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.

Keywords: caspase-1; celiac disease; enteropathy; gliadin peptides; inflammasome; innate immunity; p31-43; small intestine damage.

Figure 1

Gliadin peptide p31-43 induces small intestine pathology in a sequence-specific manner. C57BL/6 mice were injected intraluminally with native p31-43, inverted sequence peptide (IP), scrambled sequence peptide (SP), non-related peptide (NRP), or PBS. The ratio between villus length and crypt depth (V/C) (right axis) and the numbers of intraepithelial lymphocytes/100 enterocytes (left axis) were determined on H&E stained sections of proximal small intestine 16 h after administration of 10 μg (A) or 1 μg (B) peptide/mouse. IFNβ mRNA was assessed by RT-qPCR on whole proximal small intestine samples 4 h after treatment. Results shown are means ± 1 SEM for 5 mice per group. ^*p < 0.05 by ANOVA, post-test Bonferroni.

Figure 2

Structural analysis of p31-43 by circular dichroism shows polyproline II structures. (A) Circular dichroism spectra obtained using 10 μM (15.3 μg/mL), 50 μM (76.5 μg/mL), and 100 μM (153 μg/mL) solutions of peptide at 4°C. A negative band at 205 nm together with a positive one 225 nm indicates the presence a polyproline II structure, while the hypochromic shift of the bands indicates a self-assembly process. Results shown are representative of three independent experiments. (B) Circular dichroism analysis of p31-43, inverted peptides (PI) and scrambled peptides (SP) at 100 μM (153 μg/mL) at 4°C. In the scrambled peptide a negative band at 203 nm is indicative of a random secondary structure, while the negative band at around 204 nm and the positive one at 225 nm in the p31-43 and inverted peptides is indicative of a polyproline II conformation. p31-43 shows a hypochromic displacement of the negative band with respect to the inverted peptide, suggesting a self-organization process in p31-43. Results are representative of three independent experiments.

Figure 3

Oligomeric p31-43 structures characterized by TEM. Samples of p31-43 were prepared in MilliQ water and deposited on a grid covered with formvar. (A) Representative image showing the presence of oligomeric structures in a 50 μM (76.5 μg/mL) solution. (B) Size distribution of the oligomers observed in (A), showing a mean diameter of 13.6 ± 3.4 nm. (C) Representative image showing the presence of oligomers in a 100 μM (153 μg/mL) solution. (D) Size distribution of the oligomers observed in (C), showing a mean diameter of the structures is 17 ± 7 nm.

Figure 4

In-silico modeling of p31-43 aggregation. (A) Left: cartoon representation of the initial (helical) conformation used as starting conformer. For visual clarity, only Prolines are shown. Right: four different conformers arbitrarily chosen along the simulation of the isolated peptide and used to generate the initial configuration of the system containing 50 copies of p31-43 [inset indicated with 0 ms in panel (B)]. The cartoons show the conserved presence of polyproline. (B) Mean cluster sizes of aggregates formed before (bottom right) and during simulation (top left) using 50 copies of p31-43. Individual peptide copies are identified by different colors. (C) The final state is shown (left), with structural convergence within the aggregate being highlighted by superposition of individual copies within a maximum root mean square deviation of 0.1 nm, which corresponds to 41 out 50 peptides (right).

Figure 5

Oral administration of p31-43 induces caspase 1 activation and production of mature IL-1β. (A) Western blot analysis of mature IL-1β and (B) caspase-1 and pro-caspase-1 quantification in whole small intestinal tissue from p31-43 or vehicle-fed mice 16 h after treatment. (C) IL-1β/caspase 1 ratio of data shown in (A,B). (D) Representative results of Western blots are shown. Results shown are means ± 1 SEM for 5 mice and are representative of two experiments. ^**p < 0.01, ^***p < 0.001, Student's t-test.

Figure 6

p31-43 induces ASC speck formation in vitro. (A) Human THP-1 ASC speck reporter cells were preconditioned for 3 h with LPS before addition of different amounts of p31-43 for a further 16 h. Positive control cells (DNA) were transfected transiently with bacterial plasmid before addition of LPS. Cells showing ASC specks were assessed by immunofluorescence and expressed as positive cells/100 total cells. Results shown are means ±1 SEM for three replicates/group and are representative of two experiments ^**p < 0.01; ^***p < 0.0001, ANOVA. (B) Representative images of ASC speck expressing cells (green) (blue–DAPI).

Figure 7

Enteropathy induced by p31-43 requires NLRP3 and caspase 1. Morphological analysis of proximal small intestine of C57BL/6, NLRP3^−/−, and caspase 1^−/− mice, 16 h after oral administration of 20 μg p31-43 or PBS, showing mean IEL/100 enterocytes (left axis) and villus/crypt ratios (right axis). Results shown are means ± 1 SEM for 6 mice/group. ^*p < 0.05.

Figure 8

Enteropathy induced by p31-43 is prevented by inhibition of caspase 1. Villus/crypt rations (A) and intraepithelial lymphocyte counts (B) in the proximal small intestine of C57BL/6 mice fed 20 μg p31-43 or PBS that had been treated ip 30 min earlier with either 8 mg/kg of the Ac-YVAD-cmk caspase 1 inhibitor or with DMSO. Results shown are means ± 1 SEM for 5 mice/group assessed 16 h after feeding p31-43 and are representative of two experiments. ^*p < 0.05, ^**p < 0.01, ANOVA, Post-test Bonferroni.