TLR4-mediated activation of monocytes by human α S1 -casein Thorsten Saenger 1, *, Stefan Vordenbäumen 2 , Tamara Tahan 1 , Christian Nienberg 1 , Ellen Bleck 2 , Matthias Schneider 2 and Joachim Jose 1 1 Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische Wilhelms-Universität Münster, Münster, Germany. 2 Policlinic of Rheumatology, Hiller Research Unit, University Clinic, Heinrich-Heine- University Düsseldorf, Düsseldorf, Germany. * Corresponding author: Thorsten.Saenger@uni-muenster.de 1
Graphical Abstract TLR4- mediated activation of monocytes by human α S1 -casein 1 2 3 Human α S1 -casein exerts proinflammatory effects via TLR4-signaling pathway in a Phosphorylation-dependent manner 1. α S1 -casein (CSN1S1) induces no expression of cytokines in cells without TLR4 receptor. 2. Recombinant CSN1S1 exerts expression of cytokine IL- 1β, IL -6 and IL-8 concentration- and time-dependently. 3. Phosphorylation of recombinant CSN1S1 suppressed proinflammatory effects via TLR4-signaling. 2
Abstract: Human milk protein α S1 -casein (CSN1S1) was shown to be overexpressed in autoimmune diseases (osteoarthritis, benign prostatic hyperplasia, multiple sclerosis) as well as in cancer. CSN1S1 displays opioid-like activity and modulates the innate immune response of intestinal cells. Recently, it was demonstrated, that CSN1S1 induces the expression of proinflammatory cytokines (IL-1 β and IL-6) in monocytic cells via MAPK-p38 signaling [1, 2]. In this study the human TLR4 receptor, a receptor of the innate immune system, was identified as interaction partner of human CSN1S1 inducing expression of cytokines IL-1 β, IL-6 and IL-8 in human monocytic cells concentration- and time-dependently [3]. In HEK293 cells cotransfected with TLR4 human CSN1S1 (purified from Escherichia coli ) induced secretion of chemokine IL-8. In vitro flow cytometric assay confirmed CSN1S1 - TLR4 receptor interaction. Chemokine secretion as well as binding was not detected for CSN1S1 phosphorylated by protein kinase CK2 as well as denaturated CSN1S1. This supports the hypothesis, that CSN1S1 is a ligand of the TLR4 receptor exerting proinflammatory properties in phosphorylation-dependent manner. In conclusion CSN1S1 could contribute to the development of a potent immune system in breastfed offspring. Keywords: CSN1S1 / Inflammasome / Nursing / Toll-like receptor4 Literature: [1] S. Vordenbäumen, et al., BMC Immunol , 2013 , 14 , 46. [2] S. Vordenbäumen, et al., J Immunol , 2011 , 186 , 592. [3] S. Vordenbäumen, T. Saenger et al., Mol Nutr Food Res , 2016 , 60 ,1079. 3
Introduction Caseins are breast milk proteins … CSN1S1 is overexpressed in … - Nutritional source for infants - Mammary gland - Forms multimers and micelles (trace amount in breast milk) - Transport of compounds - Tissue of benign prostatic 3- , … ) (Ca 2+ , PO 4 hyperplasia patients - Calcium-sensitive - Multiples scleroses - Resource for amino acids - Rheumatiod arthritis CSN1S1 acts as … CSN1S1 may posses immunomodulatory functions -Chaperone (similar to heat shock proteins) -Tumor suppressor in breast cancer - Stimulates expression of pro- inflammatoric cytokine GM-CSF 2 -Potential tumor antigen (renal cancer) -(Ant-)agonist of κ -opioid receptors (peptides) - Induces upregulation of CD14 -Orally determined autoantigen caused - Signaling is mediated by MAPK by breast feeding (ERK, JNK, p38) 1 CSN1S1 binds to surface of monocytes Potentiell receptor could be on cell surface 4
Results and discussion 1. Inhibition of candidate receptors Inhibition of TLR4/MD2 TLR2 1 x 10 6 MonoMac6 cells/ml Incubation 24 h CSN1S1 with His-Tag from E. coli cells induced secretion IL-1 β . proinflammatory cytokine AND Inhibitor OR Stimulation of extracellular Toll-like 24 h of stimulation * * receptors of the innate immune * n .s . n .s . IL-1 β mRNA 10 3 * 10 3 response TLR2 and TLR4 is 10 2 known for the same action as 10 2 described for CSN1S1. Therefore, 10 1 n.s. 10 1 n .s . TLR2 and TLR4 signaling Upregulation of 10 0 10 0 IL- 1β m RNA pathways were inhibited by anti- control TLR4-Ab CSN1S1 CSN1S1 + TLR4-Ab Control TLR2-Ab CSN1S1 CSN1S1+ TLR2-Ab TLR2, anti-TLR4 neutralizing IL-1 β (pg/ml) 4000 4000 * * antibodies. * * n .s . * 3000 3000 2000 2000 Upregulation of 1000 1000 IL- 1β protei ein in n .s . n .s . 0 0 supernatants control TLR4-Ab CSN1S1 CSN1S1 + TLR4-Ab l b 1 b o S r A A t - 1 - n 2 2 N o R R S c L L C T T + 1 S 1 N S C Results in IL- 1β secretion no significant effect 5
Results and discussion 2. Human TLR4 prerequisite for CSN1S1-induced effects? 24 h OR IL-8 mRNA LPS as agonist TLR4 detection TLR4 - 10 4 40 HEK293 30 (TLR4-) 10 3 20 10 2 TLR4/MD2/CD14 transfected HEK293 10 HEK293 10 1 (TLR4+) (TLR4-) 0 10 0 l 1 1 0 0 0 o . 1 1 0 r 0 1 control CSN1S1 0.1 CSN1S1 1 CSN1S1 10 LPS 10 LPS 100 1 t S 1 S TLR 4 + n 1 S S o S 1 P N 1 L P c 1 N S L N C S S C C CSN1S1 binding 10 4 * ** 40 * *** TLR4+ 10 3 30 TLR4- 10 2 20 10 1 10 10 0 0 HEK293 with TLR4 l 1 1 0 0 0 control CSN1S1 0.1 CSN1S1 1 CSN1S1 10 LPS 10 LPS 100 o . 1 1 0 0 1 r t 1 S 1 S n 1 S S 1 P (TLR4+) o S N 1 P c L 1 N L N S S S C C C TLR4 was suggested as receptor, because CSN1S1-induced effects were inhibited by neutralizing anti-TLR4. To support this results, a cellular model of HEK293 cells (TLR4-) without receptor and TLR4/MD2 cotransfected HEK293 cells (TLR4+) was used. This genetically modified cells could not be monitored by IL-1 β , but showed a dose depentent secretion of IL-8. 6
Results and discussion 3. Phosphorylation of CSN1S1 24 h OR IL-8 mRNA LPS as agonist binding of CSN1S1 phosphorylated n .s . 80 ** 10 2 60 n .s . * TLR4- TLR4+ 40 10 1 20 0 10 0 control CSN1S1 P-CSN1S1 control CSN1S1 P-CSN1S1 CSN1S1 is known to be partial phosphorylated in breast milk. Therefore, phosphorylation of CSN1S1 could be a mechanism for inactivation of the proinflammatory properties. To verify this hypothesis, CSN1S1 from E. coli was phosphorylated by human protein kinase CK2. 7
Conclusions • CSN1S1-induced expression of proinflammatory cytokines requires translocation of TLR4 (not dependent on pathogen LPS, which is a common agonist of TLR4). • Posttranslational modification by phosphorylation of CSN1S1 inhibits proinflammatoric properties as well as binding to TLR4. • CSN1S1 may be a bioactive component possibly influencing development of the immune system of the new born (breast milk) as well as triggering to potential pathogens in diseases. 8
Acknowledgements Thanks to Dr. Dagmar Aichele, Sandra Kohaus and the group of Joachim Jose The authors greatfully acknowledge financial support of this study by a grant from the Hiller Rheumatology Research Foundation, Erkrath, Germany and Hiller Research Center Rheumatology of Heinrich-Heine-University Düsseldorf, Germany. 9
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