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Laboratory of thrombosis and haemostasis GIGA Cardiovascular Sciences Platelets selectively recognize bacterial DNA independently of Toll-like receptor 9. 29/11/2013 BSTH Céline Delierneux Background I Platelets Ø play a role in immunity. Background I Platelets Ø play a role in immunity. Ø express major receptors of the innate immune system. Cox, JTH, 2011 Background I Platelets Ø play a role in immunity. Ø express major receptors of the innate immune system. Ø cooperate with immune cells to clear bacteria NETs production by neutrophils Young, Plos One, 2011 Adhesion on Kupffer cells Wong, Nat Immunol, 2013 Background II Ø Recognition by immune cells of molecular features of microbes. Hacker, Immunology, 2002 Background II Ø Recognition by immune cells of molecular features of microbes. Ø In immune cells: major mechanism to discriminate pathogenic from self DNA is TLR 9 Hacker, Immunology, 2002 Background II Ø Recognition by immune cells of molecular features of microbes. Ø In immune cells: major mechanism to discriminate pathogenic from self DNA is TLR 9 Ø TLR 9 in platelets Hacker, Immunology, 2002 Background II Ø Recognition by immune cells of molecular features of microbes. Ø In immune cells: major mechanism to discriminate pathogenic from self DNA is TLR 9 Ø TLR 9 in platelets Can platelets recognize bacterial DNA? Hacker, Immunology, 2002 Background III TLR 9 agonists: CpG ODN Type A : Type B : Type C : 5’-gggggacgatcgtcgggggg-3’ 5’-tcgtcgttttgtcgttttgtcgtt-3’ 5’-tcgtcgttttcggcgc:gcgccg-3’ Results I Hirudinized PRP CpG-ODN type A, B, C cause αIIbβ3–dependent aggregation of human platelets Results I Hirudinized whole blood; N=6 CpG-ODN type A, B, C induce P-selectin exposure and fibrinogen binding Results II Type A : Type C : Washed platelets Hirudinized PRP Non CpG-ODN have similar effects, suggesting that TLR9 does not mediate the observed effects. Results III ODN PO ODN PS • Different sequences, common feature = phosphorothioate modification. • Many bacteria contain DNA that is phosphorothioate modified (PS) (enterogenic Escherichia coli, Salmonella enterica, and most Vibrio strains). Results III Hirudinized whole blood; N=6 None of ODN-PO tested activates platelets Results III Type A : Type C : Washed platelets None of ODN-PO tested induces platelet aggregation Hirudinized PRP Results IV Mice hirudinized whole blood, N=5 ODN type A and C PS activate mice platelets. Similarly as for human platelets, no effect of ODN-PO vs ODN-PS Results V TLR 9-/- MyD88-/- Mice washed platelets ODN-PS activate platelets independently of TLR pathway Results VII Mice washed platelets; N=3 Platelet activation by ODN-PS depends on αIIbβ3 signaling and on secreted ADP. Results VI Mice washed platelets; N=3 We found that ODN-PS accumulated on platelet surface. Such binding was prevented in the presence of EDTA, indicative of a role for αIIbβ3 integrin. Resting ODN Type C 30 min ODN Type C ODN Type A Results VIII Phospho Syk (Tyr 525/526) Phospho Syk (Tyr 323) Phospho Syk (Tyr 319/Tyr 352) Total Syk ODN-PS induce tyrosine phosphorylation events in platelets Conclusions Ø Platelets can selectively recognize bacterial DNA bearing phosphorothioate modification Ø Potential new mechanism of immune surveillance by platelets Ø DNA-PS induces platelet activation and aggregation via αIIbβ3 and ADP secretion Ø DNA-PS may activate αIIbβ3 integrin via disulphide bond reduction and/ or disulphide exchange Acknowledgements Team Cécile Oury, PhD Christelle Lecut, PhD Alexandre Hego Vincent Bours, MD, PhD Collaborators: Akira S., Osaka University, Japan GIGA platforms: GIGA imaging platform GIGA SPF Animal facility