Multiplexed reporter platforms for probing the bile acid- nuclear receptor interactome - Project Summary Bile acids (BAs) are core gastrointestinal (GI) metabolites with dual functions in lipid absorption and cell signaling. The past decade has seen a resurgence in BA research thanks to seminal studies – including from our lab – that established BAs as direct regulators of intestinal immune cells. Nuclear receptors (NRs) – a large family of ligand- regulated transcription factors – are a primary source of host BA receptors in the GI tract, including in immune cells. Still, only a fraction of the putative BA-NR ‘interactome’ has been explored, in part because gold-standard reporter assays needed to identify NR ligands are tedious and singleplex. Our goal is to build and use multiplex NR reporter systems capable of mapping global interactions between physiologically defined BA pools and all 48 NRs. To this end, we have developed a novel ‘multi-GI-omics’ platform that features parallel sampling of nearly 100 endogenous BA species and metabolites from across three GI sites of individual mice, and that yields high-resolution insights into dynamics of small intestinal BA reabsorption – a key and rate-limiting step in enterohepatic circulation that determines which BAs in the small intestinal lumen ultimately gain access to mucosal immune and epithelial cells in vivo. Using this approach, we described that the pool of endogenous BAs which circulates through the distal small intestine (i.e., ileum) of fasting C57BL/6 mice totals at least 0.3 moles/g and contains at least 42 discrete species. 80% of this pool is comprised of a small handful of ‘primary’ BA species synthesized in the liver [e.g., taurine-conjugated cholic acid (tCA)], and is maintained by an active transport mechanism (Asbt/Slc10a2) unique to ileal enterocytes. The remaining 20% includes mostly ‘secondary’ BAs – produced via microbial metabolism of primary BAs in the large intestine [e.g., deoxycholic acid (DCA), lithocholic acid (LCA)] – and enter the ileum via passive diffusion. Most recently, we have discovered that spontaneous ileal inflammation (i.e., ileitis) – a hallmark of human Crohn’s disease modelled experimentally in TNFARE mice – alters ileal BA absorption in ways that produce a smaller and more pro-inflammatory BA pool. In seeking to screen for putative NR-dependent mechanisms through which healthy or ileitis-associated mouse BA pools may elicit tolerogenic or inflammatory immune responses, respectively, we have performed pilot studies using a multiplex NR reporter system, called trans-FACTORIALTM available at Attagene, Inc. Appreciating both the transformative potential of multiplex NR reporter systems generally, as well as the limitations of the trans- FACTORIALTM system specifically, we have begun building a more universal NR reporter format that features DNA-barcoded fluorescent reporter genes readable by flow cytometry or RNA-seq. Our hypothesis is that using and refining multiplex NR reporter systems will lay the foundation for more broadly deciphering the BA:NR ‘interactome’, while also enhancing NR ligand and drug discovery research by us and others.
