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

Biochim Biophys Acta Gen Subj. 2019 Mar;1863(3):632-643. doi: 10.1016/j.bbagen.2018.12.007. Epub 2018 Dec 13.

Abstract

Background: The milk protein αS1-casein was recently reported to induce secretion of proinflammatory cytokines via Toll-like receptor 4 (TLR4). In this study, αS1-casein was identified as binder of theTLR4 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 αS1-casein, or LPS as control was monitored. Binding of αS1-casein to TLR4 was quantified by microscale thermophoresis (MST).

Results: αS1-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 μM and by anti-CD14-IgA. The binding constants (KD) of αS1-casein to the TLR4, MD2, and CD14 were 2.8 μM, 0.3 μM and 2.7 μM, respectively. Finally, αS1-casein showed a higher affinity to TLR4/MD2 (KD: 2.2 μM) compared to LPS (KD: 8.2 μM).

Conclusion: Human αS1-casein induced proinflammatory effects are dependent upon binding to the TLR4 ectodomain and the presence of CD14. αS1-casein displayed stronger TLR4 agonistic activity than LPS via a different mode of action.

General significance: Breast milk protein αS1-casein is a proinflammatory cytokine.

Keywords: CD14, MD2; Cofactor binding; Ecto-domain TLR4; Human α(S1)-casein; Milk protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caseins / chemistry
  • Caseins / pharmacology*
  • HEK293 Cells
  • Humans
  • Interleukin-8 / metabolism*
  • Lymphocyte Antigen 96 / chemistry
  • Lymphocyte Antigen 96 / metabolism*
  • Models, Molecular
  • Molecular Docking Simulation
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Domains
  • Protein Transport / drug effects
  • Secretory Pathway / drug effects
  • Toll-Like Receptor 4 / chemistry
  • Toll-Like Receptor 4 / metabolism*

Substances

  • Caseins
  • Interleukin-8
  • LY96 protein, human
  • Lymphocyte Antigen 96
  • Multiprotein Complexes
  • TLR4 protein, human
  • Toll-Like Receptor 4