Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders

Abstract

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4(+)Foxp3(+) regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4(+)Foxp3(+) Tregs from the CD4(+)CD25(-) population and increased the suppressor activity of naturally occurring CD4(+)CD25(+) Tregs. Conversion of T cells into Foxp3(+) Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-beta, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4(+)Foxp3(+) Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.

Fig. 1.

Probiotics increase CD4⁺Foxp3⁺ Tregs. Foxp3 levels were analyzed by real-time PCR (A) or FACS (B) in SP and MLN CD4⁺ T cells from mice fed either PBS (Cont) or IRT5 (Pro) for 20 days. (C) Levels of Foxp3⁺ cells were analyzed in CD4⁺CD25⁺ and CD4⁺CD25⁻ populations. (D) Expression levels of Treg-associated molecules were compared in MLN CD4⁺CD25⁺ Tregs from each group. GrznB, Granzyme B. (E) Suppressor activity of CD4⁺CD25⁺ Tregs was measured by T-cell proliferation assay. Tregs from each treatment group (Cont or Pro) were cocultured with responder cells (CD4⁺CD25⁻) at various ratios of Treg/responder cells. The proliferation level of responder cells alone was assigned a value of 100%. Data are the average of three independent experiments (10 mice per group); error bars indicate SD. *P < 0.05; **P < 0.005; ***P < 0.001.

Fig. 2.

Probiotics induce hyporesponsiveness in CD4 T cells. (A) Levels of cytokine expression in CD4⁺ T cells were analyzed by real-time PCR. Cytokine expression in spleen CD4⁺ T cells of Cont group mice was set at 100%. (B) Proliferation of CD4⁺ T cells from the Cont or Pro group mice was measured after stimulation with anti-CD3/anti-CD28. Data are from 10 mice per group; error bars indicate SD. Data are representative of three independent experiments. **P < 0.005; ***P < 0.001.

Fig. 3.

Probiotics induce CD11c⁺ rDCs. (A) CD11c⁺ DCs isolated from MLN of Cont or Pro group mice were stimulated with PMA/ionomycin. The levels of immunoregulatory molecules were measured by real-time PCR. (B) CD11c⁺ DCs from each group were cocultured with Do11.10 CD4⁺ T cells labeled with CFSE and ovalbumin peptide (0.3 μg/mL) in the absence (−) or presence (+) of exogenous TGF-β. (C) Levels of Foxp3⁺CD25⁻ and Foxp3⁺CD25⁺ populations were measured by FACS. (D) MLN CD11c⁺ DCs from IRT5-fed mice were cocultured with CD4⁺ T cells in the presence of ovalbumin peptide and inhibitors for rDCs. w/o, without. After coculture, alteration of the Foxp3⁺ population was measured by FACS. Data are from 10 mice per group and are the average of three independent experiments.

Fig. 4.

Probiotics suppress experimental colitis. (A) Changes in gross intestines were evaluated 3 days after induction of colitis. (B) Histological analysis after H&E staining to detect lymphocyte infiltration. (C) Real-time PCR was performed to analyze changes in the expression of proinflammatory cytokines in response to probiotics administration. Data are from 20 mice per group; error bars indicate SD. Data are representative of three independent experiments. **P < 0.005; ***P < 0.001.

Fig. 5.

Probiotics suppress experimental AD. The therapeutical efficacy of IRT5 was measured by the following criteria for AD progression: clinical score (A), lymphocyte infiltration by histological analysis (B), and total IgE levels (C). w/o, without. Bars are from 10 mice per group; error bars indicate SD. Data are representative of three independent experiments. **P < 0.005; ***P < 0.001.

Fig. 6.

Enriched CD4⁺Foxp3⁺ Tregs at inflammatory sites are associated with disease suppression. The Foxp3⁺ population from the colon of experimental colitis (A) or ear of AD (B) mice was analyzed by FACS or IHC (C) between the Pro and Cont groups. The arrows in C indicate Foxp3⁺ cells. A control reaction was performed with isotype-matched IgG antibodies (data not shown). Data are from 10 mice per group and represent the average of three independent experiments.

Fig. 7.

Increased chemoattractant expression leads to enrichment of Tregs in the inflamed region. The expression levels of chemokines in MHC class II-positive antigen-presenting cells (A) and their receptors in CD4⁺ T cells (B) were analyzed by quantitative real-time PCR. (C) CFSE-labeled T cells obtained from MLN of Cont and Pro groups were adoptively transferred to AD mice. The migration of CFSE⁺(CD4⁺)Foxp3⁺ Tregs to the ear of AD mice was analyzed by IHC. The arrows indicate CFSE⁺(CD4⁺)Foxp3⁺ Tregs. Data are from 10 mice per group; error bars indicate SD. Data are representative of three independent experiments. *P < 0.05; **P < 0.005; ***P < 0.001.