A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease
2018; Springer Nature; Volume: 38; Issue: 2 Linguagem: Inglês
10.15252/embj.2018100101
ISSN1460-2075
AutoresValeria Rachela Villella, Andrea Venerando, Giorgio Cozza, Speranza Esposito, Eleonora Ferrari, Romina Monzani, Mara Cetty Spinella, Vasileios Oikonomou, Giorgia Renga, Antonella Tosco, Federica Rossin, Stefano Guido, Marco Silano, Enrico Garaci, Yu‐Kai Chao, Christian Grimm, Alessandro Luciani, Luigina Romani, Mauro Piacentini, Valeria Raia, Guido Kroemer, Luigi Maiuri,
Tópico(s)Digestive system and related health
ResumoArticle28 November 2018Open Access Source DataTransparent process A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease Valeria R Villella Valeria R Villella European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Venerando Andrea Venerando orcid.org/0000-0003-0379-2309 Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy Search for more papers by this author Giorgio Cozza Giorgio Cozza Department of Molecular Medicine, University of Padova, Padova, Italy Search for more papers by this author Speranza Esposito Speranza Esposito European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Eleonora Ferrari Eleonora Ferrari European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Romina Monzani Romina Monzani European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Mara C Spinella Mara C Spinella European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Vasilis Oikonomou Vasilis Oikonomou Department of Experimental Medicine, University of Perugia, Perugia, Italy Search for more papers by this author Giorgia Renga Giorgia Renga Department of Experimental Medicine, University of Perugia, Perugia, Italy Search for more papers by this author Antonella Tosco Antonella Tosco Pediatric Unit, Department of Translational Medical Sciences, Regional Cystic Fibrosis Center, Federico II University Naples, Naples, Italy Search for more papers by this author Federica Rossin Federica Rossin Department of Biology, University of Rome "Tor Vergata", Rome, Italy Search for more papers by this author Stefano Guido Stefano Guido Department of Chemical, Materials and Production Engineering, Federico II University Naples, Naples, Italy Search for more papers by this author Marco Silano Marco Silano Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Roma, Italy Search for more papers by this author Enrico Garaci Enrico Garaci University San Raffaele and 21 IRCCS San Raffaele, Rome, Italy Search for more papers by this author Yu-Kai Chao Yu-Kai Chao orcid.org/0000-0002-1202-2448 Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU), Munich, Germany Search for more papers by this author Christian Grimm Christian Grimm Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU), Munich, Germany Search for more papers by this author Alessandro Luciani Alessandro Luciani Institute of Physiology CH, University of Zurich, Zurich, Switzerland Search for more papers by this author Luigina Romani Luigina Romani Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Mauro Piacentini Mauro Piacentini orcid.org/0000-0003-2919-1296 Department of Biology, University of Rome "Tor Vergata", Rome, Italy National Institute for Infectious Diseases IRCCS "L. Spallanzani", Rome, Italy Search for more papers by this author Valeria Raia Valeria Raia Pediatric Unit, Department of Translational Medical Sciences, Regional Cystic Fibrosis Center, Federico II University Naples, Naples, Italy Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer [email protected] orcid.org/0000-0002-9334-4405 Centre de Recherche des Cordeliers, Equipe11 labellisée Ligue Nationale Contrele Cancer, Paris, France Centre de Recherche des Cordeliers, INSERM U1138, Paris, France Université Paris Descartes, Paris, France Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Luigi Maiuri Corresponding Author Luigi Maiuri [email protected] orcid.org/0000-0002-4962-9016 European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Valeria R Villella Valeria R Villella European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Venerando Andrea Venerando orcid.org/0000-0003-0379-2309 Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy Search for more papers by this author Giorgio Cozza Giorgio Cozza Department of Molecular Medicine, University of Padova, Padova, Italy Search for more papers by this author Speranza Esposito Speranza Esposito European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Eleonora Ferrari Eleonora Ferrari European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Romina Monzani Romina Monzani European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Mara C Spinella Mara C Spinella European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Vasilis Oikonomou Vasilis Oikonomou Department of Experimental Medicine, University of Perugia, Perugia, Italy Search for more papers by this author Giorgia Renga Giorgia Renga Department of Experimental Medicine, University of Perugia, Perugia, Italy Search for more papers by this author Antonella Tosco Antonella Tosco Pediatric Unit, Department of Translational Medical Sciences, Regional Cystic Fibrosis Center, Federico II University Naples, Naples, Italy Search for more papers by this author Federica Rossin Federica Rossin Department of Biology, University of Rome "Tor Vergata", Rome, Italy Search for more papers by this author Stefano Guido Stefano Guido Department of Chemical, Materials and Production Engineering, Federico II University Naples, Naples, Italy Search for more papers by this author Marco Silano Marco Silano Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Roma, Italy Search for more papers by this author Enrico Garaci Enrico Garaci University San Raffaele and 21 IRCCS San Raffaele, Rome, Italy Search for more papers by this author Yu-Kai Chao Yu-Kai Chao orcid.org/0000-0002-1202-2448 Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU), Munich, Germany Search for more papers by this author Christian Grimm Christian Grimm Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU), Munich, Germany Search for more papers by this author Alessandro Luciani Alessandro Luciani Institute of Physiology CH, University of Zurich, Zurich, Switzerland Search for more papers by this author Luigina Romani Luigina Romani Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Mauro Piacentini Mauro Piacentini orcid.org/0000-0003-2919-1296 Department of Biology, University of Rome "Tor Vergata", Rome, Italy National Institute for Infectious Diseases IRCCS "L. Spallanzani", Rome, Italy Search for more papers by this author Valeria Raia Valeria Raia Pediatric Unit, Department of Translational Medical Sciences, Regional Cystic Fibrosis Center, Federico II University Naples, Naples, Italy Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer [email protected] orcid.org/0000-0002-9334-4405 Centre de Recherche des Cordeliers, Equipe11 labellisée Ligue Nationale Contrele Cancer, Paris, France Centre de Recherche des Cordeliers, INSERM U1138, Paris, France Université Paris Descartes, Paris, France Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Luigi Maiuri Corresponding Author Luigi Maiuri [email protected] orcid.org/0000-0002-4962-9016 European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy Department of Health Sciences, University of Eastern Piedmont, Novara, Italy Search for more papers by this author Author Information Valeria R Villella1, Andrea Venerando2, Giorgio Cozza3, Speranza Esposito1, Eleonora Ferrari1,4, Romina Monzani1,4, Mara C Spinella1,4, Vasilis Oikonomou5, Giorgia Renga5, Antonella Tosco6, Federica Rossin7, Stefano Guido8, Marco Silano9, Enrico Garaci10, Yu-Kai Chao11, Christian Grimm11, Alessandro Luciani12, Luigina Romani4, Mauro Piacentini7,13, Valeria Raia6,‡, Guido Kroemer *,14,15,16,17,18,19,‡ and Luigi Maiuri *,1,4,‡ 1European Institute for Research in Cystic Fibrosis, San Raffaele Scientific Institute, Milan, Italy 2Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy 3Department of Molecular Medicine, University of Padova, Padova, Italy 4Department of Health Sciences, University of Eastern Piedmont, Novara, Italy 5Department of Experimental Medicine, University of Perugia, Perugia, Italy 6Pediatric Unit, Department of Translational Medical Sciences, Regional Cystic Fibrosis Center, Federico II University Naples, Naples, Italy 7Department of Biology, University of Rome "Tor Vergata", Rome, Italy 8Department of Chemical, Materials and Production Engineering, Federico II University Naples, Naples, Italy 9Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Roma, Italy 10University San Raffaele and 21 IRCCS San Raffaele, Rome, Italy 11Department of Pharmacology and Toxicology, Faculty of Medicine, University of Munich (LMU), Munich, Germany 12Institute of Physiology CH, University of Zurich, Zurich, Switzerland 13National Institute for Infectious Diseases IRCCS "L. Spallanzani", Rome, Italy 14Centre de Recherche des Cordeliers, Equipe11 labellisée Ligue Nationale Contrele Cancer, Paris, France 15Centre de Recherche des Cordeliers, INSERM U1138, Paris, France 16Université Paris Descartes, Paris, France 17Metabolomics and Cell Biology Platforms, Institut Gustave Roussy, Villejuif, France 18Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 19Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden ‡These authors contributed equally to this work *Corresponding auhor. Tel: +33142116046; E-mail: [email protected] *Corresponding auhor. Tel: +393311313941; E-mail: [email protected] The EMBO Journal (2019)38:e100101https://doi.org/10.15252/embj.2018100101 See also: L Vachel & S Muallem (January 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31–43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31–43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease. Synopsis The molecular basis for permanent intolerance to dietary cereal proteins and excessive adaptive immune responses in the gut underlying celiac disease have remained unclear. Molecular and genetic approaches now identify the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel as major environmental stress transducer and potential therapeutic target in chronic intestinal inflammation. Constitutive CFTR deficiency in mice increases innate immune response to the gluten component gliadin. Gliadin peptide P31–43 inhibits nucleotide-binding domain-1 and ATPase activity of CFTR, impairing its function in intestinal epithelial cells. P31–43-mediated CFTR inhibition disrupts cellular proteostasis through sustained transglutaminase activation. P31–43-mediated CFTR inhibition leads to cytoskeleton disassembly, impaired endosomal trafficking, and activation of the inflammasome. Pharmaceutical CFTR potentiators rescue CTFR channel gating and protect from stress-induction in gluten-sensitive mice and celiac patient samples. Introduction The intestinal immune system is confronted with the permanent challenge to distinguish between safe and potentially harmful luminal triggers. Under physiological conditions, a finely tuned system of cellular adaptation ensures tissue homeostasis and provides the gut mucosa with the unique capacity of suppressing inflammation and promoting oral tolerance to non-self-antigens from dietary origin or commensal microbes (Kim et al, 2016). This tolerogenic response can be subverted by environmental triggers, such as viral infections (Bouziat et al, 2017), or as-yet-undefined predisposing factors, leading to immune and inflammatory responses. Celiac disease (CD), a permanent intolerance to dietary proteins from wheat, rye, and barley, occurring in ~ 1% of individuals worldwide, is a paradigm of antigen mishandling. In a subset of genetically susceptible individuals bearing the human leukocyte antigen (HLA) DQ2/DQ8, the ingestion of gluten proteins switches tolerance toward an adaptive immune response with an autoimmune component (Meresse et al, 2012; Sollid & Jabri, 2013). In the CD gut, DQ2/DQ8-restricted gluten-specific CD4+ T cells act in concert with intestinal B cells to promote the production of IgA antibodies against the self-antigen tissue transglutaminase (TG2; Meresse et al, 2012; Sollid & Jabri, 2013). However, additional factors are required to ignite an epithelial stress response with cytotoxic activation of intraepithelial CD8+ T lymphocytes, which ultimately cause villous atrophy and disease pathology (Meresse et al, 2009, 2012; Cerf-Bensussan & Meresse, 2015; Setty et al, 2015). Such stressors possibly include reovirus infections (Bouziat et al, 2017) or other yet-to-be-defined factors (Cerf-Bensussan & Meresse, 2015) that provide the danger signals for adaptive immune response to immunodominant gliadin epitopes (Maiuri et al, 2003; Meresse et al, 2009, 2012; DePaolo et al, 2011; Barone et al, 2014; Cerf-Bensussan & Meresse, 2015; Jabri & Abadie, 2015; Setty et al, 2015). However, the exact mechanisms through which gliadin can ignite a stress response are still unclear. An approximately threefold increase in the prevalence of fully diagnosed CD, as well as a ~ 4% prevalence of positive anti-TG2-IgA autoantibodies, a serological marker of CD, even in the absence of villous atrophy (Lionetti et al, 2015), has been reported in several cohorts of patients with cystic fibrosis (CF; Fluge et al, 2009; Walkowiak et al, 2010; De Lisle & Borowitz, 2013), including ours (Appendix Table S1). CF is the most frequent monogenic lethal disease worldwide (Cutting, 2015), caused by loss-of-function mutations of the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate (cAMP)-regulated anion channel that mediates chloride/bicarbonate transport across epithelia (Gadsby et al, 2006; Cutting, 2015). CF is best known for its respiratory phenotype, yet also frequently leads to intestinal problems, as CFTR protein is strongly expressed all along the intestine (Gadsby et al, 2006; Ooi & Durie, 2016). CFTR is not only an anion channel, but also orchestrates proteostasis at respiratory and intestinal epithelial surfaces, meaning that it regulates adaptation to cell-autonomous or environmental stress signals (Luciani et al, 2010b; Villella et al, 2013a,b; Ferrari et al, 2011). CFTR malfunction generates epithelial stress, early TG2 activation, inhibition of autophagy, and activation of innate immunity (Maiuri et al, 2008; Luciani et al, 2009; Luciani et al, 2010b; Villella et al, 2013a,b; Ferrari et al, 2011), features that are reminiscent of those triggered by gliadin in intestinal epithelial cells and celiac duodenal mucosa (Maiuri et al, 2003; Meresse et al, 2009; Barone et al, 2014). These considerations led us to hypothesize that CFTR might be involved in the pathogenesis of CD. Specifically, we tested the hypothesis that gliadin may induce a stress response and subvert host mucosal defenses by reducing CFTR function at the intestinal surface. Results CFTR malfunction favors gliadin responsiveness in vivo To determine whether the constitutive activation of innate immunity in the CF intestine (De Lisle & Borowitz, 2013; Nichols & Chmiel, 2015; Ooi & Durie, 2016) may favor the inflammatory and immune response to the gluten component gliadin, we administered gliadin to constitutively CFTR-deficient mice, either CFTR knock-out (B6.129P2-KOCftrtm1UNC, Cftr−/−) mice or knock-in mice harboring the most common loss-of-function F508del-CFTR mutation (Cftrtm1EUR, F508del, FVB/129, CftrF508del/F508del; Cutting, 2015). Before gliadin challenge, the small intestine from CFTR-defective mice exhibited increased TG2 protein levels (Fig 1A), high NLRP3 activation, and caspase-1 cleavage (Fig 1B) and increased levels of the pro-inflammatory cytokines IL-1β, MIP-2α, TNF-α, and IL-17A (Fig 1C; Appendix Fig S1A), but reduced IFN-γ production (De Lisle & Borowitz, 2013; Nichols & Chmiel, 2015; Fig 1C), together with cytoskeletal disassembly and increased intestinal permeability (Appendix Fig S1B and C) as compared to their wild-type (WT) controls. Consistent with the constitutive NF-κB activation in CF tissues (De Lisle & Borowitz, 2013; Nichols & Chmiel, 2015) and with the presence of an active NF-κB binding motif in the gene promoter of IL-15 (Stone et al, 2011), a master pro-inflammatory cytokine that critically contributes to breaking oral tolerance to gluten and hence to causing CD-associated pathology (Meresse et al, 2009, 2012; DePaolo et al, 2011; Cerf-Bensussan & Meresse, 2015; Jabri & Abadie, 2015; Setty et al, 2015), CFTR-defective mice showed a constitutive increase in IL-15 mRNA and protein levels in the small intestine, irrespective of differences in genetic background (P < 0.001 vs. WT mice; Fig 1D). Thus, CFTR malfunction suffices to activate the intestinal IL-15 system. IL-15 upregulation in CFTR-deficient mouse intestine was mediated by the constitutive activation of TG2, which is known to induce NF-κB activation through sequestering IK-Bα protein (Luciani et al, 2009), as IL-15 expression in the small intestine was largely reduced in CftrF508del/F508del mice backcrossed into a TG2-knock-out background (TG2−/−/CftrF508del/F508del; Fig 1E). Next, we administered gliadin to CFTR-deficient mice or their WT littermates for four consecutive weeks (5 mg/daily for 1 week and then 5 mg/daily thrice a week for 3 weeks; Galipeau et al, 2011; Papista et al, 2012; Larsen et al, 2015; Moon et al, 2016). Both FVB/129 and B6.129P2 littermates bearing WT-CFTR are not sensitive to gliadin, as oral gliadin administration failed to stimulate inflammatory response and IFN-γ production. Conversely, CFTR-mutated mice (De Stefano et al, 2014; Tosco et al, 2016) fed with gliadin greatly increased both IL-15 and IL-17A levels in their small intestine and exhibited a 3.5-fold increase in IFN-γ production (P < 0.01 vs. vehicle-treated mice; Fig 1F; Appendix Fig S1D and E). These results indicate that the constitutive stress response and innate immunity activation in CFTR-deficient intestines favor an immune response to gliadin. Figure 1. CFTR malfunction favors gliadin responsiveness in vivo A. Immunoblot with anti-TG2 or anti-β-actin as loading control in whole lysates from small intestine homogenates of CftrF508del/F508del and wild-type (CftrWT) mice (n = 5 per group) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **P < 0.01 (Student's t-test). B. Detection of NLRP3 expression (top) and caspase-1 cleavage (bottom) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del/F508del mice (n = 5) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **P < 0.01 CftrWT vs. CftrF508del/F508del (Student's t-test). C. Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n = 10). Mean ± SD of triplicates of independent pooled samples. ***P < 0.001 CftrWT vs. CftrF508del/F508del (Student's t-test). D. IL-15 mRNA (left) and protein (right) levels in small intestine homogenates from CftrF508del/F508del, Cftr−/− or their CftrWT littermates (n = 10 per group). Mean ± SD of triplicates of independent pooled samples. **P < 0.01 CftrF508del/F508del vs. CftrWT(FVB/129), or °°P < 0.01 Cftr−/− vs. CftrWT(B6.129P2) (ANOVA, Bonferroni post hoc test). E. IL-15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del/F508del or TG−/−/CftrF508del/F508del or TG−/− mice (n = 10 per group). Mean ± SD of triplicates of independent pooled samples. ***P < 0.001 vs. CftrWT, °°P < 0.01, ##P < 0.01 vs. CftrF508del/F508del (ANOVA, Bonferroni post hoc test). F. Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A, and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n = 10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **P < 0.01, ***P < 0.001 (CftrF508del/F508del vs. CftrWT mice prior gliadin challenge), °°°P < 0.001 (CftrF508del/F508del mice vs. CftrF508del/F508del mice after gliadin challenge; ANOVA, Bonferroni post hoc test). G–I. BALB/c mice (G) fed with a gluten-free diet for at least three generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for 1 week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl− secretion measured by forskolin (Fsk)-induced increase in chloride current [Isc (μA/cm2)] in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n = 3 independent experiments). Mean ± SD of samples assayed; **P < 0.01, ***P < 0.001 vs. challenged with gliadin (Student's t-test). Data information: The blots are representative of one experiment for group of treatment. Source data are available online for this figure. Source Data for Figure 1 [embj2018100101-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Gliadin inhibits CFTR function in vivo in the small intestine of gliadin-sensitive mice To determine whether gliadin may reduce CFTR function in the small intestine in vivo, we took advantage of established mouse models of gliadin sensitivity. In the first model, 10-week-old BALB/c mice were fed for three generations with a gluten-free diet and then challenged with gliadin for 4 weeks (5 mg/daily for 1 week and then 5 mg/daily thrice a week for 3 weeks), following established protocols (Galipeau et al, 2011; Papista et al, 2012; Larsen et al, 2015; Moon et al, 2016). In all tested mice, gliadin reduced CFTR function measured as the forskolin-inducible chloride current in the small intestine mounted in Ussing chambers (Fig 1G). In the second model of gluten sensitivity, we resorted to non-obese diabetic (NOD) female mice, which are prone to the development of autoimmune diseases (Maurano et al, 2005; Galipeau et al, 2011; Papista et al, 2012; Larsen et al, 2015; Moon et al, 2016). When orally administered to these mice, gliadin suppressed CFTR function (Fig 1H). Notably, we confirmed the CFTR inhibitory effects of gliadin in NOD mice transgenic for the CD-predisposing HLA molecule DQ8 (NOD-DQ8; Galipeau et al, 2011; Papista et al, 2012; Korneychuk et al, 2015; Larsen et al, 2015; Moon et al, 2016; Fig 1I). Altogether, the aforementioned results indicate that gliadin inhibits CFTR function in vivo in the small intestine of gliadin-sensitive mice. The α-gliadin LGQQQPFPPQQPY peptide (P31–43) inhibits CFTR function in intestinal epithelial cells To determine whether gliadin may perturb CFTR channel activity at the intestinal epithelial surface, we resorted to human intestinal epithelial cell lines, either Caco-2 or T84 cells, which are reportedly sensitive to gliadin or gliadin-derived peptides (Barone et al, 2014). When confluent cells were cultured for 3 h with a peptic-tryptic digest of gliadin from bread wheat (PT gliadin; 500 μg/ml; Maiuri et al, 2003; Barone et al, 2014), we noted a strong suppression of the forskolin-inducible chloride current, as compared to controls kept in medium alone (Fig EV1A). Click here to expand this figure. Figure EV1. Effects of gliadin and gliadin peptides on CFTR channel function in intestinal epithelial cells A–C. Treatment of Caco-2 cells (A, B) or T84 (C) cells with a PT gliadin (500 μg/ml) (A) or P57–68 or PGAV or P31–43 (20 μg/ml; 3 h) in the presence or absence of pre-treatment with Vrx-532. CFTR-dependent Cl− secretion measured by means of forskolin-induced (Fsk) increase in the chloride current [Isc (μA/cm2)] in cells mounted in Ussing chambers; quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc; n = 3 independent experiments). Mean ± SD of samples assayed. ***P < 0.001 vs. PT gliadin (A) or vs. P31–43 (C) or **P < 0.01 vs. P31–43 (B), °°°P < 0.01 vs. P31–43 + Vrx-532. Source data are available online for this figure. Download figure Download PowerPoint In vivo, two main α-gliadin peptides remain undigested, the 25-mer (P31–55) that is not recognized by T cells but damages the celiac intestine in vitro and in vivo (Maiuri et al, 2003; Meresse et al, 2009; Barone et al, 2014), and the 33-mer (P55–87) that is deamidated by TG2, binds to HLA-DQ2/DQ8 and induces an adaptive Th1 response (Barone et al, 2014). We challenged Caco-2 or T84 cells for 3–24 h with gliadin-derived LGQQQPFPPQQPY (P31–43) and QLQPFPQPQLPY (P57–68) peptides (20 μg/ml), which are fragments of the 25-mer and 33-mer, respectively (Maiuri et al, 2003; Meresse et al, 2009; Barone et al, 2014). These gliadin-derived peptides are capable of inducing the enterocyte stress response (P31–43) or of activating T cells in the absence of any toxic effect on epithelial cells (P57–68; Maiuri et al, 2003; Meresse et al, 2009; Barone et al, 2014). The scrambled GAVAAVGVVAGA (PGAV) peptide was used as a control. As soon as after 3 h following incubation, P31–43 (but not P57–68, P33-mer or PGAV) reduced the forskolin-inducible chloride current in Caco-2 (Figs 2A and EV1B) and T84 cell lines (Fig EV1C), although P31–43 did not affect cell viability at this time (Appendix Fig S2A), as reported (Rauhairta et al, 2011). This effect was prevented by a short (up to 20 min) pre-incubation with the CFTR potentiators VX-770 (10 μM), a Food and Drug Administration (FDA)- and European Medicines Agency (EMA)-approved drug for the treatment of CF patients bearing plasma membrane (PM)-resident CFTR mutants (Cutting, 2015) or Vrx-532 (20 μM; Figs 2A and EV1B). These CFTR potentiators allow CFTR channels to stay longer in an open conformational state and hence increase the probability of CFTR channel opening (Eckford et al, 2012; Jih & Hwang, 2013). Figure 2. P31–43 binds NBD1 and inhibits CFTR channel function A. Representative traces of CFTR-dependent Cl− secretion measured by forskolin (Fsk)-inducible chloride current [Isc (μA/cm2)] in Caco-2 cells mounted in Ussing chambers after 3 h of incubation wi
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