Bacteria‐Derived Hypoxanthine Accelerates Gastrointestinal Transit

Irritable bowel syndrome is a globally prevalent disorder categorized into constipation, diarrhea or mixed (IBS-C, IBD-D, IBS-M) based on the predominant stool form/frequency. While the gut microbiome is recognized as an important factor underlying the pathophysiology of IBS, the specific mechanisms remain an area of active investigation. In our recent longitudinal multi-omics study in IBS patients, we found bacteria-derived hypoxanthine was significantly decreased in stool from IBS-C patients. The aim of this study was to determine if IBS-C relevant molecular pathways are affected by hypoxanthine. We hypothesized that hypoxanthine acts on enterochromaffin (EC) cells to stimulate serotonin (5-HT) release, where 5-HT is an important determinant of gastrointestinal (GI) transit time. We used Ca2+ imaging in QGP cells (EC model), T84 cells (colonocyte model), primary cells and organoids from NeuroD1-cre;GCaMP5-tdTomato mice [NeuroD1 is a marker for enteroendocrine cells (EECs) which express calcium indicator GCaMP in these mice] in the absence or presence of receptor antagonists, and in ex vivo colon preparations with intact innervation from E2aCre:GCaMP6s mice (GCaMP is expressed in neuronal and non-neuronal cells), to test the effect of hypoxanthine. The physiologic relevance of hypoxanthine signaling was assessed by measuring whole gut transit time. We found hypoxanthine increased Ca2+ influx in QGP cells [using Cal520 (qualitative) and Fura-2AM (quantitative)], primary EECs and EECs within organoids (previous research showed that the majority of these EECs are EC cells), but not in T84 cells. Hypoxanthine-evoked Ca2+ response was absent in Ca2+ -free medium (n=6-8, t-test, P<0.0001), inhibited by Gαi/o -GPCR antagonist (pertussis toxin; n=8-14, t-test, P<0.01)), Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) antagonist (ML204; n=14-15, t-test, P<0.0001) and TRPC4 siRNA (n=5-21, t-test, P<0.01), and was accompanied by increased 5-HT release (measured using 5-HT biosensor). In ex vivo experiments, luminal application of hypoxanthine activated a subset of epithelial cells (likely EC cells) followed by subsets of neurons (latency to response onset, n=4, Mann-Whitney, P<0.0001), as well as movement of the imaging field, which indicates smooth muscle contraction/relaxation. Our in vitro findings were confirmed by in vivo experiments showing that germ-free mice colonized with hypoxanthine-producing bacteria had faster whole gut transit and higher stool 5-HT level compared to hypoxanthine-consuming bacteria (n=7-8, t-test, P<0.05). In summary, hypoxanthine increases Ca2+ influx specifically in EC cells by activating a signaling cascade involving a Gαi/o -GPCR and TRPC4 channels, which results in 5-HT release. The delay in activation of myenteric neurons and motor activity suggests that EC cell 5-HT release likely induces neurogenic motor patterns and may underlie the shorter GI transit time seen with increased bacterial hypoxanthine production. While the current empirically designed probiotics have proven ineffective in IBS, our findings will allow development of novel mechanism-based probiotic therapies for IBS-C using hypoxanthine-producing bacteria.