Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex

Background: The milk protein alpha(S1)-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, alpha(S1)-casein was identified as binder of the TLR4 ecto domain. Methods: IL-8 secretion 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 alpha(S1)-casein, or LPS as control was monitored. Binding of alpha(S1)-casein to TLR4 was quantified by microscale thermophoresis (MST). Results: alpha(S1)-casein induced secretion of IL-8 in TLR4(+) cells and in MM6 cells with a six-times higher final IL-8 concentration in supernatants. IL-8 secretion was inhibited by intracellular TLR4-domain antagonist TAK-242 with an IC50-value of 259.6 nM, by ecto-domain TLR4 antagonistic mianserin with 10-51 mu M and by anti-CD14-IgA. The binding constants (K-D) of alpha(S1)-casein to the TLR4, MD2, and CD14 were 2.8 mu M, 0.3 mu M and 2.7 mu M, respectively. Finally, alpha(S1)-casein showed a higher affinity to TLR4/MD2 (K-D: 2.2 mu M) compared to LPS (K-D: 8.2 mu M). Conclusion: Human alpha(S1)-casein induced proinflammatory effects are dependent upon binding to the TLR4 ecto-domain and the presence of CD14. alpha(S1)-casein displayed stronger TLR4 agonistic activity than LPS via a different mode of action. General significance: Breast milk protein alpha(S1)-casein is a proinflammatory cytokine.