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Adhesion and anti-inflammatory properties of eight strains of bifidobacteria were tested using the intestinal epithelial cell lines Caco-2, T84, and HT29. Two strains were selected for further assessment of their anti-inflammatory capacity in two murine models of colitis. In vivo results confirmed the high anti-inflammatory capacity of a Bifidobacterium bifidum strain.
Bifidobacteria are an important group of the intestinal microbiota (21). Several beneficial health effects have been claimed to be based on the presence of bifidobacteria in the colon (reviewed in reference 16), and thus, they become increasingly interesting for probiotic applications in pharmaceutical and dairy products. A promising application is their use in probiotic intervention in inflammatory bowel disease (IBD). Probiotics containing bifidobacteria have been shown to be effective in reducing the severity of inflammation in several rodent models of IBD and in patients with IBD (3, 6-9, 12, 19). Several studies have reported an upregulation of the receptors for bacterial lipopolysaccharide (LPS), CD14, and TLR4 in the intestinal epithelium in murine models of IBD and in patients with IBD (4, 13, 15), suggesting a contribution of abnormal LPS stimulation to chronic intestinal inflammation. In a previous study, we were able to show that different strains of bifidobacteria are able to inhibit LPS-induced inflammatory events in intestinal epithelial cells (IECs) (18). Besides the manufacturing criteria, shelf life and gut transit, two main characteristics are crucial for selecting potential probiotic strains: (i) the desired probiotic property, in the case of IBD, an anti-inflammatory effect, and (ii) high adhesion to the intestinal mucosa (reviewed in reference 10).
Here, we present our data from an in vitro analysis of eight strains of bifidobacteria covering the major species isolated from human fecal samples. All of the strains were tested for adhesion and anti-inflammatory effects using Caco-2, T84, and HT-29 IECs. For adhesion experiments, Caco-2, T84, and HT-29 cells were grown in 24-well tissue culture plates as described previously (17, 18). Bifidobacterium adolescentis NCC251, B. lactis NCC362, B. longum NCC2705, B. bifidum NCC189, S16, and S17, B. longum/infantis E18, and B. breve MB226 (all described previously ) were grown to stationary phase at 37°C in MRS medium supplemented with 0.5 g cysteine/liter (MRSC) in anaerobic jars using Anaerocult A (Merck). Bacteria were washed three times with phosphate-buffered saline (PBS) and resuspended at 1 × 108 CFU/ml RPMI medium (Gibco) supplemented with 1% nonessential amino acids (Gibco). One-milliliter aliquots were incubated with the IECs, i.e., a bacterium-to-cell ratio of 100:1, for 1 h. After three washings with PBS to remove nonadherent bacteria, IEC monolayers were lysed and bacterial adhesion was quantified in serial dilutions of the lysates by plate counts on MRSC agar. Adhesion was expressed as percent adherent bacteria relative to the initially added CFU. In line with previous observations of our group and others (2, 5, 17), bifidobacteria adhered to all of the IEC lines in a strain-specific manner (Fig. 1A, D, and G), with minor variations between different cell lines.
One of the early mediators of inflammatory responses in IECs is interleukin-8 (IL-8), recruiting professional immune cells to the source of IL-8 secretion (reviewed in reference 20). To determine the effect of bifidobacterial pretreatment on LPS-induced IL-8 secretion, IECs were incubated with bacteria as described for adhesion experiments and subsequently challenged with LPS (100 ng/ml; E. coli O55:B5; Sigma) in the presence of recombinant human CD14 (50 ng/ml; R&D Systems) for 16 h. IL-8 in the cell culture supernatants was quantified using the Eli-pair IL-8 enzyme-linked immunosorbent assay kit (Diaclone). Bifidobacteria were able to inhibit LPS-induced IL-8 secretion in both Caco-2 and T84 cells in a strain-dependent manner. Minor variations between the capacities of the two cell lines to inhibit LPS-induced IL-8 secretion were observed.
The central transcription factor of inflammatory mediators, including IL-8, is nuclear factor κB (NF-κB) (11). To assess whether inhibition of IL-8 secretion occurs in an NF-κB-dependent manner, a previously described convenient NF-κB-reporter system in HT-29 cells using secreted alkaline phosphatase (SEAP) (18) was employed. LPS/CD14 stimulation experiments after pretreatment with bifidobacteria were repeated as described for Caco-2 and T84 cells using HT-29 clone 34 cells, and the SEAP reporter was measured in the supernatants. Again, a strain-dependent inhibition of LPS-induced NF-κB activation was observed. Those strains that showed good inhibition of IL-8 secretion in Caco-2 and T84 cells were also able to inhibit NF-κB-dependent reporter activities, e.g., B. bifidum S17 and NCC189. By contrast, strains that failed to inhibit IL-8 secretion by Caco-2 and T84 cells upon LPS stimulation also had no effect on SEAP activity, e.g., B. longum/infantis E18 and B. breve MB226 (Fig. (Fig.1H1H).
The results of adhesion experiments and the inhibition of LPS-induced IL-8 secretion indicated that strains showing higher adhesion to IECs also had a higher anti-inflammatory capacity. This prompted us to investigate the correlation between the two characteristics in more detail. To identify promising strains for further analysis, we plotted adhesion against IL-8 secretion or SEAP reporter activity (Fig. 1C, F, and I). This revealed that, with all of the cell lines tested, B. bifidum S17 performed better than all of the other strains when both adhesion and anti-inflammatory effects (inhibition of IL-8 secretion or NF-κB activation) were taken into account. On the other hand, B. longum/infantis E18 was identified as the strain with the least promising characteristics.
These two strains were selected for further analysis of their anti-inflammatory potential in the Rag−/− transfer model of colitis. The transfer of CD4+ effector T-cell populations from wild-type C57BL/6J into congenic Rag−/− (Rag1tm1Mom) mice leads to the development of colitis mediated by Th1 cells due to a lack of mature regulatory T cells in these mice (14). Rag1−/− mice (7 to 8 weeks old, n = 6 per group) received a transfer of CD4+ CD45RBhigh T cells from wild-type C57BL/6 mice as described previously (14). Two groups received either B. bifidum S17 or B. longum/infantis E18 orally (2 × 109 CFU per animal) in PBS. Two control groups received a placebo, one of which also received a CD4+ T-cell transfer to induce colitis and the other of which served as healthy controls. Feeding with bifidobacteria and the placebo was continued three times a week, and weight was recorded for 24 days, after which all of the animals were sacrificed. Animals treated with B. bifidum S17 were protected from weight loss induced by the T-cell transfer (Fig. (Fig.2A).2A). This effect became statistically significant on day 10 of the experiment. By contrast, feeding with B. longum/infantis E18 had no effect and the weight of this group was not significantly different from that of colitic controls. Macroscopic analysis of dissected colons showed severe wall thickening and diarrhea in colitic control animals and animals treated with B. longum/infantis E18. In contrast, animals treated with B. bifidum S17 had solid fecal pellets and a normal appearance of the colon wall, as observed in healthy controls (Fig. (Fig.2B).2B). Also, these animals had normal colon weight/length ratios while animals treated with B. longum/infantis E18 showed increased colon weight/length ratios to an extent seen in colitic controls (Fig. (Fig.2C).2C). Furthermore, inflammation was assessed by scoring histological sections of colonic biopsies stained with hematoxylin and eosin (H&E) as described previously (14). Colitic control animals and mice treated with B. longum/infantis E18 displayed signs of severe colitis with a high degree of inflammatory infiltrate in the colonic mucosa, loss of goblet cells, and a disturbed mucosal architecture. By contrast, inflammation was significantly reduced in animals treated with B. bifidum S17 (Fig. 2D and E), reflected by an improvement of the inflammatory score from 2.2 to 1.3 (P = 0.003).
The anti-inflammatory effect of B. bifidum S17 was confirmed in the trinitrobenzene sulfonic acid (TNBS) model of colitis (1). Animals in the study received one oral dose of B. bifidum S17 (2 × 109 CFU per animal) or a placebo daily. On day 3, animals (7 weeks old, n = 9 or 10 per group) were deprived of food, and on day 4, two groups received an intrarectal dose of TNBS (120 mg/kg body weight) and a control group was treated with the carrier only (50% ethanol/50% PBS). Feeding of B. bifidum S17 or the placebo was continued until day 8, when all of the animals were sacrificed. Animals treated with B. bifidum S17 were partially protected from weight loss induced by TNBS treatment (Fig. (Fig.3).3). However, this effect did not reach statistical significance. Additionally, colitis was assessed in H&E-stained colonic biopsies according to a scoring system for TNBS colitis as described previously (1). Treatment with B. bifidum S17 reduced the inflammatory scores from 4.7 to 3.2, and this effect was statistically significant (P = 0.023).
The concept of probiotics has attracted increasing attention in recent years. A number of publications show anti-inflammatory effects of probiotics in mouse models or patients of IBD (3, 7, 8, 12). With the increasing interest, there is a necessity for further strains of well-documented origin and analysis of probiotic properties. Based on our in vitro data, B. bifidum S17, a highly adherent strain with potent anti-inflammatory capacity, was selected for further in vivo analysis along with B. longum/infantis E18, a nonadherent strain showing no anti-inflammatory effects. Our results demonstrate that combining adhesion assays and inflammatory readouts with different cell lines might provide a means for preselection of potential probiotic strains prior to in vivo experimentation. Further experiments with an extended range of strains will be performed to evaluate the described tools for their use as large-scale screening systems.
This study was partially funded by the German Academic Exchange Service/Federal Ministry of Education and Research (grant D/09/04778) to J.P. and C.U.R., DFG grants Ni575/6-2 and Ni575/7-1 to J.-H.N., and a Ph.D. fellowship from the Carl Zeiss Foundation to M.G.
We thank Julia Geitner for excellent technical assistance.
Published ahead of print on 12 March 2010.