Assessment of barrier function and cell junctional expression on differentiated intestinal porcine epithelial cells in response to Salmonella LPS challenge and treatment with Yeast cell wall products

Abstract Mannan rich fractions were previously demonstrated to improve junctional gene expression which can reduce leaky intestinal tracts that facilitate bacterial infection in piglets. The work here assessed MRF and other yeast mannan products’ effects on intestinal barrier function in response to bacterial challenge from Salmonella LPS. Differentiated IPEC cells were grown to 14 days until a trans-epithelial electrical resistance reading (TEER) of ~ 4500 Ohms/cm2 was reached. Prior to lipopolysaccharide (LPS) (1µg/mL) challenge, cells were pre-treated and post-treated with MRF or yeast products A, B, and C (16mg/mL). Post treated cells were lysed in RLT buffer and RNA isolated (RNeasy). RIN values above 7 were used to synthesise cDNA (SuperScript®-III). Junctional genes Occludin, Claudin3 and Tight junction protein1 (TJP1) were assessed by qPCR (Applied Biosystems 7500 Fast). TNFα proinflammatory secretion was measured by ELISA. Three independent biological replicates were performed, with One-way ANOVA carried out unless stated otherwise. TEER results showed MRF significantly recovered barrier function (5090.3±187.0, P ≤ 0.05) over the positive control (PC) (3754.2±605.8) while product A (3502.5±182.7749), B (3414.289±733.8854) and C (3938.4±491.4) demonstrated no significant improvement to the PC. MRF treated cells were significantly higher for Claudin3 gene expression (1.33±0.18, P ≤ 0.05) over the control (0.671661±0.277) and treatments A (0.53±0.16, P ≤ 0.05), B (0.91±0.18, P ≤ 0.05) and C (0.69±0.25, P ≤ 0.05). Occludin expression was significantly higher in MRF treated cells (1.09±0.01, P ≤ 0.05) over the PC (0.89±0.09, P ≤ 0.05) and treatment B (0.88±0.04, P ≤ 0.05). TJP-1 gene expression was highest in the MRF treated cells (1.30±0.41) but not significantly, compared to the PC (1.00±0.14). TNFα (pg/mL) protein secretion was significantly lower in both the MRF treated (0.0809±0.86x10-3, P ≤ 0.05) and treatment C 0.081±0.18 x10-3, P ≤ 0.05) over the PC (0.0813±0.22x10-3). MRF augmented junctional expression improving TEER readings and potentially lessened LPS intracellular leakage that led to lower proinflammatory protein secretion. The present study highlights differences in efficacy of a variety of yeast cell wall products.