Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells.

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

N-(3-oxododecanoyl)-l-homoserine lactone modulates mitochondrial function and suppresses proliferation in intestinal goblet cells.

AIMS: The quorum-sensing molecule N­(3­oxododecanoyl)­l­homoserine lactone (C12-HSL), produced by the Gram negative human pathogenic bacterium Pseudomonas aeruginosa, modulates mammalian cell behavior. Our previous findings suggested that C12-HSL rapidly decreases viability and induces apoptosis in LS174T goblet cells. MAIN METHODS: In this study, the effects of 100 µM C12-HSL on mitochondrial function and cell proliferation in LS174T cells treated for 4 h were evaluated by real-time PCR, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. KEY FINDINGS: The results showed that the activities of mitochondrial respiratory chain complexes IV and V were significantly increased (P < 0.05) in LS174T cells after C12-HSL treatment, with elevated intracellular ATP generation (P < 0.05). Flow cytometry analysis revealed significantly increased intracellular Ca2+ levels (P < 0.05), as well as disrupted mitochondrial activity and cell cycle arrest upon C12-HSL treatment. Apoptosis and cell proliferation related genes showed markedly altered expression levels (P < 0.05) in LS174T cells after C12-HSL treatment. Moreover, the paraoxonase 2 (PON2) inhibitor TQ416 (1 µM) remarkably reversed the above C12-HSL associated effects in LS174T cells. SIGNIFICANCE: These findings indicated that C12-HSL alters mitochondrial energy production and function, and inhibits cell proliferation in LS174T cells, with PON2 involvement.

Galacto-oligosaccharides may directly enhance intestinal barrier function through the modulation of goblet cells.

SCOPE: Here we have tested the hypothesis that prebiotic galacto-oligosaccharides (GOS) may enhance mucosal barrier function through direct modulation of goblet cell function. METHODS AND RESULTS: Human adenocarcinoma-derived LS174T cells, which exhibit an intestinal goblet cell-like phenotype, were used to examine the non-prebiotic effects of GOS on goblet cell functions. LS174T cells were treated with GOS, and the expression of goblet cell secretory product genes mucin 2 (MUC2), trefoil factor 3 (TFF3), resistin-like molecule beta (RETNLB) and the Golgi-sulfotransferase genes, carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), was determined by real-time quantitative RT-PCR. In addition, the abundance of CHST5, TFF3 and RETNLB was confirmed by Western blot analysis. Following treatment with GOS for 72 h, the expression of MUC2 was significantly upregulated 2-4-fold, CHST5 and RETNLB, 5-7-fold, and TFF3 2-4-fold. Western blot analysis demonstrated increased abundance of RETNLB, TFF3 and CHST5. Addition of the Th2 cytokine IL-13 along with GOS resulted in synergistic induction of RETNLB and CHST5. IL-8 secretion was not affected by GOS treatment, suggesting that the effects of GOS are not mediated through an inflammatory pathway. CONCLUSION: Collectively, the data indicate that GOS may enhance mucosal barrier function through direct stimulation of intestinal goblet cells.