LACTOBACILLUS SPP. PROTECTS THE BARRIER FUNCTIONALITY OF CACO-2 CELLS EXPOSED TO INFLAMMATORY STIMULI
Alarcón P., Mellado J., Cofré J., González M., Aguayo M., Castro E.
Facultad de medicina, Instituto de políticas públicas en salud IPSUSS, Universidad San Sebastián, Concepción, Chile
Lactobacillus bacteria are able to make contact with the immune system of the mucosae. A loss of the normal and homeostatic interaction between Lactobacillus spp. and gut cells can lead to develop several diseases, ranging from dysbiosis to chronic inflammation, and even to intestinal cancer. The Caco-2 cell line of human colon adenocarcinoma is a model for study of enterocytes. These cells differentiate into enterocytes after 15 days, and show the same morphological and functional characteristics than those obtained from humans. This study aims to determine the effects of bacterial lysates, named soluble protein homogenates (SPHs), obtained from the probiotic strains Lactobacillus gasseri LPV31 and LPV22, (extracted from healthy women’s vaginal microbiota) and Lactobacillus salivarius LPLM-O1, (extracted from healthy breast milk) in Caco-2 cells that have been exposed to inflammatory stress.
The Lactobacillus strains were subjected to mechanical disruption in order to obtain the SPH, resulting in 22 protein bands for strains LPV31 and LPV22, and 27 bands for LPLM-O1 after a 10% SDS-PAGE analysis. Three Caco-2 cell cultures (400,000/well) were differentiated to enterocytes Group 1 was stimulated with Salmonella tiphymurium lipopolysaccharide (LPS) (100 ng mL-1) for 21 hours. After this, the transepithelial electric resistance (TEER) was measured. Group 2 was stimulated with 100 ng mL-1 of LPS for 21 hours and then washed with 10 mM PBS (pH = 7.3); after that, 150 μg mL-1 of SPH of the three probiotic strains were added to three different wells in triplicate. For Group 3, Caco-2 cells were stimulated with 150 μg mL-1 of each bacterial SPH in three different wells, and 100 ng mL-1 of LPS were applied after washing.
The results showed for G1 TEER value of the cells descended by 20% in respect to cells without LPS. For G2 all three strains reduced TEER values recorded after adding the LPS. For Group 3, SPH obtained from LPLM-O1 increased TEER values over base levels, showing a better performance than the other strains. When stimulating with LPS, there are no statistically significant differences in the increase of TEER by SPHs. Also, there was no production of IL-1β, TNF-α, IL-6, and IL-10.
In Group 1, IL-8 passed its basal level, and in Group 2, it went significantly over the basal level (p<0.05) after stimulating with SPH of LPLM-O1, and in Group 3, the SPHs of both LPV31 and LPV22 reduced it, but the SPH of LPLM-O1 increased it significantly (p<0.05). When restimulating Groups 2 and 3 with LPS, IL-8 concentration is similar to basal levels.
In conclusion, it is proposed that the SPHs of probiotic strains L. gasseri LPV31 and LPV22, and of L. salivarius LPLM-O1 have a different protective effect on barrier function on Caco-2 cells that have been exposed to inflammatory stress.
Keywords: Probiotic, Inflammatory Stimuli, Lactobacillus, Caco-2 cell, Bacterial lysates
Alarcón P., et al. (2016). Lactobacillus Spp. protects the barrier funcionality of Caco-2 cells exposed to inflammatory stimuli. Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 83.). IPC2016