Amylase trypsin inhibitors activation of toll-like receptor 4 revisited: The dominance of lipopolysaccharides contamination

Amylase trypsin inhibitors (ATIs) potentially play a role in irritable bowel syndrome (IBS) and non-celiac wheat sensitivity (NCWS). These cereal-derived inhibitors are suspected to bind to the TLR4-MD2-CD14 complex and trigger intestinal pro-inflammatory responses, but confirmation through more extensive cell line studies is required. In this study, an amylase trypsin inhibitors enriched fraction (AEF) was prepared and characterized. Then, AEF binding potential to TLR4-MD2-CD14 was investigated using the human TLR4 reporter cell line HEK-Blue^TM. The method took into account the presence of lipopolysaccharides (LPS) using Polymyxin B (PMB) to block LPS binding to TLR4. Proteinase K was also used to hydrolyze AEF proteins and eliminate their induced response. The cell line experiments showed that PMB treatment of AEF reduced the binding signal by 92 %. Complete hydrolysis of the protein by Proteinase K doubled the TLR4 activation signal and might indicate that protein-LPS complexation reduced LPS's ability to activate the TLR4-receptor. These finding underline the need for future work to consider the non-protein part in cell assays, especially the LPS bias. Altogether, these results indicated that LPS activates TLR4-MD2-CD14 and challenges ATIs' intestinal inflammation capacity contributing in irritable bowel syndrome and non-celiac wheat sensitivity.