Intestinal Barrier Protection by Probiotic Lactobacillus-Associated Indoles in Caco-2 and Enteroids

Abstract

Gut bacteria generate from dietary tryptophan (trp) indole compounds, which act as potent signaling molecules intricately involved in the regulation of intestinal homeostasis and barrier integrity. Lactobacillus rhamnosus GG (LGG), a widely used probiotic, has demonstrated ability to restore the gut barrier in animal models of colitis and sepsis, as well as in children with irritable bowel syndrome. To investigate the hypothesis that LGG-derived indoles are responsible for its gut barrier-enhancing properties, we fed gnotobiotic mice trp-suffcient and trp-deficient diets. We then monocolonized them with LGG, using PBS gavaged mice as a control, and analyzed the ileal transcriptome by bulk RNA sequencing and both fecal and serum metabolomes by LC-MS. Using our novel bioinformatic tool METRCA, which correlates metabolites and gene networks, we uncovered an unexpected and substantial influence of indole spatial distribution on genes associated with gut integrity: some were significantly positively linked to tight junction (TJ) genes exclusively from the luminal side, some exclusively from the serum, and others from both. Specifically, we identified nine indoles—indole-acetamide (IAM), indole-carboxaldehyde (IAL), indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), indole-3-acrylic acid (IAR), indole-3-carbinol (ICB), indole-acetic acid (IAA), indole-3-acetonitrile (IAN), and indole-3-carboxylic acid (ICA)—that demonstrated the potential to maintain gut barrier function. To assess their impact, we used a high-throughput system that measured permeability through transepithelial electrical resistance (TEER) and FITC-dextran (4 kDa) in polarized Caco-2 cells. By luminally challenging cells with Clostridium diffcile toxins (TOX), we induced barrier disruption to model leaky gut. Intriguingly, when applied luminally, IAA, IAN, ICA, and ILA rescued the TEER of Caco-2 cells exposed to TOX, while IAM, IAL, and ILA were effective when applied basolaterally, suggesting a strong polar component to their barrier repair capacity. FITC transport revealed a significant basolateral rescue effect of IAM and luminal rescue effect of IAA and ICA, indicating that different sets of indoles are required to repair the pore and leak pathways. Furthermore, we confirmed that basolateral IAM rescued FITC permeability and induced increased expression of TJ genes Tjp1 and Ocln in enteroids challenged with lipopolysaccharides. In summary, our research highlights the potential beneficial therapeutic application of certain indoles in repairing disrupted gut barriers. NIHR01-AT010243 (NG, RF), R01DK119198 (NG), NSF 1754783 (RF). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.