Thorsten Saenger1, Stefan Vordenbäumen2, Swetlana Genich3, Samer Haidar4, Marten Schulte5, Christian Nienberg6, Ellen Bleck7, Matthias Schneider8, Joachim Jose9. 1. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: thorsten.saenger@uni-muenster.de. 2. Medical Faculty, Department of Rheumatology, Hiller Research Unit Rheumatology, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany. Electronic address: stefan.vordenbaeumen@med.uni-duesseldorf.de. 3. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: swetlana.genich@uni-muenster.de. 4. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: shaid_01@uni-muenster.de. 5. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: marten.schulte@uni-muenster.de. 6. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: christian.nienberg@uni-muenster.de. 7. Medical Faculty, Department of Rheumatology, Hiller Research Unit Rheumatology, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany. Electronic address: bleck@med.uni-duesseldorf.de. 8. Medical Faculty, Department of Rheumatology, Hiller Research Unit Rheumatology, Heinrich-Heine-University Düsseldorf, Moorenstr. 5, 40225 Düsseldorf, Germany. Electronic address: Matthias.Schneider@med.uni-duesseldorf.de. 9. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Corrensstr. 48, 48149 Münster, Germany. Electronic address: joachim.jose@uni-muenster.de.
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.
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.
Authors: Verena Spiegler; Barbara Gierlikowska; Thorsten Saenger; John N Addotey; Jandirk Sendker; Joachim Jose; Anna K Kiss; Andreas Hensel Journal: Front Pharmacol Date: 2020-06-12 Impact factor: 5.810