Influence of faecal contents from a gluten-free vs. gluten-containing diet on α-amylase/trypsin inhibitor-mediated inflammation

Introduction: Ingestion of gluten proteins triggers celiac disease (CD). We recently identified wheat a-amylase/trypsin inhibitors (ATIs) (CM3 and 0.19) as strong activators of innate immune response(s) in macrophages, monocytes and dendritic cells via toll like receptor 4. Alterations in composition of the gut microbiota have been associated with the severity of CD, suggesting that specific components of the microbiota could influence the potency of gluten peptides and ATIs as proinflammatory triggers of CD.

Objective: To evaluate the effect of faecal contents from healthy subjects or without a gluten free diet (GFD), on the inflammatory response(s) caused by ATIs.

Methods: Samples of commercial gliadin Σ and different wheat grains (#1–3) were homogenised and extracted with phosphate buffered saline and the supernatants analysed for ATIs by Western-blotting (WB). Aliquots of the supernatants, inoculated or not with faecal contents from healthy subjects, were incubated (37^oC/3h) in parallel and CM3 was quantified by reverse-phase HPLC. ATI activities were determined in a bioassay using U937 cells and production of IL-8.

Results: WB revealed the presence of ATIs in all analysed supernatants. CM3 content was 1.36±0.03mg/g in Sigma gliadin, 0.67±0.03mg/g in #1, 0.52±0.07mg/g in #2 and 0.49±0.01mg/g in #3. CM3 contents from gliadin and wheat #3 exhibited the highest degradation rate when the supernatants were inoculated with faecal contents from GFD; 21.1% vs. 7.2% and 9.6% vs. 1.8%, respectively. However, CM3 showed higher degradation from wheat #1 when inoculated with faecal contents from a normal diet (12.6% vs. 7.2%). Faecal contents from control subjects reduced ATI of wheat samples #1 and #2 without altering the inflammatory potential of #3, while faecal contents from the GFD increased the inflammatory potential of all wheat grains.

Conclusions: Wheat grains harbour ATIs which are soluble in physiological media and highly bioactive. There were significant differences in degradation rates of CM3 as a function of the wheat sample considered. Faecal contents from GFD impaired the inflammatory milieu, suggesting than alterations in gut microbiota composition could be an additional environmental factor modulating ATI's inflammatory potential.