Intestinal epithelial innate response is differentially modulated by probiotic yeast and lactic acid bacteria.

MARTIN RUMBO; ROMANIN, DAVID; GONZÁLEZ MACIEL, DOLORES; HIRIART YANINA,; GARROTE, GRACIELA

Simposio; Second International Symposium Microbes for Health; 2011

There is evidence that commensal and probiotic bacteria can modulate innate response on intestinal epithelial cells. Different pathways have been proposed to explain this activity in different microbial strains. Kefir grains are a complex symbiotic association of yeasts and lactic acid bacteria and were used as source of potentially probiotic strains for this study. Our aim was to compare the capacity of lactic acid bacteria (LAB) and yeast strains to downregulate innate response in epithelial cell lines and comparatively analyze the mechanisms responsible of this activity. A panel of 30 yeast strains and 70 lactic adic bacteria was screened using Caco-2-CCL20-luc, a Caco-2 cell line stably transfected with a luciferase reporter construction under the control of CCL20 promotor. Several Saccharomyces and Kluyveromyces strains were found to inhibit more than 90% the activation of the reporter activity elicited by flagellin (1 ug/mL), whereas any of the lactid acid bacteria was able to promote an inhibition higher than 50% in the same assay. Selected strains of yeast (CIDCA 8154 of K marxianus) were selected for further studies. Results of the inhibitory capacity were confirmed at the specific mRNA level: yeast pretreatment inhibited completely the induction of CCL20, CXCL2 and IL-8 at the mRNA level upon flagellin (1ug/mL), TNFa (50 ng/mL) or IL-1b (10 ng/mL) stimulation, while the expression of other enterocyte function-associated genes (lactase phloryzin hidrolase, CDX2, villin) was unaffected. This activity was completely dependent on yeast viability and was not related to changes in pH of the culture medium along the experiment. Pre-treatment of epithelial cells with the selected strain downregulate NF-kB-dependent transcription in Caco-2 cells transiently transfected with a NF-kB reporter construction and p65 traslocation to the nucleus observed by fluorescence microscopy. The capacity to produce radical oxigen spieces (ROS) has been associated to the cellular pathways triggered by lactic acid bacteria to mediate their anti-inflammatory capacity. We screened the panel of microorganisms by their capacity to induce ROS response in epithelial cell lines using a ROS-sensitive fluorescent dye. The ROS inducing activity of the LAB tested correlated with their capacity to modulate epithelial innate activation, whereas any of the yeast strains tested was able to induce ROS, indicating that their capacity to modulate innate response was independent of this pathway. This study shows that yeast and LAB strains isolated from kefir differ in their capacity to inhibit the intestinal epithelial innate response and in the cellular pathways that they trigger to express this capacity. Yeast strains showed much higher downregulatory capacity. Increasing our knowledge on how the anti-inflammatory activity is triggered by each group of microorganisms may allow the formulation of specific microbial combination to contribute for the management of specific intestinal homeostatic disorders.