Dietary Nanoparticles Interact with Gluten Peptides and Alter the Intestinal Homeostasis Increasing the Risk of Celiac Disease

The introduction of metallic nanoparticles (mNPs) into the diet is a matter of concern for human health. In particular, their effect on the gastrointestinal tract may potentially lead to the increased passage of gluten peptides and the activation of the immune response. In consequence, dietary mNPs could play a role in the increasing worldwide celiac disease (CeD) incidence. We evaluated the potential synergistic effects that peptic-tryptic-digested gliadin (PT) and the most-used food mNPs may induce on the intestinal mucosa. PT interaction with mNPs and their consequent aggregation was detected by transmission electron microscopy (TEM) analyses and UV–Vis spectra. In vitro experiments on Caco-2 cells proved the synergistic cytotoxic effect of PT and mNPs, as well as alterations in the monolayer integrity and tight junction proteins. Exposure of duodenal biopsies to gliadin plus mNPs triggered cytokine production, but only in CeD biopsies. These results suggest that mNPs used in the food sector may alter intestinal homeostasis, thus representing an additional environmental risk factor for the development of CeD.

A Case Study of the Response of Immunogenic Gluten Peptides to Sourdough Proteolysis

Celiac disease is activated by digestion-resistant gluten peptides that contain immunogenic epitopes. Sourdough fermentation is a potential strategy to reduce the concentration of these peptides within food. However, we currently know little about the effect of partial sourdough fermentation on immunogenic gluten. This study examined the effect of a single sourdough culture (representative of those that the public may consume) on the digestion of immunogenic gluten peptides. Sourdough bread was digested via the INFOGEST protocol. Throughout digestion, quantitative and discovery mass spectrometry were used to model the kinetic release profile of key immunogenic peptides and profile novel peptides, while ELISA probed the gluten’s allergenicity. Macrostructural studies were also undertaken. Sourdough fermentation altered the protein structure, in vitro digestibility, and immunogenic peptide release profile. Interestingly, sourdough fermentation did not decrease the total immunogenic peptide concentration but altered the in vitro digestion profile of select immunogenic peptides. This work demonstrates that partial sourdough fermentation can alter immunogenic gluten digestion, and is the first study to examine the in vitro kinetic profile of immunogenic gluten peptides from sourdough bread.

A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease

Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial (“RESET CeD”) of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 μg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.

An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease

Celiac disease (CeD) is a common autoimmune disorder induced by consuming gluten proteins found in wheat, barley, and rye. Glutens resist breakdown by gastrointestinal proteases, ultimately resulting in peptides with chemical structures that elicit inflammation in patients with CeD. Despite well-established connections between digestion-resistant glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, which have impeded our knowledge of potentially diverse peptide species that encounter the immune system. Here, we developed a novel liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. Using this protocol, we detected 679 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and 5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differed from control subjects. Our urinary peptidomic workflow provides new foundations for understanding gluten immunogenicity and enhancing CeD management. Additionally, it should promote new understandings of the uncharacterized dietary and urinary peptidomes.

Machine Learning Analysis of Naïve B-Cell Receptor Repertoires Stratifies Celiac Disease Patients and Controls

Celiac disease (CeD) is a common autoimmune disorder caused by an abnormal immune response to dietary gluten proteins. The disease has high heritability. HLA is the major susceptibility factor, and the HLA effect is mediated via presentation of deamidated gluten peptides by disease-associated HLA-DQ variants to CD4+ T cells. In addition to gluten-specific CD4+ T cells the patients have antibodies to transglutaminase 2 (autoantigen) and deamidated gluten peptides. These disease-specific antibodies recognize defined epitopes and they display common usage of specific heavy and light chains across patients. Interactions between T cells and B cells are likely central in the pathogenesis, but how the repertoires of naïve T and B cells relate to the pathogenic effector cells is unexplored. To this end, we applied machine learning classification models to naïve B cell receptor (BCR) repertoires from CeD patients and healthy controls. Strikingly, we obtained a promising classification performance with an F1 score of 85%. Clusters of heavy and light chain sequences were inferred and used as features for the model, and signatures associated with the disease were then characterized. These signatures included amino acid (AA) 3-mers with distinct bio-physiochemical characteristics and enriched V and J genes. We found that CeD-associated clusters can be identified and that common motifs can be characterized from naïve BCR repertoires. The results may indicate a genetic influence by BCR encoding genes in CeD. Analysis of naïve BCRs as presented here may become an important part of assessing the risk of individuals to develop CeD. Our model demonstrates the potential of using BCR repertoires and in particular, naïve BCR repertoires, as disease susceptibility markers.

Structural Perspective of Gliadin Peptides Active in Celiac Disease

Gluten fragments released in gut of celiac individuals activate the innate or adaptive immune systems. The molecular mechanisms associated with the adaptive response involve a series of immunodominant gluten peptides which are mainly recognized by human leucocyte antigen (HLA)-DQ2.5 and HLA-DQ8. Other peptides, such as A-gliadin P31–43, are not recognized by HLA and trigger innate responses by several routes not yet well detailed. Among the gluten fragments known to be active in Celiac disease, here we focus on the properties of all gluten peptides with known tri-dimensional structure either those locked into HLA-DQ complexes whose crystals were X-ray analyzed or characterized in solution as free forms. The aim of this work was to find the structural reasons why some gluten peptides prompt the adaptive immune systems while others do not, by apparently involving just the innate immune routes. We propose that P31–43 is a non-adaptive prompter because it is not a good ligand for HLA-DQ. Even sharing a similar ability to adopt polyproline II structure with the adaptive ones, the way in which the proline residues are located along the sequence disfavors a productive P31–43-HLA-DQ binding.

Machine learning analysis of naïve B-cell receptor repertoires stratifies celiac disease patients and controls

Celiac disease (CeD) is a common autoimmune disorder caused by an abnormal immune response to dietary gluten proteins. The disease has high heritability. HLA is the major susceptibility factor, and the HLA effect is mediated via presentation of deamidated gluten peptides by disease-associated HLA-DQ variants to CD4+ T cells. In addition to gluten-specific CD4+ T cells the patients have antibodies to transglutaminase 2 (autoantigen) and deamidated gluten peptides. These disease-specific antibodies recognize defined epitopes and they display common usage of specific heavy and light chains across patients. Interactions between T cells and B cells are likely central in the pathogenesis, but how the repertoires of naïve T and B cells relate to the pathogenic effector cells is unexplored. To this end, we applied machine learning classification models to naïve B cell receptor (BCR) repertoires from CeD patients and healthy controls. Strikingly, we obtained a promising classification performance with an F1 score of 85%. Clusters of heavy and light chain sequences were inferred and used as features for the model, and signatures associated with the disease were then characterized. These signatures included amino acid (AA) 3-mers with distinct bio-physiochemical characteristics and enriched V and J genes. We found that CeD-associated clusters can be identified and that common motifs can be characterized from naïve BCR repertoires. The results may indicate a genetic influence by BCR encoding genes in CeD. Analysis of naïve BCRs as presented here may become an important part of assessing the risk of individuals to develop CeD. Our model demonstrates the potential of using BCR repertoires and in particular, naïve BCR repertoires, as disease susceptibility markers.