Strain-Specific Effects of Bacillus velezensis Cell-Free Supernatants on Canine Epithelial and Immune Cell Responses

The probiotic potential of Bacillus velezensis is increasingly recognized in companion animal nutrition; however, the strain-specific mechanisms underlying host responses remain poorly understood. Here, we investigated metabolite-driven effects of three B. velezensis strains (LSSA01, 15AP4, and 2084) using cell-free supernatants (CFS) to decouple host responses from bacterial colonization. All findings are derived from in vitro models using cell-free supernatants (CFS), enabling assessment of metabolite-mediated effects under controlled conditions. Canine proximal epithelial (MCA-B1) cells and macrophage-like (DH82) cells were used to assess epithelial barrier regulation, cellular stress and apoptosis pathways, immune signaling, and innate effector function. LSSA01 CFS significantly enhanced transcription of epithelial junction and resilience markers, including claudin-1 (6.2-fold, p < 0.0001), ZO-1 (1.4-fold, p < 0.01), and BCL-2 (1.4-fold, p < 0.01), accompanied by an increased BCL-2/BAX ratio (p < 0.05), suggesting strengthened barrier function and enhanced expression of markers associated with cellular resilience. In contrast, 15AP4 and 2084 primarily modulated protein-level junctional organization and stress-response pathways and significantly enhanced macrophage phagocytic activity under inflammatory challenge (170-286% of control, p < 0.01-0.0001). Untargeted metabolomics revealed distinct strain-specific signatures, including marked enrichment of nucleotides such as adenosine monophosphate (31-39-fold vs. control, p < 0.0001), alongside vitamins and bioactive peptides. Together, these findings indicate that B. velezensis strains exhibit complementary, non-redundant metabolite-mediated activities on canine epithelial and immune cells, highlighting the importance of strain-resolved evaluation for mechanistic understanding and supporting future investigation, while acknowledging that in vivo validation will be important to confirm relevance for canine health applications.