Probiotic Mixture Protects Dextran Sulfate Sodium-Induced Colitis by Altering Tight Junction Protein Expressions and Increasing Tregs

Bifico is a probiotic mixture containing Bifidobacterium, Lactobacillus acidophilus, and Enterococcus. Studies support that Bifico has a protective effect in experimental colitis (IL-10-deficient and TNBS) models and in patients with inflammatory bowel disease (IBD). However, the mechanism underlying the protective effects of this mixture of probiotic bacteria remains incompletely clear. Here, we investigated the effect of Bifico on intestinal inflammation. In an in vivo experiment, dextran sulfate sodium was used to induce colitis. Bifico treatment significantly attenuated the severity of colitis in this model. Bifico increased the expression of tight junction proteins (TJs). In addition, Bifico increased the number of Tregs, but reduced the number of total CD4+ T cells in the peripheral blood. Furthermore, the expression of colonic CD4 protein was decreased while the level of forkhead box P3 (Foxp3) was upregulated. These results suggested that Bifico exerts beneficial effects on experimental colitis by increasing the expressions of TJs, upregulating the number of Tregs, and reducing the total CD4+ T cell number in both colon and peripheral blood. The intestinal damage in the pretreated + treated-Bifico-colitis group was more severe than that in only the pretreated-Bifico-colitis group. This suggested that Bifico might aggravate intestinal damage when the mucosal barrier is impaired.


Introduction
The intestinal microbiota play a role in triggering the immune system and leading to intestinal inflammation [1]. IBD patients suffer from dysbiosis, which is characterized by a decrease in diversity and abundance of some dominant commensal bacteria [2]. Some studies showed that the amounts of Lactobacillus and bifidobacteria were significantly reduced in the feces of IBD patients [3,4]. This suggests that gut flora normalization may serve as a therapeutic option for IBD patients. Bifico is a probiotic mixture, containing Bifidobacterium, Lactobacillus, and Enterococcus. In fact, evidences from both experimental studies and clinical trials have demonstrated the various therapeutic effects of Bifico on Crohn's disease, ulcerative colitis, colitis-induced and colitis-associated cancer in mice, pouchitis, diarrhea, and gastritis [5][6][7][8][9][10][11][12]. A previous study demonstrated that Bifico can increase the expression of colonic TJs and promote intestinal epithelial barrier function in interleukin-10-deficient (IL-10 KO) mice [5]. In vitro experiments also showed that the Bifico, or single probiotic strains (Bifidobacterium, Lactobacillus, or Enterococcus), increase transepithelial electrical resistance (TER) and the expression of TJs in enteroinvasive Escherichia coli-(EIEC-) treated Caco-2 monolayers. Bifico significantly inhibited the secretion of proinflammatory cytokines in EIEC-treated Caco-2 monolayers. Bifico exposure in vitro reduced bacterial invasion. Moreover, the effects of combined probiotics were more pronounced than those of single-strain probiotics. Another study reported that the anti-inflammatory effects of Bifico were related to the expansion of Tregs in mesenteric lymph nodes and disturbance of Th1/Th2 cytokines in the colonic mucosa of TNBS-induced colitis mice. However, the effect of Bifico on the Treg cells in intestinal tissue and peripheral blood has not been reported. In addition, previous animal experimental studies mainly observed the effect of Bifico as a therapeutic in active colitis [6].
The aims of this study were to observe the beneficial effect of Bifico on intestinal inflammation in dextran sodium sulfate (DSS) experimental colitis and to investigate whether the beneficial effect is associated with local and systemic immune responses. This study explored the protective effect of Bifico pretreatment on subsequent intestinal inflammation.

Materials and Methods
2.1. Animals. A total of 44 female specific-pathogen-free BALB/c mice (aged 8 to 10 weeks, weighing 20-24 g) were purchased from the Laboratory Animal Center of Nanjing Medical University (Jiangsu, China), maintained in clean cages under a 12 h light-dark cycle and conventional housing conditions, and fed with standard mouse chow. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Ethics Committee of Nanjing Medical University (Approval ID: NJMU20110312). The Bifico capsule contains Bifidobacterium, Lactobacillus, and Enterococcus living bacteria not less than 3 × 10 7 CFU (Shanghai Sine Pharmaceutical).

Measurement of Tumor Necrosis Factor (TNFα) Levels in Colonic
Tissues. Colonic tissues were homogenized in cold PBS containing a cocktail of protease inhibitors supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The levels of TNFα in colonic tissues were measured with an enzyme-linked immunoassay kit (ExCell Biology, Shanghai, China), according to the manufacturer's instructions.
2.6. Transmission Electron Microscopy (TEM). Colonic tissue samples were cut into 1 mm × 1 mm × 2 mm sections and were fixed in 2.5% glutaraldehyde for 2 h at 4°C. The sections were then postfixed in 1% osmic acid for 2 h at 4°C, washed with 0.1 M PBS, dehydrated in a graded series of acetone concentrations, embedded in a mounting medium at 37°C for 3 h, and then polymerized at 60°C for 36 h. After ultrasectioning with an ultrathin slice machine (Leica, Germany), the ultrathin sections were viewed and photographed with a JEOL-1010 TEM (Japan).

Flow Cytometric Analysis of CD4 + CD25 + Foxp3 + T Cells.
Single-cell suspensions were prepared from the peripheral blood and spleen, according to the manufacturer's protocols. Red blood cells in peripheral blood and spleen were lysed using Red Blood Cell Lysis Buffer (Beyotime, China). This step was repeated 2-3 times until no more red blood cells were visible. After that, mononuclear cells were resuspended in RPMI 1640 serum-free medium at a final cell density of 1 × 10 7 /mL. Fluorescein isothiocyanate-(FITC-) anti-mouse CD4 (RM4-5, 0.125 μg/test, eBioscience) and allophycocyanin-anti-mouse CD25 (PC61.5, 0.06 μg/test, eBioscience) were added to 100 μL of the cell suspension for 30 min, followed by the addition of 1 mL of fixation/permeabilization working solution (eBioscience) for 10 h at 4°C. Finally, the cells were incubated with phycoerythrin-(PE-) anti-mouse/rat Foxp3 (FJK, 0.5 μg/ test, eBioscience) for 30 min at 4°C in the dark. Cells labeled with rat IgG2a PE were used as the isotype negative control. All flow cytometric measurements were performed on a flow cytometer (Beckman Coulter, Krefeld, Germany).
2.11. Statistical Analysis. All images are representative of at least three independent experiments. Data are presented as the mean ± standard error of the mean (SEM). P values were calculated by one-way ANOVA followed by the Tukey post hoc test (SPSS version 21.0). P value < 0 05 was considered significant.

Administration of Bifico Reduced Colonic Levels of TNFα in Colitis
Mice. As reported previously, the colonic levels of TNFα were increased in the colitis group (P < 0 01). Meanwhile, the level of TNFα in the pretreated-Bifico-colitis group was decreased compared to the colitis group (P < 0 05). However, there was no significant difference between the colitis group and the pretreated + treated-Bifico-colitis group. Notably, the colonic levels of TNFα were slightly increased in the Bifico group, but there was no significance compared with the normal group (P = 0 934) ( Figure 3).

Alterations in Microstructures of the Colonic Epithelial
Barrier in Colitis Mice Observed by TEM. The morphology of the colonic epithelium and the cell membrane was intact, the cell junction was tight, and the villi on the cell surfaces were well-arranged in both the normal group and Bifico groups. However, in the colitis group, the integrity of the epithelial membranes was compromised, cell-cell junctions were loose, with an obvious intercellular space broadening, and villi on the cell surfaces were decreased (Figure 4(a)). The microstructure of the cell-cell junction was improved in pretreated-Bifico-colitis and pretreated + treated-Bifico-colitis groups, and some villi on the surfaces were reserved.

Expressions and Distribution of TJs in Colonic Tissues and
Epithelium Barrier. Compared with the normal group, the expressions of the TJs were significantly reduced in the colitis group (P < 0 01 for JAM-1, P < 0 001 for occludin, and P < 0 001 for claudin-4). The expression levels of these TJs in pretreated-Bifico-colitis and pretreated + treated-Bificocolitis groups were extensively increased compared with the colitis group (P 1 < 0 05 and P 2 < 0 05 for JAM-1, P 1 = 0 073 and P 2 < 0 01 for occludin, and P 1 = 0 598 and P 2 < 0 05 for claudin-4). In addition, the expression levels of occludin and claudin-4 in the pretreated + treated-Bifico-colitis group were slightly higher than those in the pretreated-Bifico-colitis group with no significant difference (P = 0 246 for occludin and P = 0 345 for claudin-4) (Figure 4(b)). There was no difference between the Bifico group and the normal group, but a scattered distribution and decreased expressions of TJs were found in colitis mice. However, the intensity and the percentage of cells stained for TJs were improved in pretreated-Bifico-colitis and pretreated + treated-Bifico-colitis groups, in comparison with mice in the colitis group (Figure 4(c)).

Bifico
Effects on Total CD4 + T Cells, CD4 Protein Expression, Tregs, and Foxp3 + Cells. The proportion of total CD4 + T cells in the peripheral blood in the colitis group was increased (colitis versus normal group: 55.10 ± 4.73 versus 34.19 ± 1.49, P < 0 01); however, compared with the colitis group, the increased proportion of total CD4 + T cells was partially reversed both in pretreated-Bifico-colitis (36.04 ± 3.99 versus 55.10 ± 4.73, P < 0 01) and pretreated + treated-Bificocolitis groups (47.90 ± 3.66 versus 55.10 ± 4.73, P = 0 556) ( Figure 5(a)). There was no significant difference in the number of splenic CD4 + T cells among different groups ( Figure 5(b)). In addition, there was a decreasing trend of the colonic CD4 protein level in the pretreated-Bifico-colitis group compared with the colitis group (P = 0 639). Meanwhile, a significant difference was shown between pretreated + treated-Bifico-colitis and colitis groups (P < 0 01) ( Figure 5(c)). In addition, the population of CD4 + CD25 +-Foxp3 + Tregs in the peripheral blood was significantly upregulated in the pretreated-Bifico-colitis and pretreated + treated-Bifico-colitis groups compared with the colitis group (P < 0 05 for both) (Figure 6(a)). However, no significant difference in the amount of CD4 + CD25 + Foxp3 + Tregs in the spleen was detected among groups ( Figure 6(b)). In fact, the number of Foxp3 + Tregs in colonic tissue was increased by the administration of Bifico in pretreated-Bifico-colitis and pretreated + treated-Bifico-colitis groups compared with the colitis group ( Figure 6(c)). The expression of Foxp3 protein was significantly decreased in the colitis group compared with the normal group (P < 0 001) as well as the pretreated-Bifico-colitis group (P < 0 05). Notably, the expressions of colonic Foxp3 protein were slightly increased in the pretreated + treated-Bifico-colitis group, but was not significant (P = 0 224) (Figure 3 versus (Figure 6(d)). However, compared with the normal group, the expression of Foxp3 protein in the Bifico group was decreased (P < 0 05).

Discussion
Some studies suggested that Bifico supplements are able to prevent colitis development in human IBD and experimental colitis [5][6][7]20]. The current study shows that the intestinal damage in the pretreated + treated-Bifico-colitis group was more severe compared with that in the pretreated-Bifico-colitis group, suggesting that Bifico might have a protective effect on colonic tissue in the intact intestinal mucosal barrier. In contrast, when the mucosal barrier is impaired, probiotics might aggravate colonic tissue damage, and this needs further research. The TJs consist of transmembrane proteins such as occludin, claudins, junctional adhesion molecules, and adaptor proteins like Zos [21]. An oral administration of Bifico has been shown to reduce colon inflammation and to protect epithelial barrier function in IL-10 KO mice [5]. Consistent with these findings, this study shows that Bifico increased the expressions of TJs and improved the microstructures in colitis mice. Previous investigators showed that Bifico treatment significantly reduced the levels of TNFα in the colon of experimental colitis mice [5,6]. Our data support these findings that Bifico may ameliorate intestinal inflammation by inhibiting TNFα production. Foxp3 is required for induction of the immune suppressive function of CD4 + Foxp3 + Tregs and maintenance of mucosal immune homeostasis by regulating the balance between CD4 + Foxp3 + Tregs and helper effector T cells. Tregs are critically involved in the prevention of human IBD and experimental colitis [6,22]. A previous study has shown that the anti-inflammatory effects of Bifico were related to the expansion of Tregs in mesenteric lymph nodes of colitis mice and the ratio of Th1/Th2 might be regulated by Tregs [6]. We found that Bifico increased the number of Tregs in the peripheral blood. However, the numbers of Tregs in the spleen among groups showed no significant differences. Meanwhile, Bifico could increase the colonic Foxp3 protein level, but decrease the colonic CD4 protein level in colitis mice. These results may indicate that the decreased frequency of Treg subsets in the peripheral blood and Foxp3 expression in the colonic tissues might destroy the intestinal immune  (3) compared to the normal group (1) (P < 0 01) and decreased in the Pretreated-Bifico-colitis group (4) and pretreated + treated-Bificocolitis group (5) (P 1 < 0 05 and P 2 = 1 00, resp.). Data are means ± SEM (n = 8-11/group). * P < 0 05 and * * P < 0 01. tolerance and activate inflammation. However, a significant investigation is required to prove these explanations. Of note, the normal mice treated with Bifico demonstrated a slightly increased TNFα level, a decreased Foxp3 protein level, and an increased CD4 protein expression in colonic tissues as well as an increased number of Tregs in the peripheral blood. It is possible that Bifico acts as a foreign antigen and triggers weak immune responses, yet this immune response may not lead to pathological inflammatory damage. This suggested that proper immune activation may enhance the mucosal defense and thus be beneficial for the promotion of the host intestinal immunity [23]. These also need to be investigated.
Our study has some strengths, as Bifico might have a protective effect on colonic tissue when the intestinal mucosal barrier is intact. In contrast, when the mucosal barrier is impaired, probiotics might aggravate colonic tissue damage. Bifico could increase the number of Tregs in the peripheral blood and have no influence in the spleen among different groups. Our study has several limitations. First, TJ expression is only an indirect reflection of the barrier function. To exactly measure the barrier function, physiologic measurements like absorption of orally administered dextran or bacteria-size particles should be performed. Second, this study explored the effects of Bifico as a whole, but functions of single probiotic strains contained in Bifico (Bifidobacterium, Lactobacillus, or Enterococcus) were not studied separately. Third, these results suggested that Bifico might exert beneficial effects on experimental colitis by upregulating the number of Tregs and reducing total CD4 + T cells in both colonic tissue and peripheral blood. Further, pathways through which Tregs and CD4 + T cells ameliorate the inflammation need to be explored. Finally, this study only explored the preventive effects of Bifico (with no major clinical application) while no study was performed to explore therapeutic effects in this model. Therefore, extensive exploration in this field is needed.

Conclusions
This study demonstrated some beneficial effect of Bifico on colitis. The potential mechanism involved in improving the expression of TJs is increasing the number of Tregs in colonic tissues and the peripheral blood, while decreasing the proportions of total CD4 + T cells in colonic tissues and the peripheral blood.