Bacillus cell‑free supernatants support canine epithelial barrier function and stress adaptation through...

Disruption of epithelial barrier function and inflammation compromise canine health and wellbeing, particularly in the gastrointestinal tract. Bacillus spp. produce bioactive components, here investigated as cell‑free supernatant (CFS), that can modulate epithelial barrier‑associated pathways. We evaluated the effects of CFS from 3 probiotic Bacillus strains on biomarkers of barrier integrity, the immune response and survival of canine proximal epithelial (MCA-B1) cells, and on the phagocytic activity of canine DH82 macrophages. CFS from Bacillus licheniformis strains BA842 and BL-21 and Bacillus velezensis strain 27 (equivalent to 1 × 107 colony-forming units [CFU]/mL per well) was applied to cells and incubated overnight. Tight junction (TJ) expression and adhesion protein molecule abundance were assessed by RT-qPCR and proteomics, respectively; cytokines and apoptosis markers were analyzed under LPS-stimulated and unstimulated conditions; metabolite abundance in CFS following incubation with MCA-B1 cells was assessed using metabolomic profiling, and phagocytic activity was determined using a fluorescein-labeled Escherichia coli uptake assay. The CFS from BA842 and BL-21 increased the expression of CLDN1 (by 2.4 ± 0.2-fold and 2.5 ± 0.2-fold, respectively, vs. control; P < 0.001), and CLDN4 (by 1.9 ± 0.2-fold and 2.3 ± 0.2-fold, respectively; P < 0.01); BA842 CFS also increased ZO-1 and E-cadherin expression (P < 0.01). The cytokine expression profile of CFS-treated cells shifted towards a more balanced phenotype; BA842- and BL-21-CFS increased IL-8 expression (6.9 and 8.5-fold vs. control, respectively; P < 0.001), and BL-21 also increased IL-12A expression (1.6-fold vs. control, respectively; P < 0.01). In addition, BA842-CFS increased BCL2 expression and BCL2L1 abundance (1.5-fold and 2.0-fold vs. control; P < 0.05) and BL-21- and 27-CFS reduced caspase-8 expression (P < 0.05). Macrophage phagocytosis increased after exposure to BA842 CFS (+194% vs. control; P < 0.01). Untargeted metabolomics identified increased Lac-Phe after 24 h in CFS-treated MCA-B1 cultures but not in tryptic soy agar, suggesting that this metabolite is associated with epithelial cell interaction rather than being a component of the growth medium. In conclusion, Bacillus-derived CFS modulated expression of TJ proteins, adhesion molecules, cytokines, and markers of apoptosis in canine epithelial cells and enhanced macrophage phagocytic activity, linked to defined metabolomic responses, providing mechanistic insight to support epithelial barrier‑relevant functions associated with canine health.