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:

Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming

Nature Biomedical Engineering volume 1, Article number: 0094 (2017) Cite this article

Subjects

Abstract

Neutrophil swarms protect healthy tissues by sealing off sites of infection. In the absence of swarming, microbial invasion of surrounding tissues can result in severe infections. Recent observations in animal models have shown that swarming requires rapid neutrophil responses and well-choreographed neutrophil migration patterns. However, in animal models, physical access to the molecular signals coordinating neutrophil activities during swarming is limited. Here, we report the development and validation of large microscale arrays of zymosan particle clusters for the study of human neutrophils during swarming ex vivo. We characterized the synchronized swarming of human neutrophils under the guidance of neutrophil-released chemokines, and measured the mediators released at different phases of human-neutrophil swarming against targets simulating infections. We found that the network of mediators coordinating human-neutrophil swarming includes start and stop signals, proteolytic enzymes and enzyme inhibitors, as well as modulators of activation of other immune and non-immune cells. We also show that the swarming behaviour of neutrophils from patients following major trauma is deficient and gives rise to smaller swarms than those of neutrophils from healthy individuals.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Human-neutrophil swarming on large-scale zymosan particle arrays.
Figure 2: The migration of neutrophils towards swarms is altered in the presence of BLT1 and BLT2 antagonists.
Figure 3: Evaluation of soluble factors guiding neutrophil migration during swarming.
Figure 4: Lipid mediators released by human neutrophils during swarming.
Figure 5: Cytokines released by human neutrophils during swarming.
Figure 6: Analysis of clinical samples during neutrophil swarming.

Similar content being viewed by others

References

  1. Ng, L. G. et al. Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events. J. Invest. Dermatol. 131, 2058–2068 (2011).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Kienle, K. & Lammermann, T. Neutrophil swarming: an essential process of the neutrophil tissue response. Immunol. Rev. 273, 76–93 (2016).

    Article  CAS  Google Scholar 

  5. Jones, C. N. et al. Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets. J. Leukoc. Biol. 100, 241–247 (2016).

    Article  Google Scholar 

  6. Malawista, S. E., de Boisfleury Chevance, A., van Damme, J. & Serhan, C. N. Tonic inhibition of chemotaxis in human plasma. Proc. Natl Acad. Sci. USA 105, 17949–17954 (2008).

    Article  CAS  Google Scholar 

  7. Lammermann, T. In the eye of the neutrophil swarm-navigation signals that bring neutrophils together in inflamed and infected tissues. J. Leukoc. Biol. 100, 55–63 (2016).

    Article  Google Scholar 

  8. Liese, J., Rooijakkers, S. H., van Strijp, J. A., Novick, R. P. & Dustin, M. L. Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation. Cell Microbiol. 15, 891–909 (2013).

    Article  CAS  Google Scholar 

  9. Kamenyeva, O. et al. Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus. PLoS Pathog. 11, e1004827 (2015).

    Article  Google Scholar 

  10. Kreisel, D. et al. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation. Proc. Natl Acad. Sci. USA 107, 18073–18078 (2010).

    Article  CAS  Google Scholar 

  11. Chtanova, T. et al. Dynamics of neutrophil migration in lymph nodes during infection. Immunity 29, 487–496 (2008).

    Article  CAS  Google Scholar 

  12. Bruns, S. et al. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA. PLoS Pathog. 6, e1000873 (2010).

    Article  Google Scholar 

  13. Jones, C. N. et al. Human neutrophils are primed by chemoattractant gradients for blocking the growth of Aspergillus fumigatus. J. Infect. Dis. 213, 465–475 (2016).

    Article  CAS  Google Scholar 

  14. Leliefeld, P. H., Wessels, C. M., Leenen, L. P., Koenderman, L. & Pillay, J. The role of neutrophils in immune dysfunction during severe inflammation. Crit. Care 20, 73 (2016).

    Article  Google Scholar 

  15. Malawista, S. E., de Boisfleury, A. C. & Naccache, P. H. Inflammatory gout: observations over a half-century. FASEB J. 25, 4073–4078 (2011).

    Article  CAS  Google Scholar 

  16. Boneschansker, L., Yan, J., Wong, E., Briscoe, D. M. & Irimia, D. Microfluidic platform for the quantitative analysis of leukocyte migration signatures. Nat. Commun. 5, 4787 (2014).

    Article  CAS  Google Scholar 

  17. Serhan, C. N. Novel lipid mediators and resolution mechanisms in acute inflammation: to resolve or not? Am. J. Pathol. 177, 1576–1591 (2010).

    Article  CAS  Google Scholar 

  18. 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  Google Scholar 

  19. Serhan, C. N. et al. Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes. J. Immunol. 176, 1848–1859 (2006).

    Article  CAS  Google Scholar 

  20. Hammerschmidt, D. E., Bowers, T. K., Lammi-Keefe, C. J., Jacob, H. S. & Craddock, P. R. Granulocyte aggregometry: a sensitive technique for the detection of C5a and complement activation. Blood 55, 898–902 (1980).

    CAS  PubMed  Google Scholar 

  21. Wagner, J. L. & Hugli, T. E. Radioimmunoassay for anaphylatoxins: a sensitive method for determining complement activation products in biological fluids. Anal. Biochem. 136, 75–88 (1984).

    Article  CAS  Google Scholar 

  22. Harvie, E. A., Green, J. M., Neely, M. N. & Huttenlocher, A. Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect. Immun. 81, 110–121 (2013).

    Article  CAS  Google Scholar 

  23. Bazzoni, F. et al. Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8. J. Exp. Med. 173, 771–774 (1991).

    Article  CAS  Google Scholar 

  24. Guerrero, A. T. et al. Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE2 . J. Leukoc. Biol. 83, 122–130 (2008).

    Article  CAS  Google Scholar 

  25. Bhaumik, P., St-Pierre, G., Milot, V., St-Pierre, C. & Sato, S. Galectin-3 facilitates neutrophil recruitment as an innate immune response to a parasitic protozoa cutaneous infection. J. Immunol. 190, 630–640 (2013).

    Article  CAS  Google Scholar 

  26. Lopez-Dee, Z., Pidcock, K. & Gutierrez, L. S. Thrombospondin-1: multiple paths to inflammation. Mediators Inflamm. 2011, 296069 (2011).

    Article  Google Scholar 

  27. Schenk, B. I., Petersen, F., Flad, H. D. & Brandt, E. Platelet-derived chemokines CXC chemokine ligand (CXCL)7, connective tissue-activating peptide III, and CXCL4 differentially affect and cross-regulate neutrophil adhesion and transendothelial migration. J. Immunol. 169, 2602–2610 (2002).

    Article  CAS  Google Scholar 

  28. Senior, R. M., Gresham, H. D., Griffin, G. L., Brown, E. J. & Chung, A. E. Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. J. Clin. Invest. 90, 2251–2257 (1992).

    Article  CAS  Google Scholar 

  29. Baltus, T. et al. Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions. J. Leukoc. Biol. 78, 435–441 (2005).

    Article  CAS  Google Scholar 

  30. Dziarski, R., Platt, K. A., Gelius, E., Steiner, H. & Gupta, D. Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)-deficient mice. Blood 102, 689–697 (2003).

    Article  CAS  Google Scholar 

  31. Bornfeldt, K. E. et al. Platelet-derived growth factor. Distinct signal transduction pathways associated with migration versus proliferation. Ann. NY Acad. Sci. 766, 416–430 (1995).

    Article  CAS  Google Scholar 

  32. Markowska, A. I., Liu, F. T. & Panjwani, N. Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response. J. Exp. Med. 207, 1981–1993 (2010).

    Article  CAS  Google Scholar 

  33. Yang, J. et al. An iron delivery pathway mediated by a lipocalin. Mol. Cell 10, 1045–1056 (2002).

    Article  CAS  Google Scholar 

  34. Garlanda, C. et al. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature 420, 182–186 (2002).

    Article  CAS  Google Scholar 

  35. Iyer, R. P., Patterson, N. L., Fields, G. B. & Lindsey, M. L. The history of matrix metalloproteinases: milestones, myths, and misperceptions. Am. J. Physiol. Heart Circ. Physiol. 303, H919– H930 (2012).

    Article  CAS  Google Scholar 

  36. Arita, M. et al. Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions. J. Biol. Chem. 281, 22847–22854 (2006).

    Article  CAS  Google Scholar 

  37. Serhan, C. N. Pro-resolving lipid mediators are leads for resolution physiology. Nature 510, 92–101 (2014).

    Article  CAS  Google Scholar 

  38. Murata, T. et al. Anti-inflammatory role of PGD2 in acute lung inflammation and therapeutic application of its signal enhancement. Proc. Natl Acad. Sci. USA 110, 5205–5210 (2013).

    Article  CAS  Google Scholar 

  39. Crea, A. E., Nakhosteen, J. A. & Lee, T. H. Mediator concentrations in bronchoalveolar lavage fluid of patients with mild asymptomatic bronchial asthma. Eur. Respir. J. 5, 190–195 (1992).

    CAS  PubMed  Google Scholar 

  40. Montuschi, P. & Barnes, P. J. Exhaled leukotrienes and prostaglandins in asthma. J. Allergy Clin. Immunol. 109, 615–620 (2002).

    Article  CAS  Google Scholar 

  41. Dalli, J. et al. Human sepsis eicosanoid and proresolving lipid mediator temporal profiles: correlations with survival and clinical outcomes. Crit. Care Med. 45, 58–68 (2017).

    Article  CAS  Google Scholar 

  42. Jones, C. N. et al. Spontaneous neutrophil migration patterns during sepsis after major burns. PLoS ONE 9, e114509 (2014).

    Article  Google Scholar 

  43. Vincent, J. L. Nosocomial infections in adult intensive-care units. Lancet 361, 2068–2077 (2003).

    Article  Google Scholar 

  44. Colas, R. A., Shinohara, M., Dalli, J., Chiang, N. & Serhan, C. N. Identification and signature profiles for pro-resolving and inflammatory lipid mediators in human tissue. Am. J. Physiol. Cell Physiol. 307, C39–C54 (2014).

    Article  CAS  Google Scholar 

  45. Reátegui, E. et al. Dataset for ‘Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming’. figsharehttp://dx.doi.org/10.6084/m9.figshare.5024567 (2017).

Download references

Acknowledgements

We thank B. Hamza and J. M. Martel of the BioMEMS Resource Center for help with microfabrication and stimulating discussions. This work was supported by a grant from the National Institute of General Medical Sciences (GM092804) and funding from the Harvard Medical School Tools and Technology Fund. Microfabrication was conducted at the BioMEMS Resource Center at Massachusetts General Hospital, supported by a grant from the National Institute of Biomedical Imaging and Bioengineering (EB002503). Work in the CNS laboratories was supported by the National Institutes of Health (GM095467 and GM38765).

Author information

Authors and Affiliations

  1. Department of Surgery, BioMEMS Resource Center, Massachusetts General Hospital, Boston, 02129, Massachusetts, USA

    Eduardo Reátegui, Fatemeh Jalali, Aimal H. Khankhel, Elisabeth Wong, Hansang Cho & Daniel Irimia

  2. Massachusetts General Hospital Cancer Center, Boston, 02129, Massachusetts, USA

    Eduardo Reátegui & Aimal H. Khankhel

  3. Harvard Medical School, Boston, 02115, Massachusetts, USA

    Eduardo Reátegui, Hansang Cho, Jarone Lee, Charles N. Serhan, Jesmond Dalli, Hunter Elliott & Daniel Irimia

  4. Department of Surgery, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Jarone Lee

  5. Department of Anesthesiology, Center for Experimental Therapeutics, Perioperative, and Pain Medicine, Brigham and Women’s Hospital, Boston, 02115, Massachusetts, USA

    Charles N. Serhan & Jesmond Dalli

  6. Image and Data Analysis Core (IDAC), Boston, 02115, Massachusetts, USA

    Hunter Elliott

  7. Shriners Burns Hospital, Boston, 02114, Massachusetts, USA

    Daniel Irimia

Authors
  1. Eduardo Reátegui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fatemeh Jalali
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aimal H. Khankhel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elisabeth Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hansang Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jarone Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Charles N. Serhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jesmond Dalli
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hunter Elliott
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel Irimia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.R. and D.I. designed the research; E.R., A.H.K., F.J., H.C. and E.W. designed and prepared the microfluidic devices; E.R. conducted the experiments; E.R., F.J., H.E., J.L. and D.I. analysed the results; C.N.S. and J.D. performed the metabololipidomic analysis; H.E. developed the biophysical model for cell swarming; and E.R. and D.I. prepared the manuscript, with significant contributions from all authors.

Corresponding author

Correspondence to Daniel Irimia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figures, tables and video captions. (PDF 2255 kb)

Supplementary Video 1

Human-neutrophil swarming on arrays of clusters of zymosan particles. (MOV 1384 kb)

Supplementary Video 2

Human-neutrophil swarming on a single cluster of zymosan particles. (MOV 2031 kb)

Supplementary Video 3

Human neutrophils do not swarm on solitary zymosan particles. (MOV 1491 kb)

Supplementary Video 4

Human-neutrophil tracking during swarming. (MOV 2644 kb)

Supplementary Video 5

Human-neutrophil tracking during swarming in the presence of LTB4 receptor. (MOV 5597 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Reátegui, E., Jalali, F., Khankhel, A. et al. Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming. Nat Biomed Eng 1, 0094 (2017). https://doi.org/10.1038/s41551-017-0094

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41551-017-0094

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