Local and systemic immune mechanisms underlying the anti-colitis effects of the dairy bacterium Lactobacillus delbrueckii

Abstract

Several probiotic bacteria have been proposed for treatment or prevention of inflammatory bowel diseases (IBD), showing a protective effect in animal models of experimental colitis and for some of them also in human clinical trials. While most of these probiotic bacteria are isolated from the digestive tract, we recently reported that a Lactobacillus strain isolated from cheese, L. delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327), also possesses anti-inflammatory effects in vitro and in vivo, demonstrating that common dairy bacteria may be useful in the treatment or prevention of IBD. Here, we studied the mechanisms underlying the protective effects of Lb CNRZ327 in vivo, in a mouse dextran sodium sulfate (DSS) colitis model. During colitis, Lb CNRZ327 modulated the production of TGF-β, IL-6, and IL-12 in colonic tissue and of TGF-β and IL-6 in the spleen, and caused an expansion of CD4+Foxp3+ regulatory T cells in the cecal lymph nodes. Moreover, a strong tendency to CD4+Foxp3+ expansion was also observed in the spleen. The results of this study for the first time show that orally administered dairy lactobacilli can not only modulate mucosal but also systemic immune responses and constitute an effective treatment of IBD.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Effect of Lb CNRZ327 on body weight and DAI in DSS-induced colitis.

A. Body weight change from day 1 to day 8. Each bar represents the average body weight change ± SEM of the 5 mice in the group (one experiment, see text for further details) as a percentage of their initial weights. B. Disease activity index (DAI) on day 8. Bars represent the mean ± SEM calculated across three independent experiments, with 5 to 10 mice per group in each experiment (combined sample sizes: 17 to 20 mice per treatment). * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 2. Colon histology after DSS-induced colitis.

A. Descending colon of the negative control group (without bacteria or DSS). B. Descending colon of the positive group control (only DSS). C. Descending colon of the experimental group (DSS plus Lb CNRZ327). A, B, and C each show representative results from three independent experiments. D. Histological score of the groups described above. Bars represent the mean ± SEM calculated across three independent experiments, with 4 or 5 mice per group in each experiment (combined sample sizes: 13 to 14 mice per treatment). *** = p<0.001.

Figure 3. Secretory IgA levels after DSS-induced colitis.

Feces were collected and total sIgA was measured by ELISA. PBS, control without bacteria or DSS; DSS, DSS only, without bacteria; 327+DSS, DSS plus Lb CNRZ327. 100 (the average amount observed in the PBS control group) corresponds to the range of 61.5 to 126 pg/mL. Bars represent the mean ± SEM calculated across three independent experiments, with 3 to 10 mice per group in each experiment (combined sample sizes: 15 to 24 mice per treatment). ** = p

Figure 4. Cytokine production in colonic tissue and spleen after DSS-induced colitis.

Cytokine concentrations in homogenized tissues are presented as a percentage of the amount observed in the PBS control group (range from different experiments given in parentheses for each cytokine) which received the value of 100. Upper panel, colon: TGF-β (100 = 69 to 131.4 pg/mL), IL-4 (100 = 70.8 to 161.2 pg/mL), IL-6 (100 = 63.2 to 159.2 pg/mL), IL-10 (100 = 72.8 to 146.2 pg/mL), IL-12 (100 = 71.4 to 168.8 pg/mL), IL-17 (100 = 83.3 to 143.4 pg/mL). Lower panel, spleen: TGF-β (100 = 80.7 to 169.7 pg/mL), IL-6 (100 = 80.1 to 134 pg/mL), IL-10 (100 = 73.1 to 143.2 pg/mL), IL-12 (100 = 53.1 to 123 pg/mL), IL-17 (100 = 68.3 to 136.2 pg/mL). PBS, control without bacteria or DSS; DSS, DSS only, without bacteria; 327+DSS, DSS plus Lb CNRZ327.Bars represent the mean ± SEM calculated across two to six independent experiments, with 2 to 10 mice per group in each experiment (combined sample sizes: 9 to 33 mice per treatment (colon) and 5 to 12 mice per treatment (spleen)). * = p

Figure 5. T lymphocyte profiles in cecal lymph nodes and spleen after DSS-induced colitis.

T cells were isolated from cecal lymph nodes (CLN, upper panel) and spleen (lower panel) at the end of the experiment, and the frequencies of CD4+CD69+ and CD4+Foxp3+ T cells, as percentage of CD4+ T cells, were assessed by flow cytometry. T-cell frequencies are presented relative to the average frequency observed in the PBS control group (range from different experiments given in parentheses for each T cell type) which received the value of 100. Upper panel, CLN: CD4+CD69+ (100 = 2.7 to 3.54), CD4+FoxP3+ (100 = 3.41 to 5.41). Lower panel, spleen: CD4+CD69+ (100 = 4.5 to 4.51), CD4+FoxP3+ (100 = 1.59 to 4.02). PBS, control without bacteria or DSS; DSS, DSS only, without bacteria; 327+DSS, DSS plus Lb CNRZ327. For CD4+CD69+ T cells, bars represent the mean ± SEM of one experiment, with 2 or 3 mice in the PBS control groups and 5 mice in each of the other groups. For CD4+FoxP3+ T cells, bars represent the mean ± SEM calculated across two independent experiments, with 3 to 5 mice per group in each experiment (combined sample sizes: 8 to 10 mice per treatment (colon) and 8 mice per treatment (spleen)). ** = p<0.01; *** = p<0.001; • = p = 0.053.

Figure 6. Correlation between relative frequencies of CD4+FoxP3 T cells in CLN and spleen.

Each point represents one mouse, placed as a function of relative CD4+FoxP3 T cell frequencies in CLN and spleen (cf legend to Fig. 5). Black dots, PBS (control without bacteria or DSS); grey triangles, DSS (DSS only, without bacteria); light grey squares, 327+DSS (DSS plus Lb CNRZ327). The grey line describes the linear relationship between the frequencies of CD4+Foxp3+ cells in the spleen and in the colon (R2 = 0.415).