Prolyl endopeptidase-degraded low immunoreactive wheat flour attenuates immune responses in Caco-2 intestinal cells and gluten-sensitized BALB/c mice.

Targeted degrading Aspergillus niger-derived prolyl endopeptidase (AN-PEP) is promising in gluten hydrolysis because it specifically cleaves the proline-rich sites in gluten. The current study aims to understand the safety aspects of AN-PEP hydrolysed low immunoreactive wheat flours by testing immune responses using cell line and animal models. In the AN-PEP hydrolysed wheat flour (AN-PEP HWF) gliadin extract, there was no increase in the levels of zonulin-1 (Zo-1) and pro-inflammatory cytokines (IL-6 and IL-8) but a significant increase was noted in the control wheat flour (CWF) gliadin-treated Caco-2 cells. The Zo-1 localization in Caco-2 cells was significantly noted in the reacted positive fluorescence cells that were treated with the control wheat flour. Further, a safety evaluation of HWF was carried out in gluten-sensitized BALB/c mice. Mouse anti-gliadin (IgG, IgA and IgE) antibodies were significantly generated in the CWF treated animals rather than the AN-PEP HWF groups. The serum pro-inflammatory (IL-1ß, IL-4, IL-6, IL-15, TNF-α and IFN-γ) markers were observed in significant levels in CWF challenged mice and a similar trend was observed in ex-vivo splenocyte cells. A small intestine histopathological sectioning revealed that there are no abnormalities or structural changes in AN-PEP HWF challenged mice.

Antigen-Specific Gut Inflammation and Systemic Immune Responses Induced by Prolonging Wheat Gluten Sensitization in BALB/c Murine Model.

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1ß, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1ß, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF-kBp50 signaling mechanism in G+ sensitized mice.