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.

  • Letter
  • Published:

Diabetes primes neutrophils to undergo NETosis, which impairs wound healing

Abstract

Wound healing is impaired in diabetes, resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins1. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed NETosis). Expression of peptidylarginine deiminase 4 (PAD4, encoded by Padi4 in mice), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4−/− mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds than in normoglycemic mice and healing was delayed. Wound healing was accelerated in Padi4−/− mice as compared to WT mice, and it was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes, in which neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.

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: Diabetes or high glucose concentrations in vitro prime human and mouse neutrophils to undergo NETosis.
Figure 2: NETs are present in the wounds of WT mice.
Figure 3: PAD4 deficiency facilitates wound repair in normoglycemic mice.
Figure 4: PAD4 deficiency or DNase 1 treatment enhances wound healing in diabetic mice.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Wang, Y. et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J. Cell Biol. 184, 205–213 (2009).

    Article  CAS  Google Scholar 

  3. Wang, Y. et al. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306, 279–283 (2004).

    Article  CAS  Google Scholar 

  4. Yipp, B.G. & Kubes, P. NETosis: how vital is it? Blood 122, 2784–2794 (2013).

    Article  CAS  Google Scholar 

  5. Martinod, K. & Wagner, D.D. Thrombosis: tangled up in NETs. Blood 123, 2768–2776 (2014).

    Article  CAS  Google Scholar 

  6. Karima, M. et al. Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis. J. Leukoc. Biol. 78, 862–870 (2005).

    Article  CAS  Google Scholar 

  7. Hanses, F., Park, S., Rich, J. & Lee, J.C. Reduced neutrophil apoptosis in diabetic mice during staphylococcal infection leads to prolonged TNFα production and reduced neutrophil clearance. PLoS ONE 6, e23633 (2011).

    Article  CAS  Google Scholar 

  8. Thomas, G.M. et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood 119, 6335–6343 (2012).

    Article  CAS  Google Scholar 

  9. Alexandraki, K.I. et al. Cytokine secretion in long-standing diabetes mellitus type 1 and 2: associations with low-grade systemic inflammation. J. Clin. Immunol. 28, 314–321 (2008).

    Article  CAS  Google Scholar 

  10. Luo, Y. et al. Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization. Biochemistry 45, 11727–11736 (2006).

    Article  CAS  Google Scholar 

  11. Li, P. et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J. Exp. Med. 207, 1853–1862 (2010).

    Article  CAS  Google Scholar 

  12. Leshner, M. et al. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol 3, 307 (2012).

    Article  Google Scholar 

  13. Alexiewicz, J.M., Kumar, D., Smogorzewski, M., Klin, M. & Massry, S.G. Polymorphonuclear leukocytes in non-insulin-dependent diabetes mellitus: abnormalities in metabolism and function. Ann. Intern. Med. 123, 919–924 (1995).

    Article  CAS  Google Scholar 

  14. Gupta, A.K., Giaglis, S., Hasler, P. & Hahn, S. Efficient neutrophil extracellular trap induction requires mobilization of both intracellular and extracellular calcium pools and is modulated by cyclosporine A. PLoS ONE 9, e97088 (2014).

    Article  Google Scholar 

  15. Rodríguez-Espinosa, O., Rojas-Espinosa, O., Moreno-Altamirano, M.M., Lopez-Villegas, E.O. & Sanchez-Garcia, F.J. Metabolic requirements for neutrophil extracellular traps (NETs) formation. Immunology 145, 213–224 (2015).

    Article  Google Scholar 

  16. Menegazzo, L. et al. NETosis is induced by high glucose and associated with type 2 diabetes. Acta Diabetol. (in the press 2014).

  17. Joshi, M.B. et al. High glucose modulates IL-6 mediated immune homeostasis through impeding neutrophil extracellular trap formation. FEBS Lett. 587, 2241–2246 (2013).

    Article  CAS  Google Scholar 

  18. Riyapa, D. et al. Neutrophil extracellular traps exhibit antibacterial activity against Burkholderia pseudomallei and are influenced by bacterial and host factors. Infect. Immun. 80, 3921–3929 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Rebecchi, I.M., Ferreira Novo, N., Julian, Y. & Campa, A. Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient. Cell Biochem. Funct. 18, 127–132 (2000).

    Article  CAS  Google Scholar 

  21. Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C. & Zychlinsky, A. Neutrophil extracellular traps: how to generate and visualize them. J. Vis. Exp., 1724 (2010).

  22. Dovi, J.V., He, L.K. & DiPietro, L.A. Accelerated wound closure in neutrophil-depleted mice. J. Leukoc. Biol. 73, 448–455 (2003).

    Article  CAS  Google Scholar 

  23. Saffarzadeh, M. et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS ONE 7, e32366 (2012).

    Article  CAS  Google Scholar 

  24. Subramaniam, M. et al. Role of endothelial selectins in wound repair. Am. J. Pathol. 150, 1701–1709 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Nachat, R. et al. Peptidylarginine deiminase isoforms 1–3 are expressed in the epidermis and involved in the deimination of K1 and filaggrin. J. Invest. Dermatol. 124, 384–393 (2005).

    Article  CAS  Google Scholar 

  26. Frenette, P.S., Mayadas, T.N., Rayburn, H., Hynes, R.O. & Wagner, D.D. Susceptibility to infection and altered hematopoiesis in mice deficient in both P- and E-selectins. Cell 84, 563–574 (1996).

    Article  CAS  Google Scholar 

  27. Martinod, K. et al. PAD4-deficiency does not affect bacteremia in polymicrobial sepsis and ameliorates endotoxemic shock. Blood 125, 1948–1956 (2015).

    Article  CAS  Google Scholar 

  28. Coudane, F. et al. Deimination and expression of peptidylarginine deiminases during cutaneous wound healing in mice. Eur. J. Dermatol. 21, 376–384 (2011).

    CAS  PubMed  Google Scholar 

  29. Herrick, S. et al. Up-regulation of elastase in acute wounds of healthy aged humans and chronic venous leg ulcers are associated with matrix degradation. Lab. Invest. 77, 281–288 (1997).

    CAS  PubMed  Google Scholar 

  30. Grice, E.A. et al. Longitudinal shift in diabetic wound microbiota correlates with prolonged skin defense response. Proc. Natl. Acad. Sci. USA 107, 14799–14804 (2010).

    Article  CAS  Google Scholar 

  31. Berends, E.T. et al. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J. Innate Immun. 2, 576–586 (2010).

    Article  CAS  Google Scholar 

  32. Farrera, C. & Fadeel, B. Macrophage clearance of neutrophil extracellular traps is a silent process. J. Immunol. 191, 2647–2656 (2013).

    Article  CAS  Google Scholar 

  33. Bodaño, A. et al. Study of DNASE I gene polymorphisms in systemic lupus erythematosus susceptibility. Ann. Rheum. Dis. 66, 560–561 (2007).

    Article  Google Scholar 

  34. Kumamoto, T. et al. Association of Gln222Arg polymorphism in the deoxyribonuclease I (DNase I) gene with myocardial infarction in Japanese patients. Eur. Heart J. 27, 2081–2087 (2006).

    Article  CAS  Google Scholar 

  35. Hakkim, A. et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc. Natl. Acad. Sci. USA 107, 9813–9818 (2010).

    Article  CAS  Google Scholar 

  36. Lewis, H.D. et al. Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat. Chem. Biol. 11, 189–191 (2015).

    Article  CAS  Google Scholar 

  37. Laakso, M. & Kuusisto, J. Insulin resistance and hyperglycaemia in cardiovascular disease development. Nat. Rev. Endocrinol. 10, 293–302 (2014).

    Article  CAS  Google Scholar 

  38. Morel, O., Jesel, L., Abbas, M. & Morel, N. Prothrombotic changes in diabetes mellitus. Semin. Thromb. Hemost. 39, 477–488 (2013).

    Article  CAS  Google Scholar 

  39. Demers, M. et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc. Natl. Acad. Sci. USA 109, 13076–13081 (2012).

    Article  CAS  Google Scholar 

  40. Brill, A. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice. J. Thromb. Haemost. 10, 136–144 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank H. Ferris for advice on diabetes protocols; L. DeVita for selection of diabetic patients; P. Forbes (The Harvard Clinical and Translational Science Center, US National Institutes of Health (NIH) Award UL1 TR001102) for statistical advice; J.E. Cabral and S. Cifuni for valuable technical support; and L. Cowan for manuscript preparation assistance. This study was supported by the American Diabetes Association (Innovation Award 7-13-IN-44 to D.D.W.), the National Heart, Lung, and Blood Institute of the NIH (R01HL102101 to D.D.W.), the National Cancer Institute (R01CA136856 to Y.W.), the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK031036 to C.R.K.) and a GlaxoSmithKline/Immune Disease Institute Alliance Fellowship (S.L.W.).

Author information

Authors and Affiliations

Authors

Contributions

S.L.W. designed the study, performed the majority of the experiments, analyzed the data and wrote the manuscript; M.D. and K.M. performed experiments and analyzed data; M.G. provided expert technical assistance; Y.W. provided Padi4−/− mice and critical discussion of the work; A.B.G. provided clinical advice and selected diabetic patients for in vitro NETosis assays; C.R.K. provided helpful suggestions on experimental design and critical reading of the manuscript; D.D.W. designed the study, supervised the project and co-wrote the manuscript.

Corresponding author

Correspondence to Denisa D Wagner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Table 1 (PDF 18613 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wong, S., Demers, M., Martinod, K. et al. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat Med 21, 815–819 (2015). https://doi.org/10.1038/nm.3887

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3887

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