Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells

Abstract

Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-β, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4(+)CD25(+) T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases.

Figure 1

Quantification of IL-8 secreted by HT-29 in culture supernatant. HT-29 cells were treated with Lp62 and S. Typhi. Levels of IL-8 secreted into the culture medium were measured. Unstimulated cultures or cultures stimulated only with Lp62 or S. Typhi i were used as controls. S: inoculated simultaneously; HI: heat-inactivated.  ^aSignificant difference from the medium (without any stimulation).  ^bSignificant difference from S. Typhi-stimulated group.  ^cSignificant difference from Lp62/S. Typhi; P < 0.05.

Figure 2

Percentage of S. Typhi adherence to HT-29 cells. HT-29 cells were treated with Lp62, and then S. Typhi was added to the culture. After incubation, the probiotic ability to inhibit pathogen binding to the epithelial cell was measured. The percentage of S. Typhi adherence was calculated in relation to the initial inoculum 1 × 10⁸. S: simultaneously inoculated; HI: heat-inactivated.  ^∗Significant difference in relation to S. Typhi-stimulated group (P < 0.05).

Figure 3

TLR-4 expression in HT-29 cells. HT-29 cells were stimulated with Lp62 and then challenged with S. Typhi. In parallel, the effect of simultaneous (S) addition of the two microorganisms was tested. HT-29 cells were labeled internally with anti-TLR-4 and analyzed by flow cytometry.  ^aStatistically different from the medium (unstimulated cell).  ^bStatistically different from the S. Typhi-stimulated group; P < 0.05.

Figure 4

J774 macrophages stimulated with Lp62 and/or LPS: J774 macrophages were stimulated with LPS and Lp62 for 2 hours. The levels of IL-1, TNF-α, and IL-17 were measured in culture supernatant by ELISA ((a), (b), and (c), resp.). The CD86 expression was analyzed by flow cytometry (d).  ^ASignificant difference compared to unstimulated cells (culture medium).  ^BSignificant difference from the control stimulated with LPS only; P < 0.05.

Figure 5

CD4⁺CD25⁺ T lymphocytes and IL-10 secretion in PBMC treated with Lp62. Cultures of peripheral blood mononuclear cells were challenged with LPS and Lp62. The proportion of CD4⁺CD25⁺ cells was determined by flow cytometry (a). IL-10 production was examined in the culture supernatant by ELISA (b).  ^AStatistical difference compared to the control without stimulation.  ^BStatistical difference from the control only stimulated with LPS; P < 0.05.