Duodenal mucosa of untreated celiac disease patients has altered expression of the GAS6 and PROS1 and the negative regulator tyrosine kinase TAM receptors subfamily.

Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.

Role of germinal center and CD39highCD73+ B cells in the age-related tonsillar involution.

BACKGROUND: The tonsils operate as a protection ring of mucosa at the gates of the upper aero-digestive tract. They show similarities with lymph nodes and participate as inductive organs of systemic and mucosal immunity. Based on the reduction of their size since puberty, they are thought to experience involution in adulthood. In this context, we have used tonsillar mononuclear cells (TMC) isolated from patients at different stages of life, to study the effect of ageing and the concomitant persistent inflammation on these immune cells. RESULTS: We found an age-dependent reduction in the proportion of germinal center B cell population (BGC) and its T cell counterpart (T follicular helper germinal center cells, TfhGC). Also, we demonstrated an increment in the percentage of local memory B cells and mantle zone T follicular helper cells (mTfh). Furthermore, younger tonsils rendered higher proportion of proliferative immune cells within the freshly isolated TMC fraction than those from older ones. We demonstrated the accumulation of a B cell subset (CD20+CD39highCD73+ cells) metabolically adapted to catabolize adenosine triphosphate (ATP) as patients get older. To finish, tonsillar B cells from patients at different ages did not show differences in their proliferative response to stimulation ex vivo, in bulk TMC cultures. CONCLUSIONS: This paper sheds light on the changing aspects of the immune cellular landscape, over the course of time and constant exposure, at the entrance of the respiratory and digestive systems. Our findings support the notion that there is a re-modelling of the immune functionality of the excised tonsils over time. They are indicative of a transition from an effector type of immune response, typically oriented to reduce pathogen burden early in life, to the development of an immunosuppressive microenvironment at later stages, when tissue damage control gets critical provided the time passed under immune attack. Noteworthy, when isolated from such histologic microenvironment, older tonsillar B cells seem to level their proliferation capacity with the younger ones. Understanding these features will not only contribute to comprehend the differences in susceptibility to pathogens among children and adults but would also impact on vaccine developments intended to target these relevant mucosal sites.

Biogeographic Variation and Functional Pathways of the Gut Microbiota in Celiac Disease.

BACKGROUND & AIMS: Genes and gluten are necessary but insufficient to cause celiac disease (CeD). Altered gut microbiota has been implicated as an additional risk factor. Variability in sampling site may confound interpretation and mechanistic insight, as CeD primarily affects the small intestine. Thus, we characterized CeD microbiota along the duodenum and in feces and verified functional impact in gnotobiotic mice. METHODS: We used 16S rRNA gene sequencing (Illumina) and predicted gene function (PICRUSt2) in duodenal biopsies (D1, D2 and D3), aspirates, and stool from patients with active CeD and controls. CeD alleles were determined in consented participants. A subset of duodenal samples stratified according to similar CeD risk genotypes (controls DQ2-/- or DQ2+/- and CeD DQ2+/-) were used for further analysis and to colonize germ-free mice for gluten metabolism studies. RESULTS: Microbiota composition and predicted function in CeD was largely determined by intestinal location. In the duodenum, but not stool, there was higher abundance of Escherichia coli (D1), Prevotella salivae (D2), and Neisseria (D3) in CeD vs controls. Predicted bacterial protease and peptidase genes were altered in CeD and impaired gluten degradation was detected only in mice colonized with CeD microbiota. CONCLUSIONS: Our results showed luminal and mucosal microbial niches along the gut in CeD. We identified novel microbial proteolytic pathways involved in gluten detoxification that are impaired in CeD but not in controls carrying DQ2, suggesting an association with active duodenal inflammation. Sampling site should be considered a confounding factor in microbiome studies in CeD.

Coexistence of apoptosis, pyroptosis, and necroptosis pathways in celiac disease.

Usually, the massive elimination of cells under steady-state conditions occurs by apoptosis, which is also acknowledged to explain the loss of enterocytes in the small intestine of celiac disease (CD) patients. However, little is known about the role of proinflammatory cell death pathways in CD. Here, we have used confocal microscopy, western blot, and RT-qPCR analysis to assess the presence of regulated cell death pathways in the duodenum of CD patients. We found an increased number of dead (TUNEL+) cells in the lamina propria of small intestine of CD patients, most of them are plasma cells (CD138+). Many dying cells expressed FAS and were in close contact with CD3+ T cells. Caspase-8 and caspase-3 expression was increased in CD, confirming the activation of apoptosis. In parallel, caspase-1, IL-1ß, and GSDMD were increased in CD samples indicating the presence of inflammasome-dependent pyroptosis. Necroptosis was also present, as shown by the increase of RIPK3 and phosphorylate MLKL. Analysis of published databases confirmed that CD has an increased expression of regulated cell death -related genes. Together, these results reveal that CD is characterized by cell death of different kinds. In particular, the presence of proinflammatory cell death pathways may contribute to mucosal damage.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1ß and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1ß by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1ß and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1ß, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

Programmed Cell Death in the Small Intestine: Implications for the Pathogenesis of Celiac Disease.

The small intestine has a high rate of cell turnover under homeostatic conditions, and this increases further in response to infection or damage. Epithelial cells mostly die by apoptosis, but recent studies indicate that this may also involve pro-inflammatory pathways of programmed cell death, such as pyroptosis and necroptosis. Celiac disease (CD), the most prevalent immune-based enteropathy, is caused by loss of oral tolerance to peptides derived from wheat, rye, and barley in genetically predisposed individuals. Although cytotoxic cells and gluten-specific CD4+ Th1 cells are the central players in the pathology, inflammatory pathways induced by cell death may participate in driving and sustaining the disease through the release of alarmins. In this review, we summarize the recent literature addressing the role of programmed cell death pathways in the small intestine, describing how these mechanisms may contribute to CD and discussing their potential implications.

Mechanisms of innate immune activation by gluten peptide p31-43 in mice.

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. Innate immunity contributes to the pathogenesis of CD, but the mechanisms remain poorly understood. Although previous in vitro work suggests that gliadin peptide p31-43 acts as an innate immune trigger, the underlying pathways are unclear and have not been explored in vivo. Here we show that intraluminal delivery of p31-43 induces morphological changes in the small intestinal mucosa of normal mice consistent with those seen in CD, including increased cell death and expression of inflammatory mediators. The effects of p31-43 were dependent on MyD88 and type I IFNs, but not Toll-like receptor 4 (TLR4), and were enhanced by coadministration of the TLR3 agonist polyinosinic:polycytidylic acid. Together, these results indicate that gliadin peptide p31-43 activates the innate immune pathways in vivo, such as IFN-dependent inflammation, relevant to CD. Our findings also suggest a common mechanism for the potential interaction between dietary gluten and viral infections in the pathogenesis of CD.

Intestinal microbiota modulates gluten-induced immunopathology in humanized mice.

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.

Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy.

Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa.

Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.

Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25(+)Foxp3(+) T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4(+) T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4(+) T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25(+)Foxp3(+) T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency.

IL-1ß blockade prevents cell death and mucosal damage of the small intestine in a model of sterile inflammation.

The intestinal mucosa is covered by a layer of epithelial cells that is constantly challenged by commensal, opportunistic, and pathogenic microorganisms, their components, and harmful compounds. Any inflammatory response to these materials must be tightly controlled to limit tissue damage and restore the integrity of the mucosal barrier. We have shown previously that production of IL-1ß via activation of the inflammasome can lead to mucosal damage in the small intestinal pathology that occurs after intragastric administration of a gluten derived peptide, p31-43. Here we show that specific inhibition of caspase-1 or NLRP3 abolishes the damage induced by p31-43, and that antibody-mediated blocking of IL-1ß inhibits the both the histological changes and the induction of apoptosis and caspase-3 activation driven by p31-43. Understanding the role of IL-1ß in sterile inflammation may help to understand chronic inflammatory pathological processes, and design new intervention strategies.

The gliadin p31-43 peptide: Inducer of multiple proinflammatory effects.

Coeliac disease (CD) is the prototype of an inflammatory chronic disease induced by food. In this context, gliadin p31-43 peptide comes into the spotlight as an important player of the inflammatory/innate immune response to gliadin in CD. The p31-43 peptide is part of the p31-55 peptide from α-gliadins that remains undigested for a long time, and can be present in the small intestine after ingestion of a gluten-containing diet. Different biophysical methods and molecular dynamic simulations have shown that p31-43 spontaneously forms oligomeric nanostructures, whereas experimental approaches using in vitro assays, mouse models, and human duodenal tissues have shown that p31-43 is able to induce different forms of cellular stress by driving multiple inflammatory pathways. Increased proliferative activity of the epithelial cells in the crypts, enterocyte stress, activation of TG2, induction of Ca2+, IL-15, and NFκB signaling, inhibition of CFTR, alteration of vesicular trafficking, and activation of the inflammasome platform are some of the biological effects of p31-43, which, in the presence of appropriate genetic susceptibility and environmental factors, may act together to drive CD.

Sterile inflammation drives multiple programmed cell death pathways in the gut.

Intestinal epithelial cells have a rapid turnover, being rapidly renewed by newly differentiated enterocytes, balanced by massive and constant removal of damaged cells by programmed cell death (PCD). The main forms of PCD are apoptosis, pyroptosis, and necroptosis, with apoptosis being a noninflammatory process, whereas the others drive innate immune responses. Although apoptosis is thought to be the principal means of cell death in the healthy intestine, which mechanisms are responsible for PCD during inflammation are not fully understood. To address this question, we used an in vivo model of enteropathy in wild-type mice induced by a single intragastric administration of the p31-43 gliadin peptide, which is known to elicit transient MyD88, NLRP3, and caspase-1-dependent mucosal damage and inflammation in the small intestine. Here, we found increased numbers of TUNEL+ cells in the mucosa as early as 2 h after p31-43 administration. Western blot and immunofluorescence analysis showed the presence of caspase-3-mediated apoptosis in the epithelium and lamina propria. In addition, the presence of mature forms of caspase-1, IL-1ß, and gasdermin D showed activation of pyroptosis and inhibition of caspase-1 led to decreased enterocyte death in p31-43-treated mice. There was also up-regulation of RIPK3 in crypt epithelium, suggesting that necroptosis was also occurring. Taken together, these results indicate that the inflammatory response induced by p31-43 can drive multiple PCD pathways in the small intestine.

Coeliac Disease Pathogenesis: The Uncertainties of a Well-Known Immune Mediated Disorder.

Coeliac disease is a common small bowel enteropathy arising in genetically predisposed individuals and caused by ingestion of gluten in the diet. Great advances have been made in understanding the role of the adaptive immune system in response to gluten peptides. Despite detailed knowledge of these adaptive immune mechanisms, the complete series of pathogenic events responsible for development of the tissue lesion remains less certain. This review contributes to the field by discussing additional mechanisms which may also contribute to pathogenesis. These include the production of cytokines such as interleukin-15 by intestinal epithelial cells and local antigen presenting cells as a pivotal event in the disease process. A subset of unconventional T cells called gamma/delta T cells are also persistently expanded in the coeliac disease (CD) small intestinal epithelium and recent analysis has shown that these cells contribute to pathogenic inflammation. Other unconventional T cell subsets may play a local immunoregulatory role and require further study. It has also been suggested that, in addition to activation of pathogenic T helper cells by gluten peptides, other peptides may directly interact with the intestinal mucosa, further contributing to the disease process. We also discuss how myofibroblasts, a major source of tissue transglutaminase and metalloproteases, may play a key role in intestinal tissue remodeling. Contribution of each of these factors to pathogenesis is discussed to enhance our view of this complex disorder and to contribute to a wider understanding of chronic immune-mediated disease.

Structural conformation and self-assembly process of p31-43 gliadin peptide in aqueous solution. Implications for celiac disease.

Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by in vitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and in silico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with ß-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased ß-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.

IL-33 Alarmin and Its Active Proinflammatory Fragments Are Released in Small Intestine in Celiac Disease.

Initially described as Th2 promoter cytokine, more recently, IL-33 has been recognized as an alarmin, mainly in epithelial and endothelial cells. While localized in the nucleus acting as a gene regulator, it can be also released after injury, stress or inflammatory cell death. As proinflammatory signal, IL-33 binds to the surface receptor ST2, which enhances mast cell, Th2, regulatory T cell, and innate lymphoid cell type 2 functions. Besides these Th2 roles, free IL-33 can activate CD8+ T cells during ongoing Th1 immune responses to potentiate its cytotoxic function. Celiac Disease (CD) is a chronic inflammatory disorder characterized by a predominant Th1 response leading to multiple pathways of mucosal damage in the proximal small intestine. By immunofluorescence and western blot analysis of duodenal tissues, we found an increased expression of IL-33 in duodenal mucosa of active CD (ACD) patients. Particularly, locally digested IL-33 releases active 18/21kDa fragments which can contribute to expand the proinflammatory signal. Endothelial (CD31+) and mesenchymal, myofibroblast and pericyte cells from microvascular structures in villi and crypts, showed IL-33 nuclear location; while B cells (CD20+) showed a strong cytoplasmic staining. Both ST2 forms, ST2L and sST2, were also upregulated in duodenal mucosa of CD patients. This was accompanied by increased number of CD8+ST2+ T cells and the expression of T-bet in some ST2+ intraepithelial lymphocytes and lamina propria cells. IL-33 and sST2 mRNA levels correlated with IRF1, an IFN induced factor relevant in responses to viral infections and interferon mediated proinflammatory responses highly represented in duodenal tissues in ACD. These findings highlight the potential contribution of IL-33 and its fragments to exacerbate the proinflammatory circuit and potentiate the cytotoxic activity of CD8+ T cells in CD pathology.

Recent Progress and Recommendations on Celiac Disease From the Working Group on Prolamin Analysis and Toxicity.

Celiac disease (CD) affects a growing number of individuals worldwide. To elucidate the causes for this increase, future multidisciplinary collaboration is key to understanding the interactions between immunoreactive components in gluten-containing cereals and the human gastrointestinal tract and immune system and to devise strategies for CD prevention and treatment beyond the gluten-free diet. During the last meetings, the Working Group on Prolamin Analysis and Toxicity (Prolamin Working Group, PWG) discussed recent progress in the field together with key stakeholders from celiac disease societies, academia, industry and regulatory bodies. Based on the current state of knowledge, this perspective from the PWG members provides recommendations regarding clinical, analytical and legal aspects of CD. The selected key topics that require future multidisciplinary collaborative efforts in the clinical field are to collect robust data on the increasing prevalence of CD, to evaluate what is special about gluten-specific T cells, to study their kinetics and transcriptomics and to put some attention to the identification of the environmental agents that facilitate the breaking of tolerance to gluten. In the field of gluten analysis, the key topics are the precise assessment of gluten immunoreactive components in wheat, rye and barley to understand how these are affected by genetic and environmental factors, the comparison of different methods for compliance monitoring of gluten-free products and the development of improved reference materials for gluten analysis.

Mucosal tissue transglutaminase expression in celiac disease.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD.

p31-43 Gliadin Peptide Forms Oligomers and Induces NLRP3 Inflammasome/Caspase 1- Dependent Mucosal Damage in Small Intestine.

Celiac disease (CD) is a chronic enteropathy elicited by a Th1 response to gluten peptides in the small intestine of genetically susceptible individuals. However, it remains unclear what drives the induction of inflammatory responses of this kind against harmless antigens in food. In a recent work, we have shown that the p31-43 peptide (p31-43) from α-gliadin can induce an innate immune response in the intestine and that this may initiate pathological adaptive immunity. The receptors and mechanisms responsible for the induction of innate immunity by p31-43 are unknown and here we present evidence that this may reflect conformational changes in the peptide that allow it to activate the NLRP3 inflammasome. Administration of p31-43, but not scrambled or inverted peptides, to normal mice induced enteropathy in the proximal small intestine, associated with increased production of type I interferon and mature IL-1ß. P31-43 showed a sequence-specific spontaneous ability to form structured oligomers and aggregates in vitro and induced activation of the ASC speck complex. In parallel, the enteropathy induced by p31-43 in vivo did not occur in the absence of NLRP3 or caspase 1 and was inhibited by administration of the caspase 1 inhibitor Ac-YVAD-cmk. Collectively, these findings show that p31-43 gliadin has an intrinsic propensity to form oligomers which trigger the NLRP3 inflammasome and that this pathway is required for intestinal inflammation and pathology when p31-43 is administered orally to mice. This innate activation of the inflammasome may have important implications in the initial stages of CD pathogenesis.