Probiotic LGG's Metabolites Repair Disrupted Gut Barrier In Vitro

The objective of this experiment is to monitor the effects of microbiota-derived metabolites of tryptophan, indoles, when applied basolaterally and luminally on a compromised tight junction system. Human IBD data of the metabolome of patients with Crohn's Disease, Ulcerative Colitis, or C Diff were compared to that of healthy control individuals and demonstrated defects in tryptophan metabolism, with deficiencies of certain indoles – exogenous metabolites of tryptophan produced by LGG. Dr. Ferraris’s lab studies the effects of tryptophan and Lactobacillus rhamnosus GG (LGG), a popular probiotic, on germ-free mice. Metabolome Transcriptomic analysis (MeTRcA) revealed that tight junctional genes had the highest enrichment score, and that indoles are positively correlated with barrier function genes such as Occludin – dependent on whether the serum or feces is being analyzed. However, functional analyses is required to demonstrate their barrier protective effects. To study these correlations further, colon carcinoma (Caco-2 BBE) cells were grown for 21 days on inserts with a semipermeable membrane to mimic the luminal and basolateral polarity of enterocytes. In the context of the correlations, the presence of metabolites in the feces was explored with luminal application, and presence in the serum with basolateral application. Following 48hr incubation of treatment with metabolites (applied luminally or basolaterally) or their DMSO solvent, application of C. difficile (CD) cell-free supernatant was used to mimic a leaky gut model by inactivating Rho enzymes, causing actin adhesion plaques to disengage. The two main readouts were transepithelial electrical resistance (TEER) and FITC-dextran. TEER reads the electrical resistance across the epithelial layer, which is a measure of paracellular ion permeability. Flux of FITC-dextran from the luminal to basolateral media measured paracellular macromolecular permeability. Cells with healthy tight junctions will have high TEER and low FITC absorbance. Luminal administration of indole-3-acetonitrile, indole-3-acetic acid, and indole-3-carboxylic acid improved TEER by 168%, 91%, and 39%, respectively, after 48 hours of incubation compared with vehicle. Treating cells with CD supernatant provoked a 46% reduction in TEER, consistent with an increased permeability of the paracellular pathway measured by FITC-dextran flux. Under an impaired barrier, the luminal application of indole-3-lactic acid, indole-3-propionic acid, indole-3-acetonitrile, indole-3-acetamide, indole-3-acetic acid, and indole-3-carboxylic acid restored TEER with efficacies ranging from 29% to 96%. Basolateral treatment of indole-3-acetamide and indole-3-carboxyaldehyde exhibited a remarkable increase in baseline TEER, with 1.3- and 1.6-fold enhancements, respectively, in comparison with the vehicle. Under CD treatment, the basolateral indole-3-lactic acid, indole-3-acetamide, and indole-3-carboxyaldehyde restored TEER by 57.5%–72.4%. FITC-dextran permeability assays were largely consistent with TEER results. Indole-3-carboxylic acid (ICA), and indole-3-acetic acid (IAA) can reduce the levels of the pro-inflammatory transcription factor NF-κB when applied luminally. Certain cocktails of high performing LGG-derived metabolites of tryptophan, including indoles, were additionally able to maintain higher TEER values under tight junctions impaired with CD toxin controls. In summary, multiple metabolites in the indole pathway provide significant rescue from intestinal permeability in a compromised tight junction model. NIHR01-AT010243 (NG, RF), R01DK119198 (NG), NSF 1754783 (RF). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.