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Keywords:

  • Bifidobacterium;
  • Lactobacillus;
  • Bacterial DNA;
  • Inflammatory bowel disease;
  • Immune modulation

Abstract

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

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1β and an increase of interleukin-10.


1Introduction

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

Inflammatory bowel diseases (IBD) are characterized clinically by chronic inflammation in the intestine. The etiology of IBD remains unclear, even if human and experimental studies indicate that genetic host susceptibility, gut mucosal immune response and enteric bacteria may contribute to the pathogenesis of IBD [1]. The importance of bacteria in sustaining inflammation in IBD is further supported by the clinical experience that antibiotics reduce disease activity. Evidence exists that in patients with active IBD there is a breakdown of normal tolerance to the resident enteric bacteria flora which leads to an excessive mucosal immune response against enteric bacteria [2]. Interleukin 10 (IL-10) is a cytokine of particular therapeutic interest in IBD since it plays a key role in the control of inflammatory responses to intestinal antigens and can restore tolerance of T cells to resident intestinal bacteria. Therapeutic benefit of IL-10 administration has been reported from experimental studies with IL-10-deficient mice [3] and in clinical trials with IBD patients [4] albeit that the clinical usefulness of IL-10 is limited for technical reasons related to the organ-specific delivery [5]. Thus, a therapeutic approach based on IL-10 which overcomes these limitations would provide great perspectives.

The important role of bacteria in the pathogenesis of IBD has suggested the possibility of preventing or treating these disorders by manipulating the intestinal microflora with probiotic treatment. Members of the genera Lactobacillus and Bifidobacterium are amongst the most common microorganisms in the human gastrointestinal tract. Evidence exists that these bacteria exert health-promoting activity because they play an important role in the control of the intestinal microflora and in maintenance of its normal state [6]. Because of their beneficial properties Lactobacillus and Bifidobacterium strains are commonly used in dairy and pharmaceutical probiotic preparations.

VSL#3 (VSL Pharmaceuticals, Ft. Lauderdale, FL, USA) contains 450 billion per sachet of a viable lyophilized bacterial mixture, including three strains of bifidobacteria (Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis), four strains of lactobacilli (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum) and one strain of Streptococcus salivarius subsp. thermophilus. In recent clinical studies, its efficacy was shown as maintenance treatment and prophylactic therapy in patients with IBD [7,8]. In addition, Ulisse et al. [9] showed an increased tissue level of IL-10 in pouch patients treated with this probiotic therapy.

The therapeutic benefit of VSL#3 was also shown by experimental studies in IL-10 knock-out mice [10,11]. Madsen et al. [11] found that an unknown, soluble factor secreted by the bacteria of this probiotic preparation enhanced epithelial barrier function and resistance to pathogenic bacterial invasion in T84 epithelial cells. In addition, sonicates – containing soluble cytoplasmic fraction – of each Lactobacillus and Bifidobacterium VSL#3 strain did not induce IL-8 production in HT29 epithelial cells [12], but stimulated IL-10 secretion in peripheral blood mononuclear cells (PBMC) (U. Helwig, personal communication). In the past few years, immune effects of unmethylated DNA sequences (CpG motifs), predominantly present in bacteria, have been described [13]. Considering the high GC content of Bifidobacterium and – to a lesser extent –Lactobacillus chromosomal DNA, we decided to examine the effect of VSL#3 bacteria DNA on IL-1β, IL-6 and IL-10 secretion and to evaluate the response induced by total bacterial DNA isolated from feces of healthy subjects before and after 2 weeks of VSL#3 treatment.

2Materials and methods

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

2.1Bacterial strains and culture conditions

The VSL#3 bacterial strains used in this study are the following: B. infantis BI07, B. breve BBSF, B. longum BL04, L. acidophilus LA14, L. delbrueckii subsp. bulgaricus LB31, L. casei LC10, L. plantarum LPT and S. salivarius subsp. thermophilus TA061, designated S. thermophilus throughout this paper. Bifidobacterium and Lactobacillus strains were grown anaerobically (Anaerobic System, Model 2028, Forma Scientific, Marietta, OH, USA) in MRS medium (Difco, Detroit, MI, USA) supplemented with 0.05%l-cysteine, at 37°C. S. thermophilus was cultured anaerobically in M17 medium (Difco) at 37°C. Enumeration of probiotic bacteria in fecal samples was carried out as previously reported [14]. Counting of the probiotic bacterial groups was performed by spreading dilutions onto plates containing the following selective agar media: LAMVAB [15] for Lactobacillus, RB [16] for Bifidobacterium and ST [17] slightly modified by adding bromocresol purple (30 mg l−1), bromocresol green (100 mg l−1) and nalidixic acid (30 mg l−1) for S. thermophilus. Plates were anaerobically incubated at 37°C for 24–48 h. Bacterial concentrations were expressed as CFU g−1 dry feces.

Specific identification of VSL#3 B. infantis and B. breve strains was performed by polymerase chain reaction (PCR)-mediated amplification using the strain-specific primers and experimental conditions previously described [14]. Quantification was carried out by direct amplification of 30–50 colonies randomly selected from the highest dilution plates of RB medium. The ratio between the number of colonies which gave positive amplicons with the strain-specific primers and the total number of colonies analyzed by PCR was calculated, and successively related to the total number of colonies grown on the highest dilution plate.

2.2DNA preparation

Isolation of genomic DNA from pure cultures of the probiotic bacteria was performed as previously described [18]. In order to obtain complete cell disruption, the method was slightly modified by prolonging the enzymatic lysis from 1 to 3 h and grinding with glass beads (150–212 μm, Sigma, St. Louis, MO, USA). Purification of total bacterial DNA from feces of two healthy donors, before (t0) and after 2 weeks of probiotic ingestion (t1), was performed using the QIAamp DNA stool kit (Qiagen, Milan, Italy). Concentration and purity of all DNA preparations were determined by measuring OD260 absorbance and OD260/280 ratio, respectively. Only DNAs with an OD260/280 ratio >1.8 were used. DNAs were also assayed for lipopolysaccharide (LPS) content using the Limulus amebocyte assay (QCL-1000, BioWhittaker, Walkersville, MD, USA). LPS content was less than 0.01 U endotoxin μg−1 DNA.

2.3Cell preparation and cell culture

For each group of experiments, PBMC were isolated from peripheral blood of four healthy volunteers by density gradient centrifugation (1.077 g ml−1) (Lymphoprep, Nycomed Pharma, Oslo, Norway). Cells were resuspended in RPMI 1640 culture medium (Life Technologies, Paisley, UK) supplemented with 10% (v/v) heat-inactivated (56°C, 1 h) fetal bovine serum, gentamicin (50 μg ml−1) (Sigma), penicillin–streptomycin (1%) and sodium pyruvate solution (0.23 mmol l−1) (Sigma) (complete medium). All compounds were purchased endotoxin tested. Cells were cultured in complete medium in a concentration of 1×106 cells ml−1 in 96-well plates (Nunc, Roskilde, Denmark) in a 5% CO2-humidified incubator at 37°C. All experiments were performed in duplicate.

2.4Stimulation experiments

Genomic DNAs from the probiotic strains were applied to PBMC at the following concentrations: 3, 6.25, 12.5, 25, 50, and 70 μg ml−1 for 24 h. For kinetics, genomic DNA of two strains, L. casei and B. breve, was applied at a concentration of 20 μg ml−1 over a period of 24, 48 and 72 h. Total bacterial genomic DNAs, isolated from feces at times t0 and t1, were applied to PBMC from three donors for 24 h. As a positive control, LPS (Sigma) at a concentration of 20 ng ml−1 was used. As a negative control, methylated DNA from fish sperm (Roche, Italy) was applied at the same concentrations used for bacterial DNA. At the indicated time points, supernatants were harvested for cytokine measurement, centrifuged and stored at −20°C until assay.

2.5Enzyme-linked immunosorbent assay (ELISA)

Culture supernatants were analyzed for IL-1β, IL-6 and IL-10 with commercially available anti-human monoclonal ‘capture’ antibodies to IL-1β (MAB601) (R&D Systems, Abingdon, UK), IL-6 (18871D) and IL-10 (18551A) (Pharmingen, San Diego, CA, USA), and specific biotinylated anti-human monoclonal ‘detection’ antibodies to IL-1β (BAF201) (R&D systems), IL-6 (18882D) and IL-10 (18562D) (Pharmingen). o-Phenylenediamine buffer/H2O2 (Sigma, Steinheim, Germany) was used as a substrate. Extravidin-peroxidase (Sigma) was applied at a concentration of 1:1000. Human recombinant IL-1β (201-LB-005) (R&D systems), human recombinant IL-6 (1966T) and IL-10 (19701V) (Pharmingen) were used as standards. The optical density values of the samples were read at 490 nm on an ELISA plate reader. The detection limit of the assay was 15.7 pg ml−1 for IL-1β and 31 pg ml−1 for IL-6 and IL-10.

2.6Statistical analysis

Experimental data are expressed as mean±S.E.M. The statistical significance of differences in mean values was determined with Student's t-test. Differences were considered to be significant at P<0.05.

3Results

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

3.1Probiotic bacterial DNA elicits IL-1, IL-6 and IL-10 production

We investigated the implication of pure genomic DNAs from VSL#3 Bifidobacterium, Lactobacillus and S. thermophilus strains in the release of the cytokines IL-1β, IL-6 and IL-10. The first encouraging results were obtained by challenging PBMC from healthy human subjects with 70 μg ml−1 of bacterial DNA (Fig. 1). This dose was chosen accordingly to previous experimental results which established 50–100 μg ml−1 as the maximal stimulatory dose range of Escherichia coli DNA [19]. To avoid stimulatory effects due to contamination of bacterial proteins or LPSs, all DNA preparations used in the experiments had purity values (OD260/280>1.8) and LPS contents (<0.01 U endotoxin μg−1 DNA) previously demonstrated to be inactive in immune stimulation [20]. Fish sperm DNA did not have stimulatory effects on PBMC (data not shown).

image

Figure 1. PBMC from four healthy donors were cultured with 70 μg ml−1 of each genomic DNA extracted from VSL#3 bacteria. IL-1β, IL-6 and IL-10 production was measured in supernatants. The cytokine response to bacterial DNAs was compared to that obtained with LPS (20 ng ml−1): *P<0.05, **P<0.01.

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Genomic DNA induced strain-specific immune effects: by comparison with LPS, genomic DNAs of VSL#3 B. infantis, S. thermophilus, L. plantarum and L. bulgaricus stimulated a pronounced IL-1β secretion, whereas B. longum, L. acidophilus and L. casei DNAs induced a significantly lower IL-1β production. DNAs of B. breve, B. infantis and S. thermophilus elicited IL-6 levels comparable to that obtained by LPS, while genomic DNAs of B. longum and Lactobacillus strains induced significantly lower IL-6 concentrations. Interestingly, DNA isolated from B. breve, B. infantis and S. thermophilus stimulated high secretion of IL-10 which exceeded the LPS-induced IL-10 level up to three-fold.

Subsequently, genomic DNAs of L. casei and B. breve were applied to PBMC at a concentration of 20 μg ml−1 for 24, 48 and 72 h (Fig. 2). The DNAs of these strains were selected as representatives for the three Bifidobacterium and four Lactobacillus strains present in the VSL#3 mixture. The magnitude of IL-1β, IL-6 and IL-10 secretion reached maximal levels after 24 h of stimulation and then remained constant. Genomic DNA of B. breve was confirmed to be a more potent inducer of interleukins than L. casei DNA and was responsible for the highest IL-10 release.

image

Figure 2. PBMC from four healthy donors were incubated with 20 μg ml−1 of genomic DNA of L. casei (closed squares) and B. breve (open squares) for 72 h. As controls medium (open circles) and LPS (20 ng ml−1) (closed circles) were used. Production of cytokines, assayed at intervals of 24 h, was compared to that induced by LPS: *P<0.05, **P<0.01.

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3.2Dose-dependent secretion of interleukins by bacterial DNA

Genomic DNA of each VSL#3 strain was then applied to the PBMC culture system at different concentrations, ranging from 3 to 70 μg ml−1, to determine which dose induced the maximum cytokine secretion (Fig. 3). This dose interval was chosen on the basis of previously described experimental results showing that cytokine production by immune cells could be detected after application of at least 3 μg ml−1E. coli DNA, and optimal stimulation required a bacterial DNA concentration >50 μg ml−1[19].

image

Figure 3. PBMC from four healthy volunteers were challenged with different doses (3–70 μg ml−1) of bacterial DNAs. IL-10 (closed circles) and IL-1β (open circles) secretion was determined after 24 h of incubation. IL-1β and IL-10 values obtained at each dose were compared: *P<0.05, **P<0.01.

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Two different patterns of cytokine response could be clearly distinguished: genomic DNAs of B. longum, S. thermophilus and Lactobacillus strains induced the highest levels of IL-1β at a concentration of 3 μg ml−1, and lower stationary levels when applied at the concentration range of 6.25–70 μg ml−1. In contrast, B. breve and B. infantis genomic DNAs showed a dose-dependent enhancement of IL-1β, reaching a plateau at concentrations of 12.5 μg ml−1. It is noteworthy that genomic DNAs of L. acidophilus, L. casei and S. thermophilus induced significantly lower levels of IL-1β than did DNAs of the other strains. The IL-10 production pattern was similar to that of IL-1 showing the highest values after challenge with 3 μg ml−1 of Lactobacillus strains, B. longum and S. thermophilus genomic DNAs, and lower stationary levels at the other doses applied. Genomic DNAs of B. breve and B. infantis induced a dose-dependent IL-10 secretion, reaching a plateau at a concentration of 12.5 μg ml−1. In comparison with the other genomic DNAs, B. breve and B. infantis DNAs appeared to be the most potent IL-10 inducers, provoking a three-fold higher IL-10 secretion. Furthermore, IL-10 production stimulated by DNAs of these two strains exceeded IL-1β production over the whole dose range. Incubation with L. casei and S. thermophilus DNAs showed a weaker but analogous response. Conversely, B. longum and L. acidophilus DNAs induced significantly higher levels of IL-10 than IL-1β only at the concentration of 3 μg ml−1. Challenge with genomic DNAs of L. plantarum and L. bulgaricus showed comparable levels of IL-1β and IL-10 at 3 μg ml−1, but at higher doses (6.25–70 μg ml−1) IL-1β exceeded significantly IL-10. The strain-related patterns, previously described, were also shown for IL-6. DNAs of B. breve and B. infantis induced in a dose-dependent way, and the other DNAs elicited the highest production at the lowest concentration (data not shown).

3.3Stimulatory effects of fecal bacterial DNA on interleukin production

Two healthy volunteers ingested for 2 weeks two sachets daily of VSL#3 (equivalent to 900 billion viable microorganisms), and feces were collected before (t0) and after the probiotic administration (t1). PBMC from three donors were challenged with a concentration of 5 μg ml−1 of total bacterial DNA extracted from feces at time t0 and t1 (Fig. 4). Fecal concentrations of lactobacilli, bifidobacteria and S. thermophilus, determined by culture techniques, showed an increase up to 3 logs after the probiotic treatment. At time t0, bifidobacterial titer was 2.5×107 CFU g−1 and increased to 4×109 CFU g−1 at time t1. t0 and t1 counts of lactobacilli were 3×105 CFU g−1 and 1.1×106 CFU g−1, while for S. thermophilus values of 7×103 CFU g−1 and 8×106 CFU g−1 were found. The availability of strain-specific primers for VSL#3 B. breve and B. infantis[14] allowed their PCR detection and quantification in the fecal samples. B. breve and B. infantis strains were detected only after probiotic treatment at concentrations of 3×108 CFU g−1 and 1×108 CFU g−1, respectively, demonstrating that the increased fecal concentration of Bifidobacterium is related to the presence of these exogenous strains. A pronounced difference in the cytokine response was observed after stimulation with t0 and t1 total bacterial DNAs extracted from feces of the two volunteers treated with VSL#3. DNA isolated from feces before the probiotic administration induced higher levels of IL-1β than IL-10, while DNA from feces after probiotic treatment enhanced the IL-10 secretion but reduced the IL-1β secretion. IL-6 production was not altered.

image

Figure 4. PBMC from three healthy donors were cultured with 5 μg ml−1 of total bacterial DNA extracted from feces of a healthy subject before and after 2 weeks of VSL#3 treatment. IL-1β (open circles) and IL-10 (closed circles) production was determined after 24 h of incubation.

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4Discussion

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

The gut represents a complex ecosystem in which the intestinal microflora and the host interact in order to maintain an immunologically balanced intestinal inflammatory response (‘physiologic inflammation’). Several clinical and experimental studies indicate that the gut inflammation may be associated with an imbalance in intestinal microflora, with a relative predominance of ‘aggressive’ bacteria and an insufficient concentration of ‘protective’ species. A recent therapeutic strategy in IBD involves manipulation of the local microenvironment by oral administration of probiotic bacteria, in order to restore the microbial balance [21]. The encouraging clinical results in IBD patients treated with the probiotic preparation VSL#3 have prompted us to elucidate the interaction between intestinal mucosa and microorganisms. Of particular interest are the results concerning the ability of VSL#3 probiotic bacteria to increase significantly the pouch tissue level of IL-10, and to reduce the proinflammatory cytokine levels [9]. Furthermore, a recent study demonstrated that the protective effect of probiotic bacteria could be mediated by release of a soluble factor(s) that alters epithelial permeability and protects against pathogenic bacterial invasion [11]. These data raise the question whether this soluble proteinaceous factor or another different cytoplasmic bacterial component(s), such as DNA, could be involved in cytokine induction and modulation. Actually, substantial evidence exists that bacterial CpG motifs induce the immune response against challenge with a wide variety of pathogens and have therapeutic activity in murine models [22] and in human clinical trials [23].

In this perspective, we first investigated the implication of pure genomic DNAs from VSL#3 Bifidobacterium, Lactobacillus and S. thermophilus strains in the release of the cytokines IL-1β, IL-6 and IL-10 using a human culture model. The results show that bacterial genomic DNA induces a remarkable strain-specific immune response. A second series of experiments was designed to study whether the beneficial immune effects observed during the VSL#3 treatment could be partly due to changes of the total bacterial DNA released by a modified microflora. In this respect, bacterial DNA extracted from feces of healthy subjects collected before and after the probiotic administration was applied to the PBMC culture model. We consider this approach more appropriate to mimic the in vivo situation than investigating the stimulatory capacity of the DNA recovered from the VSL#3 mixture, as the intestinal colonization of exogenous bacteria is not strictly related to their concentrations in the pharmaceutical formulation but strain- and host-dependent. Interestingly, these experiments showed a diverse IL-1β and IL-10 cytokine production after stimulation with bacterial DNA isolated from feces before and after probiotic treatment. Bacterial DNA from microflora before probiotic ingestion induced higher IL-1β than IL-10 secretion, contrarily, total bacterial DNA from a probiotics-enriched microflora led to higher production of IL-10 than IL-1β.

Our data suggest that genomic DNA released by exogenous bifidobacteria could provide a stimulus for mucosal IL-10 production. Taking into account that the manufacturing process and the gastroenteric passage affect the bacterial viability, the results obtained in this study may indicate a potential therapeutic effect of genomic DNA released by the dead bacteria ingested during the probiotic administration. Several articles describe different cytokine responses to CpG motifs present in bacterial DNA, some of these motifs exerting a more pronounced immunomodulatory effect than others [13]. It would be interesting to know whether the strain-specific cytokine release demonstrated in this study is related to the genomic sequence of each strain. However, the genome sequences of the VSL#3 probiotic strains have not yet been characterized and thus it is not possible to predict the redundancy of the different CpG motifs. At present, the only useful information available is the GC content. The high GC percentage reported in the literature for the Bifidobacterium species present in VSL#3 (58–61%) strongly suggests that the DNA composition may account for the cytokine response, and in particular that a high GC content favors IL-10 secretion.

To our knowledge this is the first report to show the immunomodulatory effects of genomic DNA of probiotic bacteria. The observed differences in kinetics and magnitude of IL-1β and IL-10 release in response to bacterial DNAs provide interesting information about the influence of gut microflora and/or bacterial components on the intestinal mucosal immune response.

Acknowledgements

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

K.M.L. is supported by a grant from the Fondazione del Monte di Bologna e Ravenna.

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  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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