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

Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, Malawista SE, de Boisfleury Chevance A, Zhang K, Conly J, Kubes P - Nat. Med. (2012)

Bottom Line: During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination.NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA.A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release.

View Article: PubMed Central - PubMed

Affiliation: Department of Critical Care Medicine, University of Calgary, Calgary, Alberta, Canada. 2The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

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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NETosis occurs in human abscesses due to Gram-positive bacterial infections. Five human patients presenting with Gram-positive abscesses were evaluated. Transmission electron microscopy was performed on freshly obtained clinical samples. (a) The abscesses contained intact neutrophils (PMN), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMNv), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b) A typical anuclear neutrophil is demonstrated undergoing NET formation (arrowhead) and a second cell is shown, enlarged in (c), following nuclear envelope breakdown with dispersed chromatin and nuclear vesicles. The remnants of the nuclear envelope are highlighted (arrowhead). NETs are identified by the prototypical ‘beads on a string’ appearance on EM. (d) An anuclear neutrophil with nuclear envelope breakdown decondensed and dispersed chromatin (arrowhead), with vesicles and granules fusing with the outer plasma membrane (arrowhead). (e) Late stage neutrophils that have released chromatin and granules into the extracellular space.
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Figure 5: NETosis occurs in human abscesses due to Gram-positive bacterial infections. Five human patients presenting with Gram-positive abscesses were evaluated. Transmission electron microscopy was performed on freshly obtained clinical samples. (a) The abscesses contained intact neutrophils (PMN), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMNv), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b) A typical anuclear neutrophil is demonstrated undergoing NET formation (arrowhead) and a second cell is shown, enlarged in (c), following nuclear envelope breakdown with dispersed chromatin and nuclear vesicles. The remnants of the nuclear envelope are highlighted (arrowhead). NETs are identified by the prototypical ‘beads on a string’ appearance on EM. (d) An anuclear neutrophil with nuclear envelope breakdown decondensed and dispersed chromatin (arrowhead), with vesicles and granules fusing with the outer plasma membrane (arrowhead). (e) Late stage neutrophils that have released chromatin and granules into the extracellular space.

Mentions: We investigated PMN from human subjects diagnosed with Gram-positive abscesses (Supplementary Table 1). Fresh abscess aspirates were imaged using transmission electron microscopy. In all samples, we visualized normal PMN, PMN with degraded nuclear envelopes, decondensed and dispersed chromatin (diffuse nuclei) and PMN without any intracellular DNA or chromatin (anuclear)(Fig. 5a–e). Normal PMN had abundant heterochromatin and multilobar nuclei, while NETosing cells were at various stages of nuclear envelope breakdown and NET release. Granulated cells with diffuse nuclear staining contained nuclear envelope remnants and diffuse chromatin throughout the cytoplasm however structurally intact nuclei were not present (Fig. 5b). PMN without a nucleus but still containing limited dispersed DNA and chromatin were a common finding on EM supporting our in vivo observation that PMN with diffuse nuclear staining have dissolved the nucleus and contain some free chromatin resulting in a diffuse DNA pattern of fluorescence (Fig. 5c). Of note, PMN with diffuse or completely absent nuclear material still contain granules, suggesting that they retain the necessary components to kill bacteria through conventional mechanisms.


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

Yipp BG, Petri B, Salina D, Jenne CN, Scott BN, Zbytnuik LD, Pittman K, Asaduzzaman M, Wu K, Meijndert HC, Malawista SE, de Boisfleury Chevance A, Zhang K, Conly J, Kubes P - Nat. Med. (2012)

NETosis occurs in human abscesses due to Gram-positive bacterial infections. Five human patients presenting with Gram-positive abscesses were evaluated. Transmission electron microscopy was performed on freshly obtained clinical samples. (a) The abscesses contained intact neutrophils (PMN), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMNv), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b) A typical anuclear neutrophil is demonstrated undergoing NET formation (arrowhead) and a second cell is shown, enlarged in (c), following nuclear envelope breakdown with dispersed chromatin and nuclear vesicles. The remnants of the nuclear envelope are highlighted (arrowhead). NETs are identified by the prototypical ‘beads on a string’ appearance on EM. (d) An anuclear neutrophil with nuclear envelope breakdown decondensed and dispersed chromatin (arrowhead), with vesicles and granules fusing with the outer plasma membrane (arrowhead). (e) Late stage neutrophils that have released chromatin and granules into the extracellular space.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4529131&req=5

Figure 5: NETosis occurs in human abscesses due to Gram-positive bacterial infections. Five human patients presenting with Gram-positive abscesses were evaluated. Transmission electron microscopy was performed on freshly obtained clinical samples. (a) The abscesses contained intact neutrophils (PMN), red blood cells (RBCs), activated neutrophils with vesicles in the cytoplasm (PMNv), as well as numerous anuclear neutrophils with cytoplasmic granules and nuclear vesicles (arrowheads). (b) A typical anuclear neutrophil is demonstrated undergoing NET formation (arrowhead) and a second cell is shown, enlarged in (c), following nuclear envelope breakdown with dispersed chromatin and nuclear vesicles. The remnants of the nuclear envelope are highlighted (arrowhead). NETs are identified by the prototypical ‘beads on a string’ appearance on EM. (d) An anuclear neutrophil with nuclear envelope breakdown decondensed and dispersed chromatin (arrowhead), with vesicles and granules fusing with the outer plasma membrane (arrowhead). (e) Late stage neutrophils that have released chromatin and granules into the extracellular space.
Mentions: We investigated PMN from human subjects diagnosed with Gram-positive abscesses (Supplementary Table 1). Fresh abscess aspirates were imaged using transmission electron microscopy. In all samples, we visualized normal PMN, PMN with degraded nuclear envelopes, decondensed and dispersed chromatin (diffuse nuclei) and PMN without any intracellular DNA or chromatin (anuclear)(Fig. 5a–e). Normal PMN had abundant heterochromatin and multilobar nuclei, while NETosing cells were at various stages of nuclear envelope breakdown and NET release. Granulated cells with diffuse nuclear staining contained nuclear envelope remnants and diffuse chromatin throughout the cytoplasm however structurally intact nuclei were not present (Fig. 5b). PMN without a nucleus but still containing limited dispersed DNA and chromatin were a common finding on EM supporting our in vivo observation that PMN with diffuse nuclear staining have dissolved the nucleus and contain some free chromatin resulting in a diffuse DNA pattern of fluorescence (Fig. 5c). Of note, PMN with diffuse or completely absent nuclear material still contain granules, suggesting that they retain the necessary components to kill bacteria through conventional mechanisms.

Bottom Line: During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination.NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA.A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release.

View Article: PubMed Central - PubMed

Affiliation: Department of Critical Care Medicine, University of Calgary, Calgary, Alberta, Canada. 2The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

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.

Show MeSH
Related in: MedlinePlus