Beneficial bacteria can influence host physiology through the production of bioactive compounds released during growth. In this study, we investigated the effects of cell‑free supernatants (CFS)—the secreted products of selected Bacillus strains—using canine epithelial cell and macrophage models in vitro. By focusing on bacterial CFS rather than live cells, the study aimed to examine mechanistic host cell responses under controlled experimental conditions. Exposure of epithelial cells to Bacillus‑derived CFS altered the expression of tight junction–associated and immune‑related genes, indicating modulation of cellular pathways involved in epithelial organization and immune signaling. In parallel, CFS treatment influenced macrophage function, including changes in phagocytic activity and immune responsiveness, as well as cell viability. Metabolomic analysis identified N‑lactoyl‑phenylalanine (Lac‑Phe), which showed significant changes following incubation with epithelial cells and may be relevant to cellular stress and inflammatory signaling pathways. Together, these findings provide mechanistic insight into how soluble factors produced by Bacillus strains can modulate epithelial and innate immune cell responses in vitro. The study supports the use of cell‑based models to explore host–microbe interactions mediated by bacterial CFS.

Read the full article in the Journal of Animal Science.