Porcine intestinal barrier adaptive responses to hyperosmolarity differ in relation to the in vitro environment and epithelial cell differentiation

Background The gastrointestinal tract is crucial for absorption and defence against pathogens due to a dynamic epithelium. During weaning, the pig intestine is sensitive to environmental, infectious, and osmotic stressors, which alter tight junctions (TJ), thus increasing epithelial permeability and the risk of dysfunction. The objective of the study was to investigate the intestinal epithelial barrier (IEB) response in the porcine IPEC-J2 cell line under two conditions: (1) non-stabilized monolayers and (2) stabilized monolayers, exposed to osmolarity changes. IPEC-J2 were treated for 4 h, 24 h, and 4 days with a hyperosmolar medium (500 mOsm/L) obtained by the addition of sucrose- or mannitol- supplemented media. The assessment of epithelial monolayer integrity by TEER, cell viability, nitric oxide (NO) production, and antioxidant capacity was evaluated together with the expression and localization of TJ proteins CLDN4, OCLN, and ZO-1, expression of osmolyte transporters TauT, BGT1, and SMIT, pro-inflammatory cytokines IL1α, IL8, and TNFα and intracellular signaling mediators TRIF and NF-κB. Results In IPEC-J2 non-stabilized monolayers, sucrose medium promoted functional adaptation over time (4 days), induced cell viability, and expression of the osmolyte transporters BGT1 and SMIT. In contrast, mannitol medium reduced TEER and cell viability, altered cell morphology, and impaired the ability to maintain barrier permeability due to the loss of TJp. In stabilized monolayers, sucrose medium stimulated TJp reorganization and the activation of antioxidant responses, while mannitol medium, despite increasing osmolyte transporter expression, triggered TRIF and NF-κB inflammatory expression, resulting in increased permeability and reduced TJp expression. Conclusions The results demonstrate that the IPEC-J2 barrier adaptive responses to hyperosmolar environmental stress are related to the type of osmolyte inducing the stress condition and the cell differentiation status.