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:

Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation

Nature Cell Biology volume 27, pages 931–947 (2025)Cite this article

Subjects

Abstract

Acute inflammation, characterized by a rapid influx of neutrophils, is a protective response that can lead to chronic inflammatory diseases when left unresolved. We previously showed that secretion of LTB4-containing exosomes via nuclear envelope-derived multivesicular bodies is required for effective neutrophil infiltration during inflammation. Here we report that the co-secretion of these exosomes with nuclear DNA facilitates the resolution of the neutrophil infiltrate in a mouse skin model of sterile inflammation. Activated neutrophils exhibit rapid and repetitive DNA secretion as they migrate directionally using a mechanism distinct from suicidal neutrophil extracellular trap release and cell death. Packaging of DNA in the lumen of nuclear envelope-multivesicular bodies is mediated by lamin B receptor and chromatin decondensation. These findings advance our understanding of neutrophil functions during inflammation and the physiological relevance of DNA secretion.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Nuclear DNA is packaged in the lumen of NE-MVBs in chemotaxing neutrophils.
Fig. 2: Spatial co-occurrence of nuclear DNA with NE-derived exosomes secreted from activated neutrophils.
Fig. 3: Fast and repetitive DNA secretion from the back of chemotaxing neutrophils.
Fig. 4: LBR is required for the packaging and secretion of DNA, but not exosomes, through NE-MVBs.
Fig. 5: LTB4-induced histone acetylation mediates DNA secretion.
Fig. 6: SEAD disruption impedes LTB4 temporal increase within inflamed tissue delaying neutrophil reverse migration and inflammation resolution.
Fig. 7: LTB4-PPARα signalling regulates inflammation resolution.

Similar content being viewed by others

Data availability

All the raw data and associated statistical analysis presented have been provided as ‘source data’ and ‘unprocessed western blots’ for the respective figures. Owing to the large size of high-resolution z-stack and time-lapse microscopy images, the raw microscopy images are available from the corresponding author upon reasonable request. The mass spectrometry proteomics data are available as Supplementary Table 1 and have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD061995. Source data are provided with this paper.

Code availability

All CellProfiler pipelines used in this study are made available on the CellProfiler website linked to the publication weblink and accession information, to ensure transparency and reproducibility of the analysis. The MATLAB code used to analyse under-agarose migration of neutrophils has been uploaded to a publicly available repository and can be accessed on Zenodo at https://doi.org/10.5281/zenodo.15080547 (ref. 160).

Materials availability

Requests for reagents and resources should be directed to the corresponding author C.A.P. (parentc@umich.edu).

References

  1. Németh, T., Sperandio, M. & Mócsai, A. Neutrophils as emerging therapeutic targets. Nat. Rev. Drug Discov. 19, 253–275 (2020).

    Article  PubMed  Google Scholar 

  2. Serhan, C. N. et al. Resolution of inflammation: state of the art, definitions and terms. FASEB J. 21, 325–332 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Fine, N., Tasevski, N., McCulloch, C. A., Tenenbaum, H. C. & Glogauer, M. The neutrophil: constant defender and first responder. Front. Immunol. 11, 571085 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Peiseler, M. & Kubes, P. More friend than foe: the emerging role of neutrophils in tissue repair. J. Clin. Invest. 129, 2629–2639 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Netea, M. G. et al. A guiding map for inflammation. Nat. Immunol. 18, 826–831 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Furman, D. et al. Chronic inflammation in the etiology of disease across the life span. Nat. Med. 25, 1822–1832 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Castanheira, F. V. S. & Kubes, P. Neutrophils and NETs in modulating acute and chronic inflammation. Blood 133, 2178–2185 (2019).

    Article  CAS  PubMed  Google Scholar 

  8. Wigerblad, G. & Kaplan, M. J. Neutrophil extracellular traps in systemic autoimmune and autoinflammatory diseases. Nat. Rev. Immunol. 23, 274–288 (2023).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Burn, G. L., Foti, A., Marsman, G., Patel, D. F. & Zychlinsky, A. The neutrophil. Immunity 54, 1377–1391 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Pilsczek, F. H. et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J. Immunol. 185, 7413–7425 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18, 1386–1393 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Eghbalzadeh, K. et al. Compromised anti-inflammatory action of neutrophil extracellular traps in PAD4-deficient mice contributes to aggravated acute inflammation after myocardial infarction. Front. Immunol. 10, 2313 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Schauer, C. et al. Aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines. Nat. Med. 20, 511–517 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Knopf, J., Leppkes, M., Schett, G., Herrmann, M. & Muñoz, L. E. Aggregated NETs sequester and detoxify extracellular histones. Front. Immunol. 10, 2176 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hahn, J. et al. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases. FASEB J. 33, 1401–1414 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Rodríguez-Morales, P. & Franklin, R. A. Macrophage phenotypes and functions: resolving inflammation and restoring homeostasis. Trends Immunol. 44, 986–998 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Devchand, P. R. et al. The PPARα–leukotriene B4 pathway to inflammation control. Nature 384, 39–43 (1996).

    Article  CAS  PubMed  Google Scholar 

  19. de Oliveira, S., Rosowski, E. E. & Huttenlocher, A. Neutrophil migration in infection and wound repair: going forward in reverse. Nat. Rev. Immunol. 16, 378–391 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Xu, Q., Zhao, W., Yan, M. & Mei, H. Neutrophil reverse migration. J. Inflamm. 19, 22 (2022).

    Article  CAS  Google Scholar 

  21. Lämmermann, T. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498, 371–375 (2013).

    Article  PubMed  Google Scholar 

  22. Afonso, P. V. et al. LTB4 is a signal-relay molecule during neutrophil chemotaxis. Dev. Cell 22, 1079–1091 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mandal, A. K. et al. The nuclear membrane organization of leukotriene synthesis. Proc. Natl Acad. Sci. USA 105, 20434–20439 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Arya, S. B., Chen, S., Jordan-Javed, F. & Parent, C. A. Ceramide-rich microdomains facilitate nuclear envelope budding for non-conventional exosome formation. Nat. Cell Biol. 24, 1019–1028 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Majumdar, R., Tameh, A. T., Arya, S. B. & Parent, C. A. Exosomes mediate LTB4 release during neutrophil chemotaxis. PLoS Biol. 19, 1–28 (2021).

    Google Scholar 

  26. Solovei, I. et al. LBR and lamin A/C sequentially tether peripheral heterochromatin and inversely regulate differentiation. Cell 152, 584–598 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Liokatis, S. et al. Solution structure and molecular interactions of lamin B receptor tudor domain. J. Biol. Chem. 287, 1032–1042 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Tsai, P.-L., Zhao, C., Turner, E. & Schlieker, C. The lamin B receptor is essential for cholesterol synthesis and perturbed by disease-causing mutations. eLife 5, e16011 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Polioudaki, H. et al. Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1. EMBO Rep. 2, 920–925 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gaines, P., Gotur, D., Halene, S., Olins, A. L. & Olins, D. E. Dynamic expression of the lamin B receptor and associated proteins during myeloid differentiation: insight into mechanisms of neutrophil vs. macrophage morphogenesis. Blood 112, 3546–3546 (2008).

    Article  Google Scholar 

  31. Manley, H. R., Keightley, M. C. & Lieschke, G. J. The neutrophil nucleus: an important influence on neutrophil migration and function. Front. Immunol. 9, 2867 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gaines, P. et al. Mouse neutrophils lacking lamin B-receptor expression exhibit aberrant development and lack critical functional responses. Exp. Hematol. 36, 965–976 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Toda, A., Yokomizo, T. & Shimizu, T. Leukotriene B4 receptors. Prostaglandins Other Lipid Mediat. 68–69, 575–585 (2002).

    Article  PubMed  Google Scholar 

  34. Narala, V. R. et al. Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-α agonist. J. Biol. Chem. 285, 22067–22074 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Esposito, E. et al. PPAR-α contributes to the anti-inflammatory activity of verbascoside in a model of inflammatory bowel disease in mice. PPAR Res. 2010, 917312 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Dubrac, S. & Schmuth, M. PPAR-α in cutaneous inflammation. Dermatoendocrinology 3, 23–26 (2011).

    Article  CAS  Google Scholar 

  37. Delayre-Orthez, C. et al. PPARα downregulates airway inflammation induced by lipopolysaccharide in the mouse. Respir. Res 6, 91 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Lu, P. et al. Chemical screening identifies ROCK1 as a regulator of migrasome formation. Cell Discov. 6, 51 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Saunders, C. A., Majumdar, R., Molina, Y., Subramanian, B. C. & Parent, C. A. Genetic manipulation of PLB-985 cells and quantification of chemotaxis using the underagarose assay. Methods Cell Biol. https://doi.org/10.1016/bs.mcb.2018.09.002 (2019).

  40. Heit, B. & Kubes, P. Measuring chemotaxis and chemokinesis: the under-agarose cell migration assay. Sci. STKE 2003, pI5 (2003).

    Article  Google Scholar 

  41. Deniset, J. F. & Kubes, P. Neutrophil heterogeneity: bona fide subsets or polarization states? J. Leukoc. Biol. 103, 829–838 (2018).

    Article  CAS  PubMed  Google Scholar 

  42. Gupta, S. et al. Sex differences in neutrophil biology modulate response to type I interferons and immunometabolism. Proc. Natl Acad. Sci. USA 117, 16481–16491 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Blazkova, J. et al. Multicenter systems analysis of human blood reveals immature neutrophils in males and during pregnancy. J. Immunol. 198, 2479–2488 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. McQuin, C. et al. CellProfiler 3.0: next-generation image processing for biology. PLoS Biol. 16, e2005970 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinform. 22, 433 (2021).

    Article  Google Scholar 

  46. Yousefi, S., Mihalache, C., Kozlowski, E., Schmid, I. & Simon, H. U. Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps. Cell Death Differ. 16, 1438–1444 (2009).

    Article  CAS  PubMed  Google Scholar 

  47. Lood, C. et al. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. Nat. Med. 22, 146–153 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Pendergrass, W., Wolf, N. & Poot, M. Efficacy of MitoTracker GreenTM and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytom. Part A 61A, 162–169 (2004).

    Article  Google Scholar 

  49. Neubert, E. et al. Chromatin swelling drives neutrophil extracellular trap release. Nat. Commun. 9, 3767 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  50. 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 

  51. Rohrbach, A. S., Slade, D. J., Thompson, P. R. & Mowen, K. A. Activation of PAD4 in NET formation. Front. Immunol. 3, 360 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Sollberger, G. et al. Gasdermin D plays a vital role in the generation of neutrophil extracellular traps. Sci. Immunol. 3, eaar6689 (2018).

    Article  PubMed  Google Scholar 

  53. Desai, J. et al. PMA and crystal‐induced neutrophil extracellular trap formation involves RIPK1‐RIPK3‐MLKL signaling. Eur. J. Immunol. 46, 223–229 (2016).

    Article  CAS  PubMed  Google Scholar 

  54. Stankova, J., Turcotte, S., Harris, J. & Rola-Pleszczynski, M. Modulation of leukotriene B4 receptor-1 expression by dexamethasone: potential mechanism for enhanced neutrophil survival. J. Immunol. 168, 3570–3576 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Pétrin, D., Turcotte, S., Gilbert, A.-K., Rola-Pleszczynski, M. & Stankova, J. The anti-apoptotic effect of leukotriene B4 in neutrophils: a role for phosphatidylinositol 3-kinase, extracellular signal-regulated kinase and Mcl-1. Cell Signal. 18, 479–487 (2006).

    Article  PubMed  Google Scholar 

  56. Gee, D. J. et al. Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions. Biosci. Rep. 32, 375–382 (2012).

    Article  CAS  PubMed  Google Scholar 

  57. Breitman, T. R., Collins, S. J. & Keene, B. R. Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60. Exp. Cell. Res. 126, 494–498 (1980).

    Article  CAS  PubMed  Google Scholar 

  58. Rowat, A. C. et al. Nuclear envelope composition determines the ability of neutrophil-type cells to passage through micron-scale constrictions. J. Biol. Chem. 288, 8610–8618 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Olins, A. L., Ernst, A., Zwerger, M., Herrmann, H. & Olins, D. E. An in vitro model for Pelger-Huët anomaly. Nucleus 1, 506–512 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Dusse, L. M. S. et al. Acquired Pelger–Huët: what does it really mean? Clin. Chim. Acta 411, 1587–1590 (2010).

    Article  CAS  PubMed  Google Scholar 

  61. de Oliveira, S., Boudinot, P., Calado, Â. & Mulero, V. Duox1-derived H2O2 modulates Cxcl8 expression and neutrophil recruitment via JNK/c-JUN/AP-1 signaling and chromatin modifications. J. Immunol. 194, 1523–1533 (2015).

    Article  PubMed  Google Scholar 

  62. Hamam, H., Khan, M. & Palaniyar, N. Histone acetylation promotes neutrophil extracellular trap formation. Biomolecules 9, 32 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Zhang, C. et al. Histone acetylation: novel target for the treatment of acute lymphoblastic leukemia. Clin. Epigenet. 7, 117 (2015).

    Article  Google Scholar 

  64. Ferguson, A. D. et al. Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317, 510–512 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Mancini, J. A. et al. 5‐lipoxygenase‐activating protein is an arachidonate binding protein. FEBS Lett. 318, 277–281 (1993).

    Article  CAS  PubMed  Google Scholar 

  66. Gábor, M. Models of acute inflammation in the ear. Methods Mol. Biol. 225, 129–137 (2003).

    PubMed  Google Scholar 

  67. Xu, Y., Cohen, E., Johnson, C. N., Parent, C. A. & Coulombe, P. A. Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner. PLoS Biol. 22, e3002779 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Bernut, A., Loynes, C. A., Floto, R. A. & Renshaw, S. A. Deletion of cftr leads to an excessive neutrophilic response and defective tissue repair in a zebrafish model of sterile inflammation. Front. Immunol. 11, 1733 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Wang, J. et al. Visualizing the function and fate of neutrophils in sterile injury and repair. Science 358, 111–116 (2017).

    Article  CAS  PubMed  Google Scholar 

  70. Hoste, E. et al. Epithelial HMGB1 delays skin wound healing and drives tumor initiation by priming neutrophils for NET formation. Cell Rep. 29, 2689–2701.e4 (2019).

    Article  CAS  PubMed  Google Scholar 

  71. Wong, S. L. et al. Diabetes primes neutrophils to undergo NETosis, which impairs wound healing. Nat. Med. 21, 815–819 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Serhan, C. N. & Levy, B. D. Resolvins in inflammation: emergence of the pro-resolving superfamily of mediators. J. Clin. Invest. 128, 2657–2669 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Colom, B. et al. Leukotriene B4-neutrophil elastase axis drives neutrophil reverse transendothelial cell migration in vivo. Immunity 42, 1075–1086 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Temme, S. et al. Technical advance: monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI. J. Leukoc. Biol. 95, 689–697 (2013).

    Article  PubMed  Google Scholar 

  75. Soehnlein, O., Lindbom, L. & Weber, C. Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood 114, 4613–4623 (2009).

    Article  CAS  PubMed  Google Scholar 

  76. Jiménez‐Alcázar, M. et al. Impaired DNase1‐mediated degradation of neutrophil extracellular traps is associated with acute thrombotic microangiopathies. J. Thromb. Haemost. 13, 732–742 (2015).

    Article  PubMed  Google Scholar 

  77. Li, T. et al. TBK1 recruitment to STING mediates autoinflammatory arthritis caused by defective DNA clearance. J. Exp. Med. 219, e20211539 (2022).

    Article  CAS  PubMed  Google Scholar 

  78. Malíčková, K. et al. Impaired deoxyribonuclease I activity in patients with inflammatory bowel diseases. Autoimmune Dis. 2011, 1–5 (2011).

    Article  Google Scholar 

  79. Yasutomo, K. et al. Mutation of DNASE1 in people with systemic lupus erythematosus. Nat. Genet. 28, 313–314 (2001).

    Article  CAS  PubMed  Google Scholar 

  80. Garcia, G. et al. Impaired balance between neutrophil extracellular trap formation and degradation by DNases in COVID-19 disease. J. Transl. Med. 22, 246 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Jiménez-Alcázar, M. et al. Host DNases prevent vascular occlusion by neutrophil extracellular traps. Science 358, 1202–1206 (2017).

    Article  PubMed  Google Scholar 

  82. Oved, J. H. et al. Neutrophils promote clearance of nuclear debris following acid-induced lung injury. Blood 137, 392–397 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Shimada, O. et al. Detection of deoxyribonuclease I along the secretory pathway in Paneth cells of human small intestine. J. Histochem. Cytochem. 46, 833–840 (1998).

    Article  CAS  PubMed  Google Scholar 

  84. Subramanian, B. C., Moissoglu, K. & Parent, C. A. The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis. J. Cell Sci. 131, jcs217422 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  85. Iizuka, Y. et al. Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis. FASEB J. 24, 4678–4690 (2010).

    CAS  PubMed  Google Scholar 

  86. Zinn, S. et al. The leukotriene B4 receptors BLT1 and BLT2 form an antagonistic sensitizing system in peripheral sensory neurons. J. Biol. Chem. 292, 6123–6134 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Li, G. et al. Hepatic peroxisome proliferator‐activated receptor α mediates the major metabolic effects of Wy‐14643. J. Gastroenterol. Hepatol. 33, 1138–1145 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Stienstra, R. et al. The interleukin-1 receptor antagonist is a direct target gene of PPARα in liver. J. Hepatol. 46, 869–877 (2007).

    Article  CAS  PubMed  Google Scholar 

  89. Ghosh, A. et al. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells. J. Biol. Chem. 290, 10309–10324 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Saika, A. et al. Mead acid inhibits retinol-induced irritant contact dermatitis via peroxisome proliferator-activated receptor alpha. Front. Mol. Biosci. 10, 1097955 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Xu, H. E. et al. Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARα. Nature 415, 813–817 (2002).

    Article  CAS  PubMed  Google Scholar 

  92. Briguglio, E. et al. WY-14643, a potent peroxisome proliferator activator receptor-α PPAR-α agonist ameliorates the inflammatory process associated to experimental periodontitis. PPAR Res 2010, 1–13 (2010).

    Article  Google Scholar 

  93. D’Cruz, A. A. et al. The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils. Sci. Signal 11, eaao1716 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  94. Sedej, I. et al. Extracellular vesicle‐bound DNA in urine is indicative of kidney allograft injury. J. Extracell. Vesicles 11, e12268 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Wortzel, I. et al. Unique structural configuration of EV-DNA primes Kupffer cell-mediated antitumor immunity to prevent metastatic progression. Nat. Cancer 5, 1815–1833 (2024).

    Article  CAS  PubMed  Google Scholar 

  96. Yokoi, A. et al. Mechanisms of nuclear content loading to exosomes. Sci. Adv. 5, eaax8849 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Kwon, M., Leibowitz, M. L. & Lee, J.-H. Small but mighty: the causes and consequences of micronucleus rupture. Exp. Mol. Med. 52, 1777–1786 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Lusk, C. P. & King, M. C. Rotten to the core: why micronuclei rupture. Dev. Cell 47, 265–266 (2018).

    Article  CAS  PubMed  Google Scholar 

  99. Guo, X. et al. Understanding the birth of rupture-prone and irreparable micronuclei. Chromosoma 129, 181–200 (2020).

    Article  CAS  PubMed  Google Scholar 

  100. Raab, M. et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 352, 359–362 (2016).

    Article  CAS  PubMed  Google Scholar 

  101. Mackenzie, K. J. et al. cGAS surveillance of micronuclei links genome instability to innate immunity. Nature 548, 461–465 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Sato, Y. & Hayashi, M. T. Micronucleus is not a potent inducer of the cGAS/STING pathway. Life Sci. Alliance 7, e202302424 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Tsering, T., Nadeau, A., Wu, T., Dickinson, K. & Burnier, J. V. Extracellular vesicle-associated DNA: ten years since its discovery in human blood. Cell Death Dis. 15, 668 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Jacobson, E. C. et al. Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells. BMC Biol. 16, 142 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Heo, S.-J. et al. Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues. Sci. Adv. 6, eaax5083 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Mehl, J. L. et al. Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response. iScience 25, 105528 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Denholtz, M. et al. Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program. Genes Dev. 34, 149–165 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Herrera-Uribe, J. et al. Changes in H3K27ac at gene regulatory regions in porcine alveolar macrophages following LPS or PolyIC exposure. Front. Genet. 11, 817 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Stephens, A. D. et al. Chromatin histone modifications and rigidity affect nuclear morphology independent of lamins. Mol. Biol. Cell 29, 220–233 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Popova, E. Y., Claxton, D. F., Lukasova, E., Bird, P. I. & Grigoryev, S. A. Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood. Exp. Hematol. 34, 453–462 (2006).

    Article  CAS  PubMed  Google Scholar 

  111. Barth, T. K. & Imhof, A. Fast signals and slow marks: the dynamics of histone modifications. Trends Biochem. Sci. 35, 618–626 (2010).

    Article  CAS  PubMed  Google Scholar 

  112. Hsu, B. E. et al. Immature low-density neutrophils exhibit metabolic flexibility that facilitates breast cancer liver metastasis. Cell Rep. 27, 3902–3915.e6 (2019).

    Article  CAS  PubMed  Google Scholar 

  113. Sun, R. et al. Dysfunction of low-density neutrophils in peripheral circulation in patients with sepsis. Sci. Rep. 12, 685 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  114. van Grinsven, E. et al. Immature neutrophils released in acute inflammation exhibit efficient migration despite incomplete segmentation of the nucleus. J. Immunol. 202, 207–217 (2019).

    Article  PubMed  Google Scholar 

  115. Miotto, G. et al. Insight into the mechanism of ferroptosis inhibition by ferrostatin-1. Redox Biol. 28, 101328 (2020).

    Article  CAS  PubMed  Google Scholar 

  116. Sun, Q. et al. Cholesterol mediated ferroptosis suppression reveals essential roles of coenzyme Q and squalene. Commun. Biol. 6, 1108 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Poplimont, H. et al. Neutrophil swarming in damaged tissue is orchestrated by connexins and cooperative calcium alarm signals. Curr. Biol. 30, 2761–2776.e7 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Al-Shabanah, O. A., Mansour, M. A. & Elmazar, M. M. Enhanced generation of leukotriene B4 and superoxide radical from calcium ionophore (A23187) stimulated human neutrophils after priming with interferon-α. Res. Commun. Mol. Pathol. Pharmacol. 106, 115–128 (1999).

    CAS  PubMed  Google Scholar 

  119. Kenny, E. F. et al. Diverse stimuli engage different neutrophil extracellular trap pathways. eLife 6, e24437 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  120. Pedrera, L. et al. Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics. Cell Death Differ. 28, 1644–1657 (2021).

    Article  CAS  PubMed  Google Scholar 

  121. Galluzzi, L. et al. Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25, 486–541 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  122. Pedrera, L., Ros, U. & García-Sáez, A. J. Calcium as a master regulator of ferroptosis and other types of regulated necrosis. Cell Calcium 114, 102778 (2023).

    Article  CAS  PubMed  Google Scholar 

  123. Isles, H. M. et al. Pioneer neutrophils release chromatin within in vivo swarms. eLife 10, e68755 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. 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  PubMed  PubMed Central  Google Scholar 

  125. Cahilog, Z. et al. The role of neutrophil NETosis in organ injury: novel inflammatory cell death mechanisms. Inflammation 43, 2021–2032 (2020).

    Article  PubMed  Google Scholar 

  126. Mathis, S. P., Jala, V. R., Lee, D. M. & Haribabu, B. Nonredundant roles for leukotriene B4 Receptors BLT1 and BLT2 in inflammatory arthritis. J. Immunol. 185, 3049–3056 (2010).

    Article  CAS  PubMed  Google Scholar 

  127. Tatsumi, R. et al. Stepwise phosphorylation of BLT1 defines complex assemblies with β-arrestin serving distinct functions. FASEB J. 37, e23213 (2023).

    Article  CAS  PubMed  Google Scholar 

  128. Xu, Y., Denning, K. L. & Lu, Y. PPARα agonist WY-14,643 induces the PLA2/COX-2/ACOX1 pathway to enhance peroxisomal lipid metabolism and ameliorate alcoholic fatty liver in mice. Biochem. Biophys. Res. Commun. 613, 47–52 (2022).

    Article  CAS  PubMed  Google Scholar 

  129. Paloschi, M. V. et al. Cytosolic phospholipase A2-α participates in lipid body formation and PGE2 release in human neutrophils stimulated with an l-amino acid oxidase from Calloselasma rhodostoma venom. Sci. Rep. 10, 10976 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Norris, P. C., Gosselin, D., Reichart, D., Glass, C. K. & Dennis, E. A. Phospholipase A α regulates eicosanoid class switching during inflammasome activation. Proc. Natl Acad. Sci. USA 111, 12746–12751 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Babatunde, K. A., Ayuso, J. M., Kerr, S. C., Huttenlocher, A. & Beebe, D. J. Microfluidic systems to study neutrophil forward and reverse migration. Front. Immunol. 12, 3389 (2021).

    Article  Google Scholar 

  132. Loynes, C. A. et al. PGE 2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo. Sci. Adv. 4, eaar8320 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Levy, B. D., Clish, C. B., Schmidt, B., Gronert, K. & Serhan, C. N. Lipid mediator class switching during acute inflammation: signals in resolution. Nat. Immunol. 2, 612–619 (2001).

    Article  CAS  PubMed  Google Scholar 

  134. Baig, S. et al. Baseline elevations of leukotriene metabolites and altered plasmalogens are prognostic biomarkers of plaque progression in systemic lupus erythematosus. Front. Cardiovasc. Med. 9, 3389 (2022).

    Article  Google Scholar 

  135. Bonyek-Silva, I. et al. LTB4-driven inflammation and increased expression of ALOX5/ACE2 during severe COVID-19 in individuals with diabetes. Diabetes 70, 2120–2130 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. van der Meer, A. J. et al. Systemic inflammation induces release of cell-free DNA from hematopoietic and parenchymal cells in mice and humans. Blood Adv. 3, 724–728 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  137. Cheng, S., Davis, S. & Sharma, S. Maternal‐fetal cross talk through cell‐free fetal DNA, telomere shortening, microchimerism, and inflammation. Am. J. Reprod. Immunol. 79, e12851 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  138. Linney, M. D. et al. Microbial sources of exocellular DNA in the ocean. Appl. Environ. Microbiol. 88, e02093–21 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  139. Wen, F., Curlango‐Rivera, G., Huskey, D. A., Xiong, Z. & Hawes, M. C. Visualization of extracellular DNA released during border cell separation from the root cap. Am. J. Bot. 104, 970–978 (2017).

    Article  CAS  PubMed  Google Scholar 

  140. Zhang, X., Zhuchenko, O., Kuspa, A. & Soldati, T. Social amoebae trap and kill bacteria by casting DNA nets. Nat. Commun. 7, 10938 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Liu, X. et al. PAD4 takes charge during neutrophil activation: Impact of PAD4 mediated NET formation on immune‐mediated disease. J. Thromb. Haemost. 19, 1607–1617 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  142. Cantin, A. M. DNase I acutely increases cystic fibrosis sputum elastase activity and its potential to induce lung hemorrhage in mice. Am. J. Respir. Crit. Care Med 157, 464–469 (1998).

    Article  CAS  PubMed  Google Scholar 

  143. Metersky, M. & Chalmers, J. Bronchiectasis insanity: doing the same thing over and over again and expecting different results? F1000Res 8, 293 (2019).

    Article  Google Scholar 

  144. O’Donnell, A. E., Barker, A. F., Ilowite, J. S. & Fick, R. B. Treatment of idiopathic bronchiectasis with aerosolized recombinant human DNase I. Chest 113, 1329–1334 (1998).

    Article  PubMed  Google Scholar 

  145. Ngo, A. T. P. et al. Platelet factor 4 limits neutrophil extracellular trap- and cell-free DNA-induced thrombogenicity and endothelial injury. JCI Insight 8, e171054 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  146. Mai, S. H. C. et al. Delayed but not early treatment with DNase reduces organ damage and improves outcome in a murine model of sepsis. Shock 44, 166–172 (2015).

    Article  CAS  PubMed  Google Scholar 

  147. Kremserova, S. & Nauseef, W. M. in Methods in Molecular Biology, Vol. 2087 (eds Quinn, M. & DeLeo, F.) 33–42 (Humana, 2020).

  148. Lee, J.-Y. & Lee, H.-H. A new chemical complex can rapidly concentrate lentivirus and significantly enhance gene transduction. Cytotechnology 70, 193–201 (2018).

    Article  CAS  PubMed  Google Scholar 

  149. Shechter, D., Dormann, H. L., Allis, C. D. & Hake, S. B. Extraction, purification and analysis of histones. Nat. Protoc. 2, 1445–1457 (2007).

    Article  CAS  PubMed  Google Scholar 

  150. Graham, J. M. Fractionation of subcellular organelles. Curr. Protoc. Cell Biol. 69, cb0301s69 (2015).

    Article  Google Scholar 

  151. Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles 7, 1535750 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  152. Rider, M. A., Hurwitz, S. N. & Meckes, D. G. ExtraPEG: a polyethylene glycol-based method for enrichment of extracellular vesicles. Sci. Rep. 6, 23978 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Truckenbrodt, S., Sommer, C., Rizzoli, S. O. & Danzl, J. G. A practical guide to optimization in X10 expansion microscopy. Nat. Protoc. 14, 832–863 (2019).

    Article  CAS  PubMed  Google Scholar 

  154. Gambarotto, D. et al. Imaging cellular ultrastructures using expansion microscopy (U-ExM). Nat. Methods 16, 71–74 (2019).

    Article  CAS  PubMed  Google Scholar 

  155. Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  156. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

    Article  PubMed  Google Scholar 

  157. Mastronarde, D. N. & Held, S. R. Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113 (2017).

    Article  PubMed  Google Scholar 

  158. Chen, M. et al. Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nat. Methods 14, 983–985 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  159. McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Collie, S. et al. underagarose-analysis. Zenodo https://doi.org/10.5281/zenodo.15080547 (2025).

Download references

Acknowledgements

We thank the past and present members of the Parent and Coulombe laboratories for advice and support. We thank the Platelet Pharmacology and Physiology Core at the University of Michigan for providing human blood from healthy volunteers, the proteomics resource facility and V. Basrur for assistance with mass spectrometry data acquisition and analysis, and the microscopy core facility for assistance with TEM sample processing. We also thank P. Hanson (University of Michigan) for valuable suggestions and D. Wang and D. Sinha from the Coulombe and Parent laboratories for their help with animal experiments. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This work was supported by funding from the University of Michigan School of Medicine (C.A.P.), postdoctoral (916874) (S.B.A.) and predoctoral (AWD025905) (S.P.C.) fellowship awards from an American Heart Association, a Life Sciences Institute cubed award, the Arnold and Mabel Beckmann Foundation award to the University of Michigan Cryo-EM facility and by several grant awards from the National Institutes of Health, namely T32 training program in cell and molecular biology GM145470 (S.P.C.), T32 training program in translational research GM141840 (M.F.), GM150019 (S.M.), R01AI152517 (C.A.P.) and R01AR083222 (P.A.C. and C.A.P.).

Author information

Author notes

  1. These authors contributed equally: Samuel P. Collie, Yang Xu.

Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Subhash B. Arya, Samuel P. Collie, Yang Xu, Martin Fernandez, Shyamal Mosalaganti & Carole A. Parent

  2. Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, MI, USA

    Samuel P. Collie

  3. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

    Yang Xu & Carole A. Parent

  4. Department of Biophysics, University of Michigan, Ann Arbor, MI, USA

    Martin Fernandez & Shyamal Mosalaganti

  5. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA

    Jonathan Z. Sexton

  6. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI, USA

    Jonathan Z. Sexton

  7. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA

    Shyamal Mosalaganti, Pierre A. Coulombe & Carole A. Parent

  8. Department of Dermatology, University of Michigan Medical School, Ann Arbor, MI, USA

    Pierre A. Coulombe

  9. Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA

    Pierre A. Coulombe & Carole A. Parent

Authors
  1. Subhash B. Arya
    View author publications

    Search author on:PubMed Google Scholar

  2. Samuel P. Collie
    View author publications

    Search author on:PubMed Google Scholar

  3. Yang Xu
    View author publications

    Search author on:PubMed Google Scholar

  4. Martin Fernandez
    View author publications

    Search author on:PubMed Google Scholar

  5. Jonathan Z. Sexton
    View author publications

    Search author on:PubMed Google Scholar

  6. Shyamal Mosalaganti
    View author publications

    Search author on:PubMed Google Scholar

  7. Pierre A. Coulombe
    View author publications

    Search author on:PubMed Google Scholar

  8. Carole A. Parent
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Conceptualization: C.A.P., S.B.A., P.A.C. and S.M. Methodology: S.B.A., S.P.C., Y.X., M.F. and J.Z.S. Investigation: S.B.A., S.P.C., Y.X. and M.F. Visualization: S.B.A., S.P.C. and S.M. Supervision: C.A.P., P.A.C. and S.M. Writing–original draft: S.B.A. and C.A.P. Writing–editing: S.B.A., C.A.P., S.P.C., Y.X., P.A.C. and S.M.

Corresponding author

Correspondence to Carole A. Parent.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Chromatin-like bead-on-string structures are present within MVBs in activated PMNs.

a-b. Fourfold expansion microscopy images, representative of three independent experiments, showing fixed PMNs chemotaxing towards 100 nM LTB4, immunostained post-expansion for 5LO (yellow) and FLAP (magenta), and co-stained with Vybrant™ Dil (cyan). Orange dashed line outlines cell boundary, and red inset is zoomed and presented as individual channels (grayscale invert) in panel b. c. 3D-volume rendering of the cropped NE-MVB from panel a, sectioned through the centre. The scale is 5 µm, and 1 µm in the inset. Representative of 10 images from three independent experiments. d. Histogram depicting the intensity profile of the objects along the white dashed arrow in the merged image in panel b. e. Representative immuno-TEM images of LTB4-activated PMNs stained using anti-5LO antibody, showing the presence of 5LO (electron-dense dots, gold particles) within ~200 nm ILVs present inside the MVBs. The scale is 5 µm and 200 nm in the inset. N denotes nucleus. Representative of 7 images from two independent experiments. f. Representative TEM images of PMNs migrating towards LTB4 showing the presence of chromatin-like structures in cytoplasmic MVBs alongside ILVs. The scale is 5 µm, 200 nm in the inset. N denotes nucleus. Representative of 15 images from three independent experiments. Source numerical data is available in the source data file.

Source data

Extended Data Fig. 2 NE-derived exosomes are spatially associated with secreted nuclear DNA but not migrasomes.

a. An overlay image of PMNs on fibrinogen-fibronectin coated Quantifoil grids stimulated with 100 nM LTB4 for 15 min in the presence of SYTOXgreen, plunge-frozen, and imaged on Leica Stellaris-5 cryo-confocal, showing the reflected light (grey) and extracellular DNA (green). Dashed yellow lines highlight PMNs on the grid square. Representative of two biological replicates. The scale bar is 10 μm. b. A low-magnification TEM image (6500x) of the highlighted region (red box) in panel a, is overlaid with the SYTOXgreen fluorescence signal. The cyan box indicates the area where cryo-ET data was acquired. The scale bar is 500 nm. c. A slice through the tomogram of the region highlighted (cyan box) in panel b, with insets showing bead-on-string-like structures (yellow arrowheads). The scale bar is 100 nm, and in the inset, it is 10 nm. d. Airyscan microscopy image of Hoechst 33342 (grey) stained PMN showing the extracellular distribution (white inset, zoomed below) of TSPAN4 (magenta) and 5LO (cyan). Cell is outlined in yellow, and scale is 5 µm, in the inset it is 1 µm. Representative of two biological replicates.

Source data

Extended Data Fig. 3 Unlike PMA-induced suicidal NET release LTB4-induced DNA secretion is nonlytic and is PAD4- and NOX2-independent.

a-b. Time-lapse of PMNs stained with Mitotracker red CMXros (red, mitochondria), Hoechst (blue, nuclei), and SYTOXgreen (green, extracellular DNA) (a) migrating towards LTB4 or (b) treated with 100 nM PMA. Insets in panel b show cell outline (red), nuclei (blue), and NETs (green) generated by CellProfiler. Scale is 5 μm. Images are representative of three independent experiments. See associated Supplementary Movies 3 and 5. c. Before-after aligned dot plot showing Mitotracker Red intensity in migrating PMNs before, during, and after DNA secretion within 1 hr. Total 30 data points (red circles) pooled from three independent experiments are presented as mean ± s.e.m. (black lines), with multiplicity-adjusted P values from ordinary one-way ANOVA. d. Scatter dot plot showing percentage of DNA-secreting PMNs chemotaxis towards LTB4 with or without MitoTEMPO (10 μM). Data points (red dots) from three independent experiments are presented as mean ± s.e.m. (black lines), with P value from two-tailed ratio paired t-test. e. Airyscan microscopy image of Mitotracker Red CMXros (magenta) stained PMNs chemotaxing towards LTB4, fixed and immunostained for FLAP (yellow) and Hoechst 33342 (cyan). Orange dashed line outlines cell and inset is zoomed as individual channels (invert grayscale). The 3D-volume is shown below. Images are representative of three independent experiments and scale is 5 μm. f. Graph showing nuclei area extent over time in PMNs treated with PMA or migrating towards LTB4. Dots represent mean nuclear area extent per 40,000 μm2. Blue/red dots indicate percentage of PMNs without/with SYTOXgreen staining. Thick black line represents non-linear regression. Data is representative of three independent experiments. g. Graph showing the percentage of PMNs lysed over time. Data points from three independent experiments are plotted as mean (red/blue dots) ± s.e.m. (black lines). h. Graph showing the percentage of maximum lactate dehydrogenase (LDH) activity in supernatants of PMNs treated with either DMSO, PMA (20 nM), or LTB4 (100 nM) for 2 hrs. Data points from three independent experiments (similarly coloured circles) are presented as mean ± s.e.m. (black lines), with multiplicity-adjusted P values from RM one-way ANOVA. i-j. Scatter dot plot showing percentage of DNA-secreting PMNs migrating towards LTB4 in a 40,000 μm2 observation window for 1 hr either (i) with or without PAD4 and NOX2 inhibitors, GSK484 (2 µM) and GSK2795039 (10 µM), respectively, or (j) cell death pathway inhibitors namely, Z-DEVD-FMK (apoptosis, 10 μM), ferrostatin-1 (ferroptosis, 5 μM), and GSK872 (necroptosis, 10 μM). Data points from three (or more) independent experiments (similarly coloured circles) are presented as mean ± s.e.m., with multiplicity-adjusted P values from RM one-way ANOVA (i) and mixed-effect analysis (j). See associated Supplementary Movie 6 and 7. Source numerical data is available in the source data file.

Source data

Extended Data Fig. 4 No change in cellular and nuclear morphology during and after SEADing.

a-b. Scatter dot plots showing the change in the (a) nuclear form factor and (b) cell eccentricity during DNA secretion compared to PMNs without DNA secretion. Data points (red circles) pooled from five independent experiments are plotted as mean ± s.e.m. (black lines). Multiplicity-adjusted P values obtained using ordinary one-way ANOVA for (before vs. during vs. after comparison) DNA-secreting cells and the Mann-Whitney test (before DNA secretion vs. no DNA secretion comparison) are shown. Source numerical data is available in the source data file.

Source data

Extended Data Fig. 5 LMNA KO dHL60 cells have a nuclear morphology closer to PMNs relative to SCR dHL60 cells.

a. Western blot images representative of three independent experiments, showing the levels of lamin A/C, lamin B1, lamin B2, and LBR in SCR, LMNA KO, and LMNA/LBR KO dHL60 cell lysates. GAPDH is loading control. The molecular weights (kilodaltons, kDa) are indicated on left. b. Airyscan microscopy images of PMNs, SCR, LMNA KO, and LMNA/LBR KO dHL60 cells migrating towards fMLF, fixed and stained for LBR (magenta, nuclear envelope) and Hoechst (cyan, nucleus). Presented “sum of slices” projections are representative of three independent experiments. Dashed white/black outlines indicate cell shape. Scale is 5 μm. c-f. Scatter dot plots showing (c) NE to cytoplasm LBR intensity ratio, (d) nuclei form factor, (e) NE invaginations, and (f) heterochromatin spots in dHL60 neutrophils. Data points (red circles) pooled from three independent experiments are presented as mean ± s.e.m., with multiplicity-adjusted P values from ordinary one-way ANOVA. g. Scatter plot (left) and histogram (right) showing the gating strategy used for the analysis of flow cytometry data plotted in Fig. 4e-g. Graphs are representative of DMSO-treated SCR dHL60. Source numerical data and unprocessed western blots are available in the source data file.

Source data

Extended Data Fig. 6 LMNA KO dHL60 cells exhibit a proteome profile closer to PMNs and migrate better relative to SCR dHL60 cells.

a-b. Graphs showing the fold change in the gene ontology (GO, biological process) profile of the associated (a) downregulated and (b) upregulated proteins in LMNA KO dHL60 cells relative to SCR dHL60 cells, quantified using tandem mass tagging (TMT)-based mass spectroscopy analysis. c-f. Scatter dot plots showing (c) the number of cells migrating towards fMLF for 1 hr, (d) median speed, (e) median directionality, and (f) cell eccentricity. Data is plotted as mean ± s.e.m. of 9, 8, and 5 independent experiments (red circles) for SCR, LMNA KO, and LMNA/LBR KO samples, respectively. For cell eccentricity, a total of 29, 45, and 16 data points for SCR, LMNA KO, and LMNA/LBR KO, were pooled from three independent experiments. The multiplicity-adjusted P values calculated using mixed-effect analysis (c-e) and ordinary one-way ANOVA (f) are shown. Source numerical data is available in the source data file.

Source data

Extended Data Fig. 7 rDNase I, but not RNase treatment impedes neutrophil chemotaxis in an LTB4-dependent manner.

a-b. Cell tracks of PMNs pretreated with either DMSO (vehicle) or MK886 (1 μM), migrating towards LTB4 (x-axis) in the presence or absence of rDNase I (10 U/mL) for 1 h. Graphs display the migration of 100 randomly selected tracks from each condition. The colour-coded time scale is shown on the right. Presented tracks are representative of five independent experiments. See associated Supplementary Movie 10. b-d. Bar graphs showing the (b) number of PMNs migrated towards LTB4 within 1 hr, (c) average directionality, and (d) average speed in the presence or absence of rDNase I. Data points (black circles) from five independent experiments are presented as mean ± s.e.m., with multiplicity-adjusted P values from RM one-way ANOVA. e. Bar graph showing the average directionality and speed of PMNs chemotaxing towards LTB4 in the presence or absence of RNase (10 u/mL). Data points (black dots) from five independent experiments are presented as mean ± s.e.m., with P values calculated using two-tailed student’s t-test. Source numerical data are available in the source data file.

Source data

Extended Data Fig. 8 Temporal kinetics of secreted DNA during ear inflammation.

Airyscan microscopy images of ear cryosections (20 µm thick) treated with acetone/TPA for the indicated duration in mice injected with either rDNase I or PBS showing the temporal distribution of citrullinated histone H3 (magenta) and DAPI (grey, nuclei). Presented ‘sum of slices’ projection is representative of three independent experiments. Scale is 100 µm, and 20 µm in the inset.

Source data

Extended Data Fig. 9 PLA of exosomes and secreted DNA in TPA-treated ears.

a. Schematic illustrating the antibody binding sites on FLAP and the principle of the PLA used to assess the presence of SEADs in inflamed ears. b. Airyscan microscopy images of ear cryosections (20 μm) from mice treated with TPA for 12 hrs, showing the status of PLA dots (magenta) in either FLAP- or dsDNA-only antibody conditions, used to test the nonspecific PLA signal. Sections were co-stained with DAPI (nucleus). Presented ‘sum of slices’ is representative of three independent experiments, and Scale is 100 μm. c. Airyscan microscopy images of ear cryosections (20 μm) from a mouse injected with either PBS or rDNase I and treated with TPA for 12 h, showing the status of CD63-citH3 PLA dots (magenta) in indicated conditions. Sections were co-stained with DAPI (nucleus). Zoomed insets are presented as inverted grayscale above respective conditions. Presented ‘sum of slices’ is representative of two independent experiments, and scale is 100 μm, in the inset it is 20 µm.

Source data

Extended Data Fig. 10 rDNase I-induced disruption of the SEAD-PPARα axis impedes monocyte infiltration during the resolving phase of TPA-induced ear inflammation.

a. Scatter dot plots showing the gating strategy used for quantifying Ly6G+ ICAM1high CXCR1low reverse-transmigrated neutrophils as shown in Fig. 6i. b-c. Airyscan microscopy images of ear cryosections (20 µm thick) treated with (b) either acetone/TPA for the indicated duration in mice injected with either rDNase I or PBS or (c) added topical treatment of PPARα agonist/antagonist, showing the temporal distribution of F4/80 (magenta, macrophages) and DAPI (grey, nuclei). Presented ‘sum of slices’ projection is representative of three independent experiments. Scale is 100 μm.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

List of genes with significantly altered expression in LMNA KO dHL60 relative to SCR dHL60.

Supplementary Table 2

Key resources.

Supplementary Video 1

Tomogram and segmentation of data shown in Fig. 2c,d.

Supplementary Video 2

Nonlytic, repetitive and rapid secretion of DNA from chemotaxing PMNs. PMNs stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen imaged using Airyscan microscopy. Images acquired at 30 s intervals are presented as three frames per second. Scale bar, 5 µm.

Supplementary Video 3

Phenotypes of DNA secreted from chemotaxing PMNs. PMNs stained with MitoTracker Red CMXros (mitochondria) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen imaged using confocal microscopy. Images acquired at 30 s intervals are presented as three frames per second. Scale bar, 5 µm. Cyan arrow marks DNA trails, white triangle marks ‘DNA blobs’, and hollow white triangles mark ‘attached-DNA blobs’. Right panel shows the outline of the neutrophils (red, attached to DNA; white, no DNA within 1 µm of cell membrane) as generated by object segmentation using CellProfiler. Cyan and magenta outlines denote the attached and released DNA, respectively.

Supplementary Video 4

The secretion of DNA from chemotaxing PMNs is dependent on SMase activity. PMNs stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen and either DMSO (vehicle control) or GW4869 (nSMase inhibitor) imaged using confocal microscopy. Images acquired at 30 s intervals are presented as three frames per second. Scale bar, 5 µm. Cyan arrow marks DNA trails, white triangle marks ‘DNA blobs’, and hollow white triangles mark ‘attached-DNA blobs’.

Supplementary Video 5

PMA-induced suicidal NET release in PMNs. PMNs stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) stimulated with PMA (100 nM) in the presence of SYTOXgreen and imaged using Airyscan microscopy. Images acquired at 18 s intervals are presented as three frames per second. Scale bar, 5 µm.

Supplementary Video 6

The secretion of DNA from chemotaxing PMNs is independent of PAD4 and NOX2 activity. PMNs stained with CellTrackerCMPTX (cell) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen, and either DMSO, PAD4 inhibitor and NOX2 inhibitor imaged using confocal microscopy. Images acquired at 45 s intervals are presented as one frame per second. White triangle marks ‘DNA blobs’ and hollow white triangles mark ‘attached-DNA blobs’.

Supplementary Video 7

Effect of various cell death pathway inhibitors on the secretion of DNA from chemotaxing PMNs. PMNs stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen, and either DMSO, apoptosis, ferroptosis or necroptosis inhibitors imaged using confocal microscopy. Images acquired at 30 s intervals are presented as three frames per second. Cyan arrow marks DNA trails, white triangle marks ‘DNA blobs’, and hollow white triangles mark ‘attached-DNA blobs’.

Supplementary Video 8

LBR loss inhibits DNA secretion in chemotaxing dHL60 cells. SCR, LMNA KO and LMNA/LBR KO dHL60 cells stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen imaged using confocal microscopy. Images acquired at 30 s intervals are presented as three frames per second. Hollow white triangles mark ‘attached-DNA blobs’.

Supplementary Video 9

Histone acetylation mediates the secretion of DNA from chemotaxing PMNs. PMNs stained with CellMask Orange (PM) and Hoechst 33342 (nuclei) migrating towards LTB4 in the presence of SYTOXgreen, and either DMSO, HAT or HDAC inhibitors imaged using confocal microscopy. Images acquired at 30 s intervals are presented as three frames per second. White triangle marks ‘DNA blobs’, and hollow white triangles mark ‘attached-DNA blobs’.

Supplementary Video 10

Effects of MK886 and rDNase I treatment on PMN chemotaxis. PMNs stained with Hoechst 33342 migrating towards LTB4 in the presence of either DMSO, rDNase I, MK886, or MK886 + rDNase I, were imaged using fluorescence microscopy and analysed using Trackmate on ImageJ. Images acquired every 45 s are presented at a rate of five frames per second. Circles indicate the individual cells as identified by Trackmate, and lines are colour-coded for the duration of migration from blue (earlier) to red (later). Scale bar, 200 µm.

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Arya, S.B., Collie, S.P., Xu, Y. et al. Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation. Nat Cell Biol 27, 931–947 (2025). https://doi.org/10.1038/s41556-025-01671-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41556-025-01671-4

This article is cited by

Search

Advanced 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