The Impacts of Yeast Cell Wall Mannan Rich Fraction on Porcine Intestinal Barrier Function Following Exposure to Salmonella LPS.

The epithelial cells of the gut form a monolayer that covers the intestine and has a primary role in protecting against pathogenic challenges, they have developed different mechanisms to reduce the risk of infection such as barrier function. In swine, Salmonella infections can result in disruption of the barrier functioning of the intestinal epithelium. Yeast mannan structurally resemble the receptor sites coating the intestinal epithelium to which intestinal pathogens like salmonella adhere thus preventing infection. The objective of this study was to determine if a commercial mannan rich fraction (MRF, Actigen) extracted from yeast can aid in maintenance of the intestinal epithelial barrier. Porcine intestinal epithelial cells (IPEC J2) between passages 2-18 were seeded into collagen-coated Transwells (Costar, 1.12 cm²) at a density of 2 X 10⁵ cells/well, triplicate wells for control and MRF treatment. Differentiation of monolayers was observed after 9 days in vitro as determined by measuring trans electrical resistance (TEER analysis) when a TEER reading of 20,000 ohm/m² or greater was achieved. TEER was measured daily using an epithelial volthommeter, ohm/m². On day 14 of the differentiation, cells were treated with a 1/10 dilution of MRF which was subjected to an in vitro simulated porcine digestion method, where 0.5g of MRF was digested in a volume of 14mL. On day 15 of the differentiation, cells were exposed to LPS from Salmonella (1ug/mL), membrane integrity was monitored by measuring TEER. Day 16 cells were harvested from the transwells, RNA was extracted for gene expression analysis. Control and MRF treated cells saw a drop in TEER readings following LPS exposure; 30,000 & 41,000 ohm/m² down to 9,100 & 9,000 ohm/m² respectively. The MRF treated cells recovered their TEER reading 20,500 ohm/m² following exposure to LPS (p< 0.05) where as no recovery was observed in the control cells 8,700 ohm/m². Gene expression analysis of genes associated with membrane integrity and barrier function e.g. tight junction’s protein gene and occludin were also measured. Tight junction protein gene expression levels were increased three-fold in MRF treated cells compared to control cells exposed to LPS (p< 0.05), the expression level of Occludin was also 1.5 times greater than that of the control cells. Tight junction proteins are known to play a pivotal role in barrier function; the increase in their expression following MRF supplementation together with the TEER recovery may indicate that MRF can positively impact the intestinal epithelium barrier.