Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Abstract

It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Platelet TLR4 regulates adhesion of platelets to neutrophils, but not platelet P-selectin expression or platelet aggregation.
Figure 2: LPS induces platelet-neutrophil interactions in vivo.
Figure 3: Profound neutrophil degranulation and NET formation as a result of platelet binding.
Figure 4: NETs are formed under shear forces in vitro.
Figure 5: Formation of NETs greatly enhances trapping of bacteria in vitro and in vivo.
Figure 6: LPS-stimulated platelet-neutrophil interactions damage tissue.

Similar content being viewed by others

References

  1. Bone, R.C. Sepsis, the sepsis syndrome, multi-organ failure: a plea for comparable definitions. Ann. Intern. Med. 114, 332–333 (1991).

    Article  CAS  PubMed  Google Scholar 

  2. Welbourn, C.R. & Young, Y. Endotoxin, septic shock and acute lung injury: neutrophils, macrophages and inflammatory mediators. Br. J. Surg. 79, 998–1003 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Mavrommatis, A.C. et al. Coagulation system and platelets are fully activated in uncomplicated sepsis. Crit. Care Med. 28, 451–457 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Mercer, K.W., Gail, M.B. & Williams, M.E. Hematologic disorders in critically ill patients. Semin. Respir. Crit. Care Med. 27, 286–296 (2006).

    Article  PubMed  Google Scholar 

  5. Andonegui, G. et al. Platelets express functional Toll-like receptor-4. Blood 106, 2417–2423 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Stohlawetz, P. et al. Effects of endotoxemia on thrombopoiesis in men. Thromb. Haemost. 81, 613–617 (1999).

    CAS  PubMed  Google Scholar 

  7. Klinger, M.H. & Jelkmann, W. Role of blood platelets in infection and inflammation. J. Interferon Cytokine Res. 22, 913–922 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Aslam, R. et al. Platelet Toll-like receptor expression modulates lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-α production in vivo. Blood 107, 637–641 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Ward, J.R. et al. Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor. Thromb. Haemost. 94, 831–838 (2005).

    PubMed  Google Scholar 

  10. Brinkmann, V. et al. Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Mullarkey, M. et al. Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-directed endotoxin antagonist. J. Pharmacol. Exp. Ther. 304, 1093–1102 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Lynn, M. et al. Extended in vivo pharmacodynamic activity of E5564 in normal volunteers with experimental endotoxemia [corrected]. J. Pharmacol. Exp. Ther. 308, 175–181 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Singer, G., Urakami, H., Specian, R.D., Stokes, K.Y. & Granger, D.N. Platelet recruitment in the murine hepatic microvasculature during experimental sepsis: role of neutrophils. Microcirculation 13, 89–97 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Beiter, K. et al. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. Curr. Biol. 16, 401–407 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Buchanan, J.T. et al. DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Curr. Biol. 16, 396–400 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Fuchs, T.A. et al. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol. 176, 231–241 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gross, K. et al. Bacterial clearance in the intact and regenerating liver. J. Pediatr. Surg. 20, 320–323 (1985).

    Article  CAS  PubMed  Google Scholar 

  18. Vincent, J.L., Yagushi, A. & Pradier, O. Platelet function in sepsis. Crit. Care Med. 30, S313–S317 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Ruf, A. & Patscheke, H. Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18. Br. J. Haematol. 90, 791–796 (1995).

    Article  CAS  PubMed  Google Scholar 

  20. Segal, A.W. How neutrophils kill microbes. Annu. Rev. Immunol. 23, 197–223 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Akgul, C., Moulding, D.A. & Edwards, S.W. Molecular control of neutrophil apoptosis. FEBS Lett. 487, 318–322 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Borregaard, N. & Cowland, J.B. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89, 3503–3521 (1997).

    CAS  PubMed  Google Scholar 

  23. Moraes, T.J., Zurawska, J.H. & Downey, G.P. Neutrophil granule contents in the pathogenesis of lung injury. Curr. Opin. Hematol. 13, 21–27 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Sheridan, B.C. et al. Neutrophils mediate pulmonary vasomotor dysfunction in endotoxin-induced acute lung injury. J. Trauma 42, 391–396 (1997).

    Article  CAS  PubMed  Google Scholar 

  25. Alghamdi, A.S. & Foster, D.N. Seminal DNase frees spermatozoa entangled in neutrophil extracellular traps. Biol. Reprod. 73, 1174–1181 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Frenette, P.S., Johnson, R.C., Hynes, R.O. & Wagner, D.D. Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. Proc. Natl. Acad. Sci. USA 92, 7450–7454 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Heit, B., Tavener, S., Raharjo, E. & Kubes, P. An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J. Cell Biol. 159, 91–102 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kubes, P., Kanwar, S., Niu, X.F. & Gaboury, J.P. Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells. FASEB J. 7, 1293–1299 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Lloyd, K.L. & Kubes, P. GPI-linked endothelial CD14 contributes to the detection of LPS. Am. J. Physiol. Heart Circ. Physiol. 291, H473–H481 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Ibbotson, G.C. et al. Functional α4-integrin: a newly identified pathway of neutrophil recruitment in critically ill septic patients. Nat. Med. 7, 465–470 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Reinhardt, P.H. & Kubes, P. Differential leukocyte recruitment from whole blood via endothelial adhesion molecules under shear conditions. Blood 92, 4691–4699 (1998).

    CAS  PubMed  Google Scholar 

  32. Gill, V., Doig, C., Knight, D., Love, E. & Kubes, P. Targeting adhesion molecules as a potential mechanism of action for intravenous immunoglobulin. Circulation 112, 2031–2039 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Bonder, C.S. et al. Rules of recruitment for Th1 and Th2 lymphocytes in inflamed liver: a role for α-4 integrin and vascular adhesion protein-1. Immunity 23, 153–163 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Wong, J. et al. A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature. J. Clin. Invest. 99, 2782–2790 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Hickey, M.J. et al. Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia. FASEB J. 11, 955–964 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Nishida, J., McCuskey, R.S., McDonnell, D. & Fox, E.S. Protective role of NO in hepatic microcirculatory dysfunction during endotoxemia. Am. J. Physiol. 267, G1135–G1141 (1994).

    CAS  PubMed  Google Scholar 

  37. Li, Y., Muruve, D.A., Collins, R.G., Lee, S.S. & Kubes, P. The role of selectins and integrins in adenovirus vector-induced neutrophil recruitment to the liver. Eur. J. Immunol. 32, 3443–3452 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Carvalho-Tavares, J. et al. A role for platelets and endothelial selectins in tumor necrosis factor- α-induced leukocyte recruitment in the brain microvasculature. Circ. Res. 87, 1141–1148 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank C. Gwozd for tissue processing and staining, and P. Colarusso for her technical assistance with microscopy. We would also like to acknowledge D. Brown and D. Knight for their technical support. This work was supported by grants from the Canadian Institutes of Health (CIHR) and a CIHR group grant. S.R.C. is a Heart and Stroke Foundation Canada fellow. P.K. is an Alberta Heritage Foundation for Medical Research (AHFMR) Scientist and a Canadian Research Chair recipient. A.C.M. is an AHFMR student.

Author information

Authors and Affiliations

Authors

Contributions

S.R.C., A.C.M., S.A.T., B.M. and P.K. designed the studies. S.R.C., A.C.M., S.A.T. and B.M. performed the bulk of the experiments. P.K. wrote the manuscript, and A.C.M. and S.R.C. contributed to the preparation of the manuscript. Z.G. helped with flow cytometry. M.M.K. and E.M. searched for NETs in patients and mice, respectively. K.D.P. and S.C. completed the zymography assays and G.D.S. ran the aggregometry. F.H.Y.G. and E.M.K. helped with the Richardson microscope, and E.A.-V. and R.D. produced transfected bacteria. C.J.D. provided septic human blood samples.

Corresponding author

Correspondence to Paul Kubes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

NETs within pulmonary microvasculature of an LPS-treated mouse visualized by fluorescence microscopy. (PDF 73 kb)

Supplementary Fig. 2

Sections of lung with acute interstitial pneumonitis, demonstrating intravascular NETs. (PDF 206 kb)

Supplementary Video 1 (MOV 2730 kb)

Supplementary Methods

Time-lapse of NET formation. Using darkfield and fluorescence microscopy one can see two neutrophils (orange — visualized by white light through an orange filter) begin to release DNA (green — dyed with Sytox Green) within 5 min. The DNA from both neutrophils fuses and forms one large NET. (PDF 110 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Clark, S., Ma, A., Tavener, S. et al. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat Med 13, 463–469 (2007). https://doi.org/10.1038/nm1565

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1565

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing