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Background Ultra-processed food consumption increases Crohn’s disease (CD) risk, but mechanisms remain elusive. We investigated whether aspartame affects host immunity through microbiota-derived metabolites, and whether this interaction depends on pre-existing microbial state.Methods We integrated food frequency questionnaires, fecal metagenomics, metabolomics, proteomics, and mucosal transcriptomics from 54 CD patients and 101 healthy controls in the Chinese Mainland. Dysbiosis score was calculated based on deviation from the healthy microbiome using Bray-Curtis distance. Germ-free mice received fecal microbiota transplantation from four donor subgroups (CD dysbiosis/eubiosis, Control dysbiosis/eubiosis), followed by 16-week aspartame or water intervention.Results Metagenomic and proteomic analysis identified microbiome dysbiosis in 51.9% of CD and 10.9% of controls, characterized by SCFA-producer depletion, Enterococcus enrichment, functional alterations in pathobiont-derived proteins, and elevated fecal calprotectin level (IDDF2026-ABS-0279 Figure 1. Gut microbiome dysbiosis in Crohn’s disease and healthy controls). Metabolomic analysis revealed significant dysregulation of phenylalanine and tyrosine metabolism pathway in dysbiotic group, characterized by decreased tyrosine and increased tyramine levels (IDDF2026-ABS-0279 Figure 2(A) Tyramine metabolism links dysbiosis to mucosal immune activation). These pathways are directly linked to aspartame metabolism and align with the observation of higher aspartame intake among dysbiotic individuals. Functional pathway analysis further demonstrated the activation of tyrosine-associated pathways in dysbiotic group (IDDF2026-ABS-0279 Figure 2(B) Tyramine metabolism links dysbiosis to mucosal immune activation). Notably, the level of tyrosine decarboxylase-coding gene (TyrDC), particularly from Enterococcus, was significantly elevated in dysbiotic subjects (IDDF2026-ABS-0279 Figure 2(C) Tyramine metabolism links dysbiosis to mucosal immune activation). Fecal tyramine strongly correlated with the GSVA score of mucosal Th17 cell differentiation (R=0.39, P <0.05) and NF-κB signaling pathway activity (R=0.43, P <0.01) (IDDF2026-ABS-0279 Figure 2(D) Tyramine metabolism links dysbiosis to mucosal immune activation). These metabolic-immune associations were independent of disease status, indicating that dysbiosis itself drives subclinical immune activation. Germ-free mice colonized with human-derived microbiota further validated that chronic aspartame exposure significantly disrupted microbial diversity in healthy microbiota-colonized mice (P <0.05). Dysbiotic microbiota-colonized mice exhibited reduced microbial diversity, enriched Enterococcus, and elevated levels of inflammatory markers compared to eubiotic counterparts (IDDF2026-ABS-0279 Figure 3. Chronic aspartame exposure drives disrupted microbial homeostasis and Enterococcus-associated inflammation in human microbiota-colonized mice).Conclusions We demonstrate that gut dysbiosis characterized by Enterococcus enrichment promotes mucosal immune activation through tyrDC-mediated tyramine production upon high aspartame intake. These findings establish gut microbiota as a key determinant of susceptibility to dietary exposures, providing a basis for personalized dietary recommendations based on microbial status.Abstract IDDF2026-ABS-0279 Figure 1Abstract IDDF2026-ABS-0279 Figure 2Abstract IDDF2026-ABS-0279 Figure 3