limitations, we explored metabolite-mimicry to discover compounds with potent or selective biological responses within the butyrate pathway(s). We discovered an analog, 3-chlorobutyrate (3-Cl BA), that significantly enhances epithelial barrier formation and wound healing in vitro. Mechanistically, we revealed that 3-Cl BA is a potent HDAC inhibitor. Furthermore, unlike butyrate, 3-Cl BA does not stabilize HIF and it is not used as metabolic fuel. In vivo studies in a DSS-colitis model revealed that contrary to butyrate, 3-Cl BA is protective. Studies in stem-like colonoids demonstrated that only butyrate inhibits ISC proliferation and differentiation. We demonstrate here that pharmacologic HIF stabilization inhibits colonoid differentiation and that genetic loss of HIF significantly promotes ISC differentiation. Given the fact that butyrate but not 3-Cl BA stabilizes HIF, we propose that 3-Cl BA circumvents these detrimental functional consequences. This study highlights the importance of a dual oxygen/metabolic gradient within the intestinal crypt. Additionally, it demonstrates the advantages of metabolite-mimicry in harnessing specific biological functions and reveals a promising butyrate analog protective in colitis.