Fig 2: Prevention by CFTR potentiators of P31–43 induced derangement of endosomal traffickingCaco-2 cells challenged with P57–68 or PGAV or P31–43 peptides in the presence or absence of pretreatment of VX-770 and 3-MA. A–CImmunoblot of BECN-1 (A), phosphatidylinositol-3 (PI3)-kinase (hVps34) (B), UVRAG and Rab5 (C), and ß-actin; densitometric analysis of protein levels (bottom). Mean ± SD of triplicates of independent experiments. *P < 0.05 or **P < 0.01 or ***P < 0.01 vs. P31–43, °P < 0.05 VX-770+P31–43 vs. VX-770 + 3-MA+P31–43 (ANOVA, Bonferroni post hoc test).DImmunoblot of Rab5 and Rab7 proteins in endosomal protein fractions from Caco-2 cells challenged as described; endosomal antigen-1 (EEA-1) as loading control. Densitometric analysis of immunoblot is mean ± SD of triplicates of independent experiments. *P < 0.05 vs. P31–43, °P < 0.05 vs. P31–43 + VX-770.ECaco-2 cells pre-incubated for 1 h at 4°C with S-IgA from hColostrum and then challenged with P31–43 in the presence or absence of VX-770. Confocal images of apical localization of CD71. Staining with anti-CD71 antibody and DAPI nuclear counterstaining (top) or anti-CD71 (red) and S-IgA (green) antibodies (bottom). Scale bar, 50 µm.FImmunoprecipitation of CFTR protein in endosomal protein fractions in non-reducing and non-denaturing conditions, after 15 min of challenge with P31–43 and immunoblot with HRP-streptavidin or CFTR.Data information: The blots are representative of one experiment for group of treatment.Source data are available online for this figure.

Fig 3: Mutual relationship between gliadin, TG2, and CFTR in intestinal epithelial cells AGraphical view of the in silico sampling percentage of P31–43 and of P31–43/4QA (4QA) against NBD1.BSPR analysis of 4QA peptide on rhNBD1 covalently bound to CM5 sensor chip. For further details, see Materials and Methods.C, DIncubation of Caco-2 cells with P31–43 or with modified P31–43 (P31–43-2QA 4–5th or P31–43-2QA 10–11th or P31–43/4QA mutant (4QA) peptides) for 1 h. (C) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunoblot with streptavidin-HRP or CFTR antibody (n = 3 independent experiments). (D) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase in chloride current [Isc (µA/cm2)] in Caco-2 cells mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (?Isc) in Caco-2 cells (n = 3 independent experiments). Mean ± SD of samples assayed. °°P < 0.01 vs. P31–43 challenge, ***P < 0.001 vs. Caco-2 challenged with 4QA (ANOVA, Bonferroni post hoc test).E In vitro Blue native PAGE Western blotting of enhanced binding of P31–43 to rhNBD1 in the presence of activated rTG2 that in turn promotes the formation of higher molecular weight complexes. As described in the text, only P31–43 (lanes 1, 3, 5), and not P57–68 (lanes 2, 4, 6), has the ability to form stable complexes with NBD1 and TG2 (n = 3 independent experiments).F–LIncubation of Caco-2 cells with P57–68 or P31–43, in the presence or absence of pre-treatment with TG2 inhibitor Z-DON, TG2-siRNA, BAPTA-AM, or VX-770. (F) Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunoblot with anti-isopeptide glutamime-lysine and CFTR or (G) immunoprecipitation of CFTR and then immunoblot with anti-TG2 or anti-CFTR antibodies (n = 3 independent experiments). (H and I) Effects of treatment with VX-770 and Z-DON on TG2 transamidating activity in situ. (H) In situ detection of TG2 activity in Caco-2 cells pulsed with Ca2+ by immunoblotting of the TG-catalyzed incorporation of 5-biotinamidopentylamine (BAP) and blotting with anti-biotin antibody (n = 3 independent experiments). (I) Assay of TG2 activity by immunostaining of the TG-catalyzed incorporation of monodansylcadaverine in Caco-2 cells pre-treated with VX-770 or Z-DON and then pulsed with P31–43. Scale bar, 50 µm. (J) Immunoprecipitation of CFTR and immunoblot with anti-CFTR and anti-TG2 antibodies. (K) Immunoprecipitation in non-reducing and non-denaturing conditions of PKAr2a protein and immunoblot with isopeptide glutamine-lysine and PKAr2a antibodies (n = 3 independent experiments). (L) Immunoblot of phospho-PKA (phPKA) protein; densitometric analysis of protein levels relative to ß-actin. Mean ± SD of triplicates of independent experiments. **P < 0.01 vs. P31–43, °°P < 0.001 vs. VX-770 (ANOVA, Bonferroni post hoc test).Data information: The blots are representative of one experiment for group of treatment.Source data are available online for this figure.

Fig 4: Effects of P31–43 stimulation on forskolin-activated CFTR channel ARepresentative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current [Isc (µA/cm2)] in Caco-2 cells mounted in Ussing chambers: effects of P31–43 (100 µM) and PGAV (100 µM) directly added to the bathing solution. After amiloride, the solution was directly supplied with buffer only (none, gray traces), with PGAV (green traces) or P31–43 (black traces) for 3–5 min (as detailed in Materials and Methods) and then pulsed with forskolin (Fsk; 20 µM) and finally with the CFTR inhibitor 172 (CFTRinh172). Yellow traces: after amiloride, the solution was first supplied with VX-770 (10 µM) followed by P31–43 (100 µM) and then pulsed with forskolin (Fsk) followed by CFTRinh172. Quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (?Isc) in Caco-2 cells (n = 3 independent experiments, mean ± SD). **P < 0.01 None or PGAV (gray or green traces) vs. P31–43 followed by Fsk (black traces), °°P < 0.01 P31–43 followed by Fsk (black trace) vs. VX-770 followed by P31–43 and then by Fsk (yellow trace; ANOVA, Bonferroni post hoc test).BWhole-cell CFTR current densities induced by 10 µM forskolin (Fsk) at +100 mV in Caco-2 cells w/o treatment (left), with 100 µM P31–43 peptide in the luminal solution during patch clamping (middle), or after treatment with 20 µM VX-770 (20 min), followed by an application of 100 µM P31–43 peptide in the luminal solution while patch clamping (right). Current–voltage (I–V) relationships were elicited by ramps from -100 mV to +100 mV (holding potential, -40 mV). Statistical analysis of average CFTR current densities induced by forskolin and blocked by CFTR inh-172 in cells treated as described. n = 3; ***P < 0.001 basal vs. P31–43, °°°P < 0.001 P31–43 vs. VX770 + P31–43, two-way ANOVA with Bonferroni post-test. Data are presented as means ± SEM (bottom). Source data are available online for this figure.

Fig 5: Schematic view of celiac disease pathogenesisThe P31–43 peptide from the gluten-associated protein gliadin is generated in the gut lumen and interacts with the NBD1 domain of CFTR, if the domain is in its inactive conformation. P31–43 binds to specific residues of NBD1 of inactive CFTR, competing with ATP binding, thus blocking CFTR function as an anion channel. The inhibition of CFTR disrupts cellular proteostasis through two effects (i) TG2 activation and (ii) BECN1 complex inhibition. This accessorily recruits TG2 to a tripartite complex that stabilizes P31–43 binding to CFTR, thus worsening CFTR inhibition. P31–43-mediated CFTR inhibition leads to impaired endosomal trafficking, cytoskeleton disassembly, inflammasome activation resulting in IL-1ß secretion, NF-?B activation, and consequent IL-15 production. Stressed enterocytes stimulate local inflammation and immune responses against immunodominant gliadin peptides, in particular peptide P56–88, in the context of HLA-DQ2/DQ8, thus triggering celiac disease. This pathogenic cascade may be theoretically interrupted at two levels, at the apex by CFTR potentiators that prevent P31–43 binding to CFTR (but are poorly active if they are added after P31–43 addition) or by reconstitution of cellular proteostasis by inhibiting TGase or activating the BECN1 complex.