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Keywords:

  • colitis;
  • commensal intestinal bacteria;
  • cytokines;
  • HLA-B27 transgenic rats;
  • immunomodulation;
  • prebiotics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

HLA-B27 transgenic rats develop spontaneous colitis under specific pathogen-free conditions (SPF) but germ-free rats remain disease-free, emphasizing a role for intestinal bacteria in the pathogenesis of chronic intestinal inflammation. Prebiotics are dietary substances that affect the host by stimulating growth and/or activity of potentially health promoting bacteria. The aims of this study were to investigate whether prebiotics can prevent colitis in SPF HLA-B27 rats, and secondly, to explore mechanisms of protection. SPF HLA-B27 transgenic rats received orally the prebiotic combination long-chain inulin and oligofructose (Synergy 1), or not, prior to the development of clinically detectable colitis. After seven weeks, cecal and colonic tissues were collected for gross cecal scores (GCS), histologic inflammatory scores (scale 0-4), and mucosal cytokine measurement. Cecal and colonic contents were collected for analysis of the gut microbiota by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and fluorescent in-situ hybridization (FISH), and analysis of short-chain fatty acid composition. Prebiotic treatment significantly decreased GCS and inflammatory histologic scores in the cecum and colon. Prebiotic treatment also decreased cecal IL-1β, but increased cecal TGF-β concentrations. Inulin/oligofructose altered the cecal and colonic PCR-DGGE profiles, and FISH analysis showed significant increases in cecal Lactobacillus and Bifidobacterium populations after prebiotic treatment compared with water-treated rats. In conclusion, the prebiotic combination Synergy 1 reduced colitis in HLA-B27 transgenic rats, which effect was associated with alterations to the gut microbiota, decreased tissue proinflammatory cytokines and increased immunomodulatory molecules. These results show promise for prebiotics as primary or adjuvant maintenance therapy for chronic inflammatory bowel diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The role of the gut microbiota in the pathogenesis of human inflammatory bowel diseases is well recognized.1,2 These diseases typically occur at sites with the highest concentrations of intestinal bacteria, such as the colon and terminal ileum. The role of intestinal bacteria and the genetic background of the host in the initiation and perpetuation of chronic intestinal inflammation are most convincingly demonstrated in rodent models of chronic intestinal inflammation. In these models, genetically susceptible hosts develop spontaneous colitis in the presence of intestinal bacteria, but not in the germ-free state.3

One example of a well-characterized model of chronic intestinal inflammation is the HLA-B27 transgenic rat, which develops colitis, gastritis, and arthritis in a specific pathogen-free (SPF) environment.4 However, germ-free transgenic rats, as well as nontransgenic rats colonized with SPF bacteria, do not show evidence of colitis, gastritis, and arthritis.5 Colitis and gastroduodenal inflammation in transgenic rats can be prevented with broad spectrum as well as selective antibiotics.6 Moreover, cecal bacterial overgrowth within experimental blind loops exacerbates colitis, whereas bypass of the cecum attenuates disease in this model.7Bacteroides vulgatus (B. vulgatus) preferentially induces colitis in transgenic rats after monoassociation for four weeks, whereas monoassociation with Escherichia coli (E. coli) does not cause disease.8 These findings indicate that although bacteria are crucial in the pathogenesis of colitis, not all bacteria are equal in their capacity to induce chronic intestinal inflammation.

Probiotics are “defined, live microorganisms administered in adequate amounts which confer a beneficial physiological effect on the host”.9 We and others have shown that certain probiotic bacteria such as Lactobacillus species have protective activities in several experimental models of chronic intestinal inflammation, including SPF HLA-B27 transgenic rats and SPF IL-10 deficient (IL-10−/−) mice.10,11 Probiotic cocktails have also proven efficacy in ulcerative colitis (UC)12 and refractory pouchitis.13

Prebiotics are nondigestible polysaccharides or oligosaccharides that affect the host by selectively stimulating growth and/or activity of a limited number of potentially health promoting bacteria.14 Previous studies have demonstrated that the prebiotics inulin and oligofructose selectively stimulate the growth of indigenous bifidobacteria or lactobacilli.15,16 To date, the efficacy of prebiotics as monotherapy has been limited to induced models of colitis.17,18 However, as probiotics were effective in SPF HLA-B27 transgenic rats,11 we designed this study to determine if prebiotics would have a similar degree of protection in this model. In addition, the role of immune factors, cecal and colonic short chain fatty acids, and the composition of the intestinal microbiota were studied as possible mechanisms of protection. In this study, we demonstrate that the combination of inulin and oligofructose reduced colitis. This beneficial effect was associated with alterations to the gut microbiota, a decrease in cecal proinflammatory and increased immunoregulatory cytokines.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

HLA-B27 transgenic rats (the 33-3 line on the F344 background) and their nontransgenic littermates were originally obtained from Dr. Joel D. Taurog, University of Texas Southwestern Medical Center. Rats were born and maintained in SPF housing conditions at the University of North Carolina, Chapel Hill and were fed Prolab RMH 3000 rat chow ad libitum. Presence or absence of the HLA-B27 transgene was determined by PCR using DNA isolated from tail clippings as described previously.19 All studies were approved by University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.

Experimental Design

Seven week old SPF HLA-B27 transgenic rats, prior to the development of histologic inflammation, were divided into two groups of eight rats each. The first group was administered a combination of chicory-derived long-chain inulin-type fructans and short-chain inulin fraction oligofructose in a mixture of 1:1 (Raftilose Synergy 1, Orafti, Tienen, Belgium) in their drinking water at a dose of 5 g/kg bodyweight, whereas the control group received no addition. The amount of prebiotics consumed by each rat per day was calculated by measured consumption of 30 mL of water per day. Rats were killed after seven weeks. At necropsy cecal and colonic tissue and content were snap-frozen for subsequent cytokine analysis and microbiological and short-chain fatty acid (SCFA) assessment. Mesenteric lymph node (MLN) cells were collected for cell cultures.

Gross Cecal Scores

The cecum and colon were inspected grossly in a blinded fashion for evidence of intestinal wall thickness and scored on a scale ranging from 0 to 4, using a previously validated system.11,20

Histology

Colons and ceca were fixed and stained as previously described.11,20 A validated histologic inflammatory score ranging from 0 to 4 was used for blinded evaluation of cecal and colonic inflammation.20

Preparation of Cecal Bacterial Lysates

Cecal bacterial lysates (CBL) were prepared as described previously.19 Briefly, cecal contents from several SPF HLA-B27 nontransgenic rats were homogenized and the supernatant was filtered through a 0.45 μm syringe filter. Sterility was confirmed by aerobic and anaerobic culture.

Mesenteric Lymph Node Cell Cultures

Mesenteric lymph nodes were removed from water- and prebiotic-treated HLA-B27 transgenic rats before dissecting the intestines, and single cell suspensions were prepared by gentle teasing, as previously described.19 MLN cells were washed twice and 4 × 105 cells were cultured in 96 well flat bottom microplates (Costar 3595), in 0.2 mL complete medium. Cells were stimulated with the optimal concentration of 50 μg/ml CBL, as described previously.19 Culture supernatants were collected after three days and stored at −20°C.

Cytokine Measurements in MLN Cell Cultures

Cytokines in cell culture supernatants were measured by ELISA using unlabeled capture antibodies and biotin-labeled detection antibodies, followed by horse-radish peroxidase-labeled Streptavidin.19 The concentration of each cytokine was determined by comparison to a standard curve generated using recombinant proteins. For IFN-γ, we used unlabeled polyclonal anti-IFN-γ antibody and biotin-labeled monoclonal anti-IFN-γ antibody (clone DB-1) (Biosource International, Camarillo, Calif.). For IL-10 we used unlabeled monoclonal anti-rat IL-10 antibody (clone A5-7) and biotin-labeled monoclonal anti-rat IL-10 antibody (clone A5-6) (BD Biosciences Pharmingen, San Diego, Calif.).

Cecal Cytokine Analysis

Cecal cytokines were measured as described previously.11 Briefly, frozen cecal tissues were thawed and lysed in PBS containing a cocktail of protease inhibitors.21 Tissue was then homogenized after which the homogenate was assayed for IL-1β, IL-10 and TGF-β, as described.11 IL-1β was measured by ELISA according to the manufacturer's instructions (National Institute for Biologic Standards and Controls, South Mimms, UK). IL-10 was measured as described in the previous section. TGF-β concentrations were measured after acidification and neutralization according to the manufacturer's instructions using a TGF-β specific ELISA (Promega, Madison, Wis.).

Short-Chain Fatty Acids (SCFA) Analysis in Cecal Contents

Cecal or colonic content (100 mg) was combined with 400 μL water and 100 μL of 25% phosphoric acid in a 2 mL microcentrifuge tube, mixed thoroughly, and immediately frozen. At the time of analysis, samples were thawed and 100 μL of internal standard (4-methyl-n-valeric acid at 50 μmol/ml) was added. The samples were centrifuged to obtain a clear supernatant that was placed into 1.8 mL GC vials for analysis. SCFA separation was performed using a Varian 3400 gas chromatograph equipped with a Varian 8200CX autosampler. Approximately 1 μL was injected onto a Stabilwax-DA GC capillary column (30 m × 0.53 mm ID: Restek Corp., Bellefonte PA) with an injector split ratio of 20:1. Injector and detector temperatures are set at 170°C and 190°C, respectively. Column temperature was increased from 120°C to 170°C at 10°C/min and held for 5 minutes. Data acquisition and integration were performed using a Schmadzu Class-VP Chromatography Laboratory Automated Software System.

Polymerase Chain Reaction-denaturing Gradient Gel Electrophoresis (PCR-DGGE) from Cecal and Colonic Contents

To detect changes in the composition of the large bowel microbiota of rats, we used PCR-DGGE. DNA was extracted from cecal and colonic samples by preparing a 1/10 (wt/vol) homogenate in PBS (pH 7.0). Samples were vortexed and stored at −80°C until further use. DNA extraction followed a previously described method.22 Amplification of total bacterial community DNA was carried out by targeting 16S rDNA sequences using universal bacterial primers HDA1-GC and HDA-2 and using a procedure described previously.22 The PCR products were checked by electrophoresis through a 2% agarose gel, stained with ethidium bromide (5 μg/ml) and viewed by UV transillumination. PCR amplicons were analyzed by DGGE. This was performed with a DCode apparatus (Bio-Rad, Hercules, Calif.), using a 6% polyacrylamide gel with a 30 to 55% gradient of 7.0 M urea and 40% (vol/vol) formamide that increased in the direction of the electrophoresis. Electrophoresis was carried out in 1× TAE buffer at 130 V and 60°C for 4 hours. Gels were stained with ethidium bromide solution (5 μg/ml) for 20 minutes, washed with deionised water for 20 minutes, and viewed by UV transillumination. DNA fragments of interest were cut from the polyacrylamide gel using a sterile scalpel blade after the stained gel had been photographed. DNA was eluted from the gel, cloned and sequenced as described previously.23 Sequences were compared with those in the GenBank database using the BLASTn algorithm to identify the bacterial species from which the DNA sequence originated.24

Fluorescent in-situ Hybridization

Fluorescent in-situ hybridization (FISH) was carried out as described previously.25 Briefly, 100 mg of cecal or colonic content was diluted, homogenized, fixed overnight in paraformaldehyde at 4°C, and then stored at −80°C until analysis. Total cell numbers were counted by diamidino-2-phenylindole (DAPI) staining, whereas probe Erec482 was used for the detection of cells from the Clostridium-group XIVa,26 probe BAC303 for the detection of the Bacteroides/Prevotella genera,27 and probe Bif164 for the detection of the genus Bifidobacterium.28 The probe Lab158 was used to detect lactobacilli and enterococci.29 Enterococci were discriminated from the lactobacilli using probe Efs, as described previously.29

The samples hybridized overnight at 50°C in a dark humid chamber.30 After hybridization, the slides were washed, air dried, mounted, and the fluorescent cells in the samples were counted automatically31 with a Leica DMRXA epifluorescence microscope (Leica, Wetzlar, Germany). However, when the cell number was lower than 4 × 108 cells/g wet weight, the cells were counted visually using an Olympus BH2 epifluorescence microscope.30 All results are stated as bacterial counts/g luminal content, thus normalized for the weight of luminal contents used during FISH analysis. Oligonucleotides probes were synthesized and purified by Eurogentec (Seraing, Belgium).

Statistical Analysis

Statistics were performed using the nonpaired Student t test for comparison between groups. Data are expressed as mean ± SEM, and a two-tailed P value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Gross Cecal Scores and Histology

Daily water consumption did not differ significantly between the experimental groups (data not shown). Oral administration of the prebiotic combination of long-chain inulin and oligofructose to HLA-B27 transgenic rats reduced the development of colitis. Gross cecal scores (GCS) were significantly decreased in prebiotic-treated rats (Fig. 1), whereas water-treated animals showed severe cecal macroscopic inflammation (P < 0.005). This beneficial effect of prebiotic treatment was also reflected by significantly lower histologic scores for cecum (P < 0.01) and colon (P < 0.005) (Fig. 2) in prebiotic-treated versus untreated transgenic rats. Severe mucosal and submucosal inflammation with significant crypt hyperplasia was seen in untreated transgenic rats (Fig. 3A), whereas prebiotic treatment resulted in only modest mucosal inflammation (Fig. 3B). Interestingly, all water-treated rats developed arthritis in one or more joints, whereas only 1 out 8 rats in the prebiotic group showed arthritis (data not shown).

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Figure 1. Decreased gross cecal scores (0-4) after prebiotic treatment. Specific pathogen free transgenic rats were treated for seven weeks with the prebiotic combination long-chain inulin and oligofructose Synergy 1 (mixture 1:1) or water prior to the development of histologic inflammation. Each group consisted of eight rats. Values are presented as mean ± SEM, *** P < 0.005 versus untreated transgenic rats.

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Figure 2. Decreased histologic scores after prebiotic treatment. Blinded microscopic inflammation scores from ceca and colons in specific pathogen free transgenic rats were treated for seven weeks with prebiotic Synergy 1 or water prior to the development of histologic inflammation. Each group consisted of eight rats. Values are presented as mean ± SEM, ** P < 0.01, *** P < 0.005 versus untreated transgenic rats.

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Figure 3. Decreased cecal inflammation in transgenic rats after prebiotic treatment. Representative photomicrographs of tissue sections (x40) from ceca of 14 week old specific pathogen free transgenic rats were treated for seven weeks with water (A) or prebiotic Synergy 1 (B) prior to the development of histologic inflammation. Note the extensive mucosal and submucosal inflammation as well as significant crypt hyperplasia in ceca from water-treated transgenic rats (A). Only modest mucosal inflammation was seen in transgenic rats treated with the prebiotic combination (B).

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Mucosal Cytokine Profile

Cecal IL-1β concentrations were decreased in cecal homogenates after prebiotic treatment (P < 0.05; Figure 4A). In contrast, prebiotics increased the amount of cecal TGF-β (P < 0.05; Figure 4B), whereas cecal IL-10 concentrations did not significantly differ between the groups (data not shown).

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Figure 4. Altered cecal cytokine profile after prebiotic treatment. Cecal interleukin-(IL)-1β (A) concentrations (ng/100 mg tissue) and transforming growth factor β (TGF-β) concentrations (pg/100 mg tissue) from transgenic rats treated with either water or oral prebiotic Synergy 1 prior to the development of histologic inflammation. Each group consisted of eight rats. Values are presented as mean ± SEM, * P < 0.05 versus untreated transgenic rats.

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Mesenteric Lymph Node Cell Cytokine Responses

We then analyzed the in vitro cytokine responses of MLN cells to cecal bacterial lysates (CBL), because MLN drain the inflamed cecum and proximal colon, as described.19 Stimulation with CBL induced higher interferon-γ responses in MLN cells from untreated transgenic rats compared with prebiotic-treated animals (P < 0.01; Figure 5). No differences in IL-10 production were found for CBL-stimulated MLN cells from prebiotic- versus untreated transgenic rats (data not shown).

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Figure 5. Decreased interferon-γ secretion after prebiotic treatment. Specific pathogen free transgenic rats were treated with either water or prebiotic Synergy 1 prior to the development of histologic inflammation. After seven weeks, rats were killed and mesenteric lymph nodes (MLN) were collected. MLN cells were isolated and cultured in the presence of cecal bacterial lysate at 50 μg/ml protein concentration. Interferon-γ concentrations (ng/ml) were measured in the triplicate supernatants by ELISA. Each group consisted of eight rats. Values are presented as mean ± SEM, ** P < 0.01 versus untreated transgenic rats.

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PCR-DGGE Profiles and FISH Enumeration of Cecal and Colonic Bacteria

A relatively simple microbiota profile was detected in cecal and colonic contents of the HLA-B27 rats that had been administered the prebiotic preparation (Fig. 6). Most strikingly, the profiles of untreated transgenic rats lacked a DNA fragment originating from Bifidobacterium animalis (Fig. 6, as indicated by arrow) that was present in prebiotic-treated transgenic rat profiles. This suggested that the bifidobacterial population had been influenced by prebiotic consumption. The microbiota profile in colonic contents did not differ from that of the cecum (data not shown).

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Figure 6. Altered PCR-DGGE profiles result from prebiotic treatment. Specific pathogen free transgenic rats were treated for seven weeks with either water or prebiotic Synergy 1 prior to the development of histologic inflammation. At necropsy bacterial DNA was extracted from cecal and colonic contents and PCR-DGGE was performed as described in the materials and methods section. The gel shows representative microbiota profiles generated from cecal contents derived from five water-treated and six inulin/oligofructose-treated transgenic rats. Each lane represents a single sample. Arrows indicate 16S rRNA gene fragments originating from Bifidobacterium animalis (>97% identity).

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FISH analysis showed that bifidobacteria were also present in untreated transgenic rats. However, quantitative analysis of cecal and colonic content by FISH also revealed a significant increase of bifidobacteria in prebiotic-treated versus untreated transgenic rats (Table 1), which confirmed the findings of PCR-DGGE. Additionally, FISH analysis showed an increase in Lactobacillus numbers in animals administered the prebiotic preparation (Table 1).

Table 1. Quantitative Bacteriology of Cecal Contents of HLA-B27 Transgenic Rats by Fluorescent in situ Hybridization Seven Weeks After Treatment with Either Water or Prebiotic Synergy 1 Prior to the Development of Histologic Inflammation
Bacterial Group (probe or dve)Total Bacterial Count (Dapi)Clostridium Group XIVa (Erec482)Bacteroides/Prevotella (Bac303)Bifidobacteria (Bifl64)Lactobacilli+enterococci (Lab158)Enterococci (Efs)
  1. Values represent mean ± SEM bacterial counts per gram cecal contents.aP < 0.05 versus untreated group.

Treatment× 109× 109× 108× 107× 107× 107
Water4.4 ± 1.22.6 ± 0.85.1 ± 3.08.8 ± 3.14.1 ± 0.81.7 ± 0.6
Inulin/Oligofructose12.3± 3.46.2 ± 1,98.2 ± 2.688.9 ± 32,8a26.5 ± 5.8a3.7 ± O.9

Cecal SCFA Concentrations

Analysis for the SCFA concentrations in the luminal contents of the cecum indicate that there were no significant differences between untreated and prebiotic-treated transgenic rats. Total cecal SCFA concentrations were 92 ± 17.3 (untreated) and 102 ± 37.7 (prebiotics) μmol/g, and consisted primarily of acetate (76 ± 4.9 untreated versus 82 ± 7.6% prebiotics), propionate (15 ± 3.6 untreated versus 11 ± 5.8% prebiotics), and butyrate (7 ± 1.1 untreated versus 8 ± 3.1% prebiotics). SCFA compositions did not differ significantly between colonic and cecal content (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Members of the gut microbiota do not induce disease in the normal host, whereas chronic intestinal inflammation can develop in the genetically susceptible host in response to the same intestinal bacteria.3 Manipulating the gut microbiota might be of importance to change the natural course of colitis. For example, in several models of experimental colitis, antibiotic treatment decreased the amounts of intestinal disease-inducing bacteria and prevented inflammation6,25,32 whereas increased amounts or the addition of certain strains of colonic probiotic bacteria could prevent disease.10,11,33

We showed in the present study that a combination of chicory-derived long-chain inulin and oligofructose (Synergy 1) reduced the development of intestinal inflammation in SPF HLA-B27 transgenic rats. This is the first study in which prebiotics were used as primary therapy in rodents that are genetically susceptible to chronic intestinal inflammation. The results are in agreement with findings by Schultz et al, who reported that a synbiotic preparation that contained long-chain inulin plus a probiotic cocktail of lactobacilli and bifidobacteria attenuated colitis development in SPF HLA-B27 rats.34 Our results also confirmed previous studies in induced models of colitis, in which prebiotic treatment resulted in attenuation of colitis. In rats, oral inulin decreased distal colonic lesions in dextran sodium sulfate (DSS)-induced colitis,17 and oral fructo-oligosaccharides (FOS) decreased trinitrobenzene sulfonic acid (TNBS)-induced colitis, although its effects were apparent only after 14 days of feeding.18 No effects were seen after oral administration of trans-galacto-oligosaccharide started before or during TNBS-colitis induction,35 indicating that not all prebiotic preparations are equally effective in preventing disease. Studies showing efficacy of prebiotics in IBD patients are emerging; a small study with synbiotics using Synergy 1 plus Bifidobacterium longum reported a reduced colonic inflammation in patients with UC.36 Inulin administration to UC patients with pouchitis after colectomy led to a reduction of inflammation in the pouch mucosa.37 In patients with mild to moderate ulcerative colitis, prebiotics such as germinated barley food extracts decreased clinical and endoscopic evidence of inflammation in both a small placebo-controlled trial as well as in an open-label study.38,39

Interestingly, the prebiotic combination also prevented arthritis, a common feature in HLA-B27 TG rats.4 Similar to colitis, development of arthritis is dependent on the presence of commensal intestinal bacteria.5 Protective effects of prebiotics have not been described before in experimental or rheumatoid arthritis, however administration of probiotic bacteria ameliorated induced experimental arthritis.40–41

Changes in the gut microbiota composition, especially growth promotion of endogenous beneficial bacteria induced by prebiotics,16 may contribute to colitis and arthritis prevention. In this study we analyzed intestinal microbiota changes using molecular techniques such as PCR-DGGE and FISH. PCR-DGGE is a well-established qualitative screening method to detect alterations in the composition of the intestinal microbiota. PCR-DGGE showed that there were no differences in the microbial profiles of cecal and colonic content of the same animal. However, this technique did show alterations to the intestinal microbiota profile induced by prebiotic treatment in HLA-B27 transgenic rats. Prebiotic administration to transgenic rats resulted in the detection of a 16S rRNA gene fragment that was undetectable in untreated transgenic rats. Sequence analysis revealed that this band represented Bifidobacterium animalis. Based on this qualitative finding we then performed a quantitative microflora analysis of the cecal and colonic content by FISH. Using this method we confirmed the PCR-DGGE analysis; the prebiotic combination significantly increased cecal and colonic bifidobacteria. In contrast to PCR-DGGE, FISH analysis did detect bifidobacteria in untreated transgenic rats. This result indicated that FISH was more sensitive than PCR-DGGE in detecting bifidobacteria in untreated transgenic rats and secondly, that the prebiotics had increased endogenous bifidobacteria present in transgenic rats. In addition, FISH also detected increased amounts of endogenous lactobacilli induced by prebiotics, as shown in Table 1. Changes in mucosa-associated microflora in humans after feeding this prebiotic combination were similar to the changes in luminal microbiota of the prebiotic-treated transgenic rats in the present study. Synergy 1 increased the amount of mucosa-associated bifidobacteria and lactobacilli in these individuals.42 The same bifidogenic effect in cecal contents of transgenic rats was found by PCR-DGGE after feeding probiotics plus inulin,34 however this study did not include FISH analysis. Interestingly, in this synbiotic study probiotic bacteria could not be detected in the stool, indicating that inulin likely mediated the changes in cecal microbiota. The oral administration of various probiotic bacteria has been effective in experimental colitis as well as human IBD.11,13,33 Oral administration of lactobacilli and bifidobacteria decreased colitis in TNBS-induced colitis and caused similar changes in cecal microbiota composition as those induced by FOS. Adding lactobacilli and bifidobacteria to topical butyrate solutions in rats with TNBS-induced colitis was necessary to induce a similar disease improvement as shown by oral FOS administration.18 Taken together, these studies all suggest that increased amounts of certain intestinal bacteria are associated with prevention of colitis and arthritis in the present and other studies, and may (partially) mediate the beneficial effects of prebiotic administration.

B. vulgatus and possibly other anaerobic bacteria play a pathogenic role in colitis development in transgenic rats and in human IBD.8,20 Moreover, metronidazole attenuated colitis in this model6 and is an effective adjunct therapy in Crohn's colitis.32 Probiotic bacteria are reported to suppress the growth of pathogenic intestinal bacteria. Bifidobacterium infantis suppressed growth of B. vulgatus.43 Similarly, bifidobacteria-fermented milk decreased fecal B. vulgatus concentrations in UC patients.44 Inulin or oligofructose alone also reduced luminal Clostridium difficile populations in healthy volunteers, which was associated with a bifidogenic effect.45 However, despite a trend toward a decreased intestinal population of Bacteroides/Prevotella relative to the total amount of luminal bacteria after feeding Synergy 1 in transgenic rats in the present study, this trend did not reach statistical significance. We also did not observe any significant differences in the amounts of cecal Clostridium-group XIVa between the experimental groups. These results are in agreement with those in humans; Synergy 1 fed to nonIBD patients for two weeks did not change the amount of mucosa-associated bacteroides and clostridia.42 Taken together, this would suggest that Synergy 1 and/or resident bifidobacteria and lactobacilli did not mediate protection by suppressing the growth of disease-inducing Bacteroides species or clostridia.

Prebiotics that escape digestion in the upper intestine are important sources of energy for cecocolonic bacteria. The end-products of prebiotic fermentation by these organisms lead to the production of lactate and SCFA, such as acetate, propionate and butyrate. Changes in SCFA production, especially increased luminal butyrate concentrations, can also contribute to down-regulation of pro-inflammatory cascades in models of experimental colitis and human IBD.17,39,46–47 However, to our surprise we did not detect significant differences in luminal SCFA concentrations in prebiotic- versus untreated transgenic rats. Our rats were killed around the same time of the day and did not fast, which could otherwise have explained the lack of a butyrogenic effect. These results indicate that Synergy 1 did not mediate protection by changes in luminal SCFA concentrations.

A widely accepted hypothesis for the pathogenesis of chronic intestinal inflammation, such as IBD, is an overly aggressive Th-1 mediated cytokine response to commensal bacteria. We recently showed that cecal bacteria and their products induce a pro-inflammatory cytokine response in MLN cells and cecal mucosa from colitis-susceptible transgenic rats. In contrast, the same bacteria induce anti-inflammatory cytokines, such as an increase of IL-10 and TGF-β in disease-tolerant nontransgenic littermates.19 In our current study we report that CBL-stimulated MLN cells derived from prebiotic-treated transgenic rats produced less interferon-γ than those from untreated transgenic rats. Prebiotic treatment also significantly decreased cecal IL-1β production. Decreased production of cecal IL-1β and IFN-γ in MLN from prebiotic-treated transgenic rats correlated with less cecal and colonic inflammation. Decreased production of mucosal pro-inflammatory cytokines is in agreement with probiotic treatment studies in experimental colitis10,11,33 and refractory pouchitis after colectomy for UC.13

Of major interest, we report for the first time in experimental colitis that prebiotic treatment of transgenic rats induces immunoregulatory effects, such as increased cecal TGF-β. Roller et al reported immunoregulatory effects by the same prebiotic combination in noncolitic F344 rats, in which IL-10 rather than TFG-β was increased in Peyer's patches.48 This finding indicates that prebiotics can exert immunomodulatory effects, either directly or secondarily through increase of intestinal probiotic bacteria. Indeed, probiotic Lactobacillus rhamnosus strain GG orally administered to pregnant and lactating mothers increased the amount of TGF-β in the breast milk.49 TGF-β is important for the regulation of intestinal inflammation. Colitis develops in SCID mice restored with a mixture of CD45RBhi CD4 and CD45RBlow CD4 cells that were treated with anti-TGF-β antibodies.50 Furthermore, intranasal administration of a TGF-β-containing plasmid prevented TNBS-induced colitis.51 Additionally, a milk-casein-based diet containing TGF-β decreased colitis in B27 transgenic rats.52 Therefore, increased mucosal TGF-β could mediate some of the beneficial effects of prebiotics in transgenic rats.

In conclusion, we showed in HLA-B27 transgenic rats that oral administration of the prebiotic combination long-chain inulin and oligofructose significantly reduced the development of colitis and prevented arthritis. Protection induced by these prebiotics correlated not only with changes in intestinal microbiota toward an increase of endogenous intestinal bifidobacteria and lactobacilli, but was also associated with immunomodulatory effects. Although prebiotics can mediate protective effects through probiotic bifidobacteria and lactobacilli, it is also possible that prebiotics are beneficial by direct immunosuppressive or immunomodulatory effects on the host. Dietary therapy using prebiotics forms an alternative to oral probiotic administration or can be used in combination with exogenous probiotic therapy. Our study indicates that this prebiotic combination shows potential as an easy-to-administer and cost-effective novel treatment of human inflammatory bowel diseases.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors thank Charlotte Walters of the ImmunoTechnology Core of the Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, for cytokine measurements, and Desmond McDonald, Lisa Wiltron and Sue Tonkonogy of the College of Veterinary Medicine, North Carolina State University, Raleigh, and Marga Wester, Department of Medical Microbiology, University of Groningen, The Netherlands, for their expert technical support.

This work was supported by NIH grant RO3 DK 58666, Aid of Research grant G599000767 from the Crohn's and Colitis Foundation of Canada and a grant G533000124 from the University Hospital Foundation for Dr. Dieleman, and AGIKO-grant 920-03-300 to Dr. F. Hoentjen from The Netherlands Organization for Health Research and Development, The Netherlands. Jennifer Snart and Gerald Tannock were supported by the Alberta Added Value Corporation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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