Anti-inflammatory effects of probiotic yogurt in inflammatory bowel disease patients

Abstract

Our aim was to assess anti-inflammatory effects on the peripheral blood of subjects with inflammatory bowel disease (IBD) who consumed probiotic yogurt for 1 month. We studied 20 healthy controls and 20 subjects with IBD, 15 of whom had Crohn's disease and five with ulcerative colitis. All the subjects consumed Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 supplemented yogurt for 30 days. The presence of putative regulatory T (T(reg)) cells (CD4(+) CD25(high)) and cytokines in T cells, monocytes and dendritic cells (DC) was determined by flow cytometry from peripheral blood before and after treatment, with or without ex vivo stimulation. Serum and faecal cytokine concentrations were determined by enzyme-linked immunosorbent assays. The proportion of CD4(+) CD25(high) T cells increased significantly (P = 0.007) in IBD patients, mean (95% confidence interval: CI) 0.84% (95% CI 0.55-1.12) before and 1.25% (95% CI 0.97-1.54) after treatment, but non-significantly in controls. The basal proportion of tumour necrosis factor (TNF)-alpha(+)/interleukin (IL)-12(+) monocytes and myeloid DC decreased in both subject groups, but of stimulated cells only in IBD patients. Also serum IL-12 concentrations and proportions of IL-2(+) and CD69(+) T cells from stimulated cells decreased in IBD patients. The increase in CD4(+) CD25(high) T cells correlated with the decrease in the percentage of TNF-alpha- or IL-12-producing monocytes and DC. The effect of the probiotic yogurt was confirmed by a follow-up study in which subjects consumed the yogurt without the probiotic organisms. Probiotic yogurt intake was associated with significant anti-inflammatory effects that paralleled the expansion of peripheral pool of putative T(reg) cells in IBD patients and with few effects in controls.

Fig. 1

Identification of monocytes and dendritic cells (DC) by flow cytometry. A representative example of the identification of monocytes based on the expression of (a) CD33 and (b) CD14 antigen. (c) Identification of DC as a human leucocyte antigen D-related (HLA-DR)⁺ lineage^– (CD3^–, CD56^–, CD14^–, CD19^–) population. (d) After acquiring a higher number of cells within the HLA-DR⁺ lineage^– live gate, three different dendritic cell subsets were identified on the basis of CD33 expression: myeloid CD33^high, CD33^intermed and plasmocytoid CD33^–/low.

Fig. 2

(a) A representative example of the analysis of CD4⁺ CD25⁺ cells by flow cytometer. Lymphocytes were gated according to their particular small forward-/side-scatter profile and the CD4⁺ CD25^high and CD4⁺ CD25⁺ cells identified based on the concentration of CD25 expression. (b–e) Percentage of CD4⁺ CD25^high and CD4⁺ CD25⁺ cells in peripheral blood before (0 day) and after (30 days) probiotic yogurt treatment in inflammatory bowel disease (IBD) patients and controls. Data are shown as box plots with median and 10th, 25th, 75th and 90th percentiles. *Significant (P < 0.05) increase in CD4⁺ CD25⁺ and CD4⁺ CD25^high cells in IBD patients following treatment. #The basal (0 day) percentage of CD4⁺ CD25⁺ cells was significantly higher in IBD patients than in controls (P = 0.04).

Fig. 3

The percentage of CD3⁺ CD69⁺ T cells in peripheral blood before (day 0) and after (day 30) probiotic yogurt treatment in inflammatory bowel disease (IBD) patients and controls with (+) or without (–) ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. The horizontal bars represent mean values.

Fig. 4

Correlation between the changes in the percentage of putative regulatory T cells (T_reg, CD4⁺ CD25^high) and in the percentage of tumour necrosis factor (TNF)-α- or interleukin (IL)-12-producing dendritic cells (DC) and monocytes (MC) in peripheral blood of inflammatory bowel disease (IBD) patients following treatment with the probiotic yogurt.

Fig. 5

Suppressive effect of cell-free extracts (CFE) of Lactobacillus reuteri RC-14 and L. rhamnosus GR-1 on the in vitro proliferative responses of peripheral blood mononuclear cells (PBMC). PBMC obtained from five healthy controls were cultured with (+) or without (–) PMA, ionomycin and CFE. Results are expressed as mean optical density (OD) at 575 nm +s.d., with higher OD corresponding to higher proliferation rate.

Fig. 6

Comparison of fold changes in the numbers of regulatory T cells (T_reg, CD4⁺ CD25^high), activated T cells (CD4⁺ CD25⁺) and tumour necrosis factor (TNF)-α- and interleukin (IL)-12-producing monocytes (MC) and dendritic cells (DC) in inflammatory bowel disease (IBD) patients following treatment with probiotic yogurt or unsupplemented yogurt. Individuals are indicated by connective lines. *Change following treatment with probiotic yogurt significantly different from change following treatment with unsupplemented yogurt (P < 0.05).