P043 Secretome modulation of Caco-2 cell line induced by a multi-strain probiotic

Abstract Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing 4 strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus and L. delbrueckii subsp. bulgaricus*), 3 strains of Bifidobacteria (B. longum**, B. infantis***, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. *Recently reclassified as L. helveticus. **Recently reclassified as B. longum subsp. lactis. ***Recently reclassified as B. infantis subsp. lactis. The aim of the present study was to study secreted bioactive factors to evaluate the mechanisms of action of VSL#3 to enhance intestinal epithelia. Methods Two different lots of VSL#3 ( Nutrilinea Srl, Gallarate (VA), Italy, lot #802097 and lot #802100) were used. Caco-2 cell line were treated with a conditioning media (CM) prepared using 1 g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. Caco-2 were treated with diluted CM at 1:10 and 1:25 for 24 and 48 h. Media culture for each conditions has been collected and analysed by a deeper proteomics approach. Differential protein expression in was evaluated by shotgun proteomics analysis based on nLC-HDMSE and carried out on Synapt G2-Si mass spectrometer. Protein identification and protein expression analysis were perfomed by Protein Linx Global Server (PLGS v. 3.0.3, Waters Corp). Results The analysis of supernatants from Caco-2, treated with CM, showed the presence of bacteria strain-specific proteins. Human proteins synthesised from CaCo-2 were also identified, such as caspase 1, IL8, HSP70, HSP70b, HSP90, HSP105. The production were time- and dose- dependent. In CM diluted 1:10, probiotic derived proteins have been shown to be more expressed at 24 h. Human caspase 1, IL8, HSP 70, HSP 70b, HSP 90, HSP 105 were also found up-regulated in CaCo-2 treated for 24 h with CM diluted 1:10. Conclusions This is the first time where a probiotic secretome was explored. The study on probiotic secretome is useful to understand if the probiotic was well reconstituted. Analysis of secretome from CaCo-2 treated with CM helped us to understand the mechanism by probiotics can enhance intestinal barrier: by strengthen the autophagy process, an arm of innate immunity, by overexpression of caspase 1, IL8 and HSP 70, and by HSPs dependent modulation of inflammation.