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Induction of Regulatory Dendritic Cells by Lactobacillus paracasei L9 Prevents Allergic Sensitization to Bovine β-Lactoglobulin in Mice
Induction of Regulatory Dendritic Cells by Lactobacillus paracasei L9 Prevents Allergic Sensitization to Bovine β-Lactoglobulin in Mice
Journal of Microbiology and Biotechnology. 2015. Oct, 25(10): 1687-1696
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : March 09, 2015
  • Accepted : June 18, 2015
  • Published : October 28, 2015
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About the Authors
Jing Yang
The Innovation Centre of Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
Fazheng Ren
The Innovation Centre of Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
renfazheng@cau.edu.cn
Hao Zhang
The Innovation Centre of Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
Lu Jiang
The Innovation Centre of Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
Yanling Hao
The Innovation Centre of Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
Xugang Luo
Mineral Nutrition Research Division, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China

Abstract
Supplementation with probiotics can protect against the development of food allergies by modulating the host immune response; however, the mechanisms are not fully understood. The objective of this study was to investigate the allergy-reducing effects of regulatory dendritic cells (regDCs) induced by Lactobacillus paracasei L9 (L9) in β-lactoglobulin (BLG)-sensitized mice. The L9 supplement suppressed the aberrant balance of Th1/Th2 responses to BLG in mice, via upregulation of the CD4+CD25+Foxp3+Treg cell responses. The amount of CD4+CD25+Foxp3+Treg cells in mesenteric lymph nodes increased by 51.85%. Furthermore, administration of L9 significantly induced the expression of CD103 and reduced the maturation status of DCs in mesenteric lymph nodes, Peyer’s patches, and spleen. Bone marrow-derived dendritic cells (BM-DCs) were activated by L9 in vitro , with an approximate 1.31-fold and 19.57-fold increase in expression of CD103 in CD11c+DCs and the level of IL-10 production, respectively, while the expression of CD86 did not change significantly. These data demonstrate that L9 reduced the BLG allergic sensitization, likely through regDCs mediated active suppression.
Keywords
Introduction
Food allergies are a growing health concern that can lead to serious health complications [21] . Cow’s milk allergy (CMA) is one of the earliest and most prevalent food allergies and bovine β-lactoglobulin (BLG) is the major allergen [5 , 17] . Diets based on cow’s milk play a major role in children’s nutrition, and therefore, it is important to effectively prevent and manage this food allergy.
A number of approaches have been proposed for preventing food allergies, including allergen avoidance, allergen reactivity reduction, and immune modulation. At present, the only proven therapy is a strict elimination diet [21] . For common foods, such as cow’s milk or hen’s eggs, absolute avoidance is difficult in everyday life [33] . Many modern processing techniques are used for reducing the antigenicity of food proteins by changing the structure of allergens, such as thermal treatment, glycation, enzymatic hydrolysis, and fermentation. However, allergen reactivity cannot be completely eliminated [29] . More recently, modulation of the host’s immune responses to allergens seems an ideal approach for preventing food allergies, since food allergies are characterized by activation of Thelper type 2 (Th2) T lymphocytes and reduction of T regulatory (Treg) cell activity [26] . The generation of Treg cells can suppress allergen-induced proliferative and cytokine responses in both Th1 and Th2 lymphocytes [2] . Because of this, specific allergen immunotherapy (SIT) is most effectively used for the treatment of food allergies, which results in the generation of Treg cells [3] . Dendritic cells (DCs), the most professional antigen-presenting cells, have a crucial role in the control of the adaptive immune responses by T cell activation. Different subsets of DCs have been described in the intestine of mice based on the expression of CD11c, CD11b, mPDCA, CD80, CD86, MHCII, and CD103 [30 , 31] . Regulatory dendritic cells (regDCs) that express high levels of IL-10, TGF-β, and indoleamine-2,3-dioxygenase (IDO) directly mediate the conversion of T cells into Foxp3+ Treg cells and induce immune tolerance [22] . There are two subsets of regDCs; one is immature DCs or semi-mature DCs that express reduced levels of MHCII and co-stimulatory molecules, whereas the other is CD11c+CD103+ DCs that express retinaldehyde dehydrogenase (RALDH) to metabolize vitamin A into retinoic acid [16 , 22] . Studies in mouse models indicated that oral sensitization with allergens was accompanied by a shift in intestinal DC subsets towards less CD103+ DCs [31] and this allergic response was alleviated with increased percentages of CD103+ DCs and CD4+CD25+Foxp3+ Treg cells in the mesenteric lymph nodes [26] . However, the mechanism that controls the differentiation and maturation of DCs is not well understood.
Previous studies have indicated that probiotics play crucial roles in preventing food allergies, including hydrolysis of antigenic peptides, modulation of intestinal permeability to reduce systemic penetration of antigens, as well as stimulation of epithelial cell growth and differentiation [4] . Furthermore, it is now widely accepted that probiotics, such as Lactobacillus rhamnosus GG (LGG), which has been shown to be beneficial for treating pediatric allergic disorders, is important for optimal immune homeostasis of the host [18] . It has been indicated that oral probiotics supplementation can shift Th2-dominated trends to Th1-dominated responses in murine allergy models [12 , 23] . Bacterial strain-specific induction of Foxp3+ Treg cells has also been demonstrated to be protective in respiratory and food allergy models [11 , 25] . Additionally, many probiotics can modulate the expression of cytokines and maturation surface markers in murine DCs, which are required for activation of antigen-specific T cells [37] . Lactobacillus paracasei inhibits Th2-dependent allergic responses to ovalbumin via enhancing PD-L2 expression on dendritic cells [14] , but the probiotic-associated influence of regDCs on food allergy in vivo is very poorly understood.
Lactobacillus paracasei L9 (L9) is a commensal bacterial strain originally isolated from the feces of healthy centenarians. L9 has been shown to be effective in the prevention of constipation and ulcerative colitis through regulation of chemokine and cytokine expression [36 , 38] ; however, to the best of our knowledge, the protective effect of L9 on allergies has not yet been investigated. In the current study, the effects of L9 on the modulation of intestinal and systemic antigen-specific immune responses to BLG were examined.
Materials and Methods
- Bacterial Growth
L9 (the name Lactobacillus casei LC-14 had been used before) was grown in MRS medium, supplemented with ʟ-cysteine hydrochloride (0.5 g/l), anaerobically, at 37℃. The bacteria were incubated overnight and the bacterial concentration and viability were then determined by densitometry and by CFU counts after agar plating. Bacteria were collected by centrifugation at 3,000 × g for 10 min and suspended in aseptic PBS. For in vitro experiments, L9 was adjusted to 10 10 CFU/ml in PBS and heat treated at 95℃ for 25 min, and then stored at -80℃ until use.
- Animals and Experimental Design
Three-week-old female BALB/c mice were purchased from Charles River Breeding Laboratories (Beijing, China). Mice were housed in stainless steel cages at 22-24℃ on a 12L:12D schedule under specific-pathogen-free conditions. Animals were fed a plantbased chow diet that contained no animal proteins or microbes (Charles River Breeding Laboratories). The Animal Care Committee of China Agricultural University approved these studies.
Mice were divided into three groups ( n = 8/group): sham sensitized with PBS as placebo control (CON), BLG-sensitized negative control (BLG), and L9-supplemented BLG (L9). Mice were sensitized intragastrically with BLG (1 mg/g BW; Sigma-Aldrich, St. Louis, MO, USA) plus cholera toxin (CT, 0.3 μg/g BW; Sigma-Aldrich) used as an adjuvant in a total volume of 200 μl of PBS, and administered at weekly intervals for 5 consecutive weeks. L9 was supplemented daily via gavage to L9 mice (2 × 10 9 CFU in 200 μl of PBS). At 5 weeks after the first oral gavage, all mice were fasted overnight and challenged with a high dose of BLG (50 mg/mouse) intragastrically (Fig. S1). Mice were euthanized 60 min after the last dose of BLG, blood was collected, and the spleen, mesenteric lymph node (MLN), and Peyer’s patches (PP) were removed under sterile conditions and placed in RPMI-1640 medium (Gibco, Grand Island, NY, USA).
- Lymphocytes CultureIn Vitro
The preparation of lymphocytes isolated from the spleen, MLN and PP were performed and cultured as previously described [11] . MLN and PP were scraped on metal grids and passed through 40 mm nylon cell strainers (BD Falcon, Franklin Lakes, NJ, USA). Splenocytes were collected by flushing PBS into spleens with a 1 ml syringe, and the red blood cells in splenocytes were treated with red blood cell lysis buffer (Beyotime, Nantong, China) and washed with PBS three times. Mononuclear cells, adjusted to 5 × 10 6 cells/ml, were stimulated with BLG (20 μg/ml) or media alone in 96-well plates in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM ʟ-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were cultured in a humidified incubator at 37℃ with 5% CO 2 for 60 h.
- Preparation and Activation of Bone Marrow-Derived DCs
Mouse bone marrow-derived DCs (BM-DCs) were prepared as previously described [28] , with minor modifications. Briefly, the bone marrow precursors were isolated from the femurs and tibias of mice. Cells were cultured at 5 × 10 5 /ml in culture medium with Recombinant Murine GM-CSF (20 ng/ml; Peprotech, Rocky Hill, NJ, USA) and Recombinant Murine IL-4 (10 ng/ml; Peprotech). Fresh medium was added on days 3 and 6 and BM-DCs were used on day 8. BM-DCs (10 6 cells/ml) were stimulated with BLG (20 μg/ml), in the culture medium containing heat-inactivated L9 (10 6 or 10 7 CFU/ml) for 24 h. Lipopolysaccharide (LPS; Sigma-Aldrich) at 1 μg/ml was used as the positive control.
- DC-CD4+T Cell Co-Cultures
After stimulation by L9 (BM-DCs: bacteria = 1:10) and LPS, BMDCs were harvested and pretreated with mitomycin C (25 μg/ml; Sigma-Aldrich) for 1 h to prevent proliferation. BM-DCs (10 5 cells/ml) were then cultured for 5 days with splenic CD4+ T cells (10 6 cells/ml), isolated by magnetic bead sorting (CD4+T cell isolation kit; Miltenyi, Bergisch Gladbach, Germany) from BLG mice, in the presence of BLG (20 μg/ml).
- Determination of Cytokine Levels and Immunoglobulins
Blood samples were centrifuged (3,500 × g ) for 10 min at 4℃ and serum was collected and frozen at -80℃. Serum histamine, mouse mast cell protease-1 (mMCP-1), and cytokines (IL-4, IL-10, IL-12p70, IFN-γ, and TGF-β) in the serum or culture supernatants were determined using commercial ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA; eBioscience, Vienna, Austria) following the manufacturer’s recommendations. Total serum IgE levels were measured by ELISA, according to the manufacturer's instructions (ICL Lab, Portland, OR, USA).
Levels of BLG-specific IgE, IgG, IgG1, and IgG2a were measured as previously described [28] , with minor modifications. Briefly, 96-well plates were coated with 20 μg/ml of BLG in coating buffer (0.5 M Na-bicarbonate/carbonate, pH 9.6). Serum samples were diluted 1:50 for IgE, 1:500 for IgG1 and IgG2a, and 1:1,000 for IgG, prior to being used in the assays. All other procedures, such as washing, blocking, and incubation, were done as previously described [1] .
- Flow-Cytometry Analysis
Single-cell suspensions of spleens, MLN, PP, or DCs-CD4+T cell co-cultures were preincubated with FcR blocking reagent (Miltenyi) before being stained with a Foxp3 Staining Buffer Set (eBioscience, Auburn, NY, USA). DCs were labeled with monoclonal antibodies for CD11c (FITC), CD103 (PE), MHCII (PerCP-eFluor710), CD80 (APC), or CD86 (PE-Cyanine7) (eBioscience). Cells were analyzed using a FACS Calibur flow cytometer (Becton-Dickinson), and FlowJo 7.6.1 software (TreeStar, Ashland, OR, USA) was used to analyze the data.
- Statistical Analysis
Statistical analysis of the differences among groups was evaluated with one-way ANOVA followed by Duncan’s multiplecomparison test using SPSS software (ver. 19.0; SPSS Inc., Chicago, IL, USA). Significant differences were established at the level of p < 0.05. Data are expressed as means ± standard deviations (SD).
Results
- L. paracaseiL9 Consumption Alleviated Allergic Sensitization of BLG
The most severe reactions were observed in BLG mice and significantly reduced by L9 supplementation (Fig. S2). L9 treatment decreased the BLG-induced histamine and mMCP-1 production ( Table 1 ), and reduced the serum total IgE level by 27.61%, compared with BLG mice ( Fig. 1 A). In addition, L9 down-regulated the serum level of BLG-specific IgE, IgG, and IgG1, but did not significantly change IgG2a ( Fig. 1 B). These results confirm L9 treatment prevents allergic sensitization of BLG.
Influence ofL. paracaseiL9 on cytokine levels in serum.
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Results are expressed as the mean ± SD for each group (n = 8 animals per group). Values with different superscript letters are significantly different (p < 0.05). CON: PBS, control mice; BLG: BLG + CT; L9: BLG + mice supplemented with L9. L9, Lactobacillus paracasei L9; BLG, β-lactoglobulin; CT, cholera toxin.
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Influence of L. paracasei L9 on immunoglobulin levels in serum. Serum from all mice was collected after euthanasia. (A) The level of total IgE in serum. (B) The levels of BLG-specific IgE, IgG, IgG1, and IgG2a in serum. Results are expressed as the mean ± SD for each group (n = 8 animals per group). The values with different superscript letters are significantly different (p < 0.05). CON: PBS, control mice; BLG: BLG + CT; L9: BLG mice supplemented with L9. L9, Lactobacillus paracasei L9; BLG, β-lactoglobulin; CT, cholera toxin.
- L. paracaseiL9 Supplement Induced CD4+CD25+Foxp3+ Treg Cells to Balance the Disorder of Th1/Th2
Food allergies are characterized by activation of Th2 lymphocytes and reduction of Treg cell activity. The numbers of CD4+CD25+Foxp3+ Treg cells in lymphocytes from the PP, MLN, and spleen of L9 mice were also increased by 27.68%, 51.85%, and 17.50%, respectively, with significantly decreased IL-4 and increased IFN- γ production in lymphocytes ( Figs. 2 A- 2 C). As shown in Table 1 , the BLG induced production of both Th1 and Th2 related cytokines, IL-12p70 and IL-4 were suppressed, and production of the Th1-related cytokine, IFN- γ , was increased in serum from L9 mice compared with BLG mice. In addition, L9 also significantly increased the levels of the regulatory cytokines TGF-β and IL-10 in serum or culture supernatants ( Figs. 2 D- 2 E). All these results suggest L9 is effective in inducing CD4+CD25+Foxp3+ Treg cellmediated tolerance.
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Influence of L. paracasei L9 on the number of CD4+CD25+Foxp3+ Treg cells and cytokine production. Lymphocytes isolated from PP, MLN, and spleen from all groups were determined with flow cytometric analysis. (A) Typical plots depicting percentage of CD25+Foxp3+ within CD4+ cells of lymphocytes isolated from PP, MLN, and spleen from all groups. (B-E) Levels of TGF-β, IL-10, IL-4, and IFN-γ in culture supernatants of lymphocytes from PP, MLN, and spleen were cultured with 20 μg/ml of BLG for 60 h. Results are expressed as the mean ± SD for each group (n = 4-5 animals per group). Values with different superscript letters are significantly different (p < 0.05). CON: PBS, control mice; BLG: BLG + CT; L9: BLG mice supplemented with L9. PP, Peyer’s patches; MLN, mesenteric lymph nodes; L9, Lactobacillus paracasei L9; BLG, β-lactoglobulin; CT, cholera toxin.
- L. paracaseiL9 Induced regDC and Regulatory Cytokine Production
To evaluate the possible involvement of regDCs in L9-induced hyporesponsiveness, the expression of CD103 and maturation status of DCs were analyzed. The percentages of CD103+DCs in CD11c+DCs were much lower in the PP, MLN, and spleen of BLG-sensitized mice than in CON mice and were significantly increased in L9 mice ( Fig. 3 B). There were significant decreased percentages of CD80+DCs, CD86+DCs, and MHC-II+DCs in the PP and MLN by L9 consumption, compared with BLG mice ( Figs. 3 C- 3 E). Similar results were observed in BM-DCs stimulated by L9 in vitro , with about 1.31-fold increase of CD103+DCs with the enhanced mRNA expression of RALDH2 and IDO (Fig. S3) compared with the negative control, and the expression of CD86 was not significantly changed. However, the levels of MHC-II and CD80 were increased by L9, but lower than the effect of LPS stimulation ( Figs. 4 A- 4 E).
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Influence of L. paracasei L9 on CD103 expression and maturation status of DCs in vivo. Lymphocytes isolated from PP, MLN, and spleen from all groups were determined with flow cytometric analysis. (A) Lymphocytes were analyzed by flow cytometry for CD103, CD80, CD86, and MHC-II expression in CD11c+ DCs, and CD11c-FITC was used to gate DCs (grey zone, CON; dotted line, BLG; solid line, L9). (B-E) The percentage of CD103+ DCs, CD80+ DCs, CD86+ DCs, and MHC-II+ DCs in CD11c+ DCs. Results are expressed as the mean ± SD for each group (n = 4-5 animals per group). Values within PP, MLN, or spleen with different superscript letters are significantly different (p < 0.05). CON: PBS, control mice; BLG: BLG + CT; L9: BLG mice supplemented with L9. PP, Peyer’s patches; MLN, mesenteric lymph nodes; L9, Lactobacillus paracasei L9; BLG, β-lactoglobulin; CT, cholera toxin.
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Influence of L. paracasei L9 on CD103 expression, maturation status of DCs, and cytokine production in vitro. BM-DCs from naive BALB/c mice were stimulated with BLG (20 μg/ml), and cultured with medium alone (CON), ultra-pure lipopolysaccharide from E. coli (LPS, 1 μg/ml), or Lactobacillus paracasei L9 (L9, BM-DCs: bacteria = 1:1 or 1:10) for 24 h. (A) BM-DCs were analyzed by flow cytometry for CD103, CD80, CD86, and MHC-II expression in CD11c+ DCs, and CD11c-FITC was used to gate DCs (grey zone, CON; dotted line, LPS; solid line, L9). (B-E) The percentage of CD103+ DCs, CD80+ DCs, CD86+ DCs, and MHC-II+ DCs in CD11c+ DCs at ratio of 1:10 (BM-DCs: bacteria). (F-H) Production of IL-10, TGF-β, and IL-12p70 in culture supernatant was determined. Results of three independently performed experiments are shown as the mean ± SD. Values with different superscript letters are significantly different (p < 0.05). L9 +, BM-DCs: bacteria = 1:1; L9 ++, BMDCs: bacteria = 1:10.
Furthermore, stimulation of BM-DCs by L9 increased regulatory cytokine production dose dependently, such as a 19.57-fold increase in IL-10 and a 1.85-fold increase in TGF-β production at the ratio of 1/10 (BM-DCs: bacteria), similar to LPS stimulation ( Figs. 4 F- 4 H). These results suggest L9 induces the expression of CD103 and low maturation status in DCs.
- L. paracaseiL9-Stimulated DCs Induced Foxp3 Expression
L9-stimulated DCs were incubated with CD4+ T cells from BLG mice in order to investigate the effect of L9-stimulated DCs in inducing Foxp3 expression. As shown in Fig. 5 A, Foxp3 expression was 54.27% increased, and there was significantly increased IL-10, TGF-β, and IFN- γ secretion but decreased IL-4 production in L9-stimulated BM-DCs and CD4+ T cell co-cultures, compared with CD4+ T cells incubated with unstimulated DCs ( Figs. 5 B- 5 E). These results confirm L9-stimulated DCs induce Foxp3 expression and shift the Th1/Th2 imbalance.
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Influence of L. paracasei L9-stimulated BM-DCs on Foxp3 expression in CD4+T cells and cytokine production in vitro. BM-DCs from naive BALB/c mice were stimulated with BLG (20 μg/ml), and cultured with medium alone (CON), ultra-pure lipopolysaccharide from E. coli (LPS, 1 μg/ml), or Lactobacillus paracasei L9 (L9, BM-DCs: bacteria = 1:10) for 24 h, harvested, pretreated with mitomycin C (25 μg/ml), and then washed and reincubated with CD4+ T cells from BLG mice for 5 days, in the presence of BLG (20 μg/ml). (A) The percentage of CD25+Foxp3+ within CD4+ cells. (B-E) Production of IL-10, TGF-β, and IL-12p70 in culture supernatant. Results of three independently performed experiments are shown as the mean ± SD. Values with different superscript letters are significantly different (p < 0.05).
Discussion
The present study demonstrated the protective effects of L9 on allergic inflammation in BLG-sensitized mice and investigated whether microbial induction of regDCs was associated with this protection. L9 supplementation in mice was found to reduce BLG-induced sensitization, to significantly reduce hypersensitivity scores, and to reduce histamine, mMCP-1, and total or BLG-specific serum IgE concentrations compared with BLG mice. In addition, L9 balanced the BLG-induced disorder of Th1/Th2 ( Table 1 , Figs. 1 and 2 ). These findings are in line with previous studies, which showed that probiotics suppressed CMA by manipulating the Th1/Th2 balance [6 , 35] . Recent studies have shown that Foxp3+Treg cells, induced by commensal intestinal bacteria, played an important role in regulating allergic responses and in balancing Th1/Th2 polarization [19 , 25] . In the current study, L9 significantly increased the number of CD4+CD25+Foxp3+ Treg cells in the MLN and induced high levels of TGF-β and IL-10 in the serum and lymphocyte supernatants from the MLN ( Fig. 2 ), indicating the powerful effect of probiotics on modulating the intestinal immune response. These were similar to results observed in a study of ovalbumin allergy, which showed that LGG only induced the number of CD4+CD25+Foxp3+ Treg cells and TGF-β secretion in the MLN, but not in the spleen [11] .
DCs play an important role in routing the immune responses and inducing different T cell subsets, such as the conversion from T cells to Foxp3+ Treg cells [22 , 27] . Immature DCs and CD103+DCs are important for establishing tolerance [13 , 15] . Previous studies have shown that the oral tolerance to ovalbumin or CMA was improved by high percentages of CD103+ DCs within the gut [26 , 34] . It has been reported that various strains and species of lactic acid bacteria regulated DC activation and maturation differentially and that only certain probiotic strains primed DCs for generation of Treg cells [10 , 32] . In this study, the number of CD103+DCs was increased by L9 stimulation both in vivo and in vitro ( Figs. 3 B and 4 B), and this stimulation in vitro was associated with a higher level of RALDH2 mRNA expression (Fig. S3). RALDH2 is involved in the retinoic acid-mediated introduction of Foxp+ Treg cells [25] . Previous studies have demonstrated the protective effect of CD103+ DCs on food allergies, through superantigen feeding or continuous oral antigen administration [8 , 34] . To the best of our knowledge, there have been no published studies investigating the effects of probiotic-induced CD103+DCs on CMA or food allergies.
In the current study, the percentages of CD80+DCs, CD86+DCs, and MHCII+DCs in lymphocytes of the PP and MLN were reduced in L9 mice, compared with BLG mice ( Figs. 3 C and 3 E). In constrast, MHC-II and CD80 expression levels in BM-DCs were increased by L9 in vitro ( Figs. 4 D and 4 E). However, although the L9-stimulated BM-DCs seemed to have matured, the production of TGF-β and IL-10 of BM-DCs was increased, with increased Foxp3+ expression in CD4+ T cells from BLG mice, and induced a shift in the Th1/Th2 paradigm in DC-CD4+T cell co-cultures ( Fig. 5 ). Recent studies have shown the suppressing effect of CD4+CD25+Foxp3+ Treg cells on Th1 and (or) Th2 cells [9 , 25] . Similar results were observed in a previous study, in which CD80 and CD86 were upregulated when DCs were exposed to probiotics in vitro ; however, these DCs in that study also induced the number of CD4+CD25+Foxp3+ Treg cells [20] . Other studies have shown that lactobacilli from the gut flora induced semimature DCs, which expressed high levels of MHCII and IL-10, but low levels of IL-12p70, and were actively tolerogenic via the induction of Treg cells [7 , 24] . Taken together, our data suggest that L9 includes CD4+CD25+Foxp3+ Treg cells, possibly by CD103+DCs or semi-mature DCs dependent mechanisms. To the best of our knowledge, there are few published studies investigating the tolerogenic capacity of CD103+DCs or semi-mature DCs stimulated by probiotics in food allergy models.
In summary, data from this study clearly show that oral supplementation of probiotics can reduce the development of allergic sensitization to BLG, through maintaining the intestinal and systemic immune responses. This suggests that the manipulation of CD11c+CD103+ DCs and semimature DCs, by potential probiotics, is important for preventing antigen-specific immune reactions.
Acknowledgements
We are grateful for the financial support for this work by the Ministry of Science and Technology of China (2012BAD12B08, D141100004814001), and the Beijing Municipal Commission of Education Co-constructed Program.
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