Lysosomal accumulation of gliadin p31-43 peptide induces oxidative stress and Tissue Transglutaminase mediated PPARγ downregulation in intestinal epithelial cells and coeliac mucosa

Abstract Background: An unresolved question in Coeliac disease (CD) is to understand how some toxic gliadin peptides, in particular p31-43, can initiate an innate response and lead to Tissue Transglutaminase (TG2) upregulation in coeliac intestine and gliadin sensitive epithelial cell lines. Aim : We addressed whether the epithelial uptake of p31-43 induces an intracellular pro-oxidative envoronment favoring TG2 activation and leading to the innate immune response. Methods: Time-course of intracellular delivery to lysosomes of p31-43, pα-2 or pα-9 gliadin peptides was analyzed in T84 and Caco-2 epithelial cells. The effects of peptide challenge on oxidative stress, TG2 and Peroxisome Proliferator-Activated Receptor (PPAR) γ ubiquitination and p42/44-MAP-kinase or tyrosine phosphorylation were investigated in cell lines and cultured CD biopsies with/without anti-oxidant treatment or TG2 gene silencing by immunoprecipitation, western blot, confocal microscopy and FRET analysis. Results: After 24 hours of challenge p31-43, but not pα-2 or pα-9, is still retained within LAMP1- positive perinuclear vesicles and leads to increased levels of Reactive Oxygen Species (ROS) that inhibit TG2 ubiquitination and lead to increase of TG2 protein levels and activation. TG2 induces cross-linking, ubiquitination and proteasome degradation of PPARγ. Treatment with the antioxidant EUK-134 as well as TG2 gene silencing restore PPARγ levels and reversed all monitored signs of innate activation, as indicated by the dramatic reduction of tyrosine and p42/p44 phosphorylation. Conclusion: p31-43 accumulation in lysosomes leads to epithelial activation via ROS-TG2 axis. TG2 works as a rheostat of ubiquitination and proteasome degradation and drives inflammation via PPARγ downregulation.