Polyphenolic Compounds Downregulate IL‐6 in Gut Epithelial Cells under Inflammatory Conditions

The majority of immune cells in the human body reside within gut‐associated lymphoid tissues. Gut epithelial cells serve as an important barrier between foreign substances in the lumen of the intestine and these immune cell populations. Polyphenolic compounds found in plant‐based foods are well known for their immune modulating properties; however, the bioavailability of these compounds can be limited, leaving their mode of action in question. This study examined the alterations in cytokine production in gut epithelial cells exposed to polyphenolic compounds extracted from a fruit rich in polyphenols (i.e., dried plums), under normal and inflammatory conditions. The MODE‐K epithelial cell line, which was derived from C3H/HeJ mice, was used in these studies. Cells were cultured in RPMI GlutMAX media, supplemented with 10% FBS, 1% sodium pyruvate, 1% non‐essential amino acids, 1% HEPES, and 0.1% β‐mercaptoethanol and incubated in 5% CO2/95% air‐humidified at 37° C. The polyphenol extract (DP‐PP) was prepared from dried plum powder (‘Improved French’) in 80% ethanol, followed by filtration and further purification using a Waters C‐18 column. The resulting extract was lyophilized, weighed, and re‐suspended in dimethyl‐sulfoxide (DMSO) and filtered for cell culture use. The dose‐dependent effects of DP‐PP (0, 2.5, 5, 10, 20, 40, & 80 μg/mL) and tumor necrosis factor‐alpha (TNF‐α; 0, 0.1, 1.0, & 10 ng/mL) on cell proliferation and viability were assessed over time using the methyl‐thiazolyldiphenyl‐tetrazolium (MTT) and the trypan blue exclusion assays. No significant decreases in cell growth and viability were observed with DP‐PP (0 to 40 ug/mL) and TNF‐α (0 to 1.0 ng/mL). For gene expression analyses, MODE‐K cells were plated at 1×106 cells/well. After 24 hrs the cells were treated with TNF‐α (0 or 1ng/mL) for 30‐min, followed by treatment with DP‐PP (0, 5, 10, or 20ug/mL). RNA was extracted at 2 and 24 hrs, and RT‐PCR was performed to assess gene expression of interleukin 6 (Il6) and transforming growth factor‐beta (tgfb) under normal and inflammatory conditions. The data were analyzed utilizing a factorial approach with TNF‐α and DP‐PP as factors. Main effects were observed with Il6 expression only, in that TNF‐α up‐regulated Il6 and DP‐PP attenuated this response. Under normal conditions, DP‐PP had no significant effect on Il6, but DP‐PP dose‐dependently suppressed (p<0.05) the TNF‐α‐induced increase in Il6 expression at 2 hrs. By 24 hrs, this response had normalized. DP‐PP did not affect the expression of Tgfb under normal or inflammatory conditions. Our findings indicate that DP‐PP can counteract a pro‐inflammatory state in the gut mucosa by down regulating IL‐6, which can have implications for intestinal health.