Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling

Scope: Human casein alpha S1 (CSN1S1) is a milk-derived protein, which gives rise to sustained antibody formation after breast feeding in infancy and induces the expression of proinflammatory cytokines. So far, the cellular CSN1S1 receptor is unrecognized. Our objective was to identify the receptor employed by CSN1S1 to induce proinflammatory effects. Methods and results: CSN1S1 concentration and time dependently induced expression of IL-1 beta, IL-8, and IL-6 in monocytic cells despite addition of polymyxin B, which completely abrogated LPS effects. Coincubation of monocytic cells with a neutralizing antibody to Toll-like receptor 4 (TLR4) but not to TLR2 inhibited CSN1S1-induced effects. In TLR4/MD2/CD14-cotransfected HEK293 cells CSN1S1 increased IL-8 expression, while untransfected cells were completely unresponsive. Furthermore, CSN1S1-induced IL1-beta secretion was impeded by inhibition of MyD88 and caspase-1. Flow cytometric in vitro assays confirmed binding of CSN1S1 to TLR4. Phosphorylation of CSN1S1 by casein-kinase 2 completely abolished proinflammatory cytokine induction and binding to TLR4. Conclusion: CSN1S1 is a multifunctional milk protein that exerts proinflammatory properties via TLR4 and the inflammasome pathway in a phosphorylation-dependent manner. The contribution of CSN1S1 in mother milk to the development of a potent immune system in breastfed individuals should be further assessed.