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

V. Petito*1, V. Greco2,3, L. Laterza4, C. Graziani4, L. Lopetuso1,4, F. Scaldaferri1,4, A. Urbani2,3, A. Gasbarrini1,4

1Universita' Cattolica del Sacro Cuore, Institute of Medical Pathology, Rome, Italy, 2Università cattolica del Sacro Cuore, Institute of Biochemistry and Clinical Biochemistry, Rome, Italy, 3Fondazione Policlinico A. Gemelli IRCCS, Department of Laboratory Diagnostic and Infectious Diseases, Rome, Italy, 4Fondazione Policlinico Universitario Gemelli IRCCS, Gastroenterological Area, Gastroenterological, Endocrino-Metabolical and Nefro-urological Sciences Department, Rome, Italy

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.

Conclusion

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.