Introduction: Non-coeliac wheat sensitivity (NCWS) is considerably more common than coeliac disease/wheat allergy, affecting _~13% of the population worldwide. It is characterized by gastrointestinal and extraintestinal symptoms due to an immunological pathogenesis, the activation of monocytes/macrophages, and the release of inflammatory cytokines (IL-6, IL-8, IL-1β, TNFα).

Project aims:

• To test the impact of cereal proteins on inflammatory responses in a human cell line (THP-1) in vitro.
• To test whether adherence to a gluten-free diet improved gut symptoms and inflammatory responses in participants in a “before-and-after” dietary intervention.

Methods: We tested wheat proteins such as gluten/ATIs; wheat flour; quinoa, a non-wheat control; and LPS extracted in a PBS buffer, a positive control. We characterised these via SDS gel electrophoresis and HPLC. Protein-stimulated PMA-differentiated THP-1 cell lines were analysed for cytokine release using ELISA, RT-qPCR, and Flow Cytometry.

We conducted a human study [Northumbria University ethics approval registration no. 1723] with the inclusion criteria of pre-existing mild/moderate gastrointestinal symptoms; no dietary restrictions; and no major health issues. Participants (n=15) were recruited for an ongoing, four-week intervention study of a gluten-free diet. Their gut symptomology was analysed using 24h dietary recall via intake24 to assess their dietary inflammatory index and questionnaires.

Results:

To date, the ELISA results have shown that gluten and ATI showed a significant increase over the baseline (p<0.0001), except for in regard to TNFα.

The RNA transcripts for the IL-8 cytokines were tested using RT-qPCR. The ∆∆ Cт values for ATI showed a significantly different response to that of LPS (ATI, ∆∆ Cт =1.32; LPS, ∆∆ Cт =0.90), suggesting the expression of RNA for cytokines after protein stimulation.

Flow cytometry data showed the expression of MHC class II molecules on the surface of the stimulated THP-1 cell lines, with the highest mean fluorescence intensity values for ATI compared to those for LPS (ATI_mfi =7843; LPS_mfi =6916).

Conclusions: Our in vitro findings showed inflammatory responses to specific cereal proteins. These provide a mechanistic insight into NCWS and will inform future human feeding trials.