Probiotics Supplements Reduce ER Stress and Gut Inflammation Associated with Gliadin Intake in a Mouse Model of Gluten Sensitivity

Abstract

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.

Keywords: CD; CFTR; TG2; UPR; probiotics.

Figure 1

Tissue transglutaminase 2 (TG2) and cystic fibrosis transmembrane conductance (CFTR) modulation by probiotics administration in vivo. CFTR (A) and TG2 (B) expression levels were evaluated in the small intestine of Balb/c fed third-generation gluten-free mice, treated (Glia) or not treated (CTRL) with gliadin, in the presence or absence of P1 or P2, at both mRNA (left panels) and protein (right panels) levels. Histograms represent mean ± standard deviation (SD) of triplicate sample; **** p < 0.0001; ** p < 0.01; ns = not significant; β-actin was used as loading control in the immunoblots.

Figure 2

Gliadin-mediated intestinal permeability dysregulation is restored by probiotics administration. Plasma concentration of fluorescein isothiocyanate-conjugated (FITC) fluorescence was measured 4 h after mouse gavage of a single dose of FITC-Dextran (A). Quantification of plasma concentration from n = 2 mice per group. Mean ± SD of duplicate sample. (B) Claudin 2 (upper panel), Claudin 15 (bottom left panel), and Occludin (bottom right panel) expression levels were evaluated in the small intestine of third-generation Balb/c mice fed with gluten-free diet challenged with gliadin (Glia) in the presence or absence of P1 or P2 by quantitative real-time polymerase chain reaction (qRT-PCR). Mean ± SD of triplicate sample; **** p < 0.0001; *** p < 0.001; ** p < 0.01.

Figure 3

Anti-inflammatory activity of probiotics. IL-15, IL-17a, and INFγ mRNA (A) and protein (B) levels were evaluated in the small intestine tissue homogenate of Balb/c-fed third-generation gluten-free mice challenged with gliadin (GLIA) and P1 or P2 and compared to untreated control (CTRL) by qRT-PCR and ELISA, respectively. (C) H/E staining of small intestine from mice exposed (Glia), unexposed (CTRL) to gliadin alone or in combination with probiotic formulation 1 (P1) or 2 (P2). Images are representative of three independent experiments (magnification = 20×). Mean ± SD of triplicate sample **** p < 0.0001; *** p < 0.001; ** p < 0.01.

Figure 4

Endoplasmic reticulum (ER) Stress induced by gliadin exposure was buffered by probiotics. ATF4 (A), ATF6 (B), and XBP1s (C) expression levels were evaluated in the small intestine of Balb/c-fed third-generation gluten-free diet treated (Glia) on untreated (CTRL) with gliadin in the presence or absence of P1 or P2. Mean ± SD of triplicate sample; **** p < 0.0001; *** p < 0.001; ** p < 0.01; ns = not significant.

Figure 5

Impact of probiotics on peptide (PT)-mediated ER Stress induction in vitro. Caco-2 cells were untreated (CTRL) or treated with PT-gliadin for 3 (PT 3 h) or 9 (PT 9 h) hours in the presence or absence of P1 or P2 as schematically reported in panel (A). CFTR (B), TG2 (C), ATF4 (D), ATF6 (E), and XBP1s (F) expression levels were evaluated by qRT-PCR. Mean ± SD of triplicate sample; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns = not significant.