Antigen-specific gut inflammation and systemic immune responses induced by pro-longing wheat gluten sensitization in BALB/c murine model (1)
The wheat seed contains 80% of storage proteins such as gliadins and glutenins. Gliadins are monomeric proteins with molecular weights around 28,000 –55,000, and it is soluble in aqueous alcohol. Whereas, glutenin subunit has high molecular weight (HMW) subunits (MW-67,000 – 88,000) and low molecular weight (LMW) subunits (MW- 32,000 – 35, 000). HLA DQ2 and DQ8 are the two genetic factors for Celiac disease (CD). Pathogenesis of CD results from an interaction between deaminated gliadin peptides (33 amino acid peptide) and HLA DQ2/8 molecules, which induces the activation of CD4+TH1 lymphocytes in the intestinal lamina propria and causes tissue injury.
Fifty μg of extracted proteins were separated on 12% gel. The extracted gliadins show that the separated proteins are in the range 30-40 kDa in the first 4 wells. Whereas, the animal diet (AIN-93G) have also appeared in the range of 30-40 kDa. The reacted bands and spot were observed in the wheat gliadin extract of dot-blot and immuno-blot.
Fig.1. Prepared gliadins and animal diet immunoreactivity (A) A representative SDS-PAGE profile of extracted gliadins from wheat flour. Lane 1 – Triticum aestivum; 2 – Triticum dicoccum; 3 – Refined bakery wheat flour; 4 – Commercial pellet animal diet; 5 – AIN-93G diet; 6 – commercial gluten powder; 7 – Sigma gluten powder; 8 – Bovine serum albumin. (B) Represents the dot-blot pattern of gliadins extract for its reactivity. 1- T. aestivum; 2 – T. dicoccum; 3 – refined bakery wheat flour; 4 – commercial pellet animal diet; 5 – AIN-93G diet. © Represents the immuno-blot of gliadins extract and the order has mentioned above.
The anti-IgE antibodies response was significantly increased in GL+, GT+ and G- except control groups (Control – PBS; GL+ Gliadin; GT+ Gluten; G- BSA). However, the specific anti-gliadin IgG and IgA for gluten-related disorders serological markers also found a significant increase in 93rd day of mice sera in GL+ group.
Fig.2. Specific anti-gliadin IgG, IgA & IgE reactivity of sensitized mice sera (A) Detection of serum anti-gliadin antibodies response IgG in G – and G + sensitized mice by indirect enzyme linked immune-sorbent assay (ELISA). (B) Detection of serum anti-gliadin antibodies response IgA in G – and G + sensitized mice in-direct enzyme linked immune-sorbent assay (ELISA). (C) Detection of serum anti-gliadin antibodies response IgE in G – and G + sensitized mice by indirect enzyme linked immune-sorbent assay (ELISA).
GT+ mice group showed significantly increased IL-4 levels, and it was observed as 12000 pg/ml on the 16th day of mice sera. The same pattern of results was noticed for IL-15 levels in GL+, GT+ and G-groups. IL-1β levels were significantly increased in consecutive days (37 and 59) of sensitised GL+ and GT+ group mice sera. IL-6 levels were significantly observed at 20000 pg/ml in GL+ groups and 28000 pg/ml in GT+ groups vice versa on the121st day (P< 0.05). The same pattern also observed at G- groups at 25000 pg/ml range of IL-6 level. IFN-Υ and TNF-α levels were observed the almost similar pattern in consecutive days (16 to 93), at the same time a significant increase in GL+ and GT+ mice sera on the 121st day.
Fig.3. Pro-inflammatory cytokines profile in sensitized mice sera (A-F) Detection of interleukins and cytokines levels in G – and G + sensitized mice serum by enzyme linked immune-sorbent assay (ELISA) using affymetrix ELISA kits from eBioscience.
Gluten sensitization is associated with IgG and IgA antibodies generation during the oral challenge, though mice have responded higher generation of IgE in I.P injection. food derived allergic proteins can boost the specific T cells activity in spleen and promotes intragastric food allergic model in animal system.