Bifidobacterium strains suppress in vitro the pro-inflammatory milieu triggered by the large intestinal microbiota of coeliac patients
© Medina et al; licensee BioMed Central Ltd. 2008
Received: 27 July 2008
Accepted: 03 November 2008
Published: 03 November 2008
Coeliac disease (CD) is an enteropathy characterized by an aberrant immune response to cereal-gluten proteins. Although gluten peptides and microorganisms activate similar pro-inflammatory pathways, the role the intestinal microbiota may play in this disorder is unknown. The purpose of this study was to assess whether the faecal microbiota of coeliac patients could contribute to the pro-inflammatory milieu characteristic of CD and the possible benefits of bifidobacteria.
The effect of faeces of 26 CD patients with active disease (mean age 5.5 years, range 2.1–12.0 years), 18 symptom-free coeliac disease (SFCD) patients (mean age 5.5 years, range 1.0–12.3 years) on a gluten-free diet for 1–2 years; and 20 healthy children (mean age 5.3 years, range 1.8–10.8 years) on induction of cytokine production and surface antigen expression in peripheral blood mononuclear cells (PBMCs) were determined. The possible regulatory roles of Bifidobacterium longum ES1 and B. bifidum ES2 co-incubated with faecal samples were also assessed in vitro.
Faeces of both active CD and SFCD patients, representing an imbalanced microbiota, significantly increased TNF-α production and CD86 expression in PBMCs, while decreased IL-10 cytokine production and CD4 expression compared with control samples. Active CD-patient samples also induced significantly higher IFN-γ production compared with controls. However, Bifidobacterium strains suppressed the pro-inflammatory cytokine pattern induced by the large intestinal content of CD patients and increased IL-10 production. Cytokine effects induced by faecal microbiota seemed to be mediated by the NFκB pathway.
The intestinal microbiota of CD patients could contribute to the Th1 pro-inflammatory milieu characteristic of the disease, while B. longum ES1 and B. bifidum ES2 could reverse these deleterious effects. These findings hold future perspectives of interest in CD therapy.
Coeliac disease (CD) is an enteropathy characterized by an aberrant immune response to ingested wheat-gluten proteins (gliadins) and related prolamins of rye and barley, occurring in genetically predisposed (HLA-DQ2/DQ8) individuals. The pathogenesis of CD involves interaction with genetic, immunological and environmental factors. HLA-DQ2/DQ8 molecules of antigen-presenting cells bind and present gluten peptides to lamina propria CD4+ T cells, triggering a T helper 1 (Th1) biased immune response, mainly with interferon gamma (IFN-γ) production, which enhances tumour necrosis factor alpha (TNF-α) production and plays a crucial role in damaging the intestinal mucosa [1, 2]. In addition, events leading to CD involve activation of innate immunity mediated by interleukin (IL)-15, and are characterized by expansion of intraepithelial TCRγ/δ + and CD+8 TCRα/β + lymphocytes, which are cytotoxic for epithelial cells and also contribute to tissue damage . The intestinal inflammatory milieu characteristic of CD patients depends on the pro-inflammatory cytokines produced during abnormal response to gluten, involving several intracellular signal transduction pathways, such as nuclear factor kappa (NF-κ) B, the interferon regulatory factor (IRF)-1 and signal transducer and activator of transcription [4–6]. NFκB pathway is a crucial target in the propagation of inflammatory responses triggered by cytokines (TNF-α and IFN-γ) and microbial pathogens recognised by Toll-like receptors located in intestinal epithelial and innate immune cells . IκB, a strong regulator of NFκB, is induced by lypopolysaccharide of Gram-negative bacteria, as well as by TNF-α, leading to transcription of genes that contribute to the inflammatory process. Type I interferon IRF-α, which is a cytokine produce by infected cells through the NFκB pathway, induces IFN-γ production and thereby IRF-1 expression, promoting a Th1 response in the CD small intestinal mucosa [4, 8]. Increased production of pro-inflammatory cytokines by cells of the innate immune system could also favour the recruitment of lymphocytes into the lamina propria and epithelium, contributing to full expression of the disease . These pathological mechanisms lead to typical CD lesions, characterized by a massive intraepithelial infiltration of lymphocytes, crypt hyperplasia and villous atrophy . Although CD is considered to be the commonest lifelong digestive disorder, the only therapeutic alternative available for CD patients is adherence to a strict gluten-free diet. Poor compliance and associated complications of the disease demand alternative therapeutic strategies.
There is a lack of research into the role of the intestinal microbiota in CD  despite the fact gliadin peptides and microorganisms seem to activate similar pro-inflammatory pathways. There have been recent reports of alterations in the composition of the faecal and duodenal microbiota of CD children in comparison with healthy controls [10, 11]. Bifidobacterium populations were significantly lower in faecal samples of active CD children and also tended to be lower in biopsies when compared with control subjects (, Nadal, Medina, Donat, Ribes-Koninckx, Calabuig & Sanz, unpublished). Specific Bifidobacterium strains have been acknowledged for their anti-inflammatory and regulatory properties by inducing IL-10 production and regulating the Th1/Th2 balance [12, 13]. This has led to certain strains being proposed for use as probiotics, to treat or prevent chronic inflammatory conditions like inflammatory bowel diseases but not CD [9, 14].
The aim of the present work was to assess whether alterations in microbiota of the large intestine, corresponding to children with active and non-active CD, could contribute to activate immune responses and induce the pro-inflammatory milieu associated with CD in vitro using peripheral blood-mononuclear-cells. In addition, the potential role that selected Bifidobacterium strains can play in suppressing the intestinal pro-inflammatory milieu common to these patients was evaluated, as well as their possible mechanism of action.
Subjects and faecal sampling
Altogether 64 children were included in the study: 26 CD patients with active disease (mean age 5.5 years, range 2.1–12.0 years) on a normal gluten-containing diet, 18 symptom-free coeliac disease (SFCD) patients (mean age 5.5 years, range 1.0–12.3 years) on a gluten-free diet for 1–2 years, and 20 healthy children (mean age 5.3 years, range 1.8–10.8 years) without known food intolerance. CD was diagnosed on the basis of clinical symptoms, positive serology markers (antigliadin and antitransglutaminase antibodies) and signs of severe enteropathy by duodenal biopsy examination and positive response to a gluten-free diet. SFCD patients showed negative serology markers and normal duodenal mucosal villous architecture. The children included in the study were not treated with antibiotics for at least one month before the sampling time. The study was conducted in accordance with the ethical rules of the Helsinki Declaration (Hong Kong revision, September 1989), following the EEC Good Clinical Practice guidelines (document 111/3976/88 of July 1990) and current Spanish law which regulates clinical research in humans (Royal Decree 561/1993 regarding clinical trials). Children were enrolled in the study after written informed consent obtained from their parents.
Faecal samples were collected from the three groups of children under study (2 g wet weight), diluted 10-fold in phosphate-buffered saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, [pH 7.2]) and homogenized in a Lab Blender 400 stomacher for 3 min (Seward Medical London, UK). Aliquots of this dilution were kept at -80°C for further immunologic studies.
Bacterial strains and culture conditions
The strains Bifidobacterium longum ES1 (CECT 7347) and Bifidobacterium bifidum ES2 (CECT 7365) used in the present study were isolated from faeces of healthy babies under breast-milk feeding as described elsewhere . Bifidobacteria were identified at species level by partial sequencing of the 16S rRNA gene amplified with the primers Y1 and 1401R for B. longum ES1 and 27F and 1401R for B. bifidum ES2 [16, 17] and the tuf gene amplified with primers BIF-1 and BIF-2 as described elsewhere . Additional primers (27f, Y1, 530f and U-968f) were used for sequencing in an ADN ABI 3700 automated sequencer (Applied Biosystem, Foster City, CA).
Strains were routinely grown in de Man, Rogosa and Sharpe (MRS) broth (Scharlau Chemie S.A., Barcelona, Spain) supplemented with 0.05% (w/v) cysteine (Sigma, St. Louis, MO) (MRS-C) and incubated at 37°C under anaerobic conditions (AnaeroGen; Oxoid, Basingstoke, UK) for 22 h. Cells were harvested by centrifugation (6,000 g for 15 min) during stationary growth phase, washed twice in phosphate buffered saline (PBS, 130 mM sodium chloride, 10 mM sodium phosphate, pH 7.4), and re-suspended in PBS containing 20% glycerol. Aliquots of these suspensions were frozen in liquid nitrogen and stored at -80°C until used. The number of live cells after freezing and thawing was determined by colony-forming unit (CFU) counting on MRS-C agar after 48 h incubation. These constituted the live-cell suspensions used in co-stimulating assays. For all strains tested, more than 90% cells were alive upon thawing and no significant differences were found during storage time (4 months). One fresh aliquot was thawed for every new experiment to avoid variability in the cultures between experiments.
Isolation and stimulation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood of four healthy volunteers (median age 30 years, range 24–40 years) as previously described . Briefly, PBMCs were isolated by centrifugation over a Ficoll density gradient (Amersham Biosciences, Piscataway, NJ), and adjusted to 1 × 106 cells/ml in RPMI 1640 (Cambrex, New York, USA), supplemented with 10% foetal bovine serum (FBS) (Gibco, Barcelona, Spain), 2 mM L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin (Sigma). PBMCs were incubated in 24-well flat-bottom polystyrene microtitre plates (Corning, Madrid, Spain) and stimulated by either faeces (30 μl), bifidobacterial cell suspensions (106 CFU/ml) or their combination, at 37°C under 5% CO2 for 24 h. Bifidobacterial cell suspensions were washed and re-suspended in fresh PBS prior use for PBMC stimulation. Bacterial growth was not detected during co-incubation of neither faeces or bifidobacterial cell suspensions with PBMCs as determined by colony-forming unit (CFU) counting on Wilkins-Chalgren agar for quantification of total anaerobs (Oxoid, Hampshire, England) and MRS-C agar. Purified lipopolysaccharide (LPS) from E. coli O111:B4 (Sigma, St. Louis, MO) was used at a concentration of 1 μg/ml as a positive control. Non-stimulated PBMCs were also evaluated as controls of basal cytokine production and cell-surface marker expression. To investigate the possible involvement of the NK-κB pathway on the immune effects of faeces and bifidobacteria the stimulation of PBMCs was also carried out in the presence of 20 μg/ml lactacystin (Sigma, St. Louis, MO), which is a specific inhibitor of this pathway. All reagents were tested by the E-toxate test for LPS (Sigma) and shown to be below the detection limit (2 pg/ml). Every fraction used as stimulant was assayed in duplicate. Cell-culture supernatants were collected by centrifugation, fractionated in aliquots, and stored at -20°C until cytokines were analysed.
Cytokine determinations by enzyme-linked immunosorbent assay (ELISA)
Cytokine concentrations of supernatants were measured by ELISA using the Ready SET Go! Kit (BD-Bioscience, San Diego, CA). The pro-inflammatory cytokines TNF-α and INF-γ and the regulatory cytokine IL-10 were analysed. The detection procedures were according to the manufacturer's instructions. The sensitivity of assays for each cytokine was as follows: 4 pg/ml for IFN-γ and TNF-α, and 2 pg/ml for IL-10.
PBMC surface phenotyping and flow cytometric analyses
To evaluate the effects of the faeces, bifidobacterial suspensions and the combination of both on PBMC surface antigen expression, cells of 1 ml well-culture were removed by scraping and incubated with FITC-labelled anti-human CD4, CD8 and CD86 antibodies for 30 min, according to the manufacturer's instructions (eBioscience, San Diego, CA). Then, cells were washed twice, re-suspended in ice-cold PBS and analyzed by flow cytometry on EPICS® XL-MCL flow cytometer (Beckman Coulter, Florida), setting the 0.22 μl filter that eliminates bacteria. Data were analyzed with the System II V.3 software (Beckman Coulter, Florida). Every sample was assayed in duplicate.
Statistical analyses were carried out with Statgraphics plus 5.1 software (Manugistics, Rockville, MD, USA). Significant differences between means were established by ANOVA with post hoc Fisher's least significant difference (LSD) test at P < 0.05. Data are expressed as mean and standard deviation (SD) of duplicate measures determined in four independent experiments.
Results and discussion
Cytokine patterns induced by faeces of CD patients on PBMCs
PBMC surface phenotype induced by faeces of CD patients
In contrast, expression of the co-stimulatory molecule CD86 increased significantly when PBMCs were stimulated with faeces from both active CD and SFCD patients (P < 0.001) compared with healthy controls (Fig 2C). CD86 expression levels differed after stimulation with the three faecal-sample types, with the highest to lowest levels corresponding to active CD faecal samples, followed by SFCD-patient samples, and healthy control samples, respectively. Evidently, the intestinal microbiota of both types of CD patients triggered a higher expression of the costimulatory molecule CD86, which plays a major role in initiating immune responses. CD86, together with CD80 expression on antigen-presenting cells, are essential for T-cell activation through antigen-specific stimulation, contributing to Th1 response . Stimulation of monocyte-derived dendritic cell (DC) maturation by microbial strains or derived products has also been shown to induce expression of CD86, CD83 and CD40 by both commensal and pathogenic bacteria [29, 30]. However, molecules involved in activation of Th1 cells were predominantly expressed on DC exposed to pathogens, parallel to higher pro-inflammatory cytokine production such as TNF-α . In general, Gram-negative bacteria have been shown much more effective in up-regulating maturation markers of DCs than lactic acid bacteria at lower concentrations . Gliadin is also known to induce phenotypic and functional maturation of monocytes, as well as monocyte-derived DCs . Up-regulation of surface expression of CD80, CD83, CD86 and CD40 was induced by stimulation of blood mononcytes with gliadin peptides, particularly in combination with IFN-γ . Therefore, this is the first reported evidence that gut microbiota stimulus, together with gliadin, could contribute to monocyte maturation, thereby, influencing T-cell interaction and activation.
Bifidobacteria suppress the pro-inflammatory cytokine pattern induced in PBMCs by faeces of CD patients
Regulatory IL-10 cytokine production increased significantly (P < 0.050; Fig 4C) by co-stimulation with B. longum ES1 and B. bifidum ES2 together with faeces of both patients (Fig. 4C). B. longum ES1 increased IL-10 production to a significantly higher extent than B. bifidum ES2 (P < 0.050) according to the data obtained using pure cultures of these strains. This would suggest a more powerful regulatory role for the former strain. IL-10 plays an important role in regulating the inflammatory cascade in the intestinal mucosa by its action on antigen-presenting cells via inhibition of cytokine synthesis. IL-10 inhibits the production of Th1 pro-inflammatory cytokines and particularly IFN-γ and in turn TNF-α, which is induced by IFN-γ. Mice genetically deficient in IL-10 develop chronic enterocolitis caused by an unregulated Th1 response to endogenous bacterial flora, which could be counteracted by a strain of Lactococcus lactis secreting recombinant IL-10 . IL-10 administration is also reported to exert beneficial therapeutic effects in Crohn's disease patients by intravenous administration . In the context of CD, recombinant human IL-10 has been shown to suppress Th1-mediated immune responses to gliadin in both treated and untreated coeliac mucosa via down regulation of antigen presentation, reduction of T-cell infiltration and activation, and inducing a long-lasting hyporesponsiveness in gliadin-specific T cells . However, the clinical usefulness of IL-10 is limited for technical reasons related to organ-specific delivery and, therefore, a therapeutic approach based on probiotic strains triggering IL-10 production would overcome these limitations and provide new therapeutic perspectives .
B. longum ES1 and B. bifidum ES2 co-incubated with the faeces of CD patients led to slightly lower expression of surface markers on PBMCs, particularly in the case of CD4 and CD86. The down-regulatory effects of B. bifidum ES2 seemed to be stronger than those of B. longum ES1 but none of these differences were statistically significant (data not shown).
Cytokine production but not PBMC maturation depends on NFkB pathway
CD4, CD8 and CD86 expression was slightly reduced by stimulation with every faecal sample and Bifidobacterium strains in the presence of lactacystin, but the differences were not significant (data not shown). This would suggest that a different activation mechanism, and not the NFκB pathway, mediates surface antigen expression in the assayed conditions. By contrast, supernatants of a Bifidobacterium breve strain are known to influence maturation of monocyte-derived dendritic cells by means of NFkB pathway but not survival and IL-10 production .
In summary, this is the first report that the content of the large intestine of both active and SFCD patients, representing imbalanced gut microbiota, increases pro-inflammatory cytokine production and CD86 activation marker expression in PBMCs as compared to healthy controls. Likewise it decreases anti-inflammatory IL-10 cytokine production, which reflects the Th1 pro-inflammatory milieu characteristic of CD. Moreover, Bifidobacterium strains with immunoregulatory properties have been shown to suppress the pro-inflammatory cytokine pattern induced by the altered colonic microbiota of CD patients and strengthen the immune defences of active and SFCD patients against noxious antigens from the intestinal lumen. This mechanism of action could complement the in vitro protective effects exerted by other Bifidobacterium strain against permeability changes induced by gliadin peptides .
The intestinal microbiota of CD patients could contribute to the Th1 pro-inflammatory milieu characteristic of the disease, while strains of B. longum and B. bifidum could reverse these deleterious effects. Thus, the results reported here offer novel perspectives in the therapy of CD based on immune modulation by the use of specific probiotic strains.
nuclear factor kappa
peripheral blood mononuclear cells
- SFCD patient:
symptom-free CD patient
tumour necrosis factor alpha.
This work was supported by grant AGL2007-66126-C03-01/ALI and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation (MSI). The postdoctoral scholarship to M. Medina from MSI (Spain) and the I3P scholarship to G. De Palma from CSIC (Spain) are fully acknowledged.
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