Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children

Coeliac disease (CD) is a chronic intestinal inflammatory disorder triggered by the ingestion of gluten proteins in susceptible individuals. The active phase of the disease is characterized by a pro-inflammatory intestinal milieu resulting from an aberrant immune response to dietary gluten, along with increased epithelial permeability, which may favour the traffic of luminal antigens to the submucosa [1]. In CD patients, gliadin peptides can activate either an adaptive immune response dominated by Th₁ pro-inflammatory cytokines (e.g. IFN-γ) within the mucosa or an innate immune response mediated by IL-15, both of which lead to epithelial cell killing [2]. Gliadin also activates the zonulin pathway leading to an increase in intestinal permeability [1].

The aetiology of CD is multifactorial, involving genetic and environmental factors. This disorder is strongly associated to the human leukocyte antigen genes (HLA). Approximately 95% of the patients inherit the alleles encoding for the HLA-DQ2 and HLA-DQ8 molecules, but only a small percentage develops CD [3]. Studies of identical twins have also shown that one twin did not develop CD in 25% of the cases studied [4], supporting the role played by environmental factors in the aetiology of this disorder. However, the elements leading to a breakdown in oral tolerance to gluten in predisposed individuals are as yet unknown. The gut microbiota constitutes a complex pool of antigens separated from the mucosal immunocompetent cells by just a single layer of epithelial cells. In this mucosal immune system IgA constitutes a first line of defence responsible for neutralizing noxious antigens and pathogens [5]. In fact, malfunction of immune cells of Peyer Patches in production of secretory IgA has been considered a risk factor for CD development [6]. It has also been speculated that a transient infection could promote inflammation and increase permeability of the mucosa to antigens by activating a Th₁ response with secretion of IFN-γ, the major pro-inflammatory cytokine in CD patients [7, 8]. Moreover, alterations in the intestinal microbiota composition of CD children in comparison with that of healthy controls, as well as changes in the metabolites derived from the gut microbial activity have been recently reported [9–12]. Nevertheless, the possible relationship between the gut microbiota composition and the first line of immune defence in CD patients remains uncharacterized.

Herein, the percentage of immunoglobulin-coated bacteria and the faecal microbiota composition of children with CD (untreated and treated with a gluten-free diet [GFD]) and controls were evaluated, thus shedding light on the possible associations between the intestinal bacteria and the host defences in this disorder.

This study has characterized faecal microbiology and immunoglobulin-associated features in active and non-active stages of CD in children and in age-matched controls with an aim to furthering our understanding of the interplay between the gut microbiota and the host defences in this disorder. Immunoglobulin secretions constitute a primary line of defence of the mucosal surface against noxious antigens and pathogens, and contribute to the intestinal homeostasis preventing clinical inflammation. The colon predominantly harbours IgA-secreting plasma cells (90%); moreover, 4% cells secrete IgG and 6% cells secrete IgM. A considerable percentage of faecal bacteria was coated with IgA (14.71%) in healthy individuals, whereas a lower bacterial percentage was coated with IgG and IgM, and a similar trend was observed in CD patients, as reported in other subjects [13]. Significantly lower levels of IgA-coated bacteria were detected in faecal samples of untreated and treated CD patients when compared to healthy controls. It can be speculate that these results could reflect the existence of a barrier defect in CD patients, which fails to stabilise the gut microbiota and prevent the host from the invasion of harmful antigens and pathogens. In addition, treated CD patients showed lower levels of IgG and IgM coated bacteria. In contrast, IBD patients displayed a higher percentage of immunoglobulin-coated faecal bacteria in active disease and shortly after remission, supporting the concept that the mucosal tolerance to the gut microbiota is deregulated in these patients [5].

A remarkable reduction in Gram-positive bacterial populations was characteristic of the active phase of the disease while its abundance was partially restored in patients under a GFD. In addition, a reduction in the ratio of Gram-positive to Gram-negative bacteria was found in the patients regardless of the phase of the disorder. The levels of total Gram-positive bacteria were also lower in duodenal biopsies of patients with active and inactive CD than in controls, while the proportions of total Gram-negative bacteria were over-represented particularly in biopsies of active CD patients [12]. Therefore, the results obtained first in biopsies and now in faeces from children of the same age confirm similar structural changes in the composition of the gut microbiota associated with CD. The reductions in beneficial Gram-positive bacteria could favour the residence and interactions of harmful Gram-negative bacteria within the mucosal surface of CD patients, thereby contributing to loss of gluten tolerance. Antigenic structures of Gram-negative bacteria such as flagellins and lipopolysaccharides have been related to the inflammatory responses and pathogenesis of IBD [14]. Shifts in the intestinal microbiota, characterized by increases in pro-inflammatory Gram-negative bacteria, have also been shown to aggravate murine colitis via activation of acute inflammation through Toll-like receptor signalling [15].

Of the specific bacterial groups analysed, the Bifidobacterium population was significantly reduced in faecal samples of untreated CD patients as compared with controls. Bifidobacterium populations significantly decreased or slightly decreased in faeces of IBD patients, as detected by cultural techniques and real time PCR, respectively [16]. The benefits obtained by administering some Bifidobacterium strains as part of probiotic mixtures or symbiotics (probiotics combined with prebiotics) in ulcerative colitis and pouchitis also support the notion that this bacterial group is relevant to IBD [17]. C. histolyticum, C. lituseburense and F. prausnitzii groups were present in higher proportions in healthy individuals than in CD patients; particularly, the abundance of C. histolyticum followed a similar trend to that found in biopsy specimens although the differences were not significant [12]. C. coccoides and C. leptum groups were lower in faeces of Crohn's disease and ulcerative colitis patients when determined by real-time PCR [16]. A depletion of F. prausnitzii population in faecal mucus of active Crohn's disease, but not in ulcerative colitis, has also been detected [18]. Comparative analysis of biopsy and faecal samples of IBD patients, based on genomic-library sequencing analysis, also showed reductions in Firmicutes belonging to the class Clostridia in active and in remission Crohn's disease patients as compared to healthy or ulcerative colitis groups [19, 20]. Although some studies are controversial, it appears that the presence of certain Clostridium groups and F. prausnitzii is deficient in luminal or mucosa-associated microbiotas of Crohn's disease and probably of CD patients too. These components of the microbiota are producers of butyrate, which is an important energy source for colonocytes and exerts anti-inflammatory effects, for instance by inhibiting the lipopolysaccharide-induced cytokine response [19]. In contrast, the Bacteroides-Prevotella group was found in higher proportions in untreated CD patients than in controls, as previously detected in duodenal biopsy specimens [12]. Associations between the phylum Bacteroidetes and Crohn's disease were revealed by comparative bacteriological analysis of biopsy specimens of Crohn's disease and ulcerative colitis patients by denaturing gradient gel electrophoresis (DGGE) [17]. Similar comparative analyses of the mucosal-associated microbiota by genomic-library sequencing of 16S rRNA genes showed increases in Proteobacteria and Bacteroidetes, particularly in Crohn's disease patients [19]. Nevertheless, a recent study reported that B. fragilis and B. vulgatus were found at lower levels in faeces of IBD patients when compared to those of healthy controls [16].

As Bacteroides and Bifidobacterium seem to be possible relevant bacterial groups to CD, specific percentages of IgA coating these two bacterial groups were also determined. Interestingly, the proportions of IgA-coated Bacteroides-Prevotella were higher in healthy individuals than in treated and untreated CD patients, suggesting an increased defensive response of the gut mucosal immune system to this bacterial group in healthy children than in CD patients. The combination of an increased proportion of Bacteroides-Prevotella group in faecal samples of CD patients together with a weaker defensive IgA response could explain the recurrent relationship found between Bacteroides and inflamed gut mucosa in CD [12, 21], although more direct evidence is needed to confirm this hypothesis. A higher percentage of IgA-coated Bifidobacterium than IgA-coated Bacteroides-Prevotella was detected in all groups of children, similarly to other studies [5]. This could be a consequence of an increased interaction between the gut mucosal immune system and this bacterial group [5], which contributes to mucosal tolerance towards high gut Bifidobacterium concentrations.

This study confirms that in CD patients there is an alteration in the type of faecal immunoglobulin-coated bacteria that is associated with a shift in the structure of the microbiota. In particular, increases in the relative abundance of Bacteroides-Prevotella group are paralleled to reductions in the IgA coating this group, which could suggest a reduction of of the host defences against this bacterial group. However, the possible clinical consequences of these finding are still unknown and their elucidation would require further investigations.