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

Abstract

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-1β. 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.

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

“…In a previous report, we showed by TEM that p31‐43 self‐aggregates presenting a PPII structure, in agreement with CD determinations . Subsequent NMR experiments performed by Calvanese et al confirmed that this peptide is able to adopt this structure.…”

“…We observed that wild‐type mice treated with p31‐43 presented histological alterations and increased number of intraepithelial lymphocytes, production of proinflammatory mediators and cell death. We also demonstrated that biological effects depend on the activation of the NLRP3 inflammasome . In the other hand, Villella et al .…”

“…To further evaluate the self‐assembly propensity of p31‐43 and the characteristics of the oligomers , we decided to use a variety of biophysical tools. Firstly, we used a fluorescent probe containing a boron dipyrromethane scaffold (BODIPY) .…”

“…The peptide 56‐87 from α‐gliadin fraction, called 33mer, has been extensively studied as model of T‐cell epitope because it induces a strong adaptive immune response in CeD patients , whereas the α‐gliadin peptide p31‐43 induces a non‐HLA‐mediated inflammatory response as well as several toxic effects evaluated by ex vivo and in vitro assays . Using an experimental model developed by our group, the in vivo biological effects of p31‐43 have been recently studied . We observed that wild‐type mice treated with p31‐43 presented histological alterations and increased number of intraepithelial lymphocytes, production of proinflammatory mediators and cell death.…”

“…Though biological effects triggered by p31‐43 (LGQQQPFPPQQPY) are observed very rapid upon treatment, the surface cell receptor or the mechanism of peptide entry has not been identified . In our previous study, we showed by CD and transmission electron microscopy (TEM) that p31‐43 presents a polyproline II (PPII) structure and forms oligomers which may promote the activation of the inflammasome platform . To gain structural details on p31‐43, several spectroscopic and microscopic techniques were used, as well as coarse‐grained (CG) dynamic simulation in order to understand at a molecular level, peptide interactions, and driven forces that leads to p31‐43 self‐assembly.…”