Human gut derived-organoids provide model to study gluten response and effects of microbiota-derived molecules in celiac disease.
ABSTRACT: Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium's response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.
Project description:Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk.
Project description:BACKGROUND: Celiac disease (CD) is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins. The CD-toxicity of these proteins and their derived peptides is depending on the presence of specific T-cell epitopes (9-mer peptides; CD epitopes) that mediate the stimulation of HLA-DQ2/8 restricted T-cells. Next to the thoroughly characterized major T-cell epitopes derived from the ?-gliadin fraction of gluten, ?-gliadin peptides are also known to stimulate T-cells of celiac disease patients. To pinpoint CD-toxic ?-gliadins in hexaploid bread wheat, we examined the variation of T-cell epitopes involved in CD in ?-gliadin transcripts of developing bread wheat grains. RESULTS: A detailed analysis of the genetic variation present in ?-gliadin transcripts of bread wheat (T. aestivum, allo-hexaploid, carrying the A, B and D genome), together with genomic ?-gliadin sequences from ancestrally related diploid wheat species, enabled the assignment of sequence variants to one of the three genomic ?-gliadin loci, Gli-A1, Gli-B1 or Gli-D1. Almost half of the ?-gliadin transcripts of bread wheat (49%) was assigned to locus Gli-D1. Transcripts from each locus differed in CD epitope content and composition. The Gli-D1 transcripts contained the highest frequency of canonical CD epitope cores (on average 10.1 per transcript) followed by the Gli-A1 transcripts (8.6) and the Gli-B1 transcripts (5.4). The natural variants of the major CD epitope from ?-gliadins, DQ2-?-I, showed variation in their capacity to induce in vitro proliferation of a DQ2-?-I specific and HLA-DQ2 restricted T-cell clone. CONCLUSIONS: Evaluating the CD epitopes derived from ?-gliadins in their natural context of flanking protein variation, genome specificity and transcript frequency is a significant step towards accurate quantification of the CD toxicity of bread wheat. This approach can be used to predict relative levels of CD toxicity of individual wheat cultivars directly from their transcripts (cDNAs).
Project description:The protein/peptide composition of five beer kinds, including two experimental beer-like products brewed with einkorn (Triticum monococcum), a beer labeled as "gluten-free," a traditional all-barley malt and a wheat (T. aestivum) containing beer, was characterized with HPLC-ESI MS/MS-based proteomics. To enlarge the characterization of the components, the polypeptides were fractionated according to their molecular size (cut-off 6 kDa). All the beer types contained a variety of polypeptides arising from all the gliadin subfamilies (?-/?-, ?-, and ?-gliadins) able to induce an immune response in celiac disease (CD) patients in addition to a panel of IgE-reactive food allergens. Wheat storage proteins were heavily hydrolyzed in the beer samples brewed with einkorn. The presence of gluten-like fragments, also including the 25-mer and 33-mer-like of ?-gliadin, was confirmed in beer brewed with barley and wheat malt as well as in the gluten-free beer. Both CD-toxic and allergenic peptides of all beer samples were drastically degraded when subjected to a simulated gastroduodenal (GD) digestion. After in vitro digestion, the level of gluten-like peptides assayed with the G12 competitive ELISA, was below the threshold (20 ppm) for a food to be considered as "gluten-free." A few gliadin-derived epitopes occurred in the digests of beers crafted with wheat or Norberto-ID331 line of einkorn. In contrast, digests of all barley malt and gluten-free beers did not contain detectable gluten-like epitopes, but only minor fragments of hordeins and IgE-reactive food allergens. All beer samples evoked a weak immune response on gliadin-reactive celiac T cells isolated from intestinal biopsies of celiac patients. Compared to undigested polypeptides, the response was markedly reduced by GD digestion. Although the consumption of a moderate amount of beer brewed with barley or einkorn could deliver a relatively low amount of CD-toxic epitopes, the findings of this study emphasize the urgent need of a reliable and accurate quantification of gluten epitopes in all types of beer, also including the gluten-free one, to compute realistically the contribution of beer to the overall gluten intake, which can be responsible of intestinal tissue damages in celiacs.
Project description:Celiac disease is an immune-mediated enteropathy triggered by ingestion of gluten. Although its pathogenesis has been extensively studied and the contribution from both innate and adaptive immune responses has been reported, little is still known about the contribution of macrophages to the onset or maintenance of the disease. Macrophages are extremely plastic immune cells that can be directed toward a pro- or anti-inflammatory phenotype by the surrounding microenvironment. Of note, gliadin, the most prominent causative agent of the disease, has been reported to trigger the production of pro-inflammatory cytokines in this cell population. In the present study, we aimed at investigating how the intestinal milieu and more specifically the epithelium can shape the macrophage response to gliadin. Using patient-derived organoids we showed that the intestinal epithelium derived from celiac disease donors releases anti-inflammatory factors that curb the macrophage response to gliadin. Furthermore, we uncovered that the celiac macrophages were better responders than macrophages derived from non-celiac controls. Finally, we demonstrated that IFN? released by the epithelium is in part responsible of the observed anti-inflammatory effect. Our data shed light on the cross-talk between the immune system and the epithelium and its critical role in the intestinal homeostasis. Furthermore, we provide more evidence how alterations in the innate immune machinery in celiac patients may contribute to the onset of the disease.
Project description:BACKGROUND: Wheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD. RESULTS: A 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 'unique deduced protein fragments' of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes. CONCLUSIONS: The dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.
Project description:Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available.We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces.Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody.The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients.Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.
Project description:Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission (n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints (n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the ?-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli (P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine.IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.
Project description:Celiac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities.Gluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a prokaryotic expression vector, which provides a C-terminal hexahistidine tag. ScFvs from several bacterial clones were expressed in soluble form and crude cell lysates screened for binding to PT-Gliadin by ELISA. We identified an enriched scFv motif, which showed reactivity to PT-Gliadin. One selected scFv candidate was expressed and purified to homogeneity. Polyclonal anti-PT-Gliadin IgY, purified from egg yolk of immunized chicken, served as control. ScFv binds in a dose-dependent manner to PT-Gliadin, comparable to IgY. Furthermore, IgY competitively displaces scFv from PT-Gliadin and natural wheat flour digest, indicating a common epitope of scFv and IgY. ScFv was tested for reactivity to different gastric digested dietary grain flours. ScFv detects common and khorasan wheat comparably with binding affinities in the high nanomolar range, while rye is detected to a lesser extent. Notably, barley and cereals which are part of the gluten-free diet, like corn and rice, are not detected by scFv. Similarly, the pseudo-grain amaranth, used as gluten-free alternative, is not targeted by scFv. This data indicate that scFv specifically recognizes toxic cereal peptides relevant in CD.ScFv can be of benefit for future CD treatment regimes.
Project description:Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA-gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA-gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.
Project description:Class II major histocompatibility molecules confer disease risk in Celiac disease (CD) by presenting gliadin peptides to CD4 T cells in the small intestine. Deamidation of gliadin peptides by tissue transglutaminase creates immunogenic peptides presented by HLA-DQ2 and DQ8 molecules to activate proinflammatory CD4 T cells. Detecting gliadin specific T cell responses from the peripheral blood has been challenging due to low circulating frequencies and heterogeneity in response to gliadin epitopes. We investigated the peripheral T cell responses to alpha and gamma gliadin epitopes in young children with newly diagnosed and untreated CD. Using peptide/MHC recombinant protein constructs, we are able to robustly stimulate CD4 T cell clones previously derived from intestinal biopsies of CD patients. These recombinant proteins and a panel of ?- and ?-gliadin peptides were used to assess T cell responses from the peripheral blood. Proliferation assays using peripheral blood mononuclear cells revealed more CD4 T cell responses to ?-gliadin than ?-gliadin peptides with a single deamidated ?-gliadin peptide able to identify 60% of CD children. We conclude that it is possible to detect T cell responses without a gluten challenge or in vitro stimulus other than antigen, when measuring proliferative responses.