Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis

Abstract

Background: Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis.

Methods: In the present study, we analysed the immunomodulatory effects of three L. lactis strains in vitro using intestinal epithelial cells. L. lactis NCDO 2118 was administered for 4 days to C57BL/6 mice during the remission period of colitis induced by dextran sodium sulphate (DSS).

Results: Only one strain, L. lactis NCDO 2118, was able to reduce IL-1β-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect. Oral treatment using L. lactis NCDO 2118 resulted in a milder form of recurrent colitis than that observed in control diseased mice. This protective effect was not attributable to changes in secretory IgA (sIgA); however, NCDO 2118 administration was associated with an early increase in IL-6 production and sustained IL-10 production in colonic tissue. Mice fed L. lactis NCDO 2118 had an increased number of regulatory CD4(+) T cells (Tregs) bearing surface TGF-β in its latent form (Latency-associated peptide-LAP) in the mesenteric lymph nodes and spleen.

Conclusions: Here, we identified a new probiotic strain with a potential role in the treatment of IBD, and we elucidated some of the mechanisms underlying its anti-inflammatory effect.

Keywords: Colitis; Cytokines; Lactococcus lactis; Probiotics; Regulatory T cells.

Figure 1

IL-8 levels after co-incubation of L. lactis strains with Caco-2 cells. (A)L. lactis cells. (B)L. lactis supernatant. Dash, without addition of IL-1β or bacteria; IL-1β, only IL-1β was added; GM17, only the medium was added. Bars represent the mean and the MSE of three independent experiments. *, p < 0.05.

Figure 2

Oral administration of L. lactis NCDO 2118 improved colon shortening and macroscopic score of colitis. (A) Experimental protocol. C57BL/6 mice received 2% DSS for 7 days. L. lactis NCDO 2118 was continually administered for 4 consecutive days during the remission period of colitis (arrows) between the first and second course of colitis. The control group received medium. Mice were sacrificed at days 14 and 21 (arrowheads). (B) Body weight from day 0 to day 21. (C) Colon length measured in cm. (D) Macroscopic score of colitis, including scores related to body weight, diarrhea and rectal bleeding. Bars are the mean of 6 mice/group, and the data are representative of three independent experiments; ANOVA, Tukey post-test. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 3

Oral administration of L. lactis NCDO 2118 prevented histological damage induced by colitis. Photograph (X100) of H&E-stained paraffin sections of a representative colon from control (A), DSS (B) and DSS + NCDO2118 (C) groups at day 21. (D) Histological scores of colon sections of DSS-colitis mice with or without oral administration of L. lactis. Values represent the means ± MSE (n = 6). **, p < 0.01, ***, p < 0.001.

Figure 4

Oral administration of L. lactis NCDO 2118 did not alter secretory IgA production. (A) Intestinal faeces were collected and total sIgA was measured by ELISA in mice from control, DSS and DSS + NCDO2118 groups. (B) Intestinal faeces from healthy mice were collected after 2, 3 or 4 days of L. lactis administration, and total sIgA was measured by ELISA. Bars represent the mean ± MSE of 5 mice per group. **, p < 0.01.

Figure 5

Effect of L. lactis NCDO 2118 on cytokine production by colonic cells. Colonic IL-6 (A), IL-12 (B), IFN-γ (C), IL-17 (D), TGF-β (E), IL-10 (F) and TNF-α (G) were measured by ELISA in mice from control, DSS and DSS + NCDO2118 groups. One representative result from two independent repetitions is shown. Bars represent the mean ± MSE of 5 mice per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 6

Effect of L. lactis NCDO 2118 on T cells and tolerogenic dendritic cells. The numbers of activated T cells, regulatory T cells and tolerogenic dendritic cells in chemically induced colitis were determined in the mesenteric lymph nodes (mLNs) and spleen; the cells were stained at day 21. (A) Number of CD4 + CD69+ T cells. (B) CD4 + CD25 + CD45RB^low T cells. (C) CD4 + CD25 + LAP + T cells. (D) CD11c + CD11b-CD103+ cells. Bars are the mean of 5 mice/group, and the data are representative of two independent experiments; ANOVA, Tukey post-test. **, p < 0.01, ***, p < 0.001.

Figure 7

Schematic model for the immunomodulatory effects of Lactococcus lactis NCDO 2118 in DSS-induced colitis. Orally administered Lactococcus lactis NCDO 2118 is able to induce an early increase in IL-6 production and to sustain IL-10 secretion in colonic tissue of the dextran sulphate sodium (DSS)-induced murine model of ulcerative colitis. It also increases the number of local tolerogenic dendritic cells (CD11c⁺CD11b^-CD103⁺). These cells migrate to the mesenteric lymph nodes and stimulate the expansion of CD4⁺CD25⁺LAP⁺ cells, a regulatory type of T cell (Treg), leading ultimately to downmodulation of colitis.