The integrity of the intestinal epithelial and blood-brain barriers is critical for maintaining homeostasis along the gut-brain axis as these interfaces regulate the passage of inflammatory mediators into systemic circulation and their access to the brain. Disruption of these barriers can lead to neuroinflammatory processes implicated in mood disturbances. Lacticaseibacillus rhamnosus HN001 has been associated with improved mood outcomes in clinical and preclinical studies, however the mechanistic basis for these effects is unclear. Here, we investigated whether HN001 enhances intestinal and blood-brain barrier function in a two-stage in vitro gut-brain axis model. Differentiated Caco-2 epithelial monolayers were co-cultured with HN001 (Stage-1; intestinal epithelial barrier), and the conditioned basal medium from this stage was subsequently applied to hCMEC/D3 endothelial monolayers (Stage-2; blood-brain barrier). HN001 increased transepithelial electrical resistance in Caco-2 monolayers under both aerobic and apical-anaerobic conditions. This was accompanied by transcriptional modulation of tight-junction signaling, including upregulation of OCLN and sealing claudins, and downregulation of the pore-forming claudin CLDN2. Conditioned basal medium collected from HN001-treated Caco-2 intestinal epithelial monolayers improved barrier resistance in hCMEC/D3 brain endothelial monolayers and mitigated IL-1β-induced barrier disruption. The protective effect was not accompanied by changes in IL-1β-stimulated secretion of IL-6, IL-8, MCP-1, TNF-α or IL-1β, indicating that HN001-derived signals regulate blood-brain barrier function independently of the cytokines measured here. Together, these findings indicate that HN001 supports both the intestinal barrier and the blood-brain barrier integrity within the gut-brain axis. By regulating these barrier interfaces, HN001 may reduce susceptibility to neuroinflammation, suggesting a potential mechanistic basis for its previously reported psychobiotic benefits.

The integrity of the intestinal epithelial and blood-brain barriers is critical for maintaining homeostasis along the gut-brain axis as these interfaces regulate the passage of inflammatory mediators into systemic circulation and their access to the brain. Disruption of these barriers can lead to neuroinflammatory processes implicated in mood disturbances. Lacticaseibacillus rhamnosus HN001 has been associated with improved mood outcomes in clinical and preclinical studies, however the mechanistic basis for these effects is unclear. Here, we investigated whether HN001 enhances intestinal and blood-brain barrier function in a two-stage in vitro gut-brain axis model. Differentiated Caco-2 epithelial monolayers were co-cultured with HN001 (Stage-1; intestinal epithelial barrier), and the conditioned basal medium from this stage was subsequently applied to hCMEC/D3 endothelial monolayers (Stage-2; blood-brain barrier). HN001 increased transepithelial electrical resistance in Caco-2 monolayers under both aerobic and apical-anaerobic conditions. This was accompanied by transcriptional modulation of tight-junction signaling, including upregulation of OCLN and sealing claudins, and downregulation of the pore-forming claudin CLDN2. Conditioned basal medium collected from HN001-treated Caco-2 intestinal epithelial monolayers improved barrier resistance in hCMEC/D3 brain endothelial monolayers and mitigated IL-1β-induced barrier disruption. The protective effect was not accompanied by changes in IL-1β-stimulated secretion of IL-6, IL-8, MCP-1, TNF-α or IL-1β, indicating that HN001-derived signals regulate blood-brain barrier function independently of the cytokines measured here. Together, these findings indicate that HN001 supports both the intestinal barrier and the blood-brain barrier integrity within the gut-brain axis. By regulating these barrier interfaces, HN001 may reduce susceptibility to neuroinflammation, suggesting a potential mechanistic basis for its previously reported psychobiotic benefits.

Supplementary data for the manuscript:Phimister FD, Anderson RC, Thomas DG, Farquhar MJ, Maclean P, Jauregui R, Young W, Butowski CF & . Bermingham EN (2024). Using Meta-analysis to Understand the Impacts of Dietary Protein and Fat Content on the Composition of Fecal Microbiota of Domestic Dogs (Canis lupus familiaris): A Pilot Study. MicrobiologyOpen DOI: 10.1002/mbo3.1404

Supplementary data for the manuscript:Phimister FD, Anderson RC, Thomas DG, Farquhar MJ, Maclean P, Jauregui R, Young W, Butowski CF & . Bermingham EN (2024). Using Meta-analysis to Understand the Impacts of Dietary Protein and Fat Content on the Composition of Fecal Microbiota of Domestic Dogs (Canis lupus familiaris): A Pilot Study. MicrobiologyOpen DOI: 10.1002/mbo3.1404