Download Platelets selectively recognize bacterial DNA independently of Toll

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