Alpha-Amylase/Trypsin Inhibitors, novel activators of innate immunity in ex-vivo tissue explants
Aims: We have identified wheat alpha-amylase/trypsin inhibitors (ATIs), pest resistant molecules found in grains, as potent activators of innate immunity in celiac disease and general intestinal inflammation by engaging the toll like receptor 4 (TLR4)-MD2-CD14 complex in monocytes, macrophages and dendritic cells (Junker et al, J Exp Med 2012). Precision-cut tissue slices (PCTS) are viable ex-vivo explants of tissues with a reproducible, well defined thickness, representing an organ with preserved intercellular and cell matrix interactions, similar to their natural multi-cellular environment.
Objective: To evaluate the ability of ATIs to stimulate innate immunity using precision-cut tissue slices.
Methods: ATIs were exhaustively extracted from milled and defatted samples using salt solutions and comparatively dialyzed against acidic and neutral buffers, sterile filtrated and lyophilized. ATIs and negative controls (casein or zein) were cultured overnight with individual PCTS from three different sections of the intestine (duodenum, jejunum and colon) of C57BL/6 mice on a gluten-free diet. Concentration of inflammatory cytokines and expression of mRNA transcript levels for inflammatory genes were measured.
Results: We observed that ATIs significantly stimulated the expression of inflammatory cytokines (KC/IL-8, IL-6 and TNFa) in supernatants compared to protein control at 2 mg/mL. Transcript levels of inflammatory markers (MCP-1 and KC) were also up-regulated in PCTS cultured with ATIs compared to protein controls. Furthermore, transcript levels were upregulated in a dose-dependent manner when ATIs were tested at different concentrations (0.5, 1 and 2 mg/mL).
Using PCTS we confirm the innate stimulatory activity of ATIs in a multi-cellular environment resembling the in-vivo situation;
Our results suggest that ATIs are the missing nutritional trigger of innate immunity in celiac disease as well as other autoimmune diseases.
Based on our in-vitro and ex-vivo findings we currently perform human studies.