Kefir-isolated bacteria and yeasts inhibit <i>Shigella flexneri</i> invasion and modulate pro-inflammatory response on intestinal epithelial cells

Abstract

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against <i>Shigella flexneri</i> invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in <i>S. flexneri</i> strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains <i>Saccharomyces cerevisiae</i> CIDCA 8112 or <i>Lactococcus lactis</i> subsp. <i>lactis</i> CIDCA 8221 also reduced the internalisation of <i>S. flexneri</i> into HT-29 cells although in a lesser extent than MM. Interestingly, <i>Lactobacillus plantarum</i> CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by <i>S. flexneri</i>. Regarding the pro-inflammatory response on HT-29 cells, <i>S. flexneri</i> infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of <i>S. flexneri</i>-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain <i>Kluyveromyces marxianus</i> CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of <i>S. flexneri</i> internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.