Whey Hydrolysate Modulates NF-κB Cytokine Production

In order to investigate the role of NF-κB in whey hydrolysate induced cytokine production, THP-1 monocytes were first differentiated into THP-1 macrophages as previously described [42 (link)]. First, THP-1 monocytes were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% decomplemented FBS, 2mM L-glutamine (Lonza, Verviers, Belgium), 1mM sodium pyruvate (Lonza, Verviers, Belgium), 0.05mM 2-mercaptoethanol (Scharlau, Barcelona, Spain), 60 μg/ml gentamycin sulfate (Lonza, Verviers, Belgium), and 2.2 μg/ml amphotericin B (Sigma Aldrich, Zwijndrecht, the Netherlands). Then, cells were seeded in a 24 wells plate at a concentration of 1x10⁶ cells/well (in 0.5 ml medium). To differentiate the cells into macrophages 100 ng/ml PMA was added. After 48 hours, the PMA was removed and cells were washed twice with fresh medium. Cells were cultured for another 24 hours. Then, cells were incubated with either 10 μM of the NF-κB inhibitor celastrol (Invivogen, Toulouse, France) or medium (control) for 30 minutes. Next, 2 mg/ml intact whey protein or its hydrolysates were added. Cells were incubated for 24 hours, after which the supernatant was collected. TNFα and IL-10 levels were measured in the supernatant by ELISA according to the manufacturer’s protocol (eBioscience, San Diego, USA).