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The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance to the commensal microbiota and food antigens. Different strains of probiotics possess the ability to finely regulate the activation of dendritic cells (DC), polarising the subsequent activity of T-cells. Nevertheless, information about their underlying mechanisms of action is scarce. In the present study, we investigated the immunomodulatory effects of a potentially probiotic strain,Lactobacillus rhamnosusCNCM I-4036, and its cell-free culture supernatant (CFS) on human DC challenged withEscherichia coli. The results showed that the levels of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8 and IL-12p70 were higher in the cells treated with liveL. rhamnosusthan in the cells treated with the CFS. In the presence ofE. coli, the supernatant was more effective than the probiotic bacteria in reducing the secretion of pro-inflammatory cytokines. In addition, liveL. rhamnosuspotently induced the production of transforming growth factor (TGF)-β1 and TGF-β2, whereas the CFS increased the secretion of TGF-β1. However, in the presence ofE. coli, both treatments restored the levels of TGF-β. The probiotic strainL. rhamnosusCNCM I-4036 and its CFS were able to activate the Toll-like receptor signalling pathway, enhancing innate immunity. The two treatments induced gene transcription ofTLR-9. LiveL. rhamnosusactivated the expression ofTLR-2andTLR-4genes, whereas the CFS increased the expression ofTLR-1andTLR-5genes. In response to the stimulation with probiotic/CFS andE. coli, the expression of each gene tested was notably increased, with the exception ofTNF-αandNFKBIA. In conclusion, the CFS exhibited an extraordinary ability to suppress the production of pro-inflammatory cytokines by DC, and may be used as an effective and safer alternative to live bacteria.

Numerousin vitroandin vivostudies conducted using different probiotic micro-organisms have demonstrated their ability to interfere with the growth and virulence of a variety of enteropathogens. The reported beneficial effects of the use of probiotics to complement antibiotic therapy or prevent diarrhoea or gastrointestinal infection in infants have increased in recent years. In the present study, we demonstrated the capacity of supernatants obtained from three novel probiotics (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) isolated from the faeces of breastfed infants to inhibit the growth of enterotoxigenic and enteropathogenic (EPEC) bacteria, such asEscherichia coli,SalmonellaandShigella. To assess their potential antimicrobial activity, the 17 and 24 h cell-free supernatants broth concentrates (10 × ) having 1, 2 or 4 % of the three probiotics were incubated with EPEC bacteria strains. After 17 h of co-culture, the supernatants were able to inhibit the growth ofE. coli,SalmonellaandShigellaup to 40, 55 and 81 %, respectively. However, the inhibitory capacity of some supernatants was maintained or completely lost when the supernatants (pH 3·0) were neutralised (pH 6·5). Overall, these results demonstrated thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 produce compounds that exhibited strain-specific inhibition of enterobacteria and have the potential to be used as probiotics in functional foods.