IMMUNONUTRITIONAL PROTEASE INHIBITORS FROM CEREALS PROMOTE LIVER INFLAMMATION AND DRIVEN POLARIZATION OF MACROPHAGES

Background and objectives: Innate immune Toll-like receptor (TLR)-4 has been identified to play a key role in hepatocellular cancer promotion and macrophage priming. This study aims to characterize TLR4-mediated innate and metabolic changes, caused by protease inhibitors found in the salt-soluble fraction of different cereals (TA, Triticum aestivum; AS, Avena sativa and OS, Oryza sativa), in hepatoblastoma (HB-8965®) and humanized macrophage-like (HB-8902®) cells. Methods: The salt-soluble fraction from cereals was isolated (10 mM phosphate buffer, pH 7,5, containing 0.1 M NaCI) and purified (ammonium sulphate 90% saturation) as described elsewhere. Then, isolated proteins were subjected to HPLC-RP-ESIMs/Ms characterization. Mechanistic studies were further performed using hepatoblastoma (HepG2, HB-8965®) cells and the humanized macrophage-like HB-8902® cell line. Results: All tested grains contain protease inhibitors that display a relatively high similarity between their amino acid sequences (20-40%). Taking advantage of the known specificity of major proteolytic enzymes involved in the gastrointestinal digestion it was identified a key role for pepsin allowing to solubilize up to 80% of these proteins. Protease inhibitors receive clinical attention because of their innate immunogenic potential. Protease inhibitors from cereals activated TLR4 in hepatoblastoma cells mediating innate immune responses (IL-6 and TNFa) that influence tumor cell growth and migration. A mitostress assay in hepatoblastoma cells revealed significant differences in oxygen consumption rate (OCR, pmol/min) as a function of the cereal considered according to the following treatment: TA