The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process

Abstract

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects

“…Finally, TG2 appeared to colocalize with the autophagic marker LC3 more in CD cells than in control ones. This finding could be related to the complex role of TG2 in regulating autophagy [33,34], a process that could contribute, if deranged, to CD pathogenesis [35,36].…”

Trehalose Modulates Autophagy Process to Counteract Gliadin Cytotoxicity in an In Vitro Celiac Disease Model

“…Albumin and casein (Sigma) were digested following the same protocol and were used as negative controls. PT-gliadin AlexaFluor555 labelling (GLIA-555) and purification was performed, as described previously [12], using affinity chromatography-purification G50 column (GE Healthcare, Little Chalfont, UK). The resulting labelled proteins were resuspended in PBS and labelled according to the manufacturer’s instructions with AlexaFluor555 using Alexa Fluor Microscale Labelling Kits (ThermoFisher, Waltham, MA, USA).…”

“…Particularly, the intracellular accumulation of the gliadin peptide p31-43 leads to an increase in reactive oxygen species (ROS) levels [6], and the highly immunogenic 33-mer peptide is able to form supramolecular organized immunoreactive structures resistant to low pH [7,8,9]. Although autophagy dysregulation has been largely described in different autoimmune disorders affecting the intestine, such as inflammatory bowel diseases (IBDs) [10,11], little evidence was collected in our laboratory supporting the hypothesis that autophagy could play a role in CD pathogenesis [12,13]. A major reason to thoroughly investigate autophagy in the context of CD is that this cellular process is implicated in the oxidative stress response [14] and in protein aggregates degradation [15], as well as in the maintenance of the barrier function in the epithelial cells of the small intestine [16].…”

“…According to all these considerations, autophagy modulation seems to be an interesting approach in order to counteract gliadin cytotoxicity. As already demonstrated, autophagy induction through different approaches that inhibit mTOR showed poor results as compared with other ones acting on different kinases, for example, 3-methyladenine [12], which suggests the need to test novel autophagy modulators. Recently, it has been highlighted that trehalose, raffinose, and sucrose are able to induce autophagy through an mTOR-independent mechanism [19].…”

The Emerging Role of the Autophagy Process in Children with Celiac Disease: Current Status and Research Perspectives

“…In the latest PubMed contribution, Manai and collaborators [36] reported that in Caco-2 cells, a widely used in vitro model for celiac disease studies, the administration of enzymatically digested gliadin (PT-gliadin) peptides significantly reduced the expression of the LC3-II autophagy-related marker. Furthermore, electron and fluorescent microscope analysis suggested a compromised function of the autophagosome apparatus.…”