Marta Olivares

Bifidobacterium longum CECT 7347 Modulates Immune Responses in a Gliadin-Induced Enteropathy Animal Model

Coeliac disease (CD) is an autoimmune disorder triggered by gluten proteins (gliadin) that involves innate and adaptive immunity. In this study, we hypothesise that the administration of Bifidobacterium longum CECT 7347, previously selected for reducing gliadin immunotoxic effects in vitro, could exert protective effects in an animal model of gliadin-induced enteropathy. The effects of this bacterium were evaluated in newborn rats fed gliadin alone or sensitised with interferon (IFN)-γ and fed gliadin. Jejunal tissue sections were collected for histological, NFκB mRNA expression and cytokine production analyses. Leukocyte populations and T-cell subsets were analysed in peripheral blood samples. The possible translocation of the bacterium to different organs was determined by plate counting and the composition of the colonic microbiota was quantified by real-time PCR. Feeding gliadin alone reduced enterocyte height and peripheral CD4+ cells, but increased CD4+/Foxp3+ T and CD8+ cells, while the simultaneous administration of B. longum CECT 7347 exerted opposite effects. Animals sensitised with IFN-γ and fed gliadin showed high cellular infiltration, reduced villi width and enterocyte height. Sensitised animals also exhibited increased NFκB mRNA expression and TNF-α production in tissue sections. B. longum CECT 7347 administration increased NFκB expression and IL-10, but reduced TNF-α, production in the enteropathy model. In sensitised gliadin-fed animals, CD4+, CD4+/Foxp3+ and CD8+ T cells increased, whereas the administration of B. longum CECT 7347 reduced CD4+ and CD4+/Foxp3+ cell populations and increased CD8+ T cell populations. The bifidobacterial strain administered represented between 75–95% of the total bifidobacteria isolated from all treated groups, and translocation to organs was not detected. These findings indicate that B. longum attenuates the production of inflammatory cytokines and the CD4+ T-cell mediated immune response in an animal model of gliadin-induced enteropathy.

Double-blind, randomised, placebo-controlled intervention trial to evaluate the effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed coeliac disease

Interactions between the immune system and the intestinal microbiota may play a role in coeliac disease (CD). In the present study, the potential effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed CD were evaluated. A double-blind, randomised, placebo-controlled trial was conducted in thirty-three children who received a capsule containing either B. longum CECT 7347 (10⁹ colony-forming units) or placebo (excipients) daily for 3 months together with a gluten-free diet (GFD). Outcome measures (baseline and post-intervention) included immune phenotype of peripheral blood cells, serum cytokine concentration, faecal secretory IgA (sIgA) content, anthropometric parameters and intestinal microbiota composition. Comparisons between the groups revealed greater height percentile increases (P= 0·048) in the B. longum CECT 7347 group than in the placebo group, as well as decreased peripheral CD3⁺ T lymphocytes (P= 0·004) and slightly reduced TNF-α concentration (P= 0·067). Within-group comparisons of baseline and final values did not reveal any differences in T lymphocytes and cytokines in the placebo group, while decreased CD3⁺ (P =0·013) and human leucocyte antigen (HLA)-DR⁺ T lymphocytes (P =0·029) and slightly reduced TNF-α concentration (P= 0·085) were detected in the B. longum CECT 7347 group. Comparison between the groups showed that the administration of B. longum CECT 7347 reduced the numbers of the Bacteroides fragilis group (P= 0·020) and the content of sIgA in stools (P= 0·011) compared with the administration of placebo. Although this is a first exploratory intervention with limitations, the findings suggest that B. longum CECT 7347 could help improve the health status of CD patients who tend to show alterations in gut microbiota composition and a biased immune response even on a GFD.

Modulation of phenotypic and functional maturation of dendritic cells by intestinal bacteria and gliadin: relevance for celiac disease.

DC maturation and functions are influenced by microbial and environmental stimuli, which could contribute to immune dysfunction. Here, we have investigated the role of enterobacteria (Escherichia coli CBL2 and Shigella CBD8) isolated from CD patients, bifidobacteria (Bifidobacterium longum CECT 7347 and Bifidobacterium bifidum CECT 7365), and gliadins on phenotypic and functional features of MDDCs and in coculture with Caco‐2 cells. The ultimate goal of our study is to understand the roles played by specific components of the gut microbiota in CD. Enterobacteria induced marked alterations in MDDC morphology, inducing podosome dissolution and dendrites, and activated MDDC adhesion and spreading. Enterobacteria also induced inflammatory cytokine production (IFN‐γ, TNF‐α, and IL‐12), partially resembling the gliadin‐induced Th1‐type cytokine profile. B. longum CECT 7347 and B. bifidum CECT 7365 induced minor MDDC morphological changes and activated adhesion and spreading and inflammatory cytokine production to a lesser extent compared with enterobacteria. B. longum CECT 7347 also induced lower CD86 and CD40 expression on MDDCs than the two enterobacteria. The aforementioned bifidobacterial strain also reduced gliadin‐induced IFN‐γ production and increased IL‐10 secretion when both stimuli were combined. Similar trends were detected for MDDCs cocultured with Caco‐2 cells. B. longum CECT 7347 reversed the gliadin‐reduced ZO‐1 expression in Caco‐2 cells. Thus, our results suggest that specific components of the gut microbiota may influence phenotypic and functional maturation of DCs differently and their interactions with epithelial cells. This could ultimately define the role of DCs in CD progression.

Human milk composition differs in healthy mothers and mothers with celiac disease.

AbstractPurposeTo investigate whether breast-milk composition and microbiota differ in healthy mothers and mothers with celiac disease (CD) to ultimately contribute to identify additional factors determining CD risk.MethodsBreast-milk samples from healthy mothers (nxa0=xa012) and mothers with CD (nxa0=xa012) were collected. Cytokines and secretory immunoglobulin A (sIgA) were analyzed by bead-arrays and flow cytometry and human milk oligosaccharides (HMOs) were assessed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. Breast-milk microbiota composition was analyzed by conventional and quantitative real-time PCR.ResultnBreast milk from CD mothers showed significantly lower levels of interleukin (IL) 12p70 (Pxa0<xa00.042), transforming growth factor (TGF)-β1 (Pxa0<xa00.018) and sIgA (Pxa0<xa00.003) and almost significantly lower levels of interferon (IFN)-γ (Pxa0<xa00.058). Six mothers in each group belonged to the secretor Le(a−b+) type, one to the secretor Le(a−b−) type and five to the non-secretor Le(a+b−) type. CD mothers of non-secretor Le(a+b−) type showed increased Lacto-N-tetraose content (Pxa0<xa00.042) compared with healthy mothers. CD mothers’ milk showed reduced gene copy numbers of Bifidobacterium spp. (Pxa0<xa00.026) and B. fragilis group (Pxa0<xa00.044).ConclusionCD mothers’ breast milk is characterized by a reduced abundance of immunoprotective compounds (TGF-β1 and sIgA) and bifidobacteria. The reduction in these components could theoretically diminish the protective effects of breast-feeding on the child’s future risk of developing CD.

Host genotype, intestinal microbiota and inflammatory disorders

Intestinal microbiota may influence human physiology and disease risk due to the role it plays in mediating appropriate immune responses to harmful and innocuous antigens. Colonisation of the intestine in early life seems particularly important as it is the main environmental stimulus for immune system maturation. This is a dynamic process, which depends on both environmental and genetic factors. The pathogenesis of inflammatory bowel disease, such as Crohns disease, involves genetic polymorphisms (e.g. CARD15/nucleotide-binding oligomerisation domain 2) related to an excessive inflammatory response to commensal microbiota and to its unbalanced composition. Atopic diseases have also been linked to imbalances in microbiota and to related genetic factors (e.g. TLR4 and CD14 genes), although these associations are still controversial. Research into the relationship between the genetic risk of developing celiac disease (human leukocyte antigen (HLA)-DQ2/DQ8) and the early colonisation process in infants at family risk of the disease has also reported that the HLA-DQ genotype could influence staphylococcal colonisation. Future observational studies considering both host genetics and microbiota could be critical in defining the complex host-microbe interactions and the respective role each plays in inflammatory disorders.

Oral administration of Bifidobacterium longum CECT 7347 modulates jejunal proteome in an in vivo gliadin-induced enteropathy animal model.

Celiac disease is an immune-mediated disorder triggered by gluten proteins of wheat (gliadins) and other cereals. Gliadin-mediated effects on weanling animals, sensitized or not with interferon (IFN)-γ, were investigated. Also, the influence of the co-administration of Bifidobacterium longum CECT 7347 was studied together with changes in the proteome of jejunal sections, using 2DE and MALDITOF-TOF peptide fingerprinting. Findings were compared to results for control animal groups. In the principal component analysis (PCA) of proteome pattern, two components were extracted accounting for 79.8% of variability in the expression of the identified proteins. PCA analysis clearly discriminated between the proteome of animals fed gliadins alone and those fed gliadins and B. longum simultaneously. However, the proteome patterns from animals sensitized with IFN-γ and fed gliadins together with B. longum, or alone, could not be discriminated. Gliadin feeding caused inflammatory effects as well as changes in proteins involved in intracellular ionic homeostasis, lipid turnover, cell motility and redox regulation in intestinal sections. After feeding gliadins to animals sensitized with IFN-γ, changes were also detected in proteins involved in recruitment and function of inmunocompetent cells, trophic effect on the intestine and organization of myofibers reflecting the more marked gliadin-mediated injury in jejunal sections. The administration of the bacterial strain to rats fed gliadins seemed to ameliorate the inflammation caused by gliadin feeding alone, although, in sensitized animals the co-administration of B. longum had less marked effects, which was probably due to the more extensive intestinal mucosal damage. The proteome patterns in animals administered B. longum alone did not reveal any changes reflecting impairment of jejunal functions.

Gut microbiota trajectory in early life may predict development of celiac disease

BackgroundTo investigate whether alterations in the developing intestinal microbiota and immune markers precede celiac disease (CD) onset in infants at familial risk of developing the disease.MethodsA nested case-control study was carried out as part of a larger prospective cohort study, which included healthy full-term newborns (>u2009200) with at least one first relative with biopsy-verified CD. The present study includes cases of CD (nu2009=u200910) and the best-matched controls (nu2009=u200910) who did not develop the disease after 5-year follow-up. Fecal microbiota, assessed by high-throughput 16S rRNA gene amplicon sequencing, and immune parameters were profiled at 4 and 6xa0months of age and related to CD onset.ResultsThe microbiota of infants who remained healthy showed an increase in bacterial diversity over time, characterized by increases in Firmicutes families, but not those who developed CD. Infants who subsequently developed CD showed a significant reduction in sIgA levels over time, while those who remained healthy showed increases in TNF-α correlated to Bifidobacterium spp. An increased relative abundance of Bifidobacterium longum was associated with control children while increased proportions of Bifidobacterium breve and Enterococcus spp. were associated with CD development.ConclusionThe findings suggest that alterations in the early trajectory of gut microbiota in infants at CD risk could influence the immune maturation process and predispose to CD, although larger population studies are warranted to confirm this hypothesis.

Role of Gut Microbes in Celiac Disease Risk and Pathogenesis

Celiac disease (CD) is an autoimmune disorder for which the main genetic determinant (HLA-DQ2/8) and environmental trigger (gluten) are identified, although these do not fully explain the onset of the disease. In recent years, research has been made into the role intestinal microbiota play in CD via interactions with the diet and the host immune system. The intestinal colonization of the newborn’s intestine seems to be a particularly important process since it constitutes a major stimulus for adequate development of the immune system and oral tolerance. Evidence from prospective studies in infants at risk of developing CD suggests that both breast-feeding and the HLA-DQ genotype influence the microbiota composition early in life, which could partly explain the protective role attributed to breast milk in CD development. In most studies, the microbiota of CD patients (both untreated CD and CD treated with a gluten-free diet) has also been associated with alterations in microbiota composition. The isolation of clones belonging to different bacterial groups, including bacteroides, enterobacteria, and staphylococci, has also demonstrated that the isolates from CD patients harbored higher virulence-related genes, increasing their potential pathogenicity. In addition, in vitro studies as well as animal models suggest that specific bacteria could activate pathogenic mechanisms that aggravate the deleterious effects of gluten, while others can exert a protective role. Altogether, findings indicate that gut microbiota composition and function could be one of the missing links that could help explain CD pathogenesis and risk, and thus deserve further investigation.

The gut microbiota metabolite indole alleviates liver inflammation in mice.

The gut microbiota regulates key hepatic functions, notably through the production of bacterial metabolites that are transported via the portal circulation. We evaluated the effects of metabolites produced by the gut microbiota from aromatic amino acids (phenylacetate, benzoate, p‐cresol, and indole) on liver inflammation induced by bacterial endotoxin. Precision‐cut liver slices prepared from control mice, Kupffer cell (KC)‐depleted mice, and obese mice (ob/ob) were treated with or without LPS and bacterial metabolites. We observed beneficial effects of indole that dose‐dependently reduced the LPS‐induced up‐regulation of proinflammatory mediators at both mRNA and protein levels in precision‐cut liver slices prepared from control or ob/ob mice. KC depletion partly prevented the antiinflammatory effects of indole, notably through a reduction of nucleotide‐binding domain and leucine‐rich repeat containing (NLR) family pyrin domain‐containing 3 (NLRP3) pathway activation. In vivo, the oral administration of indole before an LPS injection reduced the expression of key proteins of the NF‐KB pathway and downstream proinflammatory gene up‐regulation. Indole also prevented LPS‐induced alterations of cholesterol metabolism through a transcriptional regulation associated with increased 4β‐hydroxycholesterol hepatic levels. In summary, indole appears as a bacterial metabolite produced from tryptophan that is able to counteract the detrimental effects of LPS in the liver. Indole could be a new target to develop innovative strategies to decrease hepatic inflammation.—Beaumont, M., Neyrinck, A. M., Olivares, M., Rodriguez, J., de Rocca Serra, A., Roumain, M., Bindels, L. B., Cani, P. D., Evenepoel, P., Muccioli, G. G., Demoulin, J.‐B., Delzenne, N. M. The gut microbiota metabolite indole alleviates liver inflammation in mice. FASEB J. 32, 6681–6693 (2018). www.fasebj.org

High-fat diet induces depression-like behaviour in mice associated with changes in microbiome, neuropeptide Y, and brain metabolome.

Objectives: The biological mechanisms linking diet-related obesity and depression remain unclear. Therefore, we examined the impact of high-fat diet (HFD) on murine behaviour, intestinal microbiome, brain metabolome, neuropeptide Y (NPY) expression, and dipeptidyl peptidase-4 (DPP-4) activity. Methods: Male C57Bl/6J mice were fed an HFD (60u2005kJ% from fat) or control diet (12u2005kJ% from fat) for 8 weeks, followed by behavioural phenotyping. Caecal microbiome was analysed by 16S rDNA sequencing, brain metabolome by 1H nuclear magnetic resonance, NPY expression by PCR and immunoassay, and dipeptidyl peptidase-4 (DPP-4) activity by enzymatic assay. The effect of a 4-week treatment with imipramine (7u2005mg/kg/day) and the DPP-4 inhibitor sitagliptin (50u2005mg/kg/day) on HFD-induced behavioural changes was also tested. Results: HFD led to a depression-like phenotype as revealed by reduced sociability and sucrose preference. In the caecum, HFD diminished the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes and Cyanobacteria. In the brain, HFD modified the metabolome of prefrontal cortex and striatum, changing the relative concentrations of molecules involved in energy metabolism (e.g. lactate) and neuronal signalling (e.g. γ-aminobutyric acid). The expression of NPY in hypothalamus and hippocampus was decreased by HFD, whereas plasma NPY and DPP-4-like activity were increased. The HFD-induced anhedonia remained unaltered by imipramine and sitagliptin. Discussion: The depression-like behaviour induced by prolonged HFD in mice is associated with distinct alterations of intestinal microbiome, brain metabolome, NPY system, and DPP-4-like activity. Importantly, the HFD-evoked behavioural disturbance remains unaltered by DPP-4 inhibition and antidepressant treatment with imipramine.