Researchers exposed HT-29 cells to 1% dextran sulfate sodium, or DSS, to create an inflammatory epithelial model, and then treated the cells with 50% L. brevis cell-free supernatant for 48 hours. Viability was assessed with an MTT assay before the transcriptional analysis. The investigators profiled the expression of 84 genes involved in Toll-like receptor, or TLR, signaling using the RT² Profiler PCR Array. Differential expression was evaluated with the ΔΔCt method, and raw p-values were adjusted for multiple testing using the Benjamini–Hochberg false-discovery-rate procedure.

The most prominent response was the significant downregulation of multiple genes associated with inflammation and innate immune signaling. These included the receptors TLR2 and TLR4; the adaptor proteins MYD88 and TIRAP; the kinases IRAK1, IRAK4 and MAP3K7; and the transcription factor NFKB1. Proinflammatory cytokine genes IL1A, IL1B, IL6 and IL12A also declined, as did the chemokine CCL2 and the cyclooxygenase gene PTGS2. At the pathway level, the affected genes mapped to the MyD88-dependent and TRIF-dependent branches of TLR signaling.

Host defense-associated genes responded differently from the broadly downregulated inflammatory set. Components associated with the TRIF-dependent antiviral axis—including TLR3, TICAM1, TBK1, IRF3 and CXCL10—were also significantly downregulated. In contrast, TLR1, TLR10, TICAM2 and BTK were upregulated. Several other examined immune-response genes showed no significant change. The mix of increased, decreased and unchanged transcripts indicates that the supernatant altered specific parts of the TLR-associated gene network rather than indiscriminately shutting down every immune-related pathway.

The findings provide a cell-level view of how secreted L. brevis metabolites may influence inflammatory signaling in intestinal epithelial cells. They are consistent with attenuation of multiple inflammation-associated transcriptional responses under DSS-induced stress, while also showing that changes in host defense-related genes are context dependent. The experiment was conducted in one human intestinal epithelial cell line exposed to an inflammatory stimulus, and the results concern gene expression rather than clinical outcomes in people. The study therefore does not establish that the supernatant prevents or treats inflammatory bowel disease, nor does it define which secreted components or molecular mechanisms produced the selective effects. Its principal conclusion is limited to the observed modulation of TLR-associated transcription in DSS-stimulated HT-29 cells.

Subject of Research: Transcriptional effects of Lactobacillus brevis cell-free supernatant on TLR-associated genes in DSS-stimulated intestinal epithelial cells

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

Article References: Moghanlou, A., Rasouli, S., Asadi Lanjavani, S., Falsafi, S., & Behrouzi, A. (2026). Lactobacillus brevis cell-free supernatant attenuates inflammation-associated gene expression and differentially modulates host defense-associated genes in DSS-stimulated HT-29 cells. Molecular Biology Reports, 53(1), Article 1764. https://doi.org/10.1007/s11033-026-12890-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12890-9

Keywords: Lactobacillus brevis, cell-free supernatant, HT-29 cells, intestinal inflammation, Toll-like receptors, gene expression, innate immunity, dextran sulfate sodium, molecular biology, Lactobacillus, brevis, cell-free