The human milk protein α_S1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4)¹. In this study, the binding site of α_S1-casein to TLR4 was identified by selective inhibition of the intracellular binding domain and extracellular ecto-domain of TLR4.

For this, Interleukin 8 (IL-8) secretion was monitored after stimulation of TLR4/MD2 (myeloid differentiation factor 2)/CD14 (cluster of differentiation 14)-transfected HEK293 cells (TLR4⁺) and Mono Mac 6 cells (MM6) with recombinant α_S1-casein, or lipopolysaccharide (LPS) as control. The α_S1-casein-induced IL-8 secretion was inhibited by TAK-242, an antagonist of the intracellular binding site and mianserine, an antagonist of the extracellular binding domain. TAK-242 inhibited α_S1-casein-induced IL-8 secretion with an IC₅₀ of 259 nM and LPS-induced IL-8 secretion with an IC₅₀ of 23 nM. Mianserine was found as moderate inhibitor of the α_S1-casein-induced IL-8 secretion with an IC₅₀-range between 10-51 µM. Therefore, we suggested α_S1-casein as an inhibitor of the extracellular binding site of TLR4. These findings were supported by binding experiments using microscale thermophoresis (MST). Human α_S1-casein bound to the purified extracellular TLR4/MD2-complex with a K_D of 2.2 µM in comparison to LPS binding TLR/MD2 with a K_D of 8.7 µM. Furthermore α_S1-casein showed binding to MD2 with a K_D of 0.3 µM and CD14 with a K_D of 2.7 µM. In addition, human α_S1-casein induced IL-8 secretion via TLR4 was inhibited by inhibitory anti-CD14-IgA.

Human α_S1-casein induced proinflammatory effects by binding to the ecto-domain of TLR4 and CD14 is required as cofactor. Hence human α_S1-casein activates TLR4 in a different manner than LPS.