Abstract Background The intestinal microbiota-tryptophan metabolism axis is an important regulator in inflammatory bowel disease (IBD)1. Gut bacteria convert tryptophan into bioactive metabolites, which act on intestinal cells through the aryl hydrocarbon receptor (AHR)2. These microbially derived metabolites are known to support mucosal homeostasis2, yet their direct impact on epithelial barrier responses in Crohn’s disease (CD) compared with non-IBD under inflammatory and non-inflammatory conditions, remains underexplored. Methods Intestinal epithelial organoids were generated from caecal biopsies of CD patient and non-IBD donor and differentiated for four days. Inflammation was induced with IFN-γ and TNF-α (10 ng/mL each, 24 h). Tryptophan, tryptamine, nicotinamide, indole-3-acetic acid, and indole-3-propionic acid were applied individually 1 h before cytokine exposure and maintained throughout; non-inflamed organoids were exposed for 25 h. Working concentration of each metabolite was determined by Caco-2 screening. Total RNA was analyzed by RT-qPCR. Expression of three functional gene groups was evaluated: inflammatory (CXCL9,TNF, IL1B), AHR/xenobiotic metabolism (AHR, UGT1A1, CYP1A1), and barrier-related (TJP1, CLDN1,OCLN, MUC2, LGR5, KI67). Data were processed in RStudio, statistical significance level was set to padj. ≤ 0.05. Results In non-inflamed CD organoids, tryptophan metabolites induced AHR pathway genes such as CYP1A1 and UGT1A1, showing increases of approximately 5-8 log2FC, but did not increase barrier-related transcripts. Inflammatory stimulation strongly upregulated CXCL9, IL1B, and TNF, with increases of approximately 5-11 log2FC, while reducing barrier markers expression by about 1-2 log2FC. Metabolite co-treatment during inflammation maintained AHR activation and partially restored OCLN expression by about 0.6-0.8 log2FC. In non-IBD organoids, metabolites upregulated both AHR-related genes such as CYP1A1, with increases of approximately 5-7 log2FC, and barrier genes including CLDN1, OCLN, MUC2, and LGR5, which rose by about 1-2 log2FC. Inflammation caused weaker immune activation than in CD and moderately reduced barrier genes, while metabolite treatment partly restored OCLN expression by about 0.5-0.7 log2FC, with limited effects on other barrier markers. Although trends were consistent, not all log2FC values were statistically significant. Conclusion In conclusion, microbially derived tryptophan metabolites activate epithelial AHR signaling in both CD and non-IBD organoids, but barrier regulation differs. Therefore, our results indicate diagnosis-specific epithelial sensitivity to microbial products. Funding: European Union and the State Secretariat for Education, Research and Innovation (project: miGut-Health, no. 101095470). References: 1.Hua, X., Chen, Y., Ding, S., & Fang, J. (2025). Tryptophan metabolism and the intestinal microbiota: Implications for inflammatory bowel disease. Microbiological Research, 300, 128280. 2.Agus, A., Planchais, J., & Sokol, H. (2018). Gut microbiota regulation of tryptophan metabolism in health and disease. Cell host & microbe, 23(6), 716-724. Conflict of interest: Inčiūraitė, Rūta: No conflicts Wobbe, Marla: No conflict of interest Paškevičiūtė, Gabija: No conflict of interest Kiudelis, Vytautas: No conflict of interest Kupčinskas, Juozas: Speaker fees: Takeda, Janssen, Ferring, Abbvie, Falk, Pfizer Consultancy fees: COSMO pharma Rosenstiel, Philip: stock ownership Gerion Skieceviciene, Jurgita: No conflict of interest
Inčiūraitė et al. (Thu,) studied this question.