Intestinal Microbiota and Probiotics in Celiac Disease

MICROBIOTA AND CD

The human gastrointestinal tract is a complex and dynamic environment, sheltering a vast number and variety of commensal microorganisms (19). This balanced microecosystem provides the host a natural defense against invasion of potential pathogens. Recently, research has focused on the important role of the human intestinal microbiota in health and disease (20). Studies on the role of the gut microbiota in CD pathophysiology are still in their early stages. The main findings related to microbiota composition in CD subjects are summarized in Table 1.

CD is a common disorder in both children and adults (21). Nevertheless, our knowledge about the intestinal microbiota of adults with CD is still sparse. Indeed, studies characterizing the microbiota of adult CD patients only began in 2012 (12, 22). A year later, two studies concerning gut microbiota and CD were reported (23, 24). Studies before 2012 were conducted notably with children (13, 25 – 31). A single study of both children and adults reported a slight difference in the percentages of the main phyla between subjects and also a more diverse profile in duodenal biopsy specimens from adults (12). The Firmicutes are the most abundant bacteria in CD adults, while Proteobacteria are present mainly in CD children. Other phyla shared between CD adults and CD children belong to the Bacteroidetes and Actinobacteria. Regarding bacterial genera, CD adults harbor larger numbers of Mycobacterium spp. and Methylobacterium spp., while Neisseria spp. and Haemophilus spp. are more abundant in CD children. Future studies should focus on the similarities between children and adults with CD compared with healthy controls. If the aim is to establish causality, a specific bacterial group might be expected to be pathogenic in both adults and children.

It is still not clear whether an altered microbiota in CD patients could be the cause or the consequence of this disease. It is hypothesized that Gram-negative bacteria in genetically susceptible individuals may contribute to the loss of tolerance to gluten. If a modified microbiota is a result of this disease, the disrupted mucosa overlaid by immature enterocytes could lead to conditions favoring Gram-negative instead of Gram-positive bacterial colonization. Duodenal biopsy specimens from untreated CD children showed higher total and Gram-negative populations than did treated CD and healthy control groups. Furthermore, the counts of Gram-positive bacteria were reduced in CD children (untreated and treated) compared to controls (26). Thus, the proportions of Gram-negative and Gram-positive bacteria seem to be of importance.

The possibility that unfavorable bacteria may colonize the intestinal mucosa indicates the need to evaluate the microbiota from this site. Sampling by biopsy is an invasive method in healthy individuals, while feces still remain the easiest and most noninvasive source of data collection. Even so, the numbers of studies of CD using biopsy specimens and those using fecal samples for microbiota characterization are almost the same so far. In general, clear differences between mucosa-associated microorganisms and fecal microbiota are expected (32, 33). Indeed, Ouwehand and collaborators (33) found 4-times-higher numbers of bifidobacteria in the feces of healthy infants than in the mucosa of a group with rectal bleeding. Corroborating this finding, Di Cagno and coworkers (31) did not find bifidobacteria in biopsy specimens of CD subjects but detected them in fecal samples. In addition, those authors showed that the level microbiota diversity was higher in fecal samples than in biopsy specimens (31). In contrast, Collado et al. (28) showed a high level of correlation between the fecal and biopsy specimen levels of Bifidobacterium, Bacteroides, Staphylococcus, Clostridium coccoides, Clostridium leptum, Lactobacillus, and Escherichia coli in untreated and treated CD patients and a control group. An Akkermansia muciniphila correlation was detected only in controls. Nevertheless, the presented data suggest that the unidentified part of the microbiota, especially in the mucosa, deserves more attention.

Comparison between CD children and controls shows that their microbiota profiles differ. Higher Bacteroides counts are detected in CD children (13, 26) than in controls. Particularly, Bacteroides bacteria are an important fraction of the human gut microbiota, and some species, such as B. vulgatus and B. fragilis, have been found to exhibit proinflammatory effects (34), indicating the importance of investigations of this group at the species level. Data on the levels of Atopobium, Staphylococcus, E. coli, Eubacterium rectale-C. coccoides, the Clostridium histolyticum group, Clostridium lituseburense, and sulfate-reducing bacteria are still contradictory, as there have been reports showing increased levels in CD patients (13) or no difference (26, 28, 30) in comparison to controls. Reports regarding the characterization of the main groups containing probiotic species, such as Lactobacillus and Bifidobacterium, are of great interest. Since these bacteria are associated with protective beneficial mechanisms for the host and anti-inflammatory effects, it is expected that CD subjects would present lower lactobacillus and bifidobacterial levels. Indeed, their levels tend to be lower in CD children than in healthy controls (13). The ratio of beneficial lactobacilli and bifidobacteria to possibly harmful Gram-negative bacteria, such as Bacteroides-Prevotella and E. coli, was found to be significantly higher in controls than in CD children (26). Regarding bifidobacterial diversity in CD patients, contradictory results have been reported: lower diversity in CD children (25) and higher diversity of lactobacilli and bifidobacteria in CD adults (22) than in controls. It has been shown that levels of specific species of lactobacilli and bifidobacteria may be higher, lower, or not detected in CD patients in comparison to controls (Table 1). However, the exact value of this information still remains unclear.

Since intake of gluten is a common characteristic of this disease worldwide, it has been demonstrated that there are clear differences in microbiota composition associated with geographical location (35 – 39). To search for similarities of microbiota among CD patients from different global regions, it may be helpful to identify groups of microbes involved in the development of disease or in alleviating the symptoms of already-present CD.

It is also important to note that gluten is not an issue for CD patients only. Ideally, characterization and comparison of microbiota, genetic background, intestinal permeability, and immune function of subjects presenting CD and other gluten-related disorders, i.e., gluten ataxia, dermatitis herpetiformis, wheat allergy, and nonceliac gluten sensitivity, may advance the knowledge to elaborate treatment options adequate for each condition.

In summary, low levels of lactobacilli and bifidobacteria are the most consistent findings in CD children. Different techniques have been applied to study the mucosal and luminal microbiota of CD patients, providing quantitative (fluorescent in situ hybridization coupled with flow cytometry [FISH-FC] and real-time PCR) and qualitative (denaturing gradient gel electrophoresis [DGGE] and HITChip [human intestinal tract chip]) results. This may contribute to the lack of a consensus about the exact bacterial content in patients with CD, together with the patients' age range, specimen type (biopsy specimen or fecal sample), small number of studies, and small sample size.

PROBIOTICS AND CD

To date, the only therapy for CD is the mandatory and complete exclusion of dietary sources of grains and any food containing gluten (40, 41). However, many patients face difficulties in following a gluten-free diet (GFD). The compliance to therapy varies widely, from around 80% in patients diagnosed before 4 years of age to <40% in those diagnosed after 4 years of age (42).

Recent advances in CD pathophysiology have contributed to the development of novel and promising therapeutic solutions. Thus, many other treatments have been identified, such as genetically modified gluten, zonulin inhibitors, therapeutic vaccines, tissue transglutaminase inhibitors, and, recently, probiotics (43).

According to the FAO/WHO (44), a probiotic is defined as a “live microorganism, which when administered in adequate amounts confers a health benefit on the host.” Abnormalities in the gut microbiome in CD patients have led to the use of probiotics as a promising alternative.

The beneficial effects of probiotics on the gut health of the host can be manifested through (i) production of inhibitory substances against pathogens (hydrogen peroxide, bacteriocins, and organic acids), (ii) blockage of adhesion sites, (iii) competition for nutrients, (iv) degradation of toxin receptors, and (v) regulation of immunity (45). The molecular mechanisms of probiotic action still need to be characterized. More studies are required to assess the actions of particular probiotics against specific pathogens and disorders and to define which of these actions may benefit CD patients.

Some probiotics have been found to digest or alter gluten polypeptides. De Angelis and coworkers (37) analyzed the potential role of the specific probiotic preparation VSL#3 (a cocktail of eight strains belonging to the species Bifidobacterium breve, B. longum, B. infantis, Lactobacillus plantarum, L. acidophilus, L. casei, L. delbrueckii subsp. bulgaricus, and Streptococcus thermophilus) in decreasing the toxic properties of wheat flour during prolonged fermentation. That study found that the probiotic VSL#3 was highly effective in hydrolyzing gliadin polypeptides compared to other commercial probiotic products such as Oxadrop (B. infantis, L. acidophilus, L. brevis, and S. thermophilus), Florisia (L. brevis, L. salivarius subsp. salicinius, and L. plantarum), and Yovis (B. breve, B. infantis, B. longum, L. acidophilus, L. plantarum, L. casei, L. delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, and Enterococcus faecium). Furthermore, the activities of enzymes digesting proline-rich peptides and aminopeptidases, which regulate the hydrolysis of gliadin epitopes, were widely present in VSL#3. The other commercial probiotic products appear to lack the same ability to break down gliadin polypeptides. Interestingly, another study by De Angelis et al. (46) also reported that the capacity of VSL#3 to degrade gliadin was disabled when the probiotic strains were tested individually. The outcomes suggest that a single probiotic strain is not sufficient to degrade gliadin peptides and therefore must be used together with other strains to exert the beneficial effect against CD. The probiotic preparation VSL#3 may thus provide better effectiveness in the treatment of CD, since following a gluten-free diet is often a great challenge for patients, for instance, due to cross-contamination.

Specific lactobacillus and bifidobacterial strains have been found to improve gut health. De Palma and collaborators (30) evaluated in vitro immunomodulatory properties of B. bifidum strain IATA-ES2 and B. longum strain ATCC 15707 versus B. fragilis strain DSM2451, E. coli strain CBL2, and Shigella sp. strain CBD8 on peripheral blood mononuclear cells (PBMCs), under effects of gliadin and IFN-γ. B. bifidum strain IATA-ES2 and B. longum strain ATCC 15707 were able to induce lower levels of interleukin-12 (IL-12) and IFN-γ secretion than E. coli CBL2 and Shigella sp. CBD8. The release of TNF-α was induced by all strains tested, but its level was lower with B. bifidum IATA-ES2 than with B. fragilis DSM2451 and Shigella strain CBD8. The highest level of IL-10 secretion was observed in the presence of B. longum ATCC 15707. It seems that Gram-negative bacteria, such as E. coli CBL2 and Shigella strain CBD8, usually trigger higher levels of production of proinflammatory cytokines, which in turn contribute to the development of disease. On the other hand, B. bifidum IATA-ES2 was able to improve intestinal epithelial permeability, since it stimulated the lowest levels of production of TNF-α and IFN-γ.

Lindfors and coworkers (47) found that B. lactis exerted a protective effect on epithelial cells against cellular damage induced by gliadin incubation. Furthermore, it was observed that the addition of 10⁶ and 10⁷ CFU/ml, but not 10⁵ CFU/ml, of B. lactis was able to preserve TJ in comparison to epithelial cells maintained in the presence of gliadin alone. Administration of L. fermentum at the tested concentrations was unable to stimulate the recovery of transepithelial resistance.

Recently, a study using a gliadin-induced enteropathy animal model was developed to observe whether B. longum CECT 7347 could provide beneficial effects. The administration of B. longum CECT 7347 enhanced villus width and enterocyte height, which partially restored alterations in animals sensitized with IFN-γ and fed gliadin. In addition, it also reduced levels of TNF-α and increased levels of IL-10 synthesis, demonstrating its ability to favor an anti-inflammatory response in the gut mucosa. B. longum CECT 7347 administered to gliadin-fed animals sensitized with IFN-γ was able to moderately diminish some of the alterations in jejunal structure. This effect could apparently contribute to an improvement in the gut barrier function and prevent the translocation of gliadin to the lamina propria (48). Similar to previous work, L. casei ATCC 9595 administration was able to significantly reduce the levels of TNF-α and to repair the intestinal injury induced by gliadin in HLA-DQ8 transgenic mice under indomethacin treatment (49).

Studies regarding probiotics and CD in humans are very scarce. In a randomized, double-blind, placebo-controlled study, Smecuol and coworkers (50) evaluated the effect of the B. infantis Natren Life Start (NLS) superstrain on gut permeability, the occurrence of symptoms, and the presence of inflammatory cytokines in untreated adult CD patients. Results showed that probiotic administration was unable to modify gut barrier function, probably due to a short time of treatment or inadequate dose. After 3 weeks from the beginning of treatment with the B. infantis NLS superstrain, a marked improvement in digestion and reduction in constipation were noted. Abdominal pain and diarrheal symptom scores were also diminished although without significance. In addition, no differences in inflammatory markers were observed in either of the groups. Although there was a slight improvement in digestive symptoms, this noteworthy study demonstrates that the B. infantis NLS superstrain could be a promising tool in CD therapy.

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