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

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.

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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.

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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).

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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.

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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)

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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

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