Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Abstract

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

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Figure 1: Rapid in vivo NETosis during acute Gram-positive bacterial infections is directly visualized in vivo.
Figure 2: PMNs are viable and functional during nuclear breakdown and chromatin decondensation.
Figure 3: NET-forming PMNs show a unique crawling phenotype in vivo related to nuclear structure.
Figure 4: NETs are essential for limiting acute S. aureus dissemination.
Figure 5: NETosis occurs in human abscesses due to Gram-positive bacterial infections.
Figure 6: Immunofluorescence imaging of NETosis during human abscess formation.

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Acknowledgements

The pBT2 was a gift from R. Brückner (University of Kaiserslautern). Myd88−/− and Tlr2−/− mice were provided by S. Akira (Osaka University). T. Graf (Barcelona, Spain) provided the Tg(LysMeGFP). We thank D. Knight and C. Baddick for technical assistance and ongoing support. We acknowledge P. Colarusso and the support staff of the Snyder Institute Live Cell Imaging Facility for assisting in the image capturing and analysis (Canada Foundation for Innovation funded). We also thank the University of Calgary Flow Cytometry Facility and L. Kennedy for their assistance. We are grateful to P. Forsyth for use of the IVIS 200. We thank the physicians, nurses and support staff in the Division of Infectious Diseases in the Department of Medicine, Alberta Health Services–Calgary and Area for assistance in obtaining clinical samples. We thank E. Yung, C. Horn, K. Nelson and K. Headley from Calgary Lab Services for assistance with the electron microscopy experiments. The Canadian Institute of Health Research (CIHR) provided the operating grants to support this work. P.K. is an Alberta Innovates–Health Solutions (AIHS) Scientist, Canada Research Chair and the Snyder Chair in Critical Care Medicine. B. Petri received an AIHS postdoctoral fellowship. B. Yipp is a Clinical Scholar (Department of Critical Care Medicine, Calgary), an AIHS clinical fellow and a CIHR fellow. During a portion of this study he received salary support from the Rockefeller University by Grant Award Number UL1RR024143 from the National Center for Research Resources (NCRR), a component of the US National Institutes of Health (NIH) and NIH Roadmap for Medical Research. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the NIH.

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Contributions

B.G.Y. and B.P. designed the overall study, performed experiments, analyzed the data and wrote the manuscript, D.S. designed, performed and analyzed the transmission electron microscopy experiments, C.N.J. designed, performed and analyzed the immunofluorescence microscopy experiments, B.N.V.S. performed intravital imaging, L.D.Z. performed intravital imaging, Xenogen and dissemination experiments and analyzed the data, K.P. performed human neutrophil experiments, M.A. performed in vitro NET assays, K.W. developed the transgenic bacteria, H.C.M. helped develop the imaging analysis protocols, S.E.M. and A.d.B.C. designed and performed the human cytoplast experiments, K.Z. designed and supervised the development of the transgenic bacteria and provided expert advice on microbiology, J.C. designed the clinical experiments, supervised the acquisition of patient samples and clinical histories and provided expert advice on infectious diseases and microbiology, and P.K. provided overall supervision, analyzed the data and wrote the manuscript.

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Correspondence to Paul Kubes.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 2439 kb)

Supplementary Video 1

Live emigrated neutrophils rapidly form NETs in vivo within Gram-positive infected skin. Spinning-disk confocal intravital microscopy was performed using SYTOX Orange to visualize extracellular DNA. Neutrophils (green) chemotax through tissue NETs (red) in response to S. aureus (Xen29). PMNs do not take up the vital dye SYTOX. Neutrophils are hyperpolarized and form multiple pseudopods. White arrows demonstrate aberrant PMN crawling throughout NETs with hyperpolarization and multiple pseudopods. (MOV 1570 kb)

Supplementary Video 2

Live emigrated neutrophils rapidly release extracellular histones as a component of NETs during a Gram-positive infection. PE-conjugated histone specific antibody demonstrates extracellular histone proteins (red) as a major component of NET structure. Live PMN chemotax throughout the tissue in response to S. aureus (Xen8.1). (MOV 2306 kb)

Supplementary Video 3

Live emigrated PMN undergo NETosis while crawling during a Gram-positive infection. In vivo PMN nuclei were prelabeled with the cell-permeable dye SYTO 60. The NETosing PMN continues to crawl toward the live GFP-staphylococcus. Released NETs surround the PMN. At the conclusion of the video, we changed to Z-stack imaging, and this PMN is highlighted in Figure 2a. During the 3D imaging this PMN eventually engulfs the GFP-bacteria seen in the video. (MOV 784 kb)

Supplementary Video 4

Live PMNs form NETs while crawling in vivo. Four-color spinning-disk confocal intravital microscopy was used to visualize NET release from live PMNs in response to Gram-positive skin infection. A PMN (yellow) that is crawling while releasing NETs is circled. Extracellular DNA NETs are visualized using the cell-impermeable DNA dye SYTOX Orange. GFP-staphylococcus can be seen within this crawling PMN. A PMN with retained intracellular nuclear dye (SYTO 60, blue) can be seen crawling to the right of the highlighted NETTing PMN. This cell is not releasing NETs. (MOV 4726 kb)

Supplementary Video 5

In vivo intracellular imaging demonstrates PMNs with normal, diffuse and absent nuclei during a Gram-positive skin infection. Normal nuclei are easily distinguished by their distinct multilobar morphology. Anuclear PMNs are alive and continue to migrate. Examples are demonstrated of anuclear PMNs engorged with GFP-staphylococcus as well as anuclear PMNs devoid of bacteria. A PMN with an abnormal diffuse nucleus is highlighted for its multiple and erratic pseudopod formation and hyperpolarization. NETs are visualized within the area of anuclear PMNs. (MOV 8034 kb)

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Yipp, B., Petri, B., Salina, D. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat Med 18, 1386–1393 (2012). https://doi.org/10.1038/nm.2847

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