Effects of combined Lactococcus lactis and Escherichia coli Nissle 1917 cell-free supernatants on metabolic activity and inflammation-associated signaling in HT-29 cells.

Probiotic-derived secretory factors and metabolites have attracted considerable attention for their role in modulating host cellular responses. Here, we evaluated the effects of cell-free supernatants(CFS) derived from Lactococcus lactis and Escherichia coli Nissle 1917 (EcN), individually and in combination, on metabolic activity and selected inflammation-associated markers in the HT-29 colorectal cancer cell line. CFS were generated under different culture conditions, and mechanistic assays were performed using RPMI-derived preparations to minimize medium-related confounding effects. HT-29 cells were treated with the CFS for 24 and 48 h and metabolic activity and apoptosis were assessed using the MTT assay and Annexin V-FITC staining, respectively. Expression levels of TLR2, TLR4, IL-10, and IL-17 were analyzed using flow cytometry and qPCR, and nitric oxide (NO) production was quantified using the Griess assay. The combined CFS treatment resulted in a greater reduction in metabolic activity compared to individual treatments, without a significant increase in apoptosis. This effect was accompanied by reduced expression of TLR2, TLR4, and IL-17, along with an altered IL-10 response, and decreased NO production. These changes may reflect a shift in epithelial inflammatory signaling, as reduced TLR2 and TLR4 expression can attenuate activation of pro-inflammatory pathways such as NF-κB, while the observed changes in IL-17 and the context-dependent IL-10 response suggest broader modulation of inflammatory signaling in colorectal cancer-related contexts. However, these findings should be interpreted as preliminary observations of epithelial signaling modulation and warrant further mechanistic investigation.