Mechanism of action of Lactobacillus reuteri ATCC PTA 6475 in colon epithelial cells

Lactobacillus reuteri ATCC PTA 6475 (LR) is a well‐known probiotic that has been shown to be beneficial to the gastrointestinal (GI) system. In addition, LR has been shown to secrete beneficial immunomodulatory factors. In previous studies, we demonstrated that LR can modulate inflammatory genes in the GI tract in mice in vivo. However, the mechanisms of action of LR in the GI system during inflammatory conditions are still poorly understood. In the present study, we investigated the potential role of LR on tumor necrosis factor a (TNFa)‐induced colon epithelial cell biology, including expression of inflammatory genes. For this, we pre‐treated colon epithelial cells (SW480) with live LR, LR conditioned medium (CM), CM fractions (ranging from < 3, 3–10, 10–30, 30–50, 50–100 and >100kD) and heat inactivated CM. Subsequently, we treated the cells with TNFa (10ng/ml) for 6 or 24 hours and examined expression of inflammatory genes. We found that live LR induced IL‐8 mRNA expression in a dose dependent manner. We further identified that the less than 3KD fraction of the CM produced by LR is sufficient to induce IL‐8 at both the mRNA and proteins levels. While we observed similar effects on MIP2a, TNFa gene expression was not regulated by LR. We further found that the factors that induce IL‐8 expression are heat labile. These effects on gene expression were also observed in other intestinal epithelial cell lines including HT29 and CaCo2. The effect on gene expression was not accompanied by cell proliferation. Preliminary examination of signaling pathway activation indicated that LR‐CM small factors do not regulate IL‐8 production by activation of the MAPK or NF‐κB pathways, suggesting other mechanisms. Together, our results confirm that live LR and its secreted factors induce inflammatory genes in epithelial cells that could potentially affect intestinal inflammation without affecting cell growth.