Lactobacillus brevis cell-free supernatant attenuates inflammation-associated gene expression and differentially modulates host defense-associated genes in DSS-stimulated HT-29 cells.

Abstract

Background: Dysregulated Toll-like receptor (TLR) signaling in intestinal epithelial cells contributes to persistent mucosal inflammation in inflammatory bowel disease (IBD). Although Lactobacillus brevis has demonstrated anti-inflammatory potential, the transcriptional effects of its secreted metabolites on inflammatory and host defense-associated components of epithelial innate immune signaling remain insufficiently characterized. This study investigated the effects of L. brevis cell-free supernatant (CFS) on TLR-associated gene expression in DSS-stimulated HT-29 cells.

Methods: 1% DSS stimulated HT-29 human intestinal epithelial cells (inflammatory epithelial model) were treated with 50% L. brevis CFS (48 h) following viability assessment using MTT assay. Transcriptional profiling of 84 key genes involved in the TLR signaling pathway was performed using the RT² Profiler™ PCR Array. Differential gene expression was analyzed using the ΔΔCt method, followed by pathway-level mapping of the MyD88- and TRIF-dependent branches. Raw p-values were adjusted for multiple comparisons using the Benjamini-Hochberg FDR procedure.

Results: Treatment with L. brevis CFS was associated with significant downregulation of multiple inflammation-associated genes, including TLR2, TLR4, MYD88, TIRAP, IRAK1, IRAK4, MAP3K7, NFKB1, IL1A, IL1B, IL6, IL12A, CCL2, and PTGS2. Host defense-associated genes exhibited a non-uniform transcriptional response. Components of the TRIF-dependent antiviral axis, including TLR3, TICAM1, TBK1, IRF3, and CXCL10, were downregulated, whereas TLR1, TLR10, TICAM2, and BTK were upregulated, and several other immune-response genes showed no significant change. These findings indicate differential modulation of TLR-associated transcription rather than uniform suppression of all examined immune-related genes.

Conclusions: L. brevis CFS attenuated the expression of multiple inflammation-associated genes and differentially modulated host defense-associated genes in DSS-stimulated HT-29 cells. This non-uniform transcriptional pattern indicates selective modulation of the TLR-associated gene network rather than indiscriminate suppression of all examined immune-related genes.