Intestinal microbiota modulates gluten-induced immunopathology in humanized mice.
ABSTRACT: 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: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:Microbiota-modulating strategies, including probiotic administration, have been tested for the treatment of chronic gastrointestinal diseases despite limited information regarding their mechanisms of action. We previously demonstrated that patients with active celiac disease have decreased duodenal expression of elafin, a human serine protease inhibitor, and supplementation of elafin by a recombinant Lactococcus lactis strain prevents gliadin-induced immunopathology in the NOD/DQ8 mouse model of gluten sensitivity. The commensal probiotic strain Bifidobacterium longum NCC2705 produces a serine protease inhibitor (Srp) that exhibits immune-modulating properties. Here, we demonstrate that B. longum NCC2705, but not a srp knockout mutant, attenuates gliadin-induced immunopathology and impacts intestinal microbial composition in NOD/DQ8 mice. Our results highlight the beneficial effects of a serine protease inhibitor produced by commensal B. longum strains.IMPORTANCE Probiotic therapies have been widely used to treat gastrointestinal disorders with variable success and poor mechanistic insight. Delivery of specific anti-inflammatory molecules has been limited to the use of genetically modified organisms, which has raised some public and regulatory concerns. By examining a specific microbial product naturally expressed by a commensal bacterial strain, we provide insight into a mechanistic basis for the use of B. longum NCC2705 to help treat gluten-related disorders.
Project description:Dietary gluten causes severe disorders like celiac disease in gluten-intolerant humans. However, currently understanding of its impact in tolerant individuals is limited. Our objective was to test whether gliadin, one of the detrimental parts of gluten, would impact the metabolic effects of an obesogenic diet. Mice were fed either a defined high-fat diet (HFD) containing 4% gliadin (n = 20), or a gliadin-free, isocaloric HFD (n = 20) for 23 weeks. Combined analysis of several parameters including insulin resistance, histology of liver and adipose tissue, intestinal microbiota in three gut compartments, gut barrier function, gene expression, urinary metabolites and immune profiles in intestinal, lymphoid, liver and adipose tissues was performed. Mice fed the gliadin-containing HFD displayed higher glycated hemoglobin and higher insulin resistance as evaluated by the homeostasis model assessment, more hepatic lipid accumulation and smaller adipocytes than mice fed the gliadin-free HFD. This was accompanied by alterations in the composition and activity of the gut microbiota, gut barrier function, urine metabolome, and immune phenotypes within liver and adipose tissue. Our results reveal that gliadin disturbs the intestinal environment and affects metabolic homeostasis in obese mice, suggesting a detrimental effect of gluten intake in gluten-tolerant subjects consuming a high-fat diet.
Project description:Current understanding of T cell epitopes in coeliac disease (CD) largely derives from intestinal T cell clones in vitro. T cell clones allow identification of gluten peptides that stimulate T cells but do not quantify their contribution to the overall gluten specific T cell response in individuals with CD when exposed to gluten in vivo.To determine the contribution of a putative dominant T cell epitope to the overall gliadin T cell response in HLA-DQ2 CD in vivo.HLA-DQ2+ individuals with CD and healthy controls.Subjects consumed 20 g of gluten daily for three days. Interferon gamma (IFN-gamma) ELISPOT was performed using peripheral blood mononuclear cells (PBMC) to enumerate and characterise peptide and gliadin specific T cells before and after gluten challenge.In 50/59 CD subjects, irrespective of homo- or heterozygosity for HLA-DQ2, IFN-gamma ELISPOT responses for an optimal concentration of A-gliadin 57-73 Q-E65 were between 10 and 1500 per million PBMC, equivalent to a median 51% of the response for a "near optimal" concentration of deamidated gliadin. Whole deamidated gliadin and gliadin epitope specific T cells induced in peripheral blood expressed an intestinal homing integrin (alpha4beta7) and were HLA-DQ2 restricted. Peripheral blood T cells specific for A-gliadin 57-73 Q-E65 are rare in untreated CD but can be predictably induced two weeks after gluten exclusion.In vivo gluten challenge is a simple safe method that allows relevant T cells to be analysed and quantified in peripheral blood by ELISPOT, and should permit comprehensive high throughput mapping of gluten T cell epitopes in large numbers of individuals with CD.
Project description:Antibiotics lead to increased susceptibility to colonization by pathogenic organisms, with different effects on the host-microbiota relationship. Here, we show that metronidazole treatment of specific pathogen-free (SPF) mice results in a significant increase of the bacterial phylum Proteobacteria in fecal pellets. Furthermore, metronidazole in SPF mice decreases hind limb muscle weight and results in smaller fibers in the tibialis anterior muscle. In the gastrocnemius muscle, metronidazole causes upregulation of Hdac4, myogenin, MuRF1, and atrogin1, which are implicated in skeletal muscle neurogenic atrophy. Metronidazole in SPF mice also upregulates skeletal muscle FoxO3, described as involved in apoptosis and muscle regeneration. Of note, alteration of the gut microbiota results in increased expression of the muscle core clock and effector genes Cry2, Ror-?, and E4BP4. PPAR? and one of its important target genes, adiponectin, are also upregulated by metronidazole. Metronidazole in germ-free (GF) mice increases the expression of other core clock genes, such as Bmal1 and Per2, as well as the metabolic regulators FoxO1 and Pdk4, suggesting a microbiota-independent pharmacologic effect. In conclusion, metronidazole in SPF mice results in skeletal muscle atrophy and changes the expression of genes involved in the muscle peripheral circadian rhythm machinery and metabolic regulation.
Project description:BACKGROUND & AIMS:Heme oxygenase-1 (HO-1) and its metabolic by-product, carbon monoxide (CO), protect against intestinal inflammation in experimental models of colitis, but little is known about their intestinal immune mechanisms. We investigated the interactions among CO, HO-1, and the enteric microbiota in mice and zebrafish. METHODS:Germ-free, wild-type, and interleukin (Il)10(-/-) mice and germ-free zebrafish embryos were colonized with specific pathogen-free (SPF) microbiota. Germ-free or SPF-raised wild-type and Il10(-/-) mice were given intraperitoneal injections of cobalt(III) protoporphyrin IX chloride (CoPP), which up-regulates HO-1, the CO-releasing molecule Alfama-186, or saline (control). Colitis was induced in wild-type mice housed in SPF conditions by infection with Salmonella typhimurium. RESULTS:In colons of germ-free, wild-type mice, SPF microbiota induced production of HO-1 via activation of nuclear factor erythroid 2-related factor 2-, IL-10-, and Toll-like receptor-dependent pathways; similar observations were made in zebrafish. SPF microbiota did not induce HO-1 in colons of germ-free Il10(-/-) mice. Administration of CoPP to Il10(-/-) mice before transition from germ-free to SPF conditions reduced their development of colitis. In Il10(-/-) mice, CO and CoPP reduced levels of enteric bacterial genomic DNA in mesenteric lymph nodes. In mice with S typhimurium-induced enterocolitis, CoPP reduced the numbers of live S typhimurium recovered from the lamina propria, mesenteric lymph nodes, spleen, and liver. Knockdown of HO-1 in mouse macrophages impaired their bactericidal activity against E coli, E faecalis, and S typhimurium, whereas exposure to CO or overexpression of HO-1 increased their bactericidal activity. HO-1 induction and CO increased acidification of phagolysosomes. CONCLUSIONS:Colonic HO-1 prevents colonic inflammation in mice. HO-1 is induced by the enteric microbiota and its homeostatic function is mediated, in part, by promoting bactericidal activities of macrophages.
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:The importance of the gut microbiota (GM) in disease development has recently received increased attention, and numerous approaches have been made to better understand this important interplay. For example, metabolites derived from the GM have been shown to promote atherosclerosis, the underlying cause of cardiovascular disease (CVD), and to increase CVD risk factors. Popular interest in the role of the intestine in a variety of disease states has now resulted in a significant proportion of individuals without coeliac disease switching to gluten-free diets. The effect of gluten-free diets on atherosclerosis and cardiovascular risk factors is largely unknown. We therefore investigated the effect of a gluten-free high-fat cholesterol-rich diet, as compared to the same diet in which the gluten peptide gliadin had been added back, on atherosclerosis and several cardiovascular risk factors in apolipoprotein E-deficient (Apoe-/-) mice. The gluten-free diet transiently altered GM composition in these mice, as compared to the gliadin-supplemented diet, but did not alter body weights, glucose tolerance, insulin levels, plasma lipids, or atherosclerosis. In parallel, other Apoe-/- mice fed the same diets were treated with ampicillin, a broad-spectrum antibiotic known to affect GM composition. Ampicillin-treatment had a marked and sustained effect on GM composition, as expected. Furthermore, although ampicillin-treated mice were slightly heavier than controls, ampicillin-treatment transiently improved glucose tolerance both in the absence or presence of gliadin, reduced plasma LDL and VLDL cholesterol levels, and reduced aortic atherosclerotic lesion area. These results demonstrate that a gluten-free diet does not seem to have beneficial effects on atherosclerosis or several CVD risk factors in this mouse model, but that sustained alteration of GM composition with a broad-spectrum antibiotic has beneficial effects on CVD risk factors and atherosclerosis. These findings support the concept that altering the microbiota might provide novel treatment strategies for CVD.
Project description:OBJECTIVES:Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNF(deltaARE) mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. DESIGN:Heterozygous TNF(deltaARE) mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. RESULTS:GF-TNF(deltaARE) mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF(deltaARE) mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF(deltaARE) mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNF(deltaARE) recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF(deltaARE) mice with the human CD-related Escherichia coli LF82 did not induce ileitis. CONCLUSIONS:We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.
Project description:Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.