Canine Epithelial and Immune Cell Models Reveal Stress-Mediated Barrier and Immune Modulation by Limosilactobacillus reuteri ATCC PTA 6127 under Lipopolysaccharide Challenge

Coordinated responses of intestinal epithelial and immune cells are essential for maintaining barrier integrity and immune homeostasis in dogs, yet mechanistic understanding of probiotic‑derived metabolites remains limited due to reliance on non‑canine experimental models. Here, we investigated metabolites derived from Limosilactobacillus reuteri strain ATCC PTA6127 (Lr6127), delivered as cell-free supernatants (CFS), using canine epithelial MCA-B1 cells and macrophage-like DH82 cells subjected to lipopolysaccharide (LPS)–induced inflammatory stress. Lr6127 CFS significantly reduced epithelial permeability, decreasing FITC‑dextran leakage to 94.9 ± 1.9% relative to LPS-treated controls normalized to 100% (P < 0.001), despite no detectable transcriptional changes in tight junction, adherent junction, or mucin genes. Barrier effects were instead associated with changes in markers of cellular stress re-sponses, with heme oxygenase expression reduced from 0.9 ± 0.1 to 0.7 ± 0.1 (P < 0.05). In DH82 immune cells, Lr6127-derived metabolites suppressed LPS-induced stress and inflammatory signaling, enhanced anti-apoptotic responses as reflected by increased BCL2 expression (1.4 ± 0.1 vs. 1.0 ± 0.0; P < 0.01) and elevated BCL2/BAX ratios (P < 0.01), and reduced pro-inflammatory mediators including IL-6 and CCL2 (P < 0.05–0.001). Proteomic analysis corroborated reduced abundance of inflammatory and STAT-associated signaling proteins under LPS challenge while promoting immune readiness under resting conditions. Collectively, these results suggest that Lr6127‑derived metabolites may support epithelial barrier integrity and immune re-balancing potentially through modulation of cellular stress and inflammatory path-ways.